AU2005322640B2 - Compositions for use in identification of bacteria - Google Patents

Description

WO 2006/071241 PCT/US2005/006133 -1- COMPOSITIONS FOR USE IN IDENTIFICATION OF BACTERIA CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of priority to: U.S. Provisional Application Serial No. 60/545,425 filed February 18, 2004, U.S. Provisional Application Serial No.

60/559,754, filed April 5, 2004, U.S. Provisional Application Serial No. 60/632,862, filed December 3, 2004, U.S. Provisional Application Serial No. 60/639,068, filed December 22, 2004, and U.S. Provisional Application Serial No. 60/648,188, filed January 28, 2005, each of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT [0002] This invention was made with United States Government support under DARPA/SPO contract BAA00-09. The United States Government may have certain rights in the invention.

FIELD OF THE INVENTION [0003] The present invention relates generally to the field of genetic identification of bacteria and provides nucleic acid compositions and kits useful for this purpose when combined with molecular mass analysis.

BACKGROUND OF THE INVENTION [0004] A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al. Philos. Trans.

R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.

[0005] Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect certain pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of 00 bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of 0 0infectious disease agents and there are equally important biosafety and security concerns for Nagriculture.

100061 A major conundrum in public health protection, biodefense, and agricultural safety and security is that these disciplines need to be able to rapidly identify and characterize infectious agents, while there is no existing technology with the breadth of function to meet this need.

Currently used methods for identification of bacteria rely upon culturing the bacterium to effect isolation from other organisms and to obtain sufficient quantities of nucleic acid 0o followed by sequencing of the nucleic acid, both processes which are time and labor intensive.

10007] Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips is with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.

100081 There is a need for a method for identification of bioagents which is both specific and rapid, and in which no culture or nucleic acid sequencing is required. Disclosed in U.S. Patent Application Serial Nos: 09/798,007 Pat. Pub. 20030027135), 09/891,793 Pat.

7,217,510), 10/405,756 Pat. Pub. 20030228571), 10/418,514 Pat. Pub.

20040209260), 10/660,997 Pat. 7,226,739), 10/660,122 Pat. Pub. 20040219517), 10/660,996 Pat. 7,255,992), 10/728,486, 10/754,415 and 10/829,826 Pat. Pub.

20050266397), each of which is commonly owned and incorporated herein by reference in its entirety, are methods tor identification of bioagents (any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus) in an unbiased manner by molecular mass and base composition analysis of "bioagent identifying amplicons" which are obtained by amplification of segments of essential and conserved genes which are involved in, for example, translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins include, but are not limited to, ribosomal RNAs, ribosomal proteins, DNA and RNA polymerases, elongation factors, tRNA synthetases, protein chain initiation factors, heat shock protein groEL, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, DNA gyrases and DNA topoisomerases, metabolic enzymes, and the like.

1292052 I:JIN 100091 To obtain bioagent identifying amplicons, primers are selected to hybridize to 00 O conserved sequence regions which bracket variable sequence regions to yield a segment of nucleic acid which can be amplified and which is amenable to methods of molecular mass Sanalysis. The variable sequence regions provide the variability of molecular mass which is s used for bioagent identification. Upon amplification by PCR or other amplification methods Swith the specifically chosen primers, an amplification product that represents a bioagent identifying amplicon is obtained. The molecular mass of the amplification product, obtained by mass spectrometry for example, provides the means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent. The C1 o molecular mass of the amplification product or the corresponding base composition (which can be calculated from the molecular mass of the amplification product) is compared with a ,IC database of molecular masses or base compositions and a match indicates the identity of the bioagent. Furthermore, the method can be applied to rapid parallel analyses (for example, in a multi-well plate format) the results of which can be employed in a triangulation identification strategy which is amenable to rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent identification.

10010] The result of determination of a previously unknown base composition of a previously unknown bioagent (for example, a newly evolved and heretofore unobserved bacterium or virus) has downstream utility by providing new bioagent indexing information with which to populate base composition databases. The process of subsequent bioagent identification analyses is thus greatly improved as more base composition data for bioagent identifying amplicons becomes available.

10011] The present invention provides oligonucleotide primers and compositions and kits containing the oligonucleotide primers, which define bacterial bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify bacteria, for example, at and below the species taxonomic level.

SUMMARY OF THE INVENTION According to a first aspect of the invention there is provided a composition comprising an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 183.

According to a second aspect of the invention there is provided a method for identification of an unknown bacterium comprising: 1292052 I:JIN 00 amplifying nucleic acid from said bacterium using the composition of the first oO O aspect to obtain an amplification product; N, determining the molecular mass of said amplification product; Soptionally determining the base composition of said amplification product from said molecular mass; and Scomparing said molecular mass or base composition of said amplification product with a plurality of molecular masses or base compositions of known bacterial bioagent identifying amplicons, wherein a match between said molecular mass or base composition

IND

,I of said amplification product and the molecular mass or base composition of a member of o t0 said plurality of molecular masses or base compositions identifies said unknown bacterium.

According to a third aspect of the invention there is provided a method of determining the presence or absence of a bacterium of a particular clade, genus, species, or sub-species in a sample comprising: amplifying nucleic acid from said sample using the composition of the first aspect to obtain an amplification product; determining the molecular mass of said amplification product; optionally determining the base composition of said amplification product from said molecular mass; and comparing said molecular mass or base composition of said amplification product with the known molecular masses or base compositions of one or more known clade, genus, species, or sub-species bioagent identifying amplicons, wherein a match between said molecular mass or base composition of said amplification product and the molecular mass or base composition of one or more known clade, genus, species, or sub-species bioagent identifying amplicons indicates the presence of said clade, genus, species, or sub-species in said sample.

According to a fourth aspect of the invention there is provided a method for determination of the quantity of an unknown bacterium in a sample comprising: contacting said sample with the composition of the first aspect and a known quantity of a calibration polynucleotide comprising a calibration sequence; concurrently amplifying nucleic acid from said bacterium in said sample with the composition of the first aspect and amplifying nucleic acid from said calibration polynucleotide in said sample with the composition of the first aspect to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon; determining the molecular mass and abundance for said bacterial bioagent identifying amplicon and said calibration amplicon; and 1292052 I:JIN 0distinguishing said bacterial bioagent identifying amplicon from said calibration amplicon O based on molecular mass, wherein comparison of bacterial bioagent identifying amplicon N abundance and calibration amplicon abundance indicates the quantity of bacterium in said sample. 0012] The present invention provides primers and compositions comprising pairs of s primers, and kits containing the same for use in identification of bacteria. The primers are O designed to produce bacterial bioagent identifying amplicons of DNA encoding genes essential to life such as, for example, 16S and 23S rRNA, DNA-directed RNA polymerase d subunits (rpoB and rpoC), 1292052 I:JIN WO 2006/071241 PCT/US2005/006133 -4valyl-tRNA synthetase (valS), elongation factor EF-Tu (TufB), ribosomal protein L2 (rplB), protein chain initiation factor (infB), and spore protein (sspE). The invention further provides drill-down primers, compositions comprising pairs of primers and kits containing the same, which are designed to provide sub-species characterization of bacteria.

[0013] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 97, or a composition comprising the same; an oligonucleotide primer 20 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 451, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 97, and a second oligonucleotide primer 20 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 451.

[0014] The present invention also provides an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 127, or a composition comprising the same; an oligonucleotide primer 14 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 482, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 127, and a second oligonucleotide primer 14 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 482.

[0015] The present invention also provides an oligonucleotide primer 19 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 174, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 530, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 174, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 530.

[0016] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 310, or a composition WO 2006/071241 PCT/US2005/006133 comprising the same; an oligonucleotide primer 19 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 668, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 310, and a second oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 668.

[0017] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 313, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 670, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 313, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 670.

[0018] The present invention also provides an oligonucleotide primer 17 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 277, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 632, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 17 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 277, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 632.

[0019] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 285, or a composition comprising the same; an oligonucleotide primer 19 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 640, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 285, and a second oligonucleotide primer 19 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 640.

WO 2006/071241 PCT/US2005/006133 -6- [0020] The present invention also provides an oligonucleotide primer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 301, or a composition comprising the same; an oligonucleotide primer 21 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 656, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 301, and a second oligonucleotide primer 21 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 656.

[0021] The present invention also provides an oligonucleotide primer 18 to 35 nucleobases in length comprising 70% to 100% sequence identity with SEQ ID NO: 308, or a composition comprising the same; an oligonucleotide primer 18 to 35 nucleobases in length comprising to 100% sequence identity with SEQ ID NO: 663, or a composition comprising the same; a composition comprising both primers; and a composition comprising a first oligonucleotide primer 18 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 308, and a second oligonucleotide primer 18 to 35 nucleobases in length comprising between 70% to 100% sequence identity of SEQ ID NO: 663.

[0022] The present invention also provides compositions, such as those described herein, wherein either or both of the first and second oligonucleotide primers comprise at least one modified nucleobase, a non-templated T residue on the 5'-end, at least one non-template tag, or at least one molecular mass modifying tag, or any combination thereof.

[0023] The present invention also provides kits comprising any of the compositions described herein. The kits can comprise at least one calibration polynucleotide, or at least one ion exchange resin linked to magnetic beads, or both.

[0024] The present invention also provides methods for identification of an unknown bacterium.

Nucleic acid from the bacterium is amplified using any of the compositions described herein to obtain an amplification product. The molecular mass of the amplification product is determined.

Optionally, the base composition of the amplification product is determined from the molecular mass. The base composition or molecular mass is compared with a plurality of base compositions or molecular masses of known bacterial bioagent identifying amplicons, wherein a match between the base composition or molecular mass and a member of the plurality of base WO 2006/071241 PCT/US2005/006133 -7compositions or molecular masses identifies the unknown bacterium. The molecular mass can be measured by mass spectrometry. In addition, the prrsence or absence of a particular clade, genus, species, or sub-species of a bioagent can be determined by the methods described herein.

[0025] The present invention also provides methods for determination of the quantity of an unknown bacterium in a sample. The sample is contacted with any of the compositions described herein and a known quantity of a calibration polynucleotide comprising a calibration sequence.

Concurrently, nucleic acid from the bacterium in the sample is amplified with any of the compositions described herein and nucleic acid from the calibration polynucleotide in the sample is amplified with any of the compositions described herein to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon. The molecular mass and abundance for the bacterial bioagent identifying amplicon and the calibration amplicon is determined. The bacterial bioagent identifying amplicon is distinguished from the calibration amplicon based on molecular mass, wherein comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in the sample. The method can also comprise determining the base composition of the bacterial bioagent identifying amplicon.

BRIEF DESCRIPTION OF THE DRAWINGS [0026] Figure 1 is a represenataive pseudo-four dimensional plot of base compositions of bioagent identifying amplicons of enterobacteria obtained with a primer pair targeting the rpoB gene (primer pair no 14 (SEQ ID NOs: 37:362). The quantity each of the nucleobases A, G and C are represented on the three axes of the plot while the quantity ofnucleobase T is represented by the diameter of the spheres. Base composition probability clouds surrounding the spheres are also shown.

[0027] Figure 2 is a represenataive diagram illustrating the primer selection process.

[0028] Figure 3 lists common pathogenic bacteria and primer pair coverage. The primer pair number in the upper right hand comer of each polygon indicates that the primer pair can produce a bioagent identifying amplicon for all species within that polygon.

[0029] Figure 4 is a represenataive 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair WO 2006/071241 PCT/US2005/006133 -8number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples (labeled NHRC samples) closely match the base compositions expected for Streptococcuspyogenes and are distinct from the expected base compositions of other organisms.

[0030] Figure 5 is a represenataive mass spectrum of amplification products representing bioagent identifying amplicons of Streptococcus pyogenes, Neisseria meningitidis, and Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses and base compositions for the sense strand of each amplification product are shown.

[00311 Figure 6 is a represenataive mass spectrum of amplification products representing a bioagent identifying amplicon of Streptococcus pyogenes, and a calibration amplicon obtained from amplification of nucleic acid from a clinical sample with primer pair number 356 which targets rplB. The experimentally determined molecular mass and base composition for the sense strand of the Streptococcus pyogenes amplification product is shown.

[0032] Figure 7 is a represenataive process diagram for identification and determination of the quantity of a bioagent in a sample.

[0033] Figure 8 is a represenataive mass spectrum of an amplified nucleic acid mixture which contained the Ames strain of Bacillus anthracis, a known quantity of combination calibration polynucleotide (SEQ ID NO: 741), and primer pair number 350 which targets the capC gene on the virulence plasmid pX02 of Bacillus anthracis. Calibration amplicons produced in the amplification reaction are visible in the mass spectrum as indicated and abundance data (peak height) are used to calculate the quantity of the Ames strain of Bacillus anthracis.

DESCRIPTION OF EMBODIMENTS [0034] The present invention provides oligonucleotide primers which hybridize to conserved regions of nucleic acid of genes encoding, for example, proteins or RNAs necessary for life which include, but are not limited to: 16S and 23S rRNAs, RNA polymerase subunits, t-RNA synthetases, elongation factors, ribosomal proteins, protein chain initiation factors, cell division proteins, chaperonin groEL, chaperonin dnaK, phosphoglycerate kinase, NADH dehydrogenase, DNA ligases, metabolic enzymes and DNA topoisomerases. These primers provide the WO 2006/071241 PCT/US2005/006133 -9functionality of producing, for example, bacterial bioagent identifying amplicons for general identification of bacteria at the species level, for example, when contacted with bacterial nucleic acid under amplification conditions.

[0035] Referring to Figure 2, primers are designed as follows: for each group of organisms, candidate target sequences are identified (200) from which nucleotide alignments are created (210) and analyzed (220). Primers are designed by selecting appropriate priming regions (230) which allows the selection of candidate primer pairs (240). The primer pairs are subjected to in silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are obtained from sequence databases such as, for example, GenBank or other sequence collections (310), and checked for specificity in silico (320). Bioagent identifying amplicons obtained from GenBank sequences (310) can also be analyzed by a probability model which predicts the capability of a particular amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are stored in a base composition database (325). Alternatively, base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database (330). Candidate primer pairs (240) are validated by in vitro amplification by a method such as, for example, PCR analysis (400) of nucleic acid from a collection of organisms (410).

Amplification products that are obtained are optionally analyzed to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products (420).

[0036] Synthesis of primers is well known and routine in the art. The primers may be conveniently and routinely made through the well-known technique of solid phase synthesis.

Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed.

[0037] The primers can be employed as compositions for use in, for example, methods for identification of bacterial bioagents as follows. In some embodiments, a primer pair composition is contacted with nucleic acid of an unknown bacterial bioagent. The nucleic acid is amplified by a nucleic acid amplification technique, such as PCR for example, to obtain an amplification product that represents a bioagent identifying amplicon. The molecular mass of one strand or each strand of the double-stranded amplification product is determined by a molecular mass measurement technique such as, for example, mass spectrometry wherein the two strands of the WO 2006/071241 PCT/US2005/006133 double-stranded amplification product are separated during the ionization process. In some embodiments, the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray time of flight mass spectrometry (ESI-TOF- MS). A list of possible base compositions can be generated for the molecular mass value obtained for each strand and the choice of the correct base composition from the list is facilitated by matching the base composition of one strand with a complementary base composition of the other strand. The molecular mass or base composition thus determined is compared with a database of molecular masses or base compositions of analogous bioagent identifying amplicons for known bacterial bioagents. A match between the molecular mass or base composition of the amplification product from the unknown bacterial bioagent and the molecular mass or base composition of an analogous bioagent identifying amplicon for a known bacterial bioagent indicates the identity of the unknown bioagent.

[0038] In some embodiments, the primer pair used is one of the primer pairs of Table 1. In some embodiments, the method is repeated using a different primer pair to resolve possible ambiguities in the identification process or to improve the confidence level for the identification assignment.

[0039] In some embodiments, a bioagent identifying amplicon may be produced using only a single primer (either the forward or reverse primer of any given primer pair), provided an appropriate amplification method is chosen, such as, for example, low stringency single primer PCR (LSSP-PCR). Adaptation of this amplification method in order to produce bioagent identifying amplicons can be accomplished by one with ordinary skill in the art without undue experimentation.

[0040] In some embodiments, the oligonucleotide primers are "broad range survey primers" which hybridize to conserved regions of nucleic acid encoding RNA, such as ribosomal RNA (rRNA), of all, or at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% of known bacteria and produce bacterial bioagent identifying amplicons. As used herein, the term "broad range survey primers" refers to primers that bind to nucleic acid encoding rRNAs of all, or at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% known species of bacteria. In some embodiments, the rRNAs to which the primers hybridize are 16S and 23S rRNAs. In some embodiments, the broad range survey primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer WO 2006/071241 PCT/US2005/006133 -11pair numbers 3, 10, 11, 14, 16, and 17 which consecutively correspond to SEQ ID NOs: 6:369, 26:388, 29:391, 37:362, 48:404, and 58:414.

[0041] In some cases, the molecular mass or base composition of a bacterial bioagent identifying amplicon defined by a broad range survey primer pair does not provide enough resolution to unambiguously identify a bacterial bioagent at the species level. These cases benefit from further analysis of one or more bacterial bioagent identifying amplicons generated from at least one additional broad range survey primer pair or from at least one additional "division-wide" primer pair (vide infra). The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as "triangulation identification" (vide infra).

[0042] In other embodiments, the oligonucleotide primers are "division-wide" primers which hybridize to nucleic acid encoding genes of broad divisions of bacteria such as, for example, members of the Bacillus/Clostridia group or members of the and e-proteobacteria. In some embodiments, a division of bacteria comprises any grouping of bacterial genera with more than one genus represented. For example, the P-proteobacteria group comprises members of the following genera: Eikenella, Neisseria, Achromobacter, Bordetella, Burkholderia, and Raltsonia.

Species members of these genera can be identified using bacterial bioagent identifying amplicons generated with primer pair 293 (SEQ ID NOs: 344:700) which produces a bacterial bioagent identifying amplicon from the tufB gene of 1-proteobacteria. Examples of genes to which division-wide primers may hybridize to include, but are not limited to: RNA polymerase subunits such as rpoB and rpoC, tRNA synthetases such as valyl-tRNA synthetase (valS) and aspartyl-tRNA synthetase (aspS), elongation factors such as elongation factor EF-Tu (tufB), ribosomal proteins such as ribosomal protein L2 (rplB), protein chain initiation factors such as protein chain initiation factor infB, chaperonins such as groL and dnaK, and cell division proteins such as peptidase ftsH (hflB). In some embodiments, the division-wide primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from to 100% sequence identity with primer pair numbers 34, 52, 66, 67, 71, 72, 289, 290 and 293 which consecutively correspond to SEQ ID NOs: 160:515, 261:624, 231:591, 235:587, 349:711,240:596, 246:602, 256:620, 344:700.

[0043] In other embodiments, the oligonucleotide primers are designed to enable the identification of bacteria at the clade group level, which is a monophyletic taxon referring to a group of organisms which includes the most recent common ancestor of all of its members and WO 2006/071241 PCT/US2005/006133 -12all of the descendants of that most recent common ancestor. The Bacillus cereus clade is an example of a bacterial clade group. In some embodiments, the clade group primer pairs comprise oligonucleotides ranging in length from 13 to 35 nucleobases, each of which have from 70% to 100% sequence identity with primer pair number 58 which corresponds to SEQ ID NOs: 322:686.

[0044] In other embodiments, the oligonucleotide primers are "drill-down" primers which enable the identification of species or "sub-species characteristics." Sub-species characteristics are herein defined as genetic characteristics that provide the means to distinguish two members of the same bacterial species. For example, Escherichia coli 0157:H7 and Escherichia coli K12 are two well known members of the species Escherichia coli. Escherichia coli 0157:H7, however, is highly toxic due to the its Shiga toxin gene which is an example of a sub-species characteristic.

Examples of sub-species characteristics may also include, but are not limited to: variations in genes such as single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs). Examples of genes indicating sub-species characteristics include, but are not limited to, housekeeping genes, toxin genes, pathogenicity markers, antibiotic resistance genes and virulence factors. Drill-down primers provide the functionality of producing bacterial bioagent identifying amplicons for drill-down analyses such as strain typing when contacted with bacterial nucleic acid under amplification conditions. Identification of such sub-species characteristics is often critical for determining proper clinical treatment of bacterial infections. Examples of pairs of drill-down primers include, but are not limited to, a trio of primer pairs for identification of strains of Bacillus anthracis. Primer pair 24 (SEQ ID NOs: 97:451) targets the capC gene of virulence plasmid pX02, primer pair 30 (SEQ ID NOs: 127:482) targets the cyA gene of virulence plasmid pX02, and primer pair 37 (SEQ ID NOs: 174:530) targets the lef gene of virulence plasmid pX02. Additional examples of drill-down primers include, but are not limited to, six primer pairs that are used for determining the strain type of group A Streptococcus.

Primer pair 80 (SEQ ID NOs: 310:668) targets the gki gene, primer pair 81 (SEQ ID NOs: 313:670) targets the gtr gene, primer pair 86 (SEQ ID NOs: 227:632) targets the murl gene, primer pair 90 (SEQ ID NOs: 285:640) targets the mutS gene, primer pair 96 (SEQ ID NOs: 301:656) targets the xpt gene, and primer pair 98 (SEQ ID NOs: 308:663) targets the yqiL gene.

[0045] In some embodiments, the primers used for amplification hybridize to and amplify genomic DNA, DNA of bacterial plasmids, or DNA of DNA viruses.

WO 2006/071241 PCT/US2005/006133 13 [0046] In some embodiments, the primers used for amplification hybridize directly to ribosomal RNA or messenger RNA (mRNA) and act as reverse transcription primers for obtaining DNA from direct amplification of bacterial RNA or rRNA. Methods of amplifying RNA using reverse transcriptase are well known to those with ordinary skill in the art and can be routinely established without undue experimentation.

[0047] One with ordinary skill in the art of design of amplification primers will recognize that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction. Moreover, a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event a loop structure or a hairpin structure). The primers of the present invention may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any of the primers listed in Table 1. Thus, in some embodiments of the present invention, an extent of variation of 70% to 100%, or any range therewithin, of the sequence identity is possible relative to the specific primer sequences disclosed herein. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is otherwise identical to another nucleobase primer but having two non-identical residues has 18 of 20 identical residues (18/20 0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20 0.75 or 75% sequence identity with the 20 nucleobase primer.

[0048] Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, homology, sequence identity, or complementarity of primers with respect to the conserved priming regions of bacterial nucleic acid, is at least 70%, at least 80%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100%.

[0049] In some embodiments, the primers described herein comprise at least 70%, at least at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range therewithin) sequence identity with the primer sequences specifically disclosed herein. Thus, for example, a primer may have between 70% and WO 2006/071241 PCT/US2005/006133 -14- 100%, between 75% and 100%, between 80% and 100%, and between 95% and 100% sequence identity with SEQ ID NO: 26. Likewise, a primer may have similar sequence identity with any other primer whose nucleotide sequence is disclosed herein.

[0050] One with ordinary skill is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product of a corresponding bioagent identifying amplicon.

[0051] In some embodiments of the present invention, the oligonucleotide primers are between 13 and 35 nucleobases in length (13 to 35 linked nucleotide residues). These embodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin.

[0052] In some embodiments, any given primer comprises a modification comprising the addition of a non-templated T residue to the 5' end of the primer the added T residue does not necessarily hybridize to the nucleic acid being amplified). The addition of a non-templated T residue has an effect of minimizing the addition of non-templated A residues as a result of the non-specific enzyme activity of Taq polymerase (Magnuson et al. Biotechniques, 1996, 21, 700- 709), an occurrence which may lead to ambiguous results arising from molecular mass analysis.

[0053] In some embodiments of the present invention, primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3 rd position) in the conserved regions among species is likely to occur in the third position of a DNA triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a "universal nucleobase." For example, under this "wobble" pairing, inosine binds to U, C or A; guanine binds to U or C, and uridine binds to U or C. Other examples of universal nucleobases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053- 1056) or the purine analog 1-(2-deoxy--D-ribofuranosyl)-imidazole-4-carboxanide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).

00 100541 In some embodiments, to compensate for the somewhat weaker binding by the

O

S"wobble" base, the oligonucleotide primers are designed such that the first and second Spositions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide. Examples of these analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil which binds to adenine and propynylcytosine and phenoxazines, including G-clamp, which binds to G. Propynylated pyrimidines are described in U.S. Patent Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned and incorporated herein by reference in its entirety. Propynylated Sprimers are described in U.S Serial No. 10/294,203 Pat. Pub. 20030170680) which is also commonly owned and incorporated herein by reference in entirety. Phenoxazines are Sdescribed in U.S. Patent Nos. 5,502,177, 5,763,588, and 6,005,096, each of which is c incorporated herein by reference in its entirety. G-clamps are described in U.S. Patent Nos.

6,007,992 and 6,028,183, each of which is incorporated herein by reference in its entirety.

10055] In some embodiments, non-template primer tags are used to increase the melting temperature (T m) of a primer-template duplex in order to improve amplification efficiency. A non-template tag is at least three consecutive A or T nucleotide residues on a primer which are not complementary to the template. In any given non-template tag, A can be replaced by C or G and T can also be replaced by C or G. Although Watson-Crick hybridization is not expected to occur for a non-template tag relative to the template, the extra hydrogen bond in a G-C pair relative to a A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency for subsequent cycles of amplification when the primers hybridize to strands synthesized in previous cycles.

100561 In other embodiments, propynylated tags may be used in a manner similar to that of the non-template tag, wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer. In other embodiments, a primer contains a modified intenucleoside linkage such as a phosphorothioate linkage, for example.

[00571 In some embodiments, the primers contain mass-modifying tags. Reducing the total number of possible base compositions of a nucleic acid of specific molecular weight provides a means of avoiding a persistent source of ambiguity in determination of base composition of amplification products. Addition of mass-modifying tags to certain nucleobases of a given primer will result in simplification of de novo determination of base composition of a given bioagent identifying amplicon (vide infra) from its molecular mass.

1292052 1:JIN WO 2006/071241 PCT/US2005/006133 16 [00581 In some embodiments of the present invention, the mass modified nucleobase comprises one or more of the following: for example, 7-deaza-2'-deoxyadenosine-5-triphosphate, 5-iodo-2'- 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-bromo-2'- 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-hydroxy-2'- 4-thiothymidine-5'-triphosphate, 5-aza-2'-deoxyuridine-5'triphosphate, 5-fluoro-2'-deoxyuridine-5'-triphosphate, 06-methyl-2'-deoxyguanosine-5'triphosphate, N2-methyl-2'-deoxyguanosine-5'-triphosphate, 8-oxo-2'-deoxyguanosine-5'triphosphate or thiothymidine-5'-triphosphate. In some embodiments, the mass-modified nucleobase comprises N or 1C or both 5 N and 1

C.

[0059] In some embodiments of the present invention, at least one bacterial nucleic acid segment is amplified in the process of identifying the bioagent. Thus,,the nucleic acid segments that can be amplified by the primers disclosed herein and that provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as "bioagent identifying amplicons." The term "amplicon" as used herein, refers to a segment of a polynucleotide which is amplified in an amplification reaction. In some embodiments of the present invention, bioagent identifying amplicons comprise from about 45 to about 200 nucleobases from about 45 to about 200 linked nucleosides), from about 60 to about 150 nucleobases, from about 75 to about 125 nucleobases.

One of ordinary skill in the art will appreciate that the invention embodies compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122,123,124, 125, 126, 127, 128, 129,130,131,132,133,134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144,145, 146, 147,148, 149, 150, 151,152, 153, 154,155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, and 200 nucleobases in length, or any range therewithin. It is the combination of the portions of the bioagent nucleic acid segment to which the primers hybridize (hybridization sites) and the variable region between the primer hybridization sites that comprises the bioagent identifying amplicon. Since genetic data provide the underlying basis for identification of bioagents by the methods of the present invention, it is prudent to select segments of nucleic acids which ideally provide enough variability to distinguish each individual bioagent and whose molecular mass is amenable to molecular mass determination.

00 100601 In some embodiments, bioagent identifying amplicons amenable to molecular mass determination which are produced by the primers described herein are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible Swith a means of providing a predictable fragmentation pattern in order to obtain predictable C, 5 fragments of a length compatible with the particular mode of molecular mass determination.

Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with restriction enzymes or cleavage primers, for example. Methods of using restriction enzymes and cleavage primers are well known to those with ordinary skill in the art.

S[0061] In some embodiments, amplification products corresponding to bacterial bioagent i identifying amplicons are obtained using the polymerase chain reaction (PCR) which is a routine method to those with ordinary skill in the molecular biology arts. Other amplification methods may be used such as ligase chain reaction (LCR), low-stringency single primer PCR, and multiple strand displacement amplification (MDA) which are also well known to those with ordinary skill.

100621 In the context of this invention, a "bioagent" is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus. Examples of bioagents include, but are not limited, to cells, (including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered. In the context of this invention, a "pathogen" is a bioagent which causes a disease or disorder.

100631 In the context of this invention, the term "unknown bioagent" may mean either: a bioagent whose existence is known (such as the well known bacterial species Staphylococcus aureus for example) but which is not known to be in a sample to be analyzed, or (ii) a bioagent whose existence is not known (for example, the SARS coronavirus was unknown prior to April 2003). For example, if the method for identification of corona viruses disclosed in commonly owned U.S. Patent Serial No. 10/829,826 Pat. Pub. 20050266397), (incorporated herein by reference in its entirety) was to be employed prior to April 2003 to identify the SARS coronavirus in a clinical sample, both meanings of "unknown" bioagent are applicable since the SARS coronavirus was unknown to 1292052I_:JIN 00 science prior to April, 2003 and since it was not known what bioagent (in this case a coronavirus) was present in the sample. On the other hand, if the method of U.S. Patent Serial No. 10/829,826 Pat. Pub. 20050266397) was to be employed subsequent to April 2003 Sto identify the SARS coronavirus in a clinical sample, only the first meaning of "unknown" 5 bioagent would apply since the SARS coronavirus became known to science subsequent to April 2003 and since it was not known what bioagent was present in the sample.

I [00641 The employment of more than one bioagent identifying amplicon for identification of C a bioagent is herein referred to as "triangulation identification." Triangulation identification is S 10 pursued by analyzing a plurality of bioagent identifying amplicons selected within multiple 0 core genes. This process is used to reduce false negative and false positive signals, and enable C1 reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl.

Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.

100651 In some embodiments, the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR where multiple primers are employed in the same amplification reaction mixture, or PCR in multi-well plate format wherein a different and unique pair of primers is used in multiple wells containing otherwise identical reaction mixtures. Such multiplex and multi-well PCR methods are well known to those with ordinary skill in the arts of rapid throughput amplification of nucleic acids.

10066] In some embodiments, the molecular mass of a particular bioagent identifying amplicon is determined by mass spectrometry. Mass spectrometry has several advantages, not the least of which is high bandwidth characterized by the ability to separate (and isolate) many molecular peaks across a broad range of mass to charge ratio Thus, mass spectrometry is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular 1292052 I:JIN WO 2006/071241 PCT/US2005/006133 -19weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.

[0067] In some embodiments, intact molecular ions are generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges.

Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent identifying amplicon. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.

[0068] The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic sector, Q-TOF, and triple quadrupole.

[0069] In some embodiments, conversion of molecular mass data to a base composition is useful for certain analyses. As used herein, a "base composition" is the exact number of each nucleobase T, C and For example, amplification of nucleic acid ofNeisseria meningitidis with a primer pair that produces an amplification product from nucleic acid of 23S rRNA that has a molecular mass (sense strand) of 28480.75124, from which a base composition of A25 G27 C22 T18 is assigned from a list of possible base compositions calculated from the molecular mass using standard known molecular masses of each of the four nucleobases.

[0070] In some embodiments, assignment of base compositions to experimentally determined molecular masses is accomplished using "base composition probability clouds." Base compositions, like sequences, vary slightly from isolate to isolate within species. It is possible to manage this diversity by building "base composition probability clouds" around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A "pseudo four-dimensional plot" (Figure 1) can be used to visualize the concept of base composition probability clouds. Optimal primer design requires optimal choice WO 2006/071241 PCT/US2005/006133 ofbioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by a triangulation identification process using bioagent identifying amplicons not affected by overlap of base composition probability clouds.

[0071] In some embodiments, base composition probability clouds provide the means for screening potential primer pairs in order to avoid potential misclassifications of base compositions. In other embodiments, base composition probability clouds provide the means for predicting the identity of a bioagent whose assigned base composition was not previously observed and/or indexed in a bioagent identifying amplicon base composition database due to evolutionary transitions in its nucleic acid sequence. Thus, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition or sequence in order to make the measurement.

[0072] The present invention provides bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to identify a given bioagent.

Furthermore, the process of determination of a previously unknown base composition for a given bioagent (for example, in a case where sequence information is unavailable) has downstream utility by providing additional bioagent indexing information with which to populate base composition databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in base composition databases.

[0073] In one embodiment, a sample comprising an unknown bioagent is contacted with a pair of primers which provide the means for amplification of nucleic acid from the bioagent, and a known quantity of a polynucleotide that comprises a calibration sequence. The nucleic acids of the bioagent and of the calibration sequence are amplified and the rate of amplification is reasonably assumed to be similar for the nucleic acid of the bioagent and of the calibration sequence. The amplification reaction then produces two amplification products: a bioagent identifying amplicon and a calibration amplicon. The bioagent identifying amplicon and the calibration amplicon should be distinguishable by molecular mass while being amplified at essentially the same rate. Effecting differential molecular masses can be accomplished by choosing as a calibration sequence, a representative bioagent identifying amplicon (from a specific species of bioagent) and performing, for example, a 2 to 8 nucleobase deletion or WO 2006/071241 PCT/US2005/006133 -21insertion within the variable region between the two priming sites. The amplified sample containing the bioagent identifying amplicon and the calibration amplicon is then subjected to molecular mass analysis by mass spectrometry, for example. The resulting molecular mass analysis of the nucleic acid of the bioagent and of the calibration sequence provides molecular mass data and abundance data for the nucleic acid of the bioagent and of the calibration sequence. The molecular mass data obtained for the nucleic acid of the bioagent enables identification of the unknown bioagent and the abundance data enables calculation of the quantity of the bioagent, based on the knowledge of the quantity of calibration polynucleotide contacted with the sample.

[0074] In some embodiments, the identity and quantity of a particular bioagent is determined using the process illustrated in Figure 7. For instance, to a sample containing nucleic acid of an unknown bioagent are added primers (500) and a known quantity of a calibration polynucleotide (505). The total nucleic acid in the sample is subjected to an amplification reaction (510) to obtain amplification products. The molecular masses of amplification products are determined (515) from which are obtained molecular mass and abundance data. The molecular mass of the bioagent identifying amplicon (520) provides the means for its identification (525) and the molecular mass of the calibration amplicon obtained from the calibration polynucleotide (530) provides the means for its identification (535). The abundance data of the bioagent identifying amplicon is recorded (540) and the abundance data for the calibration data is recorded (545), both of which are used in a calculation (550) which determines the quantity of unknown bioagent in the sample.

[0075] In some embodiments, construction of a standard curve where the amount of calibration polynucleotide spiked into the sample is varied, provides additional resolution and improved confidence for the determination of the quantity of bioagent in the sample. The use of standard curves for analytical determination of molecular quantities is well known to one with ordinary skill and can be performed without undue experimentation.

[0076] In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with multiple primer pairs which also amplify the corresponding standard calibration sequences. In this or other embodiments, the standard calibration sequences are optionally included within a single vector which functions as the WO 2006/071241 PCT/US2005/006133 -22calibration polynucleotide. Multiplex amplification methods are well known to those with ordinary skill and can be performed without undue experimentation.

[0077] In some embodiments, the calibrant polynucleotide is used as an internal positive control to confirm that amplification conditions and subsequent analysis steps are successful in producing a measurable amplicon. Even in the absence of copies of the genome of a bioagent, the calibration polynucleotide should give rise to a calibration amplicon. Failure to produce a measurable calibration amplicon indicates a failure of amplification or subsequent analysis step such as amplicon purification or molecular mass determination. Reaching a conclusion that such failures have occurred is in itself, a useful event.

[0078] In some embodiments, the calibration sequence is inserted into a vector which then itself functions as the calibration polynucleotide. In some embodiments, more than one calibration sequence is inserted into the vector that functions as the calibration polynucleotide. Such a calibration polynucleotide is herein termed a "combination calibration polynucleotide." The process of inserting polynucleotides into vectors is routine to those skilled in the art and can be accomplished without undue experimentation. Thus, it should be recognized that the calibration method should not be limited to the embodiments described herein. The calibration method can be applied for determination of the quantity of any bioagent identifying amplicon when an appropriate standard calibrant polynucleotide sequence is designed and used. The process of choosing an appropriate vector for insertion of a calibrant is also a routine operation that can be accomplished by one with ordinary skill without undue experimentation.

[0079] The present invention also provides kits for carrying out, for example, the methods described herein. In some embodiments, the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon. In some embodiments, the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs. In some embodiments, the kit may comprise one or more primer pairs recited in Table 1.

[0080] In some embodiments, the kit may comprise one or more broad range survey primer(s), division wide primer(s), clade group primer(s) or drill-down primer(s), or any combination thereof. A kit may be designed so as to comprise particular primer pairs for identification of a 00 particular bioagent. For example, a broad range survey primer kit may be used initially to identify an unknown bioagent as a member of the Bacillus/Clostridia group. Another example of a division-wide kit may be used to distinguish Bacillus anthracis, Bacillus cereus and SBacillus thuringiensis from each other. A clade group primer kit may be used, for example, to 5 identify an unknown bacterium as a member of the Bacillus cereus clade group. A drill-down kit may be used, for example, to identify genetically engineered Bacillus anthracis. In some embodiments, any of these kits may be combined to comprise a combination of broad range survey primers and division-wide primers, clade group primers or drill-down primers, or any C combination thereof, for identification of an unknown bacterial bioagent.

S10081] In some embodiments, the kit may contain standardized calibration polynucleotides C for use as internal amplification calibrants. Internal calibrants are described in commonly owned U.S. Patent Application Serial No: 60/545,425 which is incorporated herein by reference in its entirety.

100821 In some embodiments, the kit may also comprise a sufficient quantity of reverse transcriptase (if an RNA virus is to be identified for example), a DNA polymerase, suitable nucleoside triphosphates (including any of those described above), a DNA ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described, above.

A kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like. A kit may also comprise reagents or other materials for isolating bioagent nucleic acid or bioagent identifying amplicons from amplification, including, for example, detergents, solvents, or ion exchange resins which may be linked to magnetic beads. A kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit.

[0083] In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.

Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning A Laboratory Manual, 1292052_1:JIN WO 2006/071241 PCT/US2005/006133 -24- 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.

EXAMPLES

[0084] Example 1: Selection of Primers That Define Bioagent Identifying Amplicons [0085] For design of primers that define bacterial bioagent identifying amplicons, relevant sequences from, for example, GenBank are obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 200 nucleotides in length and distinguish species from each other by their molecular masses or base compositions. A typical process shown in Figure 2 is employed.

[0086] A database of expected base compositions for each primer region is generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nuc. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. 1998, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs.

[0087] Table 1 represents a collection of primers (sorted by forward primer name) designed to identify bacteria using the methods herein described. The forward or reverse primer name indicates the gene region of bacterial genome to which the primer hybridizes relative to a reference sequence eg: the forward primer name 16SEC_1077_1106 indicates that the primer hybridizes to residues 1077-1106 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence represented by a sequence extraction of coordinates 4033120..4034661 from GenBank gi number 16127994 (as indicated in Table As an additional example: the forward primer name BONTA_X52066_450_473 indicates that the primer hybridizes to residues 450- 437 of the gene encoding Clostridium botulinum neurotoxin type A (BoNT/A) represented by GenBank Accession No. X52066 (primer pair name codes appearing in Table 1 are defined in Table In Table 1, Ua 5-propynyluracil; Ca 5-propynylcytosine; phosphorothioate linkage. The primer pair number is an in-house database index number.

Table 1: Primer Pairs for Identification of Bacterial Bioagents Primer For. For. Rev.

pair primer SEQ ID Rev. primer SEQ ID number name Forward sequence NO: name Reverse sequence NO: 1 16S EC 107 GTGAGATGTTGGGTTAA 1 16S EC 1175 GACGTCATCCCCACCTTCC 368 WO 2006/071241 WO 206/01241PCT/US2005!006133 7 1106 F GTCCCGTAACGAG 1195 R. TC 16SEC_1177 1SEC_108 ATGTTGGGTTAAGTCCC 1196_-lOG_1 TGACGTCATGGCOACCTTC 266 2 1100 F GC 2 IG 9. C 372 16SEC 108 ATGTTGGGTTAAGTOCC 16S EC_1177 TGACGTCATGCCCACCTTC 265 2 1100 F cc 2 11'96 lOG R C 373 163_EC_108 ATGTTGGGTTAAGTCC- 188_30_1177 TGACGTCATCCCCACCTTC 230 2 1100 F GC 2 1196 R. C 374 16SEC 108 ATGTTGGGTTAAGTCCC 16S EC_1525 263 2 1100 F GC 2 1541 R AAGGAGGTGATCCAGCC 382 16SEC_108 ATGTTGGGTTAAGTCCC 168E EC 1175 TTGACGTCATCCCCACCTT 2 2 1106 CF GCAACGAG 3 1197 R CCTC 371 16SEC 109 TTAAGTCCCGCAACGAG 158_EC_1175 TGACGTCATCCCCACCTTC 278 0 1111 2 F CGCAA 4 11Z96 R C 389 16S_3_0 EN"SEC 1175 0_1111_2_T TTTAAGTCCCGCAACGA -1196_TMOD- TTGACGTCATrCCCCACCTT 361 MOD F GCGCA.A .5 R CCTC 370 16SEC 109 TTAAGTCCCGCAACGAT 16S _EC_1175 TGACSTCATCCCCACCTTC 3 0 1111 F 000201 6 1196 R. CTC 369 16SEC 109 TGCCCCGC 168 EC 1174 GACGTCATCCCCACCTTCC 256 2 1109 F C 7 119 R TCC 367 I6SEC_110 16SEC_1174 159 0 1116 F CAAOGAGCCCAACCCTT 8 118 R TCGCCACCTTCCTCC 366 163_30119 CAAGTOATCATGGOCCT 168_EC_-1525 247 5 1213 F TA 9 1541 R. AAGGAGGTGATCCAGCC 382 16SEC 122 GCTACACACGTGCTACA 163_30_-1303 CGAGTTGCAGACTGCGATC 4 2 12E41 F ATG 10 132 3 R. CG 376 163_30_130 CGGATTGGAGTCTGCAA 168_EC_1389 232 3 1323 F OTCG 11 1407 a. GCGGCCCGTGTGTACAAG 378 16SEC 133 AAGTCGGAATCGCTAGT 165_30_-1389 2 153 F AATCG 12 1407 R GACGGGCGGTGTGTACAAG 378 16S_30_136 TACGGTGAATACGTTOC IES 30 1485 ACCTTGTTACGACTTCACC 252 7 1337 F CGGG 13 1506 R CCA 379 16S8_30_138 GCCTTGTACACACCTCC 165_T30_1494 CACGGCTACCTTGTTACGA 250 7 140D7 F CGT0 14 1513 R 0 381 16S_3C-138 CTTGTACACACCGCCCG 168 30 1525 231 9 1-407 F TO 15 1541 R AAGGAGGTGATCCAGCC 382 168_30_139 TTGTACAOA0OCGCCGT 163_301486 CCTTGTTACGACTTCACCC 251 0141 F CAlAC 16 1505 R 0380 16S_30 30_ TGAACGZTGGTGGCATG 163_30 105 TACGCATTACTCACCCGTC 6 54 FCTTAACAC 17 126 R CGC 361 16S_30_314 CACTGGAACTGAGACAC 163_30C 556 CTTTACGCCCAGTAATTCC 243 332 F GG 18 5759 R 385 168_30 38_ GTGGCATGCCTAATACA 16S_30C 101_ TTACTCACCCGTCCGCCGC 7 84 FTGCAAGTCG 19 120 R T 357 168_E0 405 TGAGTGATGAAGGCCTT 168_30_-507_ CGGCTGCTGGCAOGAAGTT 279 432 F AGGGTTGTAAA 20 527 R. AG_ 384 16S 30 49 TAACACATGCAAGTCGA 165 30 104 8 68 TF ACG 21 120 R TTACTCACCCGTCCGCC 359 165_30 49- TAACACATGCAAGTCGA 168 30 1061 275 88 F ACG 21 1078 R ACGACACGAGCTGACGAC 364 168_30 49_ TAACACATGCAAGTCGA 168_EC 880- 274 68 ACG 21 894 R CC-TACTCCCCAGGCG 380 168 30 518 CCAGCAGCCGCGGTAAT 168 80C 774_ GTATCTAATOCTGTTTGCT 244 536 F- AC 22 7959 RCCC 387 168 E0 556 CGGAATTACTGGGCGTA 168_30 683_ 226 57-5 F AAG 23 W00 R CGCATTTCACCGCTACAC 386 168_30 556 CGGAATTACTGGGCGTA 168 80C 774_ GTATOTAATCCTGTTTGCT 264 57-5 F AAG 23 7959 RCCC 387 168_30_683 GTGTAGCGGTGAAATGC 168_301303 CGAGTTGCAGACTGCGATC 273 7009 F 8 24 13233R CG 377 168 E0_693 GTGTAGCGGTGAAATGC 168_30 C774_ GTArOTAATCCTGTTTGCT 9 700 F G 24 795 R. CCC 387 16SEC_683 GTGTAGCGGTGAAATGC 168_30_880 158 700 F G 24 894 R. CGTACTCCCCAGGCG 390 168 EC_683 GTGTAC-CGGTGAAATGC 168 30 967_ 245 700 F7 G 24 985 R GGTAAGGTTCTTCGOGTTG 396 168_E0_7_3 GAGAGTTTGATCCTGGC 168_30 101 TGTTACTCACCCGTCTGCC 294 3 F TCAGAACGAA 25 122 R ACT 358 168_EC_713 AGAACZXCCGATGGCGAA 168_30 7B9 CGTGGACTACCAGGGTATC 7325 F G00 26 809 R TA 388 16SEC_713 -732_TMOD TAGAACACCGATGGCGA 16S8_EC 789_ TCGTGGACTACCAGGGTAT 346 F AGGC 27 809 TMOD R CTA 389 228 168 EC 774 1GGGAGCAAACAGGATTA 28 168 EC_ 880 CGTACTCCCCAGGCG 390 WO 2006/071241 PCT/US2005!006133 S795 F GATAC 894 R 16S_SC_785 GGATTACAGACCCTCCT 16SEC 880 11 8 06 EF AGTCC 29 897-R GGCCGTACTCCCCAGCCG 391 16SEC_785 _80_:M0D- TGGATTAGAGACCCTGG 16SS C 8ao0 347 F TAGTCC 30 -897 rM0OD R TGGCCGTACTCCCCAGGCG 392 16SEC_785 GGATTAGATACCCTGGT 16SEC_830_ 12 810 F AGTCC24CGC 31 897-2 R GGCCGTACTCCCCAGGCG 391 16SEC_789 TAGATACCCTGGTAGTC 16SEC 880_ 13 81c F CACGC 32 894 R CGTACTCCCCAGGCG 390 16SEC_789 TAGATACCCTGGTAGPC 16S EC 882_ 255 810 F CACGC 32 899-R C CGACCGTACTCCCCACG 393 16SSC_791 GATACCCTGGTAGTCCA lOSSC_-8B6- 254 812 F CACCG 33 904 R GCCTTGCGACCGTACTCCC 394 16SEC_8_2 AGACTTTGATCATGGCT 16S EC_1525 248 7 F7 CAG 34 1541 5R AAGCAGGTCATCCACCC 382 16SEC_8_2 AGAGTTTGATCATCGCT 16SEC 342 242 7 F- CAG 34 358-R ACTGCTGCCTCCCGTAG 383 lOS_EC_804 ACCACC-CCGTAAACGAT 16S_EC -909 CCCCCGTCAATTCCTTTGA 253 822 F GA 35 929 R CT 395 165SEC_937 AACCTGGAGCATCTG 16SEC_1220 ATTGTAGCACGTGTGTAGC 246 954Z F- G 35 1240O R cc 375 16SEC_960 TTCGATGCAACCAAC 16S EC_1054 ACCAGCTCACGACAGCCAT 14 q981 F- AACCT 37 10773 R G 362 16SEC_960 1OS EC_1054 -981 TM OD TTTCGD.TGCAACGCGAA 107733MOD- TACGACPACCACAGCCA 348 F GAACCT" 38 R TG 363 155SC_969 ACCCG.ZACAACCTTA 16SSC_1061 119 985 19F JC 39 1078 2P R ACCACACGAGU'C'GACCAC 364 16SS C_969 .06S SC_1061 985 F- ACGCGAAGAACCTTACC 39 1078 R ACGACACGAGCTGACGAC 364 16SEC 969 LOS EC_1389 272 985 F- ACGCCPACAACCTTACC 40 14067 Ri GACGGGCGGTGTGTACAAC 378 16SEC 9,71 CAACAACCTTACCAG LOSEC 1043 ACAACCATGCACCACCTGT 344 99C F7 GTC 41 1062 -R C 360 16S EC_972 1-63SEC 10654 120 985 2-P P CGAACAAU'U'TTALC 42 1075 2P' R ACACCAGUaCaGAC 3655 16S_'9EC_972 lO6S SC_1064 121 g8o CGAAGPACCTTACC 42 1075 R ACACGAGCTOAC 305 23SBRS_11 TGCGCGGAAGATGTAAC 23SBRM_117 TCGCAGGCTTACAI9ACGC 1073 10 1129 F SGG 43 -6 1201 5 TCTCCTA 39*7 233_BRM_51 TGCATACAAACAGTCGG 23S_3594 616 TCGGACTCGCTTTCGCTAC 1074 5 536 F- AGCCT 44 0 35 R C 396 233_S AAACTAGATAACAGTAG 23S_6 BS5-21 241 68 -44 F ACATCAC 45 R GTGCGCCCTTTCTAACIT 399 233SEC_160 TACCCCAAACCGACACA 23SEC_1686 235 2 1520 OF GG 45 1 70d3 R CCTTCTCCCGAAGTTACG 402 23SEC_168 CCGTAACTTCGGGAGAA 235_SC_1828 236 5 1703-F GG 47 1842 R CACCGGGCAGGCGPC 403 233S C_182 CTGACACCTGCCCGGTG 233Sc_1905 0 1843 F C 48 1924 R GACCGTTATAGTTACGGCC 404 233SEC_182 233S C_1906 6_1543_TMSO TCTCACACC;TCCCGGT 192 4_TMOD TGACCGTTATAGTTACGGC 349 DE FCC 49 R C 405 23SSC 182 23S SC_1929 CCGACAACCAATTTCGCTA 237 7 1843-F GACGCCTGCCCGGTGC 5) -194Z9 R cc 407 23SEC 183 ACCTGCCCAGTGCTGGA 235_SC_1919 249 1 18 49-F AG 51 1936 5R TCGCTACCTTAGGACCGT 406 23S_EC_-187 GGGAACTGAAACATCTA 23SSC 242_ 234 207 F AGTA 52 2565R TTCGCTCGCCGCTAC 408 233SEC_23 23S SC 115 233 37 F GGTGGATGCCTTGGC 53 1305R- GGGTTTCCCZATTCGG 401 23S SC 243 AAGCTACTCCGGGGATA 23SSC_2490 ACCCACATCGACCTCCCA 238 4 24Z56 F ACAGGC 54 2511 R AAC 409 233 SC 258 TAGAACGTCGCGAGACA 233SC_2058 AGTCCATCCCGGTCCTCTC 257 0 2607-F GTTCG 55 2077 R G 411 23S SC 259 CACAGTTCCGTCCCTAT 23S SC_2653 239 9 2618 6F C 56 2069 R CCGGTCCTCTCGTACTA 410 233 SC 204 CTGTCCCTAGTACGAGA 23S SC_2751 GTTTCATGC'TAGATGCTT 18 5 2Z69-2 F GGACCGG 57 276 7 R TCAGC 417 23SEC_264 TCTGTCCCTAGTACGAG 23SSC_2744 17 5 2869 9' AGCACCCC 53 _2761 R TGCTTACATGCTTTCACC 414 233 EC 204 CTGTTCTTAGTACGAGA 23S SC_2745 TTCGTGCTTAGATGCTTTC 118 0 2607-F GGACC 59 276 5 R AG 415 360 233 SC 204 ,TCTCTTCTTAGTACGAG I60 233 SC 2745 1TTTCGTGCTTAGATGCTTT 416 WO 2006/071241 PCT/US2005!006133 6_2667_TMO AGGACC _2765_TMOD- CAG D F P.

23SEC_265 CTAGTACGAGAGGACCG 23SEC_2741 ACFTAGATGCTTTCAGCGG 14-7 2 2T69 F G 61 -2760 R T 413 239 EC_265 23S EC 2737 TTAkGATGCTTTCAGCACPT 240 3 2669 F TAGTACGAGAGGACCGG 62 275 8 BR ATC 412 23SEC_493 GGGGAGTGAAAGAGATC 23SBC_551_ ACAAAAGGCACGCCATCAC 518 2-F CTGAAACCG 63 571 2 R cc 418 239_IC 493 GGGGAGTGAAAGA2ATC 239 EC 551_ ACAAAAGGTACGCCGTCAC 19 -518 F- CTCAAACCG 63 571-R cc 419 23SEC_971 CGAGAGGGAAACAACCC 23SEC_1059 21 9 92 F7 AGACC 64 1077 R TGGCTGCTTCTAAGCCAAC 400

AD_MLST-

1 1- ARMLST-il- 01F037_120 TCGTGCCCGCA.ATTTGC 01F007_1266 TATGCCGGGTAGTGCAAT 1158 2 1225 F ATAAAGZ 65 1296 R CCA PTCTTCTAG 420

AB_MLST-

11- ABMMST-il- 01F007 120 TCGTGCCCCCAATTTGC 01F007_1299 1159 2 122S F ATAAAGC 65 1316 B TGCACCTGCGGTCGAGCG 421

ADMLST-

11- ADMLsT-11- OIF007_123 TTGTAGCACAGCAAGGC CIF007_1335 TGCCATCCATAATCACGCC 1160 4 1264-F AAATTTCCTGAAAC 66 1362 B ATACTGACM 422

AB_MLST-

1 1_ ABMLST-il- 01F007_132 TAGGTTTACGTCAGTAT 01F007_1422 TGCCAGTTTCCACATTTCA 1161 7 1356 F GGCGTGATTATGG 67) 1448 B CGTTCGTG 423

ADMLST-

11- ABMLST-11- 01F007 -134 TCGTGATTATGGATGGC 01F007_1470 TCGCTrGAGTGTAGTCATG 1162 5 1369 F AACGTGAA 68 1494 R ATTGCG 424

ABMLST-

11- ABMIST-il- 01F007_135 TTATGGATGCCAACGTG CliOO7_1470 TCGCTTGAGTGTAGTCATG 1163 1 1375-F AAACGCGT 69 1494 BR ATTGCG 424

ADMIST-

11- AE MLSTr-11- 01F007_138 TCTTTGCCATTGAAGAT 01F007_1470 TCGCTTrCAGTGTAGTCATG 1164 7 1412 F GACTTAAGC 70 1494 R ATTGCG 424

ADMLST-

11- ABMLST-11- 01F007 -154 TACTAGCGGTAAGCTTA 01F007_1656 TGAGTCGGGTTCACTTTAC 1165 2 1569 F AACAAGATTGC '71 1680 R CTGGCA 425

AB_MIST-

11- ABMIST-li- 01F007 -1596 TTGCCAATGATATTCGT 01F007_1656 TGAGTCGGGTTCACTTTAC 1166 6 1593 F TGGTTACCAAG 72 1680 R CTGGCA 425

ADMIST-

OIF007 -161 TCGGCGAAATCCGTATT 01F0W7_1731 TACCGGAAGCACCAGCGAC 117 1 1638 F CCTGAAAATGA 73 1757 R ATTAATAG 427 AB_MLST- I 11- ABMIST-1l- 0IF007_172 TACCACTATTAATGTCG 01F007_1790 TGCAACTCAATACATTGCA 1168 6 1752 F CTGGTGCTTC 74 1821 R GTAAGTTATAAGC 428

AB_MIST-

I1- TTATAACTTACTGCAAT ABMIST-il- OIFOOI-179 CTATTCAGTTGCTTGGT 01F007 1876 TGAATTATCCAAGAAGTGA 1169 2 1026 F G '75 1909 R. TCAATTTTCTCACGA 429 AB_MLST-

I

I1- TTATAACTTACTGCAAT ABDMST-il- 01F007 179 CTATTCAGTTGCTTGGT 01F007_1895 TGCCGTAACTAACATAAGA 1170 2 1826 F G 75 1927 R GAATTATGCAAGAA 430

ABMLST-

11- ABMIST-il- 01F007_185 TATTGTTTCAAATGTAC 01F007 291 TCACAGGTTCTACTTCATC 1152 214 F- AAGGTGAAGTGCG 76 324 B AATAATTTCCATTGC 432

ADMLST-

11- ABMIST-il- OIF007 197 TGGTTATGTACCAAATA 01F007_2D97 TGACGGCATCGATACCACC 1171 0 2002 F CTTTGTCTGAAGATGG 77 2118 R GTC 431

ADMIST-

11- ADMIST-li- 01F007_-206 TGAAGTGCGTGATGATA 0IF007_318 TCCCCAAAA.ACTCCCCTT 1154 239 F TCGATGCACTTGA7GTA 78 344_R TTCACAGG 433 WO 2006/071241 WO 206/01241PCT/US2005!006133 -28

ABMIST-

11- ABMLST-11- 0TF007_260 TGGAACGTTATCAGGTG 01F007_364_ TTGCAATCGACATATCCAT 1153 289 F CCCCAAAAATTCG 79 393 R TTCACCATGCC 434

ABMLST-

11- ABMLST-11- 01F007_522 TCGGTTTAGTAAAAGAA 01F007_587 TTCTGCTTGAGGAATAGTG 1155 552 F7 CGTATTGCTCAACC 80 610 R CGTGG 435

ABMLST-

11- ASMLST-11- OIF007 -547 TCAACCTrGACTGCGTGA 0:F007_656_ TACGTTCTACGATTTCTTC 1156 571 F ATGGTTGT 81 686 R -ATCAGGTACATC 436

ABMLST-

11- ABMLST-li- 01F007_601 TCAAGCAGAAGCTTTGG OIF007 710 TACAACGTGATAAACACGA 1157 62-7 F AAGAAGAAGG 82 736 R CCAGAAGC 437 AS_14167- 11- ABMIST-il- OIF007 2 TGAGAT-GCTGAAiCATT 01F007_169_ TTGTACATTTGAAACAATA 1151 91 F TAATGC"GATTGA 83 203 R TGCATGACATGTGAAT 426 ASD -FRT 1_ TTGCTTAAAGTTGGTTT ASDFRT -86_ TGAGATGTCGAAAAAAACG 1100 29 F TATTGGTTGGCG 84 116 R TTGGCAAAATAC 439 ASD FRT_43 TCAGTTTTAATGTCTCG ASDFRT_129 TCCATATTGTTGCIATAAAA 1101 76 F TATGATCCAATCAAAAG 85 156 R CCTGTTGGC 438 ASPS ES 40 GCACAACCTGCGGCTGC ASPS -EC_521 291 5 422 F G 86 5385R ACGGCACGAGGTAGTCGC 440 BONTAX520 66_450_ 473 TCTAGTAATAATAGGAC BONTAX5206 TAACCATTTCGCGTAAGAT 485 F- CTCAGC 87 6 517 539 R TSAA 441 BONTAX520 T*tU*CATAATAATAG BONTA X5206 66_450_473 GA*Ua-U*U*C'S*U'AG 6_517_539P TAACCA*Ca*Ca*C'*0oSC 486 P F 5 87 R GTAAGA*C'*C'*UAA 441 SONTA X520 66_538_552 13ONTA -x5206 481 F TATGGCTCTACTCAA 88 6 647 660 R TGTTACTGCTGGAT 443 BOSTAX520 BONTAX5206 66 5389 552 TA*CaGGC*Ca*U'*CaA 6_647_660P GC*AU'CGU* 402 P, F 81*'A BE RGGAT 443 BONTA X520 66_591_620 TGAGTCACTTGAAGTTG BONTA_-X5206 TCATGTGCTAATGTTACTG 487 F ATACAAATCCTCT 89 6 644 671- R CTGGATCTG 442 BONTAX520 66 701 720 GAATAGCAATTAATCCA BONTAX5206 483 F AAT 90 6 '759 775 R TTACTTCTAACCCACTC 444 BONTAX520 BONTAX5206 66_-701_-720 GAA*CaAG*UaAA* Ca*C 6_759 775P TTA* U*Ca*Ca*Ua*CaAA* 44 484 P F aX5* C *Ua*UaM JA9J 90 R Ua*Ua*UaA*Ua*CaC 44 CAFi AF053 CAFI AF0539 947 3j3407 TCAGTTCCGTTATCGCC 47 33i494_33 TGCGGGCTGGTTCAACAAG; 774 33430 F ATTGCAT 91 514 R AG 445 CAFiAF053 CAF1_AF0539 947 353435 TGGAACTATTGCAACTG 47_33499_33 776 33457 F CTAATG 92 517 R TGATGCGGGCTGGTTCAAc 446 CAMiAF053 CAF1 AF0539 947-3515 TCACTCTTACATATAAG 47_33595_33 TCCTGTTTTATAGCCGCCA 775 33541 F GAAGGCGCTC 93 621 R AGAGTAAG 447 CAFI_ AF053 CAFI_-AF0339 947_33687 TCAGGATGGAAATAACC 47 33755_33 TCAAGGTTCTCACCGTTTA '777 33716 F ACAATTCACTAC 94 782 R CCTTAGGAG 448 GTTATTTAGCACTCGTT CAPC-BA-100 TGAATCTTGAAACACCATA 22 4 131 F TTTAATCAGCC 95 205 A CGTAACG 449 CAPO BA 11 ACTCGTTTTTAATCAGC CAPC BA_185 TGAATCTTGAAACACCATA 23 4 133 F CCG 96 205 R CG 450 CAPCBA -27 GATTATTGTTATCCTGT SAPSBA_-349 GTAACCCTTOTCTTTGAAT 24 4 303' F TATGCCATTTGAG 97 376 R TGTATTTGC 451 CAPCBA_27 4_303 -TROD TGATTATTGTTATCCTG SAPSBA_349 TGTAACCCTTGTCTTTGAA 350 F TTATGCCATTTGAG 98 376 TMOD R TTGTATTTGC 452 CAPCBA_27 TTATTGTTATCCTGTTA SAPSBA_358 GGTAACCSTTGTCTTTGAA 6 2 96 F- TGCC 99 377 R T 453 SAPCBA 28 GTTATCCTGTTATGCCA SAPSBA 361 26 1 301 F TTTG 100 378 A TGGTAACCCTTGTCTTTG 454 CAPCSA_-31 SCGTGGTATTGGA:3TTA CAPS_BA_361 27 5 334Z F TTG 101 378 R TGGTAACCSTTGTCTTTG 454 1053 SrCJSTJ 10 TTGAGGGTATGCACCGT 102 CJST CJ 116 1TCCSSTCATGTTTAAATGA 456 WO 2006/071241 WO 206/01241PCT/US2005!006133 29 1110 F CTTTTTGATTCTTT 6 1198 R. TCAGGATAAAAAGC CJST CJ 12 AGTTATAA.ACACGGCTT CJST CJ 134 TCGGTTTAAGCTCTACATG 1063 68 1299F TCCTATGGCTTATCC 103 9 13-79 R. ATCGTAAGGATA 457 CJST CJ 12 TGGCTTATCCAAATTTA CJST cJ7 140 TTTGCTCATGATCTGCATG 1050 90 1320 F GATCGTGGTTTTAC_ 104 6 143§3 R AAGCATAAA 458 CJ7ST CJ_16 TTATCGTTTGTGGAGCT CJSTcJ 172 VGCAATGTGTGCTATGTCA 1058 43 1670 F AGTGCTTATGC 105 4 1752 R. GCAAAAAGAT 459 CJST CJ 16 TGCTCGAGTGATTGACT CJSTCJ 177 TGAGCGTGTGGAAAAGGAC 1045 68 1700 F TTGCTAAATTTAGAGA 106 4 1799 R. TTGGATG 460 CJSTCJ_16 TGATTTTGCTAAATTTA CJST CJ 179 TATGTGTAGTTGAGCTTAC 1064 80 1113 F GAGAAATTGCGGATGAA 107 5 1822 R TACATGAGC 461 CJSTCJ_18 TCCCAATTAATTCTGCC CJSTCJ_198 TGGTTCTTACPTGCTTTGC 1056 80 1910 F ATTTTTCCAGGTAT 108 -1 2011 R. ATAAACTTTCCA 462 OJST CJ 20 TCCCGGACTTAAI'ATCA CJST CJ_-214 TCGATCCGCATCACCATCA 1054 60 2090 F ATGAAAATTGTGGA 109 8 2174 R AAAGCAAA 463 CJSTCJ_21 TGCGGATCGTTTGGTGG CJST CJ -224 TCCACACTGGATTGTAATT 1059 65 2194 F TTGTAGATGAAAA 110 7 2278 R. TACTGTTCTTT 464 CJST CJ 21 TCGTTTGGTGGTGGTAG CJST_CL7 228 TCTCTTTCAAAGCACCATT 1046 71 2197 F ATGAAAAAGG ill 3 2313 R GC-CATTATAGT_ 465 CJSTCJ_-21 TAGATGAAAAGGGCGAA CJST_CJ_228 TGAATTCTTTCAA-AGCACC 1057 85 2212 F GTGGCTA-ATGG 112 3, 2316 R ATTGCTCATTATAGT 466 CJST CJ 26 TGCCTAGAAGATCTTAA CJST_-CJ -275 TTGCTGCCATAGCAAAGCC 1049 36 266SF? AAATTTCCGCCAACTT 113 3 2777 R TACAGC 467 CJSTCJ_26 TCCCCAGGACACCCTGA CJST -CJ 276 TGTGCTTTTTTTCCTGCCA 1362 78 2703 F AATTTCAAC 114 0 2787 R. TAGCAAAGC 468 CJSTCJ_28 TGGCATTTOTTATGAAG CJST CJ_-296 TGCTTCAAAACGCATTTTT 1065 57 2887 F CTTGTTCTTTAGCA 115 5 299 8 R. ACATTTTCGTTAAAG 469 CJST CJ 28 TGAAGCTTGTTCTTTAG CJST -CJ 297 TCCTCCTTGTGCCTCAAAA 1055 69 28 9SF CAGGACTTCA 116 9 3007 ii CGCATTTTTA 470 CJST CJ_32 TTTGATTTTACGCCGTC CJST_CJ_-335 rCAAAGAACCCGCACCTAA 1051 67 3293 F CTCCAGGTCG 117 6 3385 R TTOATCATTTA 471 CJSTCJ 36 TCCTGTTATCCCTGAAG CJST -CJ_-443 TACAACTGGTTCAAAAACA 1061 0 393 F- TAGTTAATCAAGTTTGT 118 477 R. TTAAGCTGTAATTGTC -473

TCCTGTTATCOCTGAAG

CJSTCJ_36 TAGTTAATCAAGTTTGT OJST_-CJ_-44'2 TCAACTGGTTCAAAAACAT 1048 0 394 F T 119 476 R. TAPAGTTGTAATTGTCC 472

TAGGCGAAGATATACAA

CJSTCJ 5- AGAGTATTAGAAGCTAG CJST_CJ_104 TCCCTTATTTTTCTTTCTA 1052 39 F- A 120 137 R. CTACCTTCCOATAAT 455 CJST CJ 58 TCCAGGACAAATGTATG CJST -CJ_-663 TTCATTTTCTGGTCCAAAG 1047 4 61i F AAAAATGTCCAAGAAG 121 692 9. TAAGCAGTATC -474 CJSTCJ_59 TGAAABAATGTCCAzAGAA CJET_CJ_711 TCCCGAACAATGAGTTGTA 1060 9 632 F GCATAGCAAAAAAAGCA 122 743 R. TCAACTATTTTTAC 475 CTXAVBC_1 'CTTATGCCAAGAGGAC CTXA_-VBC_-19 TGCCTAACAA.ATCCCGTCT 1096 17 142 F7 AGAGTGAGT 123 4 218 R GAGTTC 476 CTXAVBC 3 TGTATTAGGGGCATACA CTXA VBC_44 TGTCATCAAGCACCCCAAA 1397 51 377 F GTCCTCATCC 124 1 466 R. ATGAACT 477 CYA-BA-105 GAAAGAGTTCGGATTGG CYA BA_1112 28 5 1072 F G 125 1 130 R TGTTGACCATGCTTCTTAG 479 CYABA_134 ACAACGAAGTACAATAC CYA_-BA 126 CTTCTACATTTTTAGCCAT 277 9 1370 F AAGAC 126 1447 Ri CAC 480 CYA BA 135 CGAAGTACAATACAAGA CYABA -1448 TC-TTAACGGCTTCAAGACC 3 1379 F CAAAAGAAGG 127 1467 i c 4B2 CYABA_135 CYA PA 1448 3_1379TMO TCGAAGTACAATACAAG 1467 89.00 TTGTTAACGGCTTCAAGAC 351 D0 F ACAAAAGAAGG 128 9. cc; 483 CYABA 135 ACAATACAAGACAAAAG CYA_-9BA -1447 31 9 137 9 F AAGG 129 1461 R CGGCTTCAAGACCCC 481 CYABA_914 CAGGTTTAGTACCAGAA CYA BA-999- ACCACTTTTAATAAGGTTT 32 937 F CATGCAG 130 1026 P. GTAGCTAAC 484 CYABA_916 GGTTTAGTACCAGAACA CYA BA 1003 CCACTTTTAATAAGGTTTG 33 93Z5 F TGC 131 105 9. TAGC 478 DNAEBC_42 CGGCGIACTTCAACGAC DNAYKEC_503 CGCGGTCGGCTCGTTGATG 115 8 449 F- AGCCA 132 522P-R A 485 GALEFRT_1 TTATCAGCTAGACCTTT GALE_FRT_24 TCACCTACAGCTTTAAAGO 1102 68 29 9 F TAGGTAAAGCTAAGC 133 1 269 P. CAGCAAAATS 486 GALE FRT 3 TCCAAGGTACAOTAAAC GALEFRT -39 TCTTCTGTAAnAGGGTGGTT 1104 08 3359 F TTACTTGAGCTAATG 134 0 42-2 R TATTATTCATCCCA 487 GALEFRT 8 TCAAAAAGCCCTAGGTA GALEFRT_90 TAGCCTTGGCAACATCAGC 1103 34 865 F AAGAGATTCCATATC 135 1 925 R AAAACT 488 GLTARKP 1 TCCGTTCTTACAAATAG GLTA RKP_11 TTGGCGACGGTATACCCAT 1092 023 1055 F CAATAGAACTTGAAGC 136 29 1156 P. AGCTTTATA 489 GITARKP_1 043_1072_2 TGGAGCTTGAAGCTATC GLTARKP_11 TGAACATTTGCGACGGTAT 1093 F GCTCTTAAAGATG ,137 38 1162 P. ACCCAT 490 WO 2006/071241 WO 206/01241PCT/US2005!006133 GLTDRKP_1 043_1072_3 TGGAACTTGAAGCTCTC GLTA RKP 11 TGTGAACATTTGCGACGGT 1094 F GCTCTTAAAGATG 138 38 1164 R ATACCCAT 492 GLTARKP_-1 TGGGACTTGAAGCTATC GLTARH? 11 TGAACATTTGCGACGGTAT 1090 043 1072 F GCTCTTAAAGATG- 139- 38 1162 R ACCCAT 491 GLTARH?_4 TCTTCTCATCCTATGGC GLTA RK?_49 TGGTGGGTATCTTAGCAAT 1091 00 428B F7 TATTATGCTTGC 140 9 529 R CATTCTAAT.AGC 493 GLTA_RKP_4 TCTTCTCATCCTATGGC GLTA -RK?_50 TGCGATGGT.AGGTATCTTA 1095 00 428 F TATTATGCTTGC 140 5 534 R GCAATCATTCT 494 GROLEC_21 GGTGAAAGAAGTTGCCT GROJLEC 328 TTCAGGTCCATCGGGTTCA 224 9 202 F CTAAAGC 141 350R TGCC 496 GROLEC_49 ATGGACAAGGTTGGCAA GROL EC_577 TAGCCGCGGTCGAATTGCA 280 6- 518 -F GGAAGG 142 596-R T 49E GROLEC -51 AAGGAAGGCGTGATCAC GROL_-EC_571 CCGCGGTCGAATTGCATGC 281 1 5 36 F CGTTGAAGA 143 593 R CTTC 497 GROLEC_94 TGGAAGArCTGGGTCAG GROLEC_103 CAATCTGCTSACGGATCTG 220 1 959 F GC 144 9 1080 R AGO 495 GYRAAF1005 GYRA, AF100 TOTGCCCGTGTOGTTGG 57_119 142 TCGAACCGAAGTTACCCTG 924 557 4 23 F TGA 145 R ACCAT 499 GYRAAF100 GYRAAF1O05 557_7 0_-94 TCCATTGTTCGTATGGC 57_-178 201 TGOCAGCL'TAGTCATACGG 925 F TCAAGACT 146 R ACTTC 500 GYRB A130087 700_-19_40 TCAGGTGGCTTACACGG 00_1l1 140 TATTGCGGATCACCATGAT 9265 F CGTAG 147 R GATATTCTTGC 501 GYRB ABOO8 GYRBA30087 700_2 65_29 TCTTTCTTGAATGCTGG 00_36 9 395 TCGTTGAGATGGTTTTTAO 927 2 F TOTACCTATCC 148 R CTTCGTTG 502 GYRBAB008 GYRSAB0087 700 3 -68_-39 TCAACGAAGGT11AAAAC 00_-46494 TTTGTGAAACAGCGAACAT 928 4 F CATCTCAACG 149 R TTTCTTGGTA 503 GYRB ABOOB GYRBAO087 700_477 50 TGTTCGCTGTTTCACAA 00_611_632- TCACCOGCZ\TCACACCAG 925 4 F ACAACATTCCA 150 R TCA 504 GYRB ASOOB GYROAB0087 700_760_78 TACTTACI'TGAGAATCC 00_86 2 888 TCCTGCAATATCTAATGCA 949 7 F ACAAGCTGCAA 151 2 -R CTCTTACG 505 GYROABOOB GYRO ABO307 700_760_78 TACTTACTTGAGAATCC 00_8-62 888 ACCTGCAATATCTAATGCA 930 7 F- ACAAGCTGCAA 151 R CTCTTAOG 506 HFLB EC 10 TGGCGAACCTGGTGAAC H-FLEC_114 CTTTCGCTTTCTCGAACTC 222 82 1102 F GAAGC 152 4 1 168 -R AACCAT 507 HOPSCU 11 TAGTTGCTCAAACAGCT HEWSCJ 157 TCCCTAATAGTAGAAATAA 1128 3 134 F GGGCT 153 188- H TGCATCAGTAGC 509 HEJBCJ 76 TCCCGGAGCTTTTATGA HEJFB Ca 134 TAGCCCAGCTGTTTGAGCA.

1130 102 F CTAAAGCAGAT 154 135 R ACT 508 HUBCJ _76 TCCOGGAGCTTTTATGA HUPB_Ca_ -157 TCCCTAATAGTAGAAATAA.

1129 102 F CTAAAGCAGAT 154 188 H CTGCATCAGTAGC 510 ICD CXB 17 TCGCCGTGGAAAAATCC I CD CXB_224 TAGCCTTTTCTCCGGCGTA 1079 6 19 8 F- TACGCP 155 247 R GATCT 512 ICD OHS_92 TTCCTGACCGACCCATT IODOHS_172 TAGGATTTTTCCACGGCGG 1078 120 F ATTCOOTTTATC 156 194i H CATC 510 ICD OHS_93 TCCTGACCGACCCATTA ICDOHS_172 TAGGATTTTTCCACGGCGG 1077 120 F TTCCCTTTATC 157 194 R CATC 511 INFBSEC_11 GTCGTGAAAACGAGCTG INFOEC_117 221 03 1124 F GAAG 158 4 1191 H CATGATGGTCACAACCGG 513 IRFB EC 13 TGCGTTTACCGCAATGC INESEO 141 964 47 1367 F GTGC 159 4 1432 R TCGGCATCACGCCGTCGTC 514 INFBEC_13 TGCTCGTGGTGCACAAG INFO EC_143 TGCTGCTTTCGCATGGTTA 34 65 1393 F TAACGGATATTA 160 9 146 7 P ATTGCTTCAA 515 INFBEC_13 INFOEC_143 65_1393_TM TTGCTCGTGGTGCACAA. 9_146 7_TE00 TTGCTGOTTTOGCATGGTT 352 00 F GTAACGGATATTA 161 iP AATTGCTTCAA 516 INFSEC_19 CGTCAGGGTAAATTCCG INFO EC_203 AACTTCGCCTTCGGTCATG 223 69 1994 F TGAAGTTAA 162 8 2058 R TT 517 INV U22457 1558_ -1581 TGGTAACAGAGCCTTAT INV 0 22457_ TTGCGTTGCAGATTATCTT 781 F AGGCGCA 163 1619 1643 R TACCAA 518 INV_022457 TGGCTCCTTGGTATGAC INV 022457- TGTTAAGTGTGTTGCGGCT 778 515 539 F TCTGCTTC 164 571_598 R GTCTTTATT51 ISV 022457 TGCTGAGGCCTGGACCG INV_022457 TCACGCGACGAGTGCCATC 779 699 724 F ATTATTTAC 165 753-776 R CATTG 520 INVU22457 TTATTTACCTGCACTCC INVu22457 TPACCCAAAGCTGAAAGCT 780 834 858 F CACAACTG 166 942 966 R TTACTG 521 WO 2006/071241 WO 206/01241PCT/US2005!006133 -31- IPAI SGF 1 TCCTTGACCGCCTTTCC IFAHSGF_-17 TTTTCCAGCCATGCAGCGA 1106 13 134 F GATAC 167 2 191 R C 522 IPAHSGF_2 TGAGGACCGTGTCGCGC 18 IPAHSGF_-30 TCCTTCTGATGCCTGATGG 52 1105 58 277 F- TCA 68 1 327 R ACCAGGAG52 IPAH SGF -4 TCAGACCATGCTCGCAG IPAHSGF_5252 1107 62 486 F AGAAACTT 169 2 540 R TGTCACTCCCGACACGCCA 52 IS1111ANC IS1111A NCO 0029713686 TCAGTATGTATCCACCG 02971_6929 TAAACGTCCGATACCAATG 1080 6 6891-F TAGCCAC-EC 170 6954 B GTTCGCTC 525 IS1111ANC IS1111ANCO 002971_-745 TGGGTGACATTCATCAA 02971 R 7529 TCAACAACACCTCCTTATT 52 1081 6 7483 F TTTCATCGTTC 171 7554 RCCCACTC52 LEFBA_103 LEFBA_-R1119 AT CATGAC 52 3 10652-F TCAAGAAGAAAAAGAGC 172 1135R GAACATGAC 52 LEFEBA -103 CAAGAAGAAAAAGAGCT LEF _BA -1119 AGATAAAGAATCACGAATA 52 36 6 10666 F TCTA7AAAGAATAC 173 -1149 R TCAATTTGTASC52 LEFBA_756 AGCTTTTGCATATTATA LEFBA 843- TCTTCCAAGGATAGATTTA 53 37 781 F TCGAGCCAC 174 872 R TTTCTTGTI'CG53 LEF BA_756 -781_TMOD- TAGCTTTTGCATATTAT LEF_-BA -843_ FTCTTCCAAGGATAGATTT 353 F ATCGAGCCAC 175 872 TMOD R ATTTCTTGTTCG -531 LEFBA_758 CTTTTGCATATTATATC LEFBA 843- AGGATAGATTTATTTCTTG 38 778 F GAGO 176 865 R TTCG 529 LEF _5BA -795 TTTACAGCTTTATG7-AC LEFBA 883- 3 39 813 F CG 177 900 R TCTTGACAGCATCCGTTG 53 LEFBA_883 LEFBA 939 CAGATAAAGAATCGCTCCA 899 F CAACGGATGCTGGCAAG 178 958 R G 533 L.L NC00314 LL_-NC003143 3 2366996 TGTAGCCGCTAAGCACT 2367073 23 TCTCATCCCGATATTACCG 53 782 2367019 F ACCATCC 179 67097 R CCATGA53 LLNC00314 LLNC003143 3 2 367172 TGGACGGCATCACGATT 2367249_23 TGGCAACAGCTCAACACCT 783 23§67194 F CTCTAC 180 672-71 R TTGG53 MECA TY1405 TAGA ATAC MECA -Y14051 1_3645 367 3AGAATAC 369063719 TGATCCTGAATGTTTATAT 878 0-F AACGTCCGA 181 R CTTS2AACGCCT 536 MECA Y1405 MECA Y14051 1 -3774 38 TAAAACAAACTACGGTA 382835 877 23F ACATTGATCCA 182 P.TCAT53 MECAY1405 mECA_-Y14051 1_4507_453 TCAGGTACTGCTATCCA 455594581_ TGGATAGACGTCATATGAA 879 07F CCCTCAA 183 R GGTGTGCT 538 MECA Y1405 MECAY14051 1 -4510 -453 TGTACTGCTATCCACCC 4586 461 TTCTC5A3CA9 880 O F TCAA 1E4 BCATACA53 MECAY1405 MECAY14051 1 4520 453 4590_4600P54 882 OP F TUUUU'''A 185 R CaAtj'C'tUACaGtUaUaA 54 MECA Y1405 mECA_-Y14051 1_-4520_-453 4600_-4610P T 4 883 Op F TU'UaAUatUaUC'UaAA 185 R CaACaC'UaC'C'U'C 4 MECA Y1405 MECAY14051 1_4669 469 TCACCAGGTTCAACTCA 47654793_ TAACCACCCCAAGATTTAT 881 8BF AAAAATATTAACA 186 R CTTTTTGCCA 542 MECIAY140 NECIAY1405 51_-3315_-33 TTACACATATCGTGAGC 1_33673393 TGTGATATGGAGGTGTAGA 54 876 41 F AATGAACTGA 187 R AGGTGTTA54 OMPAAY485 OMPA AY4852 227_272 30 TTACTCCATTATTGCTT 27 3 4 398 GAGCTGCGCCAACGAATA 3 914 1 F GGTTACACTTTCC 188 R ATCGTC 544 OMPAAY485 OMPAAY4352 227_3511 33 TACACANACAATGGCGGT 27 -42 4 -453_ TACGTCGCCTTTAACTTGG 916 5 F AAAGATGG 189 R TTATATTCAGC 545 OMPA AY485 OMPAAY4B52 227_39_4 TGCGCAGCTCTTGGTAT 27_-492 519 TGCCGTAACATAGAAGTTA 915 1 F- CGAGTT 190 R CCGTTGATT 546 OMPA AY485 OMPA AY4852 227_41I5_44 TGCCTCGAAGCTGAATA 27_-514 -546 TCGGGCCTAGTTTTTAGTA 917 1 F TAACCAAGTT 191 R ATAAATCAGAAGT 547 OMPA AY485 OMPAAY4852 227_494_52 TCAACGGTAACTTCTAT 27 -56E9 -596 TCGTCGTATTTATAGTGAC 918 0OF GTTACTTCTG 192 R CAGCACCTA 548 OMPAAY485 om2AAY4852 227_-551_-57 TCAAGCCGTACGTATTA 27_658_680_ TTTAAGCGCCAGAAAGCAC 919 7 F TTAGGTGCTrG 193 R -CAAC50 WO 2006/071241 WO 206/01241PCT/US2005!006133 -32 OMPAAY4 852 227_555_58 TCCGTACGTATTATTAG 27_6 35 -662 TCAACACCAGCGTTACCTA 920 1 F GTGCTGGTCA 194 R AAGTACCTT 549 OMPAAY485 OMPA AY4852 227J_56_58 TCGTACGTATTATTAGG 27_659 683 TCGTTTAAGCGCCAGAAAG 921 3 F TGCTGGTCACT 195 R CACCAA 551 OMPA AY485 OMPA AY4852 227_657_67 TGTTGGTGCTTTCTGGC 27 7 9 765 TAAGCCAGCAAGAGCTGTA 922 9 F GCTTAA 196 R TAGTTCCA 552 OMPA AY485 OMPAAY4852 227_60 68 TGGTGCTTTCTGGCGCT 27_78 6 807 TACAGGAGCAGCACGCTTC 923 3 F- TAAACGA 197 R AAG 553 aMPBRKP_1 TCTACTGATTTTGGTAA 01MP KP_12 TAGCAGCAAAAGTTATCAC 1088 192 1221 F TCTTGCAGCACAG 198 88 1315 R ACCTGCAGT 554 OMPBRKP_3 TGCAAGTGGTACTTCAA 0MBS AP_35 TGGTTGTAGTTCCTGTAGT 1089 417 3440 F CATGGGG 199 20 3550 R TGTTGCATTAAC 555 OMPBAMP_8 TTACAGGAAGTTTAGGT 0MPG RKP 97 TCCTGCAGCTCTACCWGCT 1087 60 890 F- GGTAATCTAAAAGG 200 2 996 R CCATTA 556 PAGBA_122 CAGAATCAAGTTCCCAG PAGBA 190 CCTGTAGTAGAAGAGGTAA 41 142 F GGG 201 209- C 558 FAG BA_123 AGAATrCAAGTTCCCAGG FAG_BA.187 CCCTGTAGTAGAAGAGGTA 42 14-5 Y GGTTAC 202 210 R ACCAC 557 PAGBA 269 AATCTGCTATTTGGTCA FAGBA 326 43 27F GG 203 344 A GATTATCAC-CGGAAGTAG 559 PAGBA_655 GAAGGATATACGGTTGA FAG_BA 755_ 44 675 F TGTC 204 772 PR CCGTGCTCCP.TTTTTCAG 560 FAGBA_753 TCCTGAAAAATGGAGCA FAGBA -849 TCGGATAAGCTGCCACAAG 4 5 -777 F CGG 2059 868-R G 561 PAGBA '763 TGGAGCACGGCTTCTGA FAGBA 849 TCGGATAAGCTGCCACAAG 46 7 81 F TC 206 868 R G 562 PARCX9581 9_123 147 GGCTCAGCCATTTAGTT PARCX95CI9 TCGCTCAGCAATAATTCAC 912 F7 ACCGCTAT 207 232-260 R TATAAGCCGA 566 PARC X9581 TCAGCGCGTACAGTGGG PARC X95819 TTCCCCTGACCTTCGATTA 913 9 43 63 F TGAT 208 143 17C R AASGATAGC 563 PARCX9581 TGGTGACTCGGCATGTT PARC X95819 GGTATAACGCATCGCAGCA 911 9 87 110 F ATGAAGC 2C9 192 219 R AAAGATTTA 564 PARCX9581 TGGTGACTCGGCATGTT PARCx95819 TTCGGTATAACGCATCGCA 910 9 87 110 F ATCAACC 209 201 222 R GCA 565 9PLAAF0539 PLAAF'05394 45_-7186_-72 TTATACCGGAAACTTCC 5_7257_7280 TA2VGCGATACTGGCCTGC 773 11 F CGAAAGGAG 210 R AAGTC 567 PTA_jAF0539 IPLAAF05394 45_7377_74 1TGACATCCGGCTCACGT 5_7447462 TGTAAATTCCGCAAAGACT 770 02 F TATTATGGT 211 RTTGGCATTAG 568 PLAAF0539 FLAAF05394 45_-7382_74 TCCGGCTCACGTTATTA 5_7482_7502 1GGTCTGAGTACCTCCTTT 771 04 F TGGTAC 212 R GC 569 PLAAF0539 rLA AF05394 45_7481_75 TGCAAAGGAGGTACTCA 5_75 39_7562 'ATTGGAAATACCGGCAGC '772 03 F GACCAT 213 R ATCTC 570 RECAAF251 FECAAF2514 469169 19 TGACATGCTTGTCCGTT 69_-27-7300 TGGCTCATAAGACGCGCTT 909 0 F CAGGC 2'4 R GrTAGA 572 RECA AF251 AECAAF2514 469_Z3 68 TGGTA-A'TGTGCCTTCA 69_-140_-163 TTCAAGTGCTTGCTCACCA 908 F TTGATGCTG 215 A TTGTC 571 ANASEPSOP TGGCACGGCCATCTCCG RNASEP BDP_ TCGTTTCACCCTGTCATGC 1072 574 5'92 F TG 216 616 635; R CC 573 ABASE? BEB TGCGGWTAGGGAGCTTG RNASEPBEN_ TCCGATAAGCCGGATTCTG 1070 580 599 F AGC 217 665 686 R TGC 574 ANAEEP SEN TCCTAGAGGAATGGCTG RNASEP BEN TGCCGATAAGCCGGATTCT 1071 816 637 F CCACG 218 665 687 R GTGC 575 RNASEPBAN TACCCCAGGGAAAGTGC RNASEPBBM_ TCTCTTACCCCACCCTTTC 1112 325 34Z7 F CACAGA 219 402 428 R ACCCTTAC 576 ANASEF BPM TAAACCCCATCGGGAGC RNASEPBAN_ TGCCTCGTGCAACCCACCC 1172 461 4868 F AAGACCGAATA 220 542 561 2 P. G 57T? RNASEP B514 TAAACCCCATCGGGAGC RNASEF 5884- TGCCTCGCGOAACCTACCC 1111 461 468 F AAGACCGAATA 220 542 561 AR G 57E ANASEP 86 GAGGAAAGTCCATGCTC ENASEP_BS_3 GTAAGCCATGTTTTGTTCC 258 43 61 F- GC 221 63 384 R ATC 579 RNASEPBS GAGGAAAGTCCATGCTC ANASEPS_3 GTAAGCCATGTTTTGTTCC 259 43 61 F GC 221 63 384 R ATC 578 ABASE? BS GAGGAAAGTCAGT RIASEPEc_3 258 43 61 GO 22 45_362 R ATAAGCCGGGTTCTGTCG 581 WO 2006/071241 PCT/US2005!006133 R8IASEP BS_ GAGGAAAGTCCATGCTC RNASEP_SA_3 ATAAGCCATGTTCTGTTCC 258 43 61 F GC 221 58 379 R ATC 584 RNASEP CLB TAAGGATAGTGCAACAG RNASEP CLE TTTACCTCGCCTTTCCACC 1076 459 4867 F AGATATACCGCC 222 498 522 R CTTACC 57 9 RNASEP CLB TAAGGATAGTGCAACAG RNASEPCLB_ TGCTCTTACCTCACCTTC 1075 459 48 7 F AGATATACCGCC 222 498 526 R CACCCTTACC 580 RNASEPEC RNASEP BS 3 GTAGCCATGTTTTGTTCC 258 61 77 F GAGGAAAGTCCGCGCTC 223 63 384 R ATC 578 RNASEP_EC_ RNASEP EC -3 258 61 77 F GAGGAAAGTCCGGGCTC 223' 45 362 R ATAAGCCGGGTTC GTCG 581 RNASEP EC RNASEPEC_3 260 61 77 F GAGGAAAGTCCGGGCTC 223 45 362-R ATAAGCCGGGTTCTGTCG 581 RNASEP BC- RNASEP SA_3 ATAAGCCATGTTCTGTTCC 258 61 77 F GAGGAAAGTCCGGGCTC 223 58 379 R ATC 584 RNASEPRKP TCTAAATGGTCGTGCAG RNASEP R.P_ TCTATAGAGTCCGGACTTT 1085 264 2867 F 2TGCGTG 224 295 321 R CCTCGTGA 582 RNASEP RKP TGGTAAGAGCGCACCGG RNASEP RKP TCAAGCGATCTACCCGCAT 1082 419 448 F TAAGTTGGTAACA 225 542 565 R TACAA 583 RNASEPRKP TAAGAGCGCACCGGTAA RNASEP RB.P TCAAGCGATCTACCCGCAT 1083 422 443 F GTTGG 226 542 565 R. TACAA 583 RNASB RKP TGCATACCGGTAAGTTG 2NASEPRKP TCAAGCGATCTACCCGCAT 108 6 426 448 F GCAACA 22*7 542 565 R TACAA 583 RNASEPRKP TCCACCAAGAGCAAGAT RNASEP RKP TCAAGCGATCTACCCGCAT 1084 466 491 F CAAATA9GC 228 542 565 R. TACI4A 583 RNASEPSA- GAGGAAAGTCCATCCTC RNASEP_-BS_3 GIAAGCCATGTTTTGTTCC 258 31 49 F AC 229 63 384 R ATC 578 RNASBPA GAGGAAAGTCCATGCTC BBASBP EC 3 258 31 49 Fi AC 229 45 362 R ATAAGCCGGGTTCTGTCG 581 RNASSP SA GACCAAACTCCATGCTC RNASEPSA_3 ATAAGCCATGTTCTGTTC 258 31 49 F AC 229 58 379 R ATC 584 RNASBPSA GAGGAAAGTCCATGCTC PNASEP_SA_-3 ATAAGCCATGTTCTGTTCC 262 31 49 F AC 229 58 379 R ATC 584 RNASEP VBC TCCGCGGAGTTGACTCG SNASEP VBC TGACTTTCCTCCCCCTTAT 1098 331 349 F GT 230 388 414 R Cl\GTCTCC 585 GACCTACAGTAAGAGGT RPBODEC_739 TCCAAGTGCTGGTTTACCC 66 0 679 F TCTGTAATGAACC 231 762 R CATGG 591 RPLB 0_67-9_TMOD TGACCTACAGTAAGAGG RPLB-EC_739 TTCCAAGTGCTGGTTTACC 356 F TTCTGTAATGAACC 232 762 TMOD B CCATGG 592 RPLB-EC-66 TGTAATGAACCCTAATG RPLB_-EC_-735 CCAAGTGCTGGTTTACCCC 73 9 698 F ACCATCCACACGG 233 761 R ATGGAGTA 586 RPLB BC_67 TAATGAACCCTAATGAC RPLB BC 737 TCCAAGTGCTGGTTTACCC 74 1 700 F CATCCACACGGTG 234 762-R CATGGAG 590 RFLB EC 68 CATCCACACGGTGGTEGG RPLBEC 736 GTGCTGGTTTACCCCATGC 67 8 710 F- TGAAGC- 235 757 R AGT 587 ROLE EC 68 CATCCACACGGTGGTGG RFLBB C_743 TGTTPTGTATCCAAGTGCT 8 710 F TGAAGG 235 771 R GGTTTACCCC 593 RPLEBC_68 8_710_-TMOD TCATC;CACACGGTGGTG RPLBEC_736 TGTGCTGGTTTACCCCATG 357 F GTGAAGG 236 757 TMOD R GAGT 588 ROLEBC_69 TCCACACGGTGGTGGTG RPLB BC-737 TGTGCTGGTTTACCCCATG 449 0 7 10 F AAGG 237 758 R GAG 509 ROB BC 13 GACCACCTCGGCAACCG RPOBEC -143 113 36 1353 F T 23B '8 1455 R TTCGCTCTCGGCCTGGCC 594 RPOB BC 15 TCAGCTGTCGCAGTTCA BPOOSBC_163 TCGTCGCGGIACTTCGAAGC 963 27 1549 F TGGACC 239 0 1649 R, C 595 RPOB BC 18 TATCGCTCAGGCGAACT RPODBEC_190 GCTGGATTCGCCTTTGCTA 72 45 18666 F CCAAC 240 9 1929 B CG 596 ROOBBC_18 RPOBBC_190 45_266_ TM TTATCGCTCAGGCGAAC 9_1929TMOD TGCTGGATTCGCCTTTGCT 359 00 F TCCAAC 241 RACG 597 TCGTTCCTGGAACACGA ROOB_BC_-204 TTGACGTTGCATGTTCGAG 962 05 2027 F TGACGC 242 1 2064 R CCCAT 598 RPOB BC_37 TCAACAACCTCTTGGAG RPOB BC_383 TTTCTTGAAGAGTATGAGC 69 62 3790 F GTAAAGCTCAGT 243 6 38Z5 P TGCTCCGTAAG 600 RPOBC-37 CTTGGAGGTAAGTCTCA ROODBEC_382 CGTATA.AGCTGCACCATAA i11 75 3803 F TTTTGGTGGGCA 244 9 3858 R GCTTGTAATGC 599 RPOB BC_37 TGGGCAGCGTTTCGGCG RPOB EC 386 TGTCCGACTTGACGGTTAG 940 98 38621 F AAATGGA 245 2 3869 2f R CATTTCCTG 604 RPOBEC 37 TGGGCAGCGTTTCSGCG ROD BC 386 TGTCCGACTTGACGGTCAG 939 98 38 21 F AAATGGA 245 2 38869 R CATTTCCTG 605 ROODBEC 37 GGGCAGCGTTTCGGCGA RPOB BC 386 GTCCGACTTGACGGTCAAC 289 99 3821 F AATGGA 246 2 3 888 *9 ATTTCCTG 602 RPOBBC_37 TGGGCAGCGTTTCGGCG RPOBBC_386 TGTCCGACTTGACGGTCAA 362 99 38 21 TM IAAATGGA 245 2 3888 TMOD CATTTCCTG 603 WO 2006/071241 WO 206/01241PCT/US2005!006133 34- 0OD F 9 9.909 EC 38 CAGCGTTTCG9.CGAAAT RPOB EC_386 CGACTTGACGGTTAACATT 288 02 3821 F GA 247 2 3885 R TCCTG 601 P9C EC 18_-10452 CAAAACTTATTAGGTAA P9C EC_109 TCAAGCGCCP.TCTCTTTCG 48 F GCGTGTTGACT 248 5 112 4 2 P. GTAATCCACAT 610 9.9CC EC 10 CAAAACTTATTAGGTAA RPCC EC 109 TC-AAGCGCCATTTCTTTTG 47 18 1045-F GCCTTTCACT 248 5 112 4 GTIAAACCACAT 611 CGTGTTGACTATTCGGG 9.2CCEC_109 ATTCAAGAGCCATTTCTTT 68 36 1060 F GCGTTCAG 249 7 1126 R. TGGTAAACCAC 622 9.9CCEC_11 TAAGAAGCCGGAAACCA PCCEC_213 GGCGCTTGTACTTACCGCA 49 4 140 F TCAACTACCG 250 232 P. C 617 RPOCEC_12 ACCCAGTGCTGCTGAAC 9POCEC_129 GTTCAAATGCCTGGATACC 227 56 1277 F CGTGC 251 5 1315 P. CA 613 9.9CCEC_13 CGCCGACTTCGACGGTG PCCEC 143 292 74 1393 F ACC 252 7 145 5 R GAGCATCAGCGTGCGTGCT 614 RPC CMC_13 RPCCEC_143 74_1393_TM TCGCCGACTTCGACGGT 7_1455 11403 TCACCATCAGCGTGCGTGC 364 OD F GACO 253 R T 615 9.9CC EC 15 TG9.CCC9AAAGAAGCTG RPCEC_162 ACGCGGGCATGCAGAGATG 229 84 1T04 F AGCG 254 3 1643 9 cc 616 RPOCCMC21 TCAGGA9.TCGTTCAACT RP9CCEC_222 TTACGCCATCACGCCACGC 978 45 2175 F CGATCTACATGATG 255 8 2247 R A 622 9.9CC EC 21 CAGCAGTCcGTTCAACTC RPOCCEC_222 290 46 2174 F GATCTACATGAT 256 7 224 5 Ri ACGCCATCAGGCCACGCAT 620 RPOCCEC_21 RP9CCMC222 46_-2174_TM TCAGGAGTCGTTCAACT 7_224Z5_TMCD TACCCCATCAGGCCACGCA 363 OD Fl CGATCTACATGAT 257 R T 621 9.9CCC21 78_2196-2 TCATTCCCGTGCCCGTG RP9C MC_222 TTGGCCATCAGACCACGC-A 51 F GT 258 5 224629. TAC 618 RPOC EC_21 TGATTCTGGTGCCCGTG RP9CCMC222 TTGGCCATCAGGCCACGCA 78 2196 F GT 259 5 2246 P. TAG 619 9.9CC EC 22 18 22412 CTTGCTGGTATGCGTCG 9.9CC EC_231 CGCACCATGCGTAGAGATG 53 F -TCTGATG 260 3 233 7 2 R. AAGTAC 623 RPOC-EC-22 CTGGCAGCTATGCGTGG RP9CCEC_231 CGCACCGTGGG'TGAGATG 52 18 2241 F TCTGATG 261 3 233 7 R AAGTAC 624 9.9CCMC22 9.9CC EC213 1 18 -2241 TM LCTGGCAGGTATGCGTG 3_2337-- 140D TCGCACCCTGGGTTGAGAT 354 OD F GTCTGATG 262 R GAAGTAC 625 RPOCEC_22 TGGTPATGCGTGGTCTGA 9.9CC EC_232 TG-CTAGACCTTTACGTGCA 958 23 2243 F TCGC 263 9 2352 9. CCGTG 626 P.POC MC 23 TGCTCGTAAGGGTCTGG RP9CC MC239 TICTAGACGACGGGTCAGG 960 34 2-357 F CGGATAC 264 C 2403 R TA1ACC 627 CGTCGTGTAATTAACCG RP9C CMC_865 ACGTTTTTCGTTTTGAACG 8 833 2 F TAACAACCG 265 891 R. ATAATGCT 629 9.900 EC 80 CGTCGGGTGATTAACCG 9.9CC MC_865 GITTTTCGTTGCCTACGAT 54 8 833 F TAACAACCG 266 889 9. GATOTIC 628 P9CCMC91 TATTGGACAACGGTCGT RPC CMC100 TTACCGAGCAGGTTCTGAC 961 7 9389 F- CGCGG 267 9 103'4 P. GGAAACG 607 RP00_EC 91 TCTGGATAACGGTCGTC 9.9CC MC_100 TCCAGCAGGTTCTGACGGA 959 8 9386 F GCGG 268 9 1031 P. AACG 606 PCCEC 99 CAAAGCTAAGCAAGGAC RP9CCEC_103 CGAACGGCCAGAGTAGTCA 57 3 1019 2 F GTPTCCGTCA 269 6 1059 2 R. ACACG 608 P9C EC 99 CAA.AGC-TAAGCAAGGTC RPOCCC103 CGAACGGCCTGAGTAGTCA 56 3 101I9 F GTTTCCGTCA 270 6 1059 9. ACACG 609 SP101_-SPET 9.ACCTTAATTGGAAAGA 59101_SPETl CCTACCCAACGTTCACCAA 11 1 29 F 1AACCCPAGAAGT 271 1 92 1169. GGGCAG 676 SP101_SPET SF101_SPETI 11_1_2f9_TM TAACCTTAATTGGAAAG 1 92_116_TM TCCTACCCAACGTTCACCA 446 0D F AAACCCAAGAAGT 272 CD R. AGGGCAG 677 SP101_SPET 59101_SPETl 11_-1154_-11 CAATACCGCAACAGCGG 1_1251_1277 GACCCCAACCTGGCCTTTT 79 9 TGGCTTGGG 273 R. GTCGTTGA 630 9101 SPET SP101 SPETI 11_315l-4_-11 TCAATACCGCAACAGCG 1 1251_1277 TGACCCCAACCTGGCCTTT 424 79 11400 F GTGGCTTGGG 274 TMCD P. TGTCGTTGA 631 SPICl SPET 11_118_ -147 GCTGGTGAAAATAACCC SF101_SPETI TGTGGCCGATTTCACCACC 76 F AGATGTCGTCTTC 275 1 213 238 P. TGCTCCT 644 SPlClSPET SP101_SPETI 11_116_147 TGCTGGTGAAAATAACC 1_213_238_T TTGTGGCCGATTTCACCAC 425 TMCD F CAGATGTCGTCTTC 276 MCOD P. CTGCTCCT 645 86 59101 S9ET CCCAAAAAAATCCAGCT 1277 SPl1l SPETI AAACTATTTTTTTAGCTAT 632 WO 2006/071241 WO 206/01241PCT/US2005!006133 11_1314_13 -ATTAGC 1_1403_1431 ACTCGAACAC 36 F R SF101 SFET SP101 SPETI 11_1314_13 TCGCAAAAAATCCAGC 1 1403§ 1431 TAAACTATTTTTTTAGCTA 426 36 TI4OD F WATTAGC 278 YMOD R TACTCGAACAC 633 SF101_SPET SP101_SPETi 11_14-08_14 CGAGTATAGCTAAAAAA 1_1486_1515 GGATAATTGGTCGTAACAA 87 37 F ATAGTATGACA 279 R GGGATAGTGAG 634 SF101 SFET SF101 SPETi 11_1408_14 TCGAGTATAGCTAAAAA 1_1486_1515 TGGATAATTGGTCGTAACA 4 27 37 TMOD F AATAGTTTATGACA 280 TM4OD iF AGGGATAGTGAG 635 SF101 SPET SF101_SPETI 11_1688_17 CCTATATTAATCGTTTA 1_1783_1808 ATATGATTATCATTGAACT 88 16 F CAGAAACTGGCT 281 R. GCGGCCG 636 SP101_SFET SF101 SPET1 11_168_17 TCCTATATTAATCGTTT 1 1783§ 1808 TATATGATTATCATTGAAC 428 16 TMCD F ACAGAAACTGGCT 282 TMOD F TGCGGCCG 637 SF101_SFET SP101 -SPET.

11_1711_17 CTGGCTAAAACTTTGGC 1 1808 1835 GCGTGACGACCTTCTTGAA 89 33 F AACGGT 283 R TTGTAATCA 638 SF101_SPEW SP101_-SFST1 11_1711_17 TCTGGCTAAAACTTTGG 1_1808_1835 TGCGTGACGACCTTCTTGA 429 33 TMOD F _CAACGGT 264 TMOD Ri ATTGTAATCA 639 SP101_SPET SF101_SFOT 11_1807_18 ATGATTACAATTCAAGA 1_1901_1927 TTGGACCTGTAATCAGCPG 35 F AGGTCGTCACGC 285 R AATACTGG 640 SF101_SPET SF101_-SPETi 11_1807_18 TATGATTACAATTCAAG 1_1901_1927 TTTGGACCTGTAATCAGCT 430 35 TMOD F AAGGTCGTCACGC 286 TMOD F GAATACTGG 641 SP101_SPET SF101_SFST1 11_196 7_19 TAACGGTTATCATGGCC 1_2062_2083 ATTGCCCAGAAATCAAATC 91 91-F AGATGSG 287 R ATC 642 SF101_SPET SF101_SFET1 11_1967_19 TTAACGGTTATCATGGC 1 2062 -2083 TATTGCCAGAAATCAAAT 431 91 TMOD CCAGATGGG 288 W1400 R CATC 643 11_216_243 AGCACG1'GGTGAAATCG SF101_SFET1 TGCCACTTTGACAACTCCT 77 GCCACAI'GATT 289 1 308-333 R GTTGCTG 654 SF101_SPET SF101_-SFET1 11_216_243 TAGCAGGTGGTGAAATC I1_308_-333_-T TTGCCACTTTGACAACTCC 432 TMOD F GGCCACATGATT 290 OD R TGTTGCTG 655 SF101_SFET SF101_SPETi 11_2260 22 CAGAGACCGTTTTATCC 1_2375_2397 TCTGGGTGACCTGGTGTTT 92 83 F tATCAGC 291 R TAGA 646 SF101 SFET SF101_SPETi 11_22160_22 TCAGAGACCGTTTTATC 1_2375_2397 TTCTGGGTGACCTGGTGTT 433 83 TMOD F CTATCAGC 292 TMOD R TTAGA 647 SF101_SPEW SF101_-SPETi I1 2375 23 TCTAAAACACCAGGTCA 1_2470_2497 AC-CTGCTAGATGAGCTTCT 93 99-F CCCAGEAAG 293 RGCCATGGCC 648 SF101_SPET SF101_SPETi 11_2375_23 TTCTAAAACACCAGGTC 1_'2470_-2497 TAGCTGCTAGATGAGCTTC 434 99 T840D F ACCCAGAAG 294 T MOD R TGCCATGGCC 649 SF101 SPET SP101 SFETi 11_2468_24 ATGGCCATGGCAGAAGC 1_2543 2570 CCATA.AGGTGACCGTCACC 94 87 F TCA 295 -R ATTCAAAGC 650 SF101_SPEW SF101_SPE;Ti 11_-24j8_24 TATGGCCATGGCAGAAG 1 2543 2570 TCCATAAGGTCACCGTCAC 435 87 TMOD F CTCA 296 TMO R CATTCAAAGC 651

SPICISPEW

11_266_ 295 CTWGTACTTGTGGCTCA SF101_SPETI GCTGCTTTGATGGCTGAAT 78 F7 CACGGCTGTTTGG 297 1 355 380 R CCCCTTC 661 SF101_SPEW SF101_SFET1 11_266_295 TCTTGTACTTGTGGCTC -!_355_380_T TGCTGCTTT(;ATGGCTGAA 436 TMOD F ACACGGCTGTTTGG 298 MOD R. TCCCCTTC 662 SF101_SPEW SF101 -SPETi 11_-2961_29 ACCATGACAGAAGGCAT 1_3023_3045 GGAATTTACCAGCGATAGA 84 F TTTGACA 299 F. CACC 652 SF101_SPEW SP101_SFET1 11_2961_ 29 T2CATGACAGAAGGCA 1_3023_3045 TGGAATTTACCAGCGATAG 437 84 TMOD F TTACA 300 TMOD R ACACC 653 SF101_SPET SF101_SEEi 11_3075 31 GATGACTWTTTAGCTAA 1_3168 3196 AATCGACGACCAWCTTGGA 96 03 F TGGTCAGGCAGC 331. R AAGATTTCTE 656 438 SF101 SPEW TGATGACTTAGCTA I332 SF101 SPET1 WF-AAWCGAOGACCATCTTGG 657) WO 2006/071241 WO 206/01241PCT/US2005!006133 -36 1 1_3168_3].Sb J1.iL.I.u 11_3075_31 ATGGTCA 3GCAGC 13168319

AAAGATTTCTC

SP101_SPET SF101_SF271 11_3085_31 TAGCTAATGGTCAGGCA 1_3170_3194 TCGACGACCATCTTGGAAA 448 04 F GCC 303 R GATTTC 658 sF101_SPET 11_322344 GTCAAAGTGGCACGTTT SP101_SPETI 79 F ACTGGC 304 1 423 441 R ATCCCCTGCTTCTGCTGCC 665 SP101 SPET SP101_SPETi 11_322344 TGTCAAAGTGGCACGTT 1_423_441_T TATCCCCTGCTTCTGCTGC 439 TMOD F TACTGGC 305 MOD R. C 666 SF101_SPET SF101_SPETi 11_-3386_34 AGCGTAAAGGTGAACCT 1_3480_3506 CCAGCAGTTACTGTCCCCT 97 03 F T 306 R CATCTTTG 659 SF101_SPET SP101_SPETi 11 338 6 34 TAGCGTAAAGGTGAACC 1_3480 3506 TCCAGCAGTTACTGTCCCC 440 03-TMOD-F TT 307 TMOD 9. TCATCTTTG 660 SF101_SPET SF101_SPEll 11_3511_35 CCTTCAGGAATCAATGA 1_-3605_ 3629 GGGTC-TACACCTCCACTTG 98 35 F TGGAGCAG 308 R. CATAAC 663 SF101 SPET SF101_SPETl 11_3511 35 TGCTTCAGGAATCAATS 1_3605_3629 TGGGTCTACACCTGCACTT 441 35 TMOD F ATGGAGCAG 309 TMOD R~ GCATAAC 664 SP101 SPET 11_358_387 GGGGATTCAGCCATCAA SF101_-SPETi CCAACCTTTTCCACAACAG F AGCAGCTATTGAC 310 1 448 473 R. AATCAGC 668 SF101_SF51 S2101_-SPET1 11_358_387 TGGGGATTCAGCCATCA 1_448_473_T TCCAACCTTTTCCACAACA 442 TMOD F, 3AGrCACCTATTGAC 311 MOD R GAATCAGC 669 SP101_5951 11_364 385 TCAGCCATCAAAGCAGC 59101_SPEll TACCTTTTCCACAACAGAA 447 -F TATTG 312 1 448 471 R. TCAGC 667 SP101_SPET 11_600_629 CCTTACTTCGPACTkTG SF101_SFET1 CCCATTTTTTCACGCATGC 81 F AATCTTTTgGAAG 313 1 686 714 R TGAAAATATC 670 SF101._SFET SF101_SPETl 11_600_629 TCCTTACTTCGAACTAT 1_686_714_T TCCCATTTTTTCACGCATG 443 TMOD F GAATCTTTTGGAAG 314 EMOD R. CTGAAAATATC 671 SP101 SPET 11_658_ 684 GGGGATTGATATCACCG SF101_SPETi GATTGGCGATAAAGTGATA 82 F ATAAGAAGAA 315 1 756 784 R. TTTTCTAAAA 672 82101_SPET SF101_-SFE127 11_6586 684 TGGGGATTGATATCACC 1 7)56_'784_T TGAkTTGGCGATAAAGTGAT 444 TROD F GATAAGAAGAA 316 M0CD R- ATTTTCTAAAA 673 SF101_SFET 11_776_801 TCGCCAATCAAAACTAA 52101_SPETl GCCCACCAGAAAGACTAGC 67 83 F GGGAATGGC 317 1 871 896 R AGGATAA67 SF101 SPET SF101_SPETi 11 776 801 TTCGCCAATCAAAACTA 1 871_896 _P TGCCCACCAGAAAGACTAG 445 TMOD F AGGGAATGGC 318 MOD R CAGGATAA 675 SP101_SF51 SF101_SPETI 11 893_8921 GGGCAACAGCAGCGGAT 1_988_1012_ CATGACAGCCAAGACCTCA 84 F- TGCGATTCCGCG 319 R CCCACC 678 SP101 SPET 59101_-SPETl 11_893_921 TGGGCAACAGCAGCGGA 1_988 1012 TCATGACAGCCAAGACCTC 423 'IMOD F TTGCGATTGCGCG 320 IMOD R ACCCACC 679 SSPEBA_11 TCAAGCAAACGCACAAT SSPEBA -196 TTGCACGTCTGTTTCAGTT 706 4 137 F CAGAAGC 321 222 R. GCAAATTC 683 SSFE BA_11 TCAAGCAAACGCACAAC SSPEBA 196 TtGCACGTU C OTTTCAGT 612 4 137P F aU'AGAGC 321 2229 R7 TGCAAATTC 684 5525_BA_11 CAAGCAAACGCACAATC SSPE_-BA_-197 TGCACGTCTGTTTCAGTTG 58 5 137 F AGAAGC 322 222 R. CAAATTC 686 S825_BA_11 5_137TMlOD TCAAGCAAACS3CACAAT SSPEBA-197 TTGCACGTCTGTTTCAGTT 355 F CRGAAC-C 321 222 TMOD R. CCAAATTC 687 5595_BA_12 SSFEBA_-197 TCTGTTTCAGTTGCAAATT 215 1 137 F AACGCACAATCAGAAGC 323 216 R C 685 S8P8_BA_12 TGCACAATCAGAAGCTA SSFE -BA -202 TTTCACAGCATGCACGTCT 699 3 153 F AGAAAGCGCAAGCT 324 231 R. GTTTCAGTTGC 688 SSFEBA_14 TGCAAGCTTCTGGTGCT 8525_-BA -242 TTGTGATTCTTTTGCAGCT 704 6 1 68 F AGCATT 325 267 R. GATTGTG 689 SSHE BA 15 TGCTTCTGGTGCTAGCA SSPEBA-243 TGATTGTTTTGCAGCTGAT 702 0 168 F- TT 326 264 R. TGT 691 5525_BA_15 TGCTTCTGGC'GU'C;AG 5525_BA_243 TGATTGTTTTGU AGUaTGA 0168P F UAT 326 264P R C'C'GT 691 WO 2006/071241 WO 206/01241PCT/US2005!006133 -37 SSPEBA 15 SSPE_-BA_-243 700 -6 168 F 5GGTGCTAGCATT 327 255 R TGCQAGCTGATTGT 690 SSPEBA 15 SSPE BA_243 608 -6 168? F TGGCaGUaCaAGtUaATT 327 255? R7 TGUAGU'TGAC'C'GT 690 SSPEBA_63 TGCTAGTTATGGTACAG SSPE_-BA_-163 TCATAACTAGCATTTGTGC 705 89 Fi AGTTTGCGAC 32 191 R TTTGAATGCT 682 S SPESA_72 TGGTACAGAGTTTGCGA SSPE B A_-163 TCATTTGTGCTTTGAATGC 703 89SF C 329 182 R T 661 SSPEBA_72 TGGTAUaAGAGCaC'C aG SSPE BA_-163 TCATTTGTGCC'C'C'GAAC 611 89P F UaGAC 329 182T R 'GUJ'T 681 SSPEBA_163 701 89 F TACAGAGTTTGCGAC 330 17? 7 R TGTGCTTTGAATGCT 680 SSPEBA_75 TAU'AGAGCaC'CCQG-J'G SSPE_-BA_-163 609F 89P F AC 330 177i R TGTGCC'C'CaGAAC'GU'T 680 TOXRVBC_1 TCGATT1AGGCAGCAACG TCXRVBC -22 TTCAAAACCTTGCTCTCGC _1099 3 5 150 F AAAGCCG 331 1 24 R CAI4ACAA 692 TFRPEA-Y094 TRPE_-AY0943 355_1 064 1 TCGACCTTTGGCAGGAA 5E 1171_119 TACATCGTTPQGCCCAAGA 905 086 F CTAGAQ 332 6 R TCAATCA 693 TRPZ AY094 TREAY0943 355_-1278_1 TCAAATC-TACAAG2GA 55_1392 141 TCCTCTTTTCAC-AGGCTCT 904 303 F AGTGCGTGA 333 8-R AC~TQATC 694 TRPE AY094 TRPEAY0943 355_1445_1 TGATGGCATGC;TGAAA 55_1551_158 TATTTGGGTTCATTCCAC 903 471 F TGGATATGTC 334 .0 Ri TCAGATTCTGG 695 TFRPEAY094 TRPE AY0943 355_1 467_1 ATGTCGATTGCAATCCG 55 15 69 -159 TGCGCGAGCTTTTATTTGG 902 491 F TACTTGTG 335 2 RGTTTC 696 TRPE AY094 TRPEAY0943 355_66 68 GTGCATGCGGATACAGA 55_769791 TTCAAAATGCGGAGGCGTA 69 906 8 F GCAGAG 336 R TGTG69 TRPE AY094 TRPEAY0943 355_757 77 TGCAAGCGCGACCAQAT 55_-864 -883 TGCCCAGGTACAACCTGCA 907 6 F AG337 R T 698 TUFBEC 22 GCACTATGCACACTAG TUFB_-EQ_284 TATAGCACCATCCATCTGA 114 5 251 F ATTGTCCTGG 338 309 RP GCGGCAC 706 TUJFB-EC-23 TTGACTGCQQAGGTCAC TUFBEQ_283 GCCGTCCATTTGAGCAGCA 9 259 2 -F GQTG 339 303 2 R cc 704 TtJFB EC 23 TAGACTGCCCAGGACAC TUFBEC_283 GCCGTCCATCTGAGCAGCA 59 9 259 F GCTG 340 303 R cc 705 TUFB-EQ 25 TGCACGCCGACTATGTT TUFB EC_337 TATGTGCTCACGAGTTTGC 942 1 278 F AAGAACATGAT 341 360 R GGCAT -707 TEJFB EC 27 TGATCAftGGTGTGCT T(JFB_-EC_-337 TGGATGTGCTCACGAGTCT 941 5 2 99 F QAGATGGA 342 362 R GPTGAT 708 AAGAkCGACCTGCACGGG TUFBEC_849 117 7 774 F C 343 967 R GCSCTCCACGTCTTCACGC 709 TUFB-EQ 95 CCACAQGCCGTTCTTCA TOEB_EQ_103 GGCATQACCATTTCCTTGT 293 7 979 F AQAACT 344 4 1058 R TUFBECQ_103 7_972_-TIOD TCCACACGCCGTTCTTC 4_1059_-T1400 TGGCATCACCATTTCCTTG 367 F AACAACT 345 R TCCTTCG 701 TUFB EC_97 AACTACCGTCCTQAGTT TUFBBEQ 104 GTTGTCACCAGGCATTACC 62 6 1000 2 F CTCTTCC 346 5 1068 2 R ATTTC 702 TUFBEQ 91 AACTACCGTCCGCAGTT TUFBEC 104 GTTGTCGCCAGGCATAACC 61 6 1000 F QTAQTTQQ 347 5 1060a R ATTTC '703 TUFB-EQ 98 CCACAGTTCTACTTCQG TUFBECQ103 TCQAGGQATTACQATTTCT 63 5 1012 F TATACTGAQG 348 3 1062 R. AQTQQTTCTGG 699 VALSEQ_11 CGTGGCGGCGTGGTTAT VALSECQ_119 ACGAACTGGATGTCGCQGT 225 05 1124 F QUA 349 5 12-14 R T 710 VALS EQ_11 CGTGGCGGCGTGGTTAT VALSEC_-119 CGGTACGAACTGGATGTCG 71 05 1124 F CGA 349 5 1218 R. CCGTT- 711 VAL.SEC_11 VALS_EC -119 05_1124 TM TCGTGGCGGCGTGGTTA 5_1218_TMOD TCGGTACGAACTGGATGTC 358 00 F TQGA 350 P. GCCGTT 712 VALS EQ 11 TAPGCTGACCGACCAGT VAISEQ_123 TTCGCGCATCCAGGAGAAG 965 28 1151 F GGTACGT 351 1 1257 R TAQATGTT 713 VALSEC 18 CGAQGQGQTGQGQTTCA V -ALSEQ 192 GQGTTQQAQAGCTTGTTGQ 112 33 1850 F Q 352 0 1943 R. AGAAG 714 VALS EQ 19 CTTQTGCAACAAGQ. TGT VALSTEQ_-194 TQGQAGTTQATQAGQAQGA 116 20 19 43 F GGAAQGQ 353 8 1970 R AGOG 715 VALSEQ_61 AQQGAGQAAGGAGACCA VALS_-ECQ 705 TATAAQGQAQATQGTQAGG 295 0 649 F- GC 354 727 R GTGA 71E WAAA z9692 TQTTGQTQTTTCGTGAG WAAA _Z96925 CAAGQGGTTTGQQTCAAAT 931 5 2 29 F TTQAGTAAATG 355 115 138 R AGTCA 717 932 WPAA Z9692 TGATTGGTTTQ~aTG 5 WA 992 GCCGGQGQQG 1 WO 2006/071241 PCT/US2005/006133 -38- 286 311 TGTTTCAGT 394 412 R [0088] Primer pair name codes and reference sequences are shown in Table 2. The primer name code typically represents the gene to which the given primer pair is targeted. The primer pair name includes coordinates with respect to a reference sequence defined by an extraction of a section of sequence or defined by a GenBank gi number, or the corresponding complementary sequence of the extraction, or the entire GenBank gi number as indicated by the label "no extraction." Where "no extraction" is indicated for a reference sequence, the coordinates of a primer pair named to the reference sequence are with respect to the GenBank gi listing. Gene abbreviations are shown in bold type in the "Gene Name" column.

Table 2: Primer Name Codes and Reference Sequences Organism Extraction Primer Reference Extracted gene or entire name GenBank coordinates of gi gene code Gene Name gi number number SEQ ID NO: 16S rRNA (16S Escherichia 719 ribosomal RNA coli 16S EC gene) 16127994 4033120..4034661 23S rRNA (23S Escherichia 720 ribosomal RNA coli 23S EC gene) 16127994 4166220..4169123 capc (capsule Bacillus Complement 721 CAPC BA biosynthesis gene) anthracis 6470151 (55628..56074) cya (cyclic AMP Bacillus Complement 722 CYA BA gene) anthracis 4894216 (154288..156626) dnaK (chaperone Escherichia 723 DNAK EC dnaK gene) coli 16127994 12163..14079 groL (chaperonin Escherichia 724 GROL EC groL) coli 16127994 4368603..4370249 hflb (cell Escherichia 725 division protein coli Complement HFLB EC peptidase ftsH) 16127994 (3322645..3324576) infB (protein Escherichia 726 chain initiation coli Complement INFB EC factor infB gene) 16127994 (3310983..3313655) lef (lethal Bacillus Complement 727 LEF BA factor) anthracis 21392688 (149357..151786) pag (protective Bacillus 728 PAG BA antigen) anthracis 21392688 143779..146073 rplB (50S Escherichia 729 ribosomal protein coli RPLB EC L2) 16127994 3449001..3448180 rpoB (DNA-directed Escherichia 730 RNA polymerase coli Complement RPOB EC beta chain) 6127994 4178823..4182851 rpcC (DNA-directed Escherichia 731 RNA polymerase coli RPOC EC beta' chain) 16127994 4182928..4187151 SP1OIET Concatenation SPET 1 comprising: Artificial 732 1 Sequence* 15674250 gki (glucose partial gene Complement kinase) sequences of (1258294..1258791) Streptococcus gtr (glutamine pyogenes complement transporter (1236751..1237200) protein) murl (glutamate 312732..313169 racemase) mutS (DNA mismatch complement WO 2006/071241 PCT/US2005!006133 -39repair protein) (1787602..1788007) xpt (xanthine 930977..931425 phosphoribosyl transferase) yqiL (acetyl-CoA- 129471.. 129903 acetyl transferase) tkt 1391844. .1391386 (transketolase) sspE (small acid- 733 soluble spore Bacillus SSPE BA protein) anthracis 30253828 226496..226783 tufB (Elongation Escherichia 734 TUEB BC factor Tu) ccli 16127994 4173523..4174707 valS (Vayl-tRNA Escherichia Complement 735 VALS EC synthetase) coli 16127994 (4481405..4478550) asps (Aspartyl- Escherichia 16127994 complement(1946777.. 736 ASPS EC tRNA synthetase) coli 1948546) 2996286 No extraction CAFlAF caf1 (capsular Yersinia GenBenk coordinates 053947 protein cafl) psstis used INV U22 Yersinia 1256565 74..3772 737 457 inv (invasin) pestis Y. pestis specific 16120353 No extraction chromosomal genes GenBank coordinates LLNCO0 difference Yersinia used 3143 region pestis DONTAX BoNT/A (neurotoxin Clostridium 40381 77..3967 738 52066 type A) botulinum 2791983 No extraction 739 MECAYl mecA methicillin Staphylococcus GenBank coordinates 4051 resistance gene aureus used trpE (anthranilate 20853695 No extraction TRPE AY synthase (large Acinetobacter GenBank coordinates 094355 component)) baumanli used 740 9965210 No extraction RECA AF recA (recombinase Acinetobacter Geneank coordinates 2514Z9 A) baumanii used 741 4240540 No extraction GYRAAF gyrA (DNA gyrase Acinetobacter GenBank coordinates 100597 subunit A) baumanii used 742 4514436 No extraction GYRB AB gyr (DNA gyrase Acinetobactr GenBank coordinates 008700 subunit B) baumanli used 743 waaA (3-ceoxy-D- 2765828 No extraction WAAAZ9 manno-octuLosonic- Acinetobacter GenBank coordinates 6925 acid transferase) baumanil used 744 Concatenation comprising: Artificial Sequence* partial gene tkt sequences of 1569415..1569873 (transketolase) Campylobacter CJSTCJ jejuni glyA (serine 367573..368079 hydroxymethyltrans ferase) 15791399 gltA (citrate complement synthase) (1604529. .1604930) espA (aspartate 96892..97168 ammonia lyase) 745 ginA (glutamine complement synthase) (657609..658085) pgm (phosphoglycerate 327773..328270 mutase) WO 2006/071241 PCT/US2005!006133 uncA (ATP 112163..112651 synthetase alpha chain) RNASEP_ Rase P Bordetella 33591275 Complement BDP (ribonuclease P) pertussjs (3226720..3227933) 746 RNASEP RNase P Burkholderia 53723370 Complement BKM (ribonuclease P) mallei (2527296..2528220) 747 ONASEP_ RNas6 P Bacillus 16077068 Complement BS (ribonuclease P) subtilis (2330250..2330962) 748 RNASEP RNase P Clostridium 18308982 Complement CLB (ribonuclease P) pefringens (2291757..2292584) 749 ENASEP_ MNae P Escherichia 16127994 Complement EC (ribonuclease P) coli (3267457..3268233 750 RNASEP RNase P Rickettsia 15603881 complement(605276..6 RKP (ribonuclease P) prowvzekii 06109) 751 FNASEP_ PNase P Staphylococcus 15922990 coeplement(1559869..

SA (ribonuclease P) aureus 1560651) 752 RNASEP M3ase P Vibrio 15640032 complemet(2580367..

VBC (ribonuclease P) cholerae 2581452) 753 icd (isocitrate Coxiella 29732244 complement (1143867..

ICD CXB dehydrogenase) burnetii 1144235) 754 multi-locus Acinetobacter 29732244 IS1llA insertion baumannii IS111LA element No extraction ompA (outer Rickettsia 40287451 OMPAAY membrane protein prowvzekil 485227 A) No extraction 755 ompE (outer R!ckettsia 15603881 OMPB RK membrane protei prowazekii complement(881264..8 P B) 86195) 756 GLTARK gltA (citrate Vibrio 15603801 complement(1062547..

P synthase) cholerae 1063857) 757 toxR Francisella 15640032 TOXR VE (transcription tularensis complement(1047143..

C regulator toxR) 1048024) 758 asd (Aspartate Franciselia 56707187 semialdehyde tularensis complement(438608..4 ASD FRT dehydrogenase) 39702) 759 GALE FR galE (UDP-glucose Shigella 56707187 T 4-epimerase) flexneri 809039..810058 760 IpAH-SG ipaH (invasion Campylobacter 30061571 F plasmid antigen) jejuni 2210775..2211614 761 Coxiella complement(849317..8 hupB (DNA-binding hurnetii 15791399 49819) HUPB CJ protein Hu-bete) 762 Concatenation comprising: Artificial 763 Sequence* partial gene sequences of Acinetobacter baumannii trpE (anthranilate synthase component

I))

adk (adenyLate Sequenced in-house ABMLST kinase) mutY (adenine glycosylase) fumC (fumarate hydratase) efp (elongation factor p) ppa (pyrophosphate phosphohydratase WO 2006/071241 PCT/US2005/006133 -41- [0089] Note: These artificial reference sequences represent concatenations of partial gene extractions from the indicated reference gi number. Partial sequences were used to create the concatenated sequence because complete gene sequences were not necessary for primer design.

The stretches of arbitrary residues "N"s were added for the convenience of separation of the partial gene extractions (100N for SP101_SPET11 (SEQ ID NO: 732); 50N for CJST_CJ (SEQ ID NO: 745); and 40N for AB_MLST (SEQ ID NO: 763)).

[0090] Example 2: DNA isolation and Amplification [0091] Genomic materials from culture samples or swabs were prepared using the DNeasy® 96 Tissue Kit (Qiagen, Valencia, CA). All PCR reactions are assembled in 50 pl reactions in the 96 well microtiter plate format using a Packard MPII liquid handling robotic platform and MJ Dyad® thermocyclers (MJ research, Waltham, MA). The PCR reaction consisted of 4 units of Amplitaq Gold®, lx buffer II (Applied Biosystems, Foster City, CA), 1.5 mM MgClz, 0.4 M betaine, 800 gM dNTP mix, and 250 nM of each primer.

[0092] The following PCR conditions were used to amplify the sequences used for mass spectrometry analysis: 95C for 10 minutes followed by 8 cycles of 95C for 30 seconds, 48C for seconds, and 72C for 30 seconds, with the 48C annealing temperature increased 0.9C after each cycle. The PCR was then continued for 37 additional cycles of 95C for 15 seconds, 56C for seconds, and 72C for 20 seconds.

[00931 Example 3: Solution Capture Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads [0094] For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, [pl of a 2.5 mg/mL suspension of BioClon amine terminated supraparamagnetic beads were added to 25 to 50 [tl of a PCR reaction containing approximately 10 pM of a typical PCR amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed 3x with 50mM ammonium MeOH or 100mM ammonium bicarbonate/50% MeOH, followed by three more washes with 50% MeOH. The bound PCR amplicon was eluted with 25mM piperidine, imidazole, 35% MeOH, plus peptide calibration standards.

WO 2006/071241 PCT/US2005/006133 -42- [0095] Example 4: Mass Spectrometry and Base Composition Analysis [0096] The ESI-FTICR mass spectrometer is based on a Bruker Daltonics (Billerica, MA) Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer that employs an actively shielded 7 Tesla superconducting magnet. The active shielding constrains the majority of the fringing magnetic field from the superconducting magnet to a relatively small volume. Thus, components that might be adversely affected by stray magnetic fields, such as CRT monitors, robotic components, and other electronics, can operate in close proximity to the FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT operating system. Sample aliquots, typically 15 pl, were extracted directly from 96-well microtiter plates using a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, NC) triggered by the FTICR data station. Samples were injected directly into a 10 [l sample loop integrated with a fluidics handling system that supplies the 100 1l /hr flow rate to the ESI source.

Ions were formed via electrospray ionization in a modified Analytica (Branford, CT) source employing an off axis, grounded electrospray probe positioned approximately 1.5 cm from the metalized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary was biased at 6000 V relative to the ESI needle during data acquisition. A countercurrent flow of dry N 2 was employed to assist in the desolvation process. Ions were accumulated in an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode, prior to injection into the trapped ion cell where they were mass analyzed.

Ionization duty cycles 99% were achieved by simultaneously accumulating ions in the external ion reservoir during ion detection. Each detection event consisted of 1M data points digitized over 2.3 s. To improve the signal-to-noise ratio 32 scans were co-added for a total data acquisition time of 74 s.

[0097] The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOFTM. Ions from the ESI source undergo orthogonal ion extraction and are focused in a reflectron prior to detection. The TOF and FTICR are equipped with the same automated sample handling and fluidics described above. Ions are formed in the standard MicroTOFTM ESI source that is equipped with the same off-axis sprayer and glass capillary as the FTICR ESI source.

Consequently, source conditions were the same as those described above. External ion accumulation was also employed to improve ionization duty cycle during data acquisition. Each detection event on the TOF was comprised of 75,000 data points digitized over 75 ps.

00 [0098] The sample delivery scheme allows sample aliquots to be rapidly injected into the electrospray source at high flow rate and subsequently be electrosprayed at a much lower flow rate for improved ESI sensitivity. Prior to injecting a sample, a bolus of buffer was Sinjected at a high flow rate to rinse the transfer line and spray needle to avoid sample C s contamination/carryover. Following the rinse step, the autosampler injected the next sample and the flow rate was switched to low flow. Following a brief equilibration delay, data acquisition commenced. As spectra were co-added, the autosampler continued rinsing the syringe and picking up buffer to rinse the injector and sample transfer line. In general, two

(NO

C syringe rinses and one injector rinse were required to minimize sample carryover. During a C 10 routine screening protocol a new sample mixture was injected every 106 seconds. More Srecently a fast wash station for the syringe needle has been implemented which, when

C

i combined with shorter acquisition times, facilitates the acquisition of mass spectra at a rate of just under one spectrum/minute.

[0099] Raw mass spectra were post-calibrated with an internal mass standard and deconvoluted to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides. Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 500 molecules per well for the ribosomal DNA-targeted primers and 100 molecules per well for the protein-encoding gene targets. Calibration methods are commonly owned and disclosed in U.S. Provisional Patent Application Serial No. 60/545,425.

10100] Example 5: De Novo Determination of Base Composition of Amplification Products using Molecular Mass Modified Deoxynucleotide Triphosphates [0101] Because the molecular masses of the four natural nucleobases have a relatively narrow molecular mass range (A 313.058, G 329.052, C 289.046, T 304.046 See Table a persistent source of ambiguity in assignment of base composition can occur as follows: two nucleic acid strands having different base composition may have a difference of about 1 Da when the base composition difference between the two strands is G A (-15.994) combined with C T 15.000). For example, one 99-mer nucleic acid strand having a base composition of A 27

G

30

C

21

T

21 has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A 26

G

3 1

C

22

T

20 has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a 1292052 I:JIN WO 2006/071241 PCT/US2005/006133 -44molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor.

[0102] The present invention provides for a means for removing this theoretical 1 Da uncertainty factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural nucleobases. The term "nucleobase" as used herein is synonymous with other terms in use in the art including "nucleotide," "deoxynucleotide," "nucleotide residue," "deoxynucleotide residue," "nucleotide triphosphate or deoxynucleotide triphosphate (dNTP).

[0103] Addition of significant mass to one of the 4 nucleobases (dNTPs) in an amplification reaction, or in the primers themselves, will result in a significant difference in mass of the resulting amplification product (significantly greater than 1 Da) arising from ambiguities arising from the G A combined with C T event (Table Thus, the same the G A (-15.994) event combined with 5-Iodo-C T (-110.900) event would result in a molecular mass difference of 126.894. If the molecular mass of the base composition A 27

G

3 0 5-Iodo-C 21

T

21 (33422.958) is compared with A 26

G

31 5-Iodo-C 22

T

20 (33549.852) the theoretical molecular mass difference is +126.894. The experimental error of a molecular mass measurement is not significant with regard to this molecular mass difference. Furthermore, the only base composition consistent with a measured molecular mass of the 99-mer nucleic acid is A 2 7

G

30 Iodo-C 21

T

21 In contrast, the analogous amplification without the mass tag has 18 possible base compositions.

Table 3: Molecular Masses of Natural Nucleobases and the Mass-Modified Nucleobase lodo-C and Molecular Mass Differences Resulting from Transitions Nucleobase Molecular Mass Transition a Molecular Mass A 313.058 -9.012 A 313.058 -24.012 A 313.058 A-->5-Iodo-C 101.888 A 313.058 15.994 T 304.046 9.012 T 304.046 -15.000 T 304.046 T-->5-Iodo-C 110.900 T 304.046 25.006 C 289.046 24.012 C 289.046 15.000 WO 2006/071241 PCT/US2005/006133 C 289.046 40.006 414.946 5-Iodo-C-->A -101.888 414.946 5-Iodo-C-->T -110.900 414.946 5-Iodo-C-->G -85.894 G 329.052 -15.994 G 329.052 -25.006 G 329.052 -40.006 G 329.052 G-->5-odo-C 85.894 [0104] Example 6: Data Processing [0105] Mass spectra ofbioagent identifying amplicons are analyzed independently using a maximum-likelihood processor, such as is widely used in radar signal processing. This processor, referred to as GenX, first makes maximum likelihood estimates of the input to the mass spectrometer for each primer by running matched filters for each base composition aggregate on the input data. This includes the GenX response to a calibrant for each primer.

[0106] The algorithm emphasizes performance predictions culminating in probability-ofdetection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database is used to define the mass base count matched filters. The database contains the sequences of known bacterial bioagents and includes threat organisms as well as benign background organisms. The latter is used to estimate and subtract the spectral signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. the maximum likelihood process is applied to this "cleaned up" data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.

[0107] The amplitudes of all base compositions of bioagent identifying amplicons for each primer are calibrated and a final maximum likelihood amplitude estimate per organism is made based upon the multiple single primer estimates. Models of all system noise are factored into this two-stage maximum likelihood calculation. The processor reports the number of molecules of each base composition contained in the spectra. The quantity of amplification product WO 2006/071241 PCT/US2005/006133 -46corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction.

[0108] Example 7: Use of Broad Range Survey and Division Wide Primer Pairs for Identification of Bacteria in an Epidemic Surveillance Investigation [0109] This investigation employed a set of 16 primer pairs which is herein designated the "surveillance primer set" and comprises broad range survey primer pairs, division wide primer pairs and a single Bacillus clade primer pair. The surveillance primer set is shown in Table 4 and consists of primer pairs originally listed in Table 1. This surveillance set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Primer pair 449 (non-T modified) has been modified twice. Its predecessors are primer pairs 70 and 357, displayed below in the same row. Primer pair 360 has also been modified twice and its predecessors are primer pairs 17 and 118.

Table 4: Bacterial Primer Pairs of the Surveillance Primer Set Primer Forward Primer Name Forward Reverse Primer Name Reverse Target Gene Pair Primer Primer Nn. (SEQ ID (SEQ ID NO:)

NO:)

346 16S EC 713 732 TMOD F 27 165 EC 789 809 TMOD_R 389 16S rRNA 16S EC 713 732 F 26 16S EC 789 839 388 16S rRNA 347 16S_EC 785 806 TMOD F 30 16 SEC 880_897 TMODR 392 16S rRNA 11 16S EC 785 806 F 29 16S EC 880 897 R 391 16S rRNA 348 16S EC 960 981 TMOD F 38 16S EC 1054_1073_TMODR 363 16S rRNA 14 16S EC 960 981 F 37 16S EC 1054 1073 R 362 16S rRNA 349 23SEC 1826 1843 TMOD F 49 23SEC 1906_1924_TMOD R 405 23S rRNA 16 23S EC 1826 1843 F 48 23S EC 1906 1924 R 404 23S rRNA 352 INFB EC 1365_1393_TMOD_F 161 INFB_EC_1439_1467_TMODR 516 infB 34 INFB EC 1365 1393 F 160 INFB EC 1439 1467 R 515 infB 354 RPOC EC_2218_2241 TMOD F 262 RPOC EC 2313_2337_TMODR 625 rpoC 52 RPOC EC 2218 2241 F 261 RPOC EC 2313 2337 R 624 rpoC 355 SSPE BA 115 137 TMOD F 321 SSPEBA 197 222 TMODR 687 sspE 58 SSPE BA 115 137 F 322 SSPE BA 197 222 R 686 sspE 356 RPLB-EC 650 579 TMOD F 232 RPLB EC 739 762 TMODR 592 rplB 66 RPLB EC 650 679 F 231 RPLB EC 739 762 R 591 rplB 358 VALS EC 1105 1124 TMOD F 350 VALS_EC_1195_1218_TMODR 712 valS 71 VALS EC 1105 1124 F 349 VALS EC 1195 1218 R 711 valS 359 RPOB EC 1845 1866 TMOD F 241 RPOB EC_1909 1929 TMOD R 597 rpoB 72 RPOB EC 1845 1866 F 240 RPOB EC 1909 1929 R 596 rpoB 360 23S EC 2646 2667 TMOD F 60 235 EC 2745 2765_TMODR 416 23S rRNA 118 23_ EC 2646 2667_F 59 23S EC 2745_2765_R 415 23S rRNA 17 23S EC 2645 2669 F 58 23S EC 2744 2761 R 414 23S rRNA WO 2006/071241 PCT/US2005/006133 -47- 361 16S_EC_1090_1111 2 TMODF 5 16S EC 1175_1196_TMOD R 370 16S rRNA 3 16S EC 1090 1111 2 F 6 16S EC 1175 1196 R 369 16S rRNA 362 RPOBEC_3799_3821 TMODF 245 RPOB EC 3862_3888_TMOD R 603 rpoB 289 RPOB EC 3799 3821 F 246 RPOB EC 3862 3888 R 602 rpoB 363 RPOCEC_2146 2174 TMOD_F 257 RPOC EC 2227_2245_TMOD_R 621 rpoC 290 RPOC EC 2146 2174 F 256 RPOC EC 2227 2245 R 620 rpoC 367 TUFB EC 957 979 TMOD_F 345 TUFB EC 1034_1058_TMOD R 701 tufB 293 TUFB EC 957 979 F 344 TUFB EC 1034 1058 R 700 tufB 449 RPLB EC 690 710 F 237 RPLB EC 737 758 R 589 rplB 357 RPLB_EC_688_710_TMOD_F 236 RPLB EC 736 757 TMOD R 588 rplB 67 RPLB EC 688 710 F 235 RPLB EC 736 757 R 587 rplB [0110] The 16 primer pairs of the surveillance set are used to produce bioagent identifying amplicons whose base compositions are sufficiently different amongst all known bacteria at the species level to identify, at a reasonable confidence level, any given bacterium at the species level. As shown in Tables 6A-E, common respiratory bacterial pathogens can be distinguished by the base compositions ofbioagent identifying amplicons obtained using the 16 primer pairs of the surveillance set. In some cases, triangulation identification improves the confidence level for species assignment. For example, nucleic acid from Streptococcus pyogenes can be amplified by nine of the sixteen surveillance primer pairs and Streptococcus pneumoniae can be amplified by ten of the sixteen surveillance primer pairs. The base compositions of the bioagent identifying amplicons are identical for only one of the analogous bioagent identifying amplicons and differ in all of the remaining analogous bioagent identifying amplicons by up to four bases per bioagent identifying amplicon. The resolving power of the surveillance set was confirmed by determination of base compositions for 120 isolates of respiratory pathogens representing different bacterial species and the results indicated that natural variations (usually only one or two base substitutions per bioagent identifying amplicon) amongst multiple isolates of the same species did not prevent correct identification of major pathogenic organisms at the species level.

[0111] Bacillus anthracis is a well known biological warfare agent which has emerged in domestic terrorism in recent years. Since it was envisioned to produce bioagent identifying amplicons for identification of Bacillus anthracis, additional drill-down analysis primers were designed to target genes present on virulence plasmids of Bacillus anthracis so that additional confidence could be reached in positive identification of this pathogenic organism. Three drilldown analysis primers were designed and are listed in Tables 1 and 5. In Table 5 the drill-down set comprises primers with T modifications (note TMOD designation in primer names) which WO 2006/071241 PCT/US2005/006133 -48constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

Table 5: Drill-Down Primer Pairs for Confirmation of Identification of Bacillus anthracis Primer Forward Primer Name Forward Reverse Primer Name Reverse Target Gene Pair Primer Primer No. (SEQ ID (SEQ ID NO:)

NO:)

350 CAPC BA 274 303 TMODF 98 CAPC_BA_349_376_TMOD_R 452 capC 24 CAPC BA 274 303 F 97 CAPC BA 349 376 R 451 capC 351 CYA BA 1353_1379 TMODF 128 CYA BA 1448_1467 TMODR 483 cyA CYA BA 1353 1379 F 127 CYA BA 1448 1467 R 482 cyA 353 LEF BA 756 781 TMOD F 175 LEF_BA_843 872_TMOD_R 531 lef 37 LEF BA 756 781 F 174 LEF BA 843 872 R 530 lef [0112] Phylogenetic coverage of bacterial space of the sixteen surveillance primers of Table 4 and the three Bacillus anthracis drill-down primers of Table 5 is shown in Figure 3 which lists common pathogenic bacteria. Figure 3 is not meant to be comprehensive in illustrating all species identified by the primers. Only pathogenic bacteria are listed as representative examples of the bacterial species that can be identified by the primers and methods of the present invention. Nucleic acid of groups of bacteria enclosed within the polygons of Figure 3 can be amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand comer of each polygon. Primer coverage for polygons within polygons is additive. As an illustrative example, bioagent identifying amplicons can be obtained for Chlamydia trachomatis by amplification with, for example, primer pairs 346-349, 360 and 361, but not with any of the remaining primers of the surveillance primer set. On the other hand, bioagent identifying amplicons can be obtained from nucleic acid originating from Bacillus anthracis (located within 5 successive polygons) using, for example, any of the following primer pairs: 346-349, 360, 361 (base polygon), 356, 449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350, 351 and 353 (fifth polygon). Multiple coverage of a given organism with multiple primers provides for increased confidence level in identification of the organism as a result of enabling broad triangulation identification.

[0113] In Tables 6A-E, base compositions of respiratory pathogens for primer target regions are shown. Two entries in a cell, represent variation in ribosomal DNA operons. The most predominant base composition is shown first and the minor (frequently a single operon) is indicated by an asterisk Entries with NO DATA mean that the primer would not be expected to prime this species due to mismatches between the primer and target region, as determined by theoretical PCR.

WO 2006/071241 PCT/US2005!006133 -49- Table 6A Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 346, 347 and 348 Primer 346 Primer 347 Primer 348 organism Strain [A G C T] [A G C T) [A G C T] Klebsiella [29 32 25 13) [23 38 28 26) [26 32 28 pneucfniae MGH78578 [29 31 25 13]* (23 37 28 26J* [26 31 28 CO-92 Biovar [29 30 28 29) Yersinia pestis rientalis [29 32 25 13] [22 39 28 26) [30 30 27 293* P12 (Biovar Yersinia pestis Medievalis) [29 32 25 13] [22 39 28 26) [29 30 28 29] [29 30 28 29) Yersinia pestis 91001 [29 32 25 13) [22 39 28 263 [30 30 27 291* Haemophilus influenzae KW20 [28 31 23 17) [24 37 25 273 [29 30 28 29] Pseudomonas [26 36 29 24] aeruglnosa PACI [30 31 23 15) [27 36 29 231* [26 32 29 29] Pseudoinonas fluorescens PfC-1 [30 31 23 15) [26 35 29 251 [28 31 28 29) pseudoinonas putida KT2440 [30 31 23 151 [28 33 27 27) [27 32 29 28) Legionelo pneumophila Philadelphia-i [30 30 24 15) [33 33 23 27] [29 28 28 31) Francisella tularensis schu 4 [32 29 22 16) [28 38 26 26] [25 32 28 31] Bordetella pertussis Tohama i [30 29 24 16] [23 37 30 24) [30 32 30 26] Burklholderia [27 36 31 24) cepacla L2315 [29 29 27 14] [27 32 26 29] [20 42 35 19]* Burkholderia pseudomallei K96243 [29 29 27 14) [27 32 26 29] [27 36 31 24) Neisserla FA 1090, ATCC gonorrhoeae 700825 [29 28 24 18] [27 34 26 28) [24 36 29 27] Neisseria meningitides MC58 (serogroup B) [29 28 26 16] [27 34 21 27) [25 35 30 26) Neisseria meningitides serogroup C, FAM18 [29 28 26 16) [27 34 27 27] [25 35 30 26] Nelsseria meningitides 22491 (serogroup A) [29 28 26 16] [27 34 27 273 [25 35 30 26] Chlainydophila pneumoniae TW-183 [31 27 22 19] NO DATA [32 27 27 29) Chlamydophila pneumoniae AR39 [31 27 22 19] NO DATA [32 27 27 29] Chlamydophila pneumoniae CWLC29 [31 27 22 19) NO DATA [32 27 27 29] Chlamydophila pneumoniae J130 [31 27 22 19] NO DATA [32 27 27 29] Corynebacteriun diphtheriaef NCTC13129 [29 34 21 151 [22 38 31 251 [22 33 25 34) Mycobacterlzu avium k10 [27 36 21 15] [22 37 30 28] [21 36 27 Mycobacteriun avium 104 [27 36 21 15] [22 37 30 28] [21 36 27 Mycobacteriun tuberculosis CSU#93 [27 36 21 15) [22 37 30 28) [21 36 27 Mycoba cteri um tuberculosis CDC 1551 [27 36 21 15] [22 37 30 28) [21 36 27 Mycohacterium tuberculosis 137Rv (lab strain) [27 36 21 15] 122 37 30 283 [21 36 27 Mycoplasma pneuzoniae M129 [31 29 19 20] NO DATA NO DATA Staphylococcus [30 29 30 29) aureus MRSA252 [27 30 21 21) [25 35 3D 26] [29 31 30 29]* Staphylococcus [30 29 30 29] aureus MSSA476 [27 30 21 213 [25 35 30 26] [30 29 29 Staphylococcus [30 29 30 29] aurecs CCL [27 30 21 21) [25 35 30 26] [30 29 29 301* Staphylococcus [30 29 30 29) aurous M50 [27 30 21 21) [25 35 30 26] [30 29 29 Staphylococcus [30 29 30 29] aureus 24162 [27 30 21 21] :25 35 30 26] [30 29 29 301* WO 2006/071241 PCT/US2005/006133 staphylococcus [30 29 30 29] aureus N315 [27 30 21 21] [25 35 30 26] [30 29 29 Staphylococcus [25 35 30 26] [30 29 30 29] aureus NCTC 8325 [27 30 21 21] [25 35 31 261* [30 29 29 Streptococcus [24 36 31 agalactiae NEM316 [26 32 23 18] [24 36 30 26]* [25 32 29 Streptococcus equi NC 002955 [26 32 23 181 [23 37 31 25] [29 30 25 32] Streptococcus pyogenes MGASB232 [26 32 23 18] [24 37 30 25] [25 31 29 31] Streptococcus pyogenes MGAS315 [26 32 23 181 [24 37 30 25] [25 31 29 31] Streptococcus pyogenes SSI-1 [26 32 23 18] [24 37 30 25] [25 31 29 31] Streptococcus pyogenes MGAS10394 [26 32 23 18] [24 37 30 25] (25 31 29 31] Streptococcus pyogenes Manfredo (M5) [26 32 23 18] [24 37 30 25] [25 31 29 31] Streptococcus pyogenes SF370 (Ml) [26 32 23 18] [24 37 30 25] [25 31 29 311 Streptococcus pneumoniae 670 [26 32 23 18] [25 35 28 28] [25 32 29 Streptococcus pneumoniae R6 [26 32 23 18] [25 35 28 28] [25 32 29 Streptococcus pneumoniae TIGR4 [26 32 23 181 [25 35 28 28] [25 32 30 29] Streptococcus gcordonii NCTC7868 [25 33 23 18] [24 36 31 25] [25 31 29 31] Streptococcus [25 32 29 mitis NCTC 12261 [26 32 23 18] [25 35 30 26] [24 31 35 291* Streptococcus mutans UA159 [24 32 24 19] [25 37 30 24] [28 31 26 31] Table 6B Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 349, 360, and 356 Primer 349 Primer 360 Primer 356 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella pneumoniae MGH78578 [25 31 25 22] [33 37 25 271 NO DATA CO-92 Biovar [25 31 27 Yersinia pestis Orientalis [25 32 26 20]* [34 35 25 28] NO DATA P12 (Biovar [25 31 27 Yersinia pestis Mediaevalis) [25 32 26 20]* [34 35 25 28] NO DATA Yrsinia pestis 91001 [25 31 27 20] [34 35 25 28] NO DATA Haemophilus influenzae KW20 [28 28 25 20] [32 38 25 27] NO DATA Pseudomonas [31 36 27 27] aeruginosa PAO1 [24 31 26 20] [31 36 27 28]* NO DATA Pseudomonas [30 37 27 28] fluorescens PfO-1 NO DATA [30 37 27 28] NO DATA Pseudomonas putida KT2440 [24 31 26 20] [30 37 27 28] NO DATA Legionella pneumophila Philadelphia-i [23 30 25 23] [30 39 29 24] NO DATA Francisella tularensis schu 4 [26 31 25 19] [32 36 27 27] NO DATA Bordetella pertussis Tohama I [21 29 24 18] [33 36 26 27] NO DATA Burkholderia capacia J2315 [23 27 22 20] [31 37 28 26] NO DATA Burkholdleria pseudomallei K96243 [23 27 22 20] [31 37 28 26] NO DATA Neisseria gonorrhocae FA 1090, ATCC 700825 [24 27 24 17] [34 37 25 26] NO DATA Neisseria meningitidis MC58 (serogroup B) [25 27 22 18] [34 37 25 26] NO DATA Neisseria meningitidis serogroup C, FAM18 [25 26 23 18] [34 37 25 26] NO DATA Neisseria Z2491 (serogroup A) [25 26 23 18] [34 37 25 26] NO DATA WO 2006/071241 PCT/US2005/006133 51 meningitidis Chlamydophila pneumoniae TW-183 [30 28 27 18] NO DATA NO DATA Chlamydophila pneumoniae AR39 [33 28 27 18] NO DATA NO DATA Chlamydophila pneumoniae CWL029 [3D 28 27 18] NO DATA NO DATA Chlamydophila pneumoniae J138 [30 28 27 18] NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA [29 40 28 25] NO DATA Mycobacterium avium k10 NO DATA [33 35 32 22] NO DATA Mycobacterium avium 104 NO DATA [33 35 32 22] NO DATA Mycobacterium tuberculosis CSU#93 NO DATA [30 36 34 22] NO DATA Mycobacterium tuberculosis CDC 1551 NO DATA [30 36 34 22) NO DATA Mycobacterium tuberculosis H37Rv (lab strain) NO DATA [30 36 34 22] NO DATA Mycoplasma pneumoniae M129 [28 30 24 19] [34 31 29 28] NO DATA Staphylococcus aureus MRSA252 [26 30 25 20] [31 38 24 29] [33 30 31 27] Staphylococcus aureus MSSA476 [26 30 25 20] [31 38 24 29] [33 30 31 27] Staphylococcus aureus COL [26 30 25 20] [31 38 24 29] [33 30 31 27] Staphylococcus aureus Mu50 [26 30 25 201 [31 38 24 29] [33 30 31 27] Staphylococcus aureus MW2 [26 30 25 20] [31 38 24 29] [33 30 31 271 Staphylococcus aureus N3.5 [26 30 25 20] [31 38 24 29] [33 30 31 27] Staphylococcus aureus NCTC 8325 [26 30 25 20] [31 38 24 29] [33 30 31 27] Streptococcus agalactiae NEM316 [28 31 22 20] [33 37 24 28] [37 30 28 26] Streptococcus equi NC 002955 [28 31 23 19] [33 38 24 27] [37 31 28 Streptococcus pyogenes MGAS8232 [28 31 23 19] [33 37 24 26] [38 31 29 23] Streptococcus pyogenes MGAS315 [28 31 23 19] [33 37 24 28] [38 31 29 23] Streptococcus pyogenes SSI-1 [28 31 23 19] [33 37 24 28] [38 31 29 23] Streptococcus pyogenes MGAS10394 [28 31 23 19] [33 37 24 28] [38 31 29 23] Streptococcus pyogenes Manfredo (M5) [28 31 23 19] [33 37 24 28] [38 31 29 23] Streptococcus [28 31 23 191 pyogenes SF370 [28 31 22 201* [33 37 24 28] [38 31 29 23] Streptococcus pneumoniae 670 [28 31 22 20] [34 36 24 28] [37 30 29 Streptococcus pneumoniae R6 [28 31 22 20] [34 36 24 28] [37 30 29 Streptococcus pneumoniae TIGR4 [28 31 22 20] [34 36 24 28] [37 30 29 Streptococcus gordonii NCTC7868 [28 32 23 20] [34 36 24 28] [36 31 29 Streptococcus [28 31 22 mitis NCTC 12261 [29 30 22 20]* [34 36 24 28] [37 30 29 Streptococcus mutans UA159 [26 32 23 22] [34 37 24 27] NO DATA WO 2006/071241 PCT/US2005/006133 -52- Table 6C Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 449, 354, and 352 Primer 449 Primer 354 Primer 352 Organism Strain [AG C T] [A G C T] [A G C T] Klebsiella pneumoniae MGH78578 NO DATA [27 33 36 26] NO DATA CO-92 Biovar Yersinia pestis Orientalis NO DATA [29 31 33 291 [32 28 20 KIMS P12 (Biovar Yersinia pestis Mediaevalis) NO DATA [29 31 33 29] [32 28 20 Yersinia pestis 91001 NO DATA [29 31 33 29] NO DATA Haemophilus influenzae KW20 NO DATA [30 29 31 32] NO DATA Pseudomonas aeruginosa PAO1 NO DATA [26 33 39 24] NO DATA Pseudomonas fluorescens PfO-1 NO DATA [26 33 34 29] NO DATA Pseudomonas putida KT2440 NO DATA [25 34 36 27] NO DATA Legionella pneumophila Philadelphia-1 NO DATA NO DATA NO DATA Francisella tularensis schu 4 NO DATA [33 32 25 32] NO DATA Bordetella pertussis Tohama I NO DATA [26 33 39 24] NO DATA Burkholderia cepa cia J2315 NO DATA [25 37 33 27] NO DATA Burkholderia pseudomallei K95243 NO DATA [25 37 34 26] ND DATA Neisseria gonorrhoeae FA 1090, ATCC 700825 [17 23 22 10] [29 31 32 30] NO DATA Neisseria meningitidis MC58 (sergroup B) NO DATA [29 30 32 31] NO DATA Neisseria meningitidis serogroup C, FAM18 NO DATA [29 30 32 31] NO DATA Neisseria meningitidis Z2491 (serogroup A) NO DATA [29 30 32 31] NO DATA Chlamydophila pneumoniae TW-183 NO DATA NO DATA NO DATA Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA Mycobacterium avium k10 NO DATA NO DATA NO DATA Mycobacterium avium 104 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CSU#93 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CDC 1551 NO DATA NO DATA NO DATA Mycobacteri umn tuberculosis H37Rv (lab strain) NO DATA NO DATA NO DATA Mycoplasma pneumoniae M129 NO DATA NO DATA ND DATA Staphylococcus aureus MRSA252 [17 20 21 17] [30 27 30 35] [36 24 19 26] Staphylococcus aureus MSSA476 [17 20 21 17] [30 27 30 35] [36 24 19 26] Staphylococcus aureus COL [17 20 21 17] [30 27 30 35] [35 24 19 27] Staphylococcus aureus Mu50 [17 20 21 17] [30 27 30 35] [36 24 19 26] Staphylococcus aureus MW2 [17 20 21 17] [30 27 30 351 [36 24 19 26] WO 2006/071241 PCT/US2005/006133 -53- Staphylococcus aureus N315 [17 20 21 17] [30 27 30 35] [36 24 19 26] Staphylococcus aureus NCTC 8325 [17 20 21 17] [30 27 30 35] [35 24 19 27] Streptococcus agalactiae NEM316 [22 20 19 14] [26 31 27 38] [29 26 22 281 Streptococcus equi NC 002955 [22 21 19 131 NO DATA NO DATA Streptococcus pyogenes MGAS9232 [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes MGAS315 [23 21 19 121 [24 32 30 36] NO DATA Streptococcus pyogenes SSI-1 [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes MGAS10394 [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pyogenes Manfredo (M5) [23 21 19 12] [24 32 30 36] ND DATA Streptococcus pyogenes SF370 (Ml) [23 21 19 12] [24 32 30 36] NO DATA Streptococcus pneumoniae 670 [22 20 19 14] [25 33 29 35] [30 29 21 Streptoccus pneumoniae R6 [22 20 19 14] [25 33 29 35] [30 29 21 Streptococcus pneumoniae TIR4 [22 20 19 14] [25 33 29 35] [30 29 21 Streptococcus gordonii NCTC7868 [21 21 19 14] NO DATA [29 26 22 28] Streptococcus mitis NCTC 12261 [22 20 19 14] ]26 30 32 34] NO DATA Streptococcus mutans UA159 NO DATA NO DATA NO DATA Table 6D Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 355, 358, and 359 Primer 355 Primer 358 Primer 359 Organism Strain [A G C T] [A G C T] [A G C T] Klebsiella pneumoniae MGH78578 NO DATA [24 39 33 20] [25 21 24 17] CO-92 Biovar Yersinia pestis Orientalis NO DATA [26 34 35 21] [23 23 19 22] 912 (Biovar Yersinia pestis Mediaevalis) NO DATA [26 34 35 21] [23 23 19 22] Yersinia petis 91001 NO DATA [26 34 35 21] [23 23 19 22] Haemophilus influenzae KW20 NO DATA NO DATA NO DATA Pseudomonas aeruginosa PAO1 NO DATA NO DATA NO DATA Pseudomonas fluorescens Pf0-1 NO DATA NO DATA NO DATA Pseudomonas putida KT2440 ND DATA [21 37 37 21] NO DATA Legionella pneumophila Philadelphia-1 ND DATA NO DATA NO DATA Francisella tularensis schu 4 ND DATA NO DATA NO DATA Bordetella pertussis Tohama I NO DATA NO DATA NO DATA Burkholderia cepacia J2315 NO DATA NO DATA NO DATA Burkholderia pseudomallei K96243 NO DATA NO DATA NO DATA Neisseria gonorrhoeae FA 1090, ATCC 700825 NO DATA NO DATA NO DATA Neisseria meningitidis MC58 (serogroup B) NO DATA NO DATA NO DATA Neisseria meningitidia serogroup C, FAM168 NO DATA NO DATA NO DATA WO 2006/071241 PCT/US2005/006133 -54- Neisseria meningitidis Z2491 (serDgroup A) NO DATA NO DATA NO DATA Chlamydophila pneumoniae TW-183 NC DATA NO DATA NO DATA Chlamydophila pneumoniae AR39 NO DATA NO DATA NO DATA Chlamydophila pneumoniae CWL029 NO DATA NO DATA NO DATA Chlamydophila pneumoniae J138 NO DATA NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA Mycobacterium avium k10 NO DATA NO DATA NO DATA Mycobacterium avium 104 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CSU#93 NO DATA NO DATA NO DATA Mycobacterium tuberculosis CDC 1551 NO DATA NO DATA NO DATA Mycobacterium tuberculosis H37Rv (lab strain) NO DATA NO DATA NO DATA Mycoplasma pneumoniae M129 NO DATA NO DATA NO DATA Staphylococcus aureus MRSA252 NO DATA NO DATA NO DATA Staphylococcus aureus MSSA476 NO DATA NO DATA NO DATA Staphylococcus aureus COL NO DATA NO DATA NO DATA Staphylococcus aureus Mu50 NO DATA NO DATA NO DATA Staphylococcus aureus MW2 NO DATA NO DATA NO DATA Staphylococcus aureus N315 NO DATA NO DATA NO DATA Staphylococcus aureus NCTC 8325 NO DATA NO DATA NO DATA Streptococcus agalactiae NEM316 NO DATA NO DATA NO DATA Streptococcus equi NC 002955 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS8232 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS315 NO DATA NO DATA NO DATA Streptococcus pyogenes SSI-1 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS10394 NO DATA NO DATA NO DATA Streptococcus pyogenes Manfredo (M5) NO DATA NO DATA NO DATA Streptococcus pyogenes SF370 (MI) NO DATA NO DATA NO DATA Streptococcus pneumoniae 670 NO DATA NO DATA NO DATA Streptococcus pneumoniae R6 NO DATA NO DATA NO DATA Streptococcus pneumoniae TIGR4 NO DATA NO DATA NO DATA Streptococcus gordonii NCTC7868 NO DATA NO DATA NO DATA Streptococcus mitis NCTC 12261 NO DATA NO DATA NO DATA Streptococcus mutans UA159 NO DATA NO DATA NO DATA WO 2006/071241 PCT/US2005/006133 Table 6E Base Compositions of Common Respiratory Pathogens for Bioagent Identifying Amplicons Corresponding to Primer Pair Nos: 362, 363, and 367 Primer 362 Primer 363 Primer 367 Organism Strain [A G C T] [A G C TI [A G C T] Klebsiella pneumoniae MGH78578 [21 33 22 161 [16 34 26 26] NO DATA CO-92 Biovar Yersinia pestis Orientalis [20 34 18 20] NO DATA NO DATA P12 (Biovar Yersiia pestis Mediaevalis) [20 34 18 20] NO DATA NO DATA.

Yersinia pestis 91001 [20 34 18 20] NO DATA NO DATA paemophilus influenzae KW20 NO DATA NO DATA NO DATA pseudomnonas aeruginosa PAO1 [19 35 21 17] [16 36 28 22] NO DATA Pseudomonas fluorescens Pf0-i NO DATA [18 35 26 23] NO DATA Pseudomonas putida KT2440 NO DATA [16 35 28 23] NO DATA Legionella pneumophila Philadelphia-i NO DATA NO DATA NO DATA Francisella tularensis schu 4 NO DATA NO DATA NO DATA Bordetella pertussis Tohama I [20 31 24 17] [15 34 32 21] [26 25 34 19] Burkholderia cepacia J2315 [20 33 21 18] [15 36 26 25] [25 27 32 Burkholderia pseudomallei K96243 [19 34 19 20] [15 37 28 22] [25 27 32 Neisseria gonorrhoeae FA 1090, ATCC 700825 NO DATA NO DATA NO DATA Neisseria meningitidis MC58 (serogroup B) NO DATA NO DATA NO DATA Neisseria meningitidis serogroup C, FAM18 NO DATA NO DATA NO DATA Neisseria meningitidis Z2491 (serogroup A) NO DATA NO DATA NO DATA Chlamydophila pneumoniae TW-183 NO DATA NO DATA NO DATA Chlamraydophile pneumoniae AR39 NO DATA NO DATA NO DATA Chlamydophila pneumoniae CWLO29 NO DATA NO DATA NO DATA Chlanlydophila pneumoniae J130 NC DATA NO DATA NO DATA Corynebacterium diphtheriae NCTC13129 NO DATA NO DATA NO DATA Mycobacterium avium kl0 [19 34 23 16] NO DATA [24 26 35 19] Mycobacterium avium 104 [19 34 23 16] NO DATA [24 26 35 19] Mycobacteriumn tuberculosis CSU#93 [19 31 25 17] NO DATA [25 25 34 Mycobacterium tuberculosis CDC 1551 [19 31 24 18] NO DATA [25 25 34 Mycobacterium tuberculosis H37Rv (lab strain) [19 31 24 18] NO DATA [25 25 34 Mycoplasma pneumoniae M129 NO DATA NO DATA NO DATA Staphylococcus aureus MRSA252 NO DATA NO DATA NO DATA Staphylococcus aureus MSSA476 NO DATA NO DATA NO DATA Staphylococcus aureus COL NO DATA NO DATA NO DATA Staphylococcus aureus Mu50 NO DATA NO DATA NO DATA Staphylococcus aureus MW2 NO DATA NO DATA NO DATA Staphylococcus N315 NO DATA NO DATA NO DATA WO 2006/071241 PCT/US2005/006133 -56aureus Staphylococcus aureus NCTC 8325 NO DATA NO DATA NO DATA Streptococcus agalactiae NEM316 NO DATA NO DATA NO DATA Streptococcus equi NC 002955 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS8232 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS315 NO DATA NO DATA NO DATA Streptococcus pyogenes SSI-1 NO DATA NO DATA NO DATA Streptococcus pyogenes MGAS10394 NO DATA NO DATA NO DATA Streptococcus pyogenes Manfredo (M5) NO DATA NO DATA NO DATA Streptococcus pyogenes SF370 (Ml) NO DATA NO DATA NO DATA Streptococcus pneumoniae 670 NO DATA NO DATA NO DATA Streptococcus pneumoniae R6 [20 30 19 23] NO DATA NO DATA Streptococcus pneumoniae TIGR4 [20 30 19 23] NO DATA NO DATA Streptococcus gordonii NCTC7868 NO DATA NO DATA NO DATA Streptococcus mitis NC"C 12261 NO DATA NO DATA NO DATA Streptococcus mutans UA159 NO DATA NO DATA NO DATA [0114] Four sets of throat samples from military recruits at different military facilities taken at different time points were analyzed using the primers of the present invention. The first set was collected at a military training center from November 1 to December 20, 2002 during one of the most severe outbreaks of pneumonia associated with group A Streptococcus in the United States since 1968. During this outbreak, fifty-one throat swabs were taken from both healthy and hospitalized recruits and plated on blood agar for selection of putative group A Streptococcus colonies. A second set of 15 original patient specimens was taken during the height of this group A Streptococcus -associated respiratory disease outbreak. The third set were historical samples, including twenty-seven isolates of group A Streptococcus, from disease outbreaks at this and other military training facilities during previous years. The fourth set of samples was collected from five geographically separated military facilities in the continental U.S. in the winter immediately following the severe November/December 2002 outbreak.

[0115] Pure colonies isolated from group A Streptococcus-selective media from all four collection periods were analyzed with the surveillance primer set. All samples showed base compositions that precisely matched the four completely sequenced strains of Streptococcus pyogenes. Shown in Figure 4 is a 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair 00 number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of 0 other organisms.

(0116] In addition to the identification of Streptococcus pyogenes, other potentially pathogenic organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid was amplified by primer pair number 349 (SEQ ID NOs: 49 and 405) exhibited signals of bioagent identifying amplicons with molecular masses that were found to C 10 correspond to analogous base compositions of bioagent identifying amplicons of SStreptococcus pyogenes (A27 G32 C24 TI8), Neisseria meningitidis (A25 G27 C22 TI8), and

C

1 Haemophilus injluenzae (A28 G28 C25 T20) (see Figure 5 and Table 6B). These organisms were present in a ratio of 4:5:20 as determined by comparison of peak heights with peak height of an internal PCR calibration standard as described in commonly owned U.S. Patent Application Serial No: 60/545,425 which is incorporated herein by reference in its entirety.

101171 Since certain division-wide primers that target housekeeping genes are designed to provide coverage of specific divisions of bacteria to increase the confidence level for identification of bacterial species, they are not expected to yield bioagent identifying amplicons for organisms outside of the specific divisions. For example, primer pair number 356 (SEQ ID NOs: 232:592) primarily amplifies the nucleic acid of members of the classes Bacilli and Clostridia and is not expected to amplify proteobacteria such as Neisseria mentngitidis and Haemophilus injluenzae. As expected, analysis of the mass spectrum of amplification products obtained with primer pair number 356 does not indicate the presence of Neisseria meningitidis and Haemophilus injluenzae but does indicate the presence of Streptococcus pyogenes (Figures 3 and 6, Table 6B). Thus, these primers or types of primers can confirm the absence of particular bioagents from a sample.

10118] The 15 throat swabs from military recruits were found to contain a relatively small set of microbes in high abundance. The most common were Haemophilus influenza, Neisseria meningitides, and Streptococcus pyogenes. Staphylococcus epidermidis, Moraxella cattarhalis, Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus were present in fewer samples. An equal number of samples from healthy volunteers from three different geographic locations, were identically analyzed. Results indicated that the healthy volunteers have bacterial 1292052 1:JIN WO 2006/071241 PCT/US2005/006133 -58flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the viridans group streptococci parasangunis, S. vestibularis, S. mitis, S. oralis and S.

pneumoniae; data not shown), and none of the organisms found in the military recruits were found in the healthy controls at concentrations detectable by mass spectrometry. Thus, the military recruits in the midst of a respiratory disease outbreak had a dramatically different microbial population than that experienced by the general population in the absence of epidemic disease.

[0119] Example 8: Drill-down Analysis for Determination of emm-Type of Streptococcus pyogenes in Epidemic Surveillance [0120] As a continuation of the epidemic surveillance investigation of Example 7, determination of sub-species characteristics (genotyping) of Streptococcus pyogenes, was carried out based on a strategy that generates strain-specific signatures according to the rationale of Multi-Locus Sequence Typing (MLST). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced (Enright et al. Infection and Immunity, 2001, 69, 2416-2427).

In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced. In the present investigation, bioagent identifying amplicons from housekeeping genes were produced using drill-down primers and analyzed by mass spectrometry. Since mass spectral analysis results in molecular mass, from which base composition can be determined, the challenge was to determine whether resolution of emm classification of strains of Streptococcus pyogenes could be determined.

[0121] An alignment was constructed of concatenated alleles of seven MLST housekeeping genes (glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murl), DNA mismatch repair protein (mutS), xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyl transferase (yqiL)) from each of the 212 previously emm-typed strains of Streptococcus pyogenes. From this alignment, the number and location of primer pairs that would maximize strain identification via base composition was determined. As a result, 6 primer pairs were chosen as standard drill-down primers for determination of emm-type of Streptococcus pyogenes.

These six primer pairs are displayed in Table 7. This drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

WO 2006/071241 PCT/US2005/006133 -59- Table 7: Group A Streptococcus Drill-Down Primer Pairs Primer Forward Primer Name Forward Primer Reverse Primer Name Reverse Primer Target Gene Pair No. (SEQ ID NO:) (SEQ ID NO:) SP101 SPET11 358 387 SP101_SPET11 448 442 TMOD F 311 473_TMOD_R 669 gki SP101 SPET11 358 387 310 SP101_SPET11 448 668 gki F 473 TMOD R SP101 SPET11 600 629 SP101_SPET11 686 443 TMOD F 314 714_TMOD_R 671 gtr 81 SP101_SPET11 600 629 313 SP101_SPET11 686 670 gtr F 714 SP101 SPET11 1314 133 SP101_SPET11_1403 426 6 TMOD F 278 1431_TMOD_ 633 murl 86 SP101 SPET11 1314 133 277 SP101_SPET11_1403 632 murl 6F 1431 R SP101 SPET11 1807 183 SP101_SPET11_1901 430 5 TMOD F 286 _1927_TMOD_R 641 mutS SP101 SPET11_ 107_183 285 SP101 SPET11_1901 640 mutS F 1927 R i SPET11 3075 310 SP101_SPET11_3168 438 3 TMOD F 302 _3196_TMOD_R 657 xpt 96 SP101 SPET11 3075 310 301 SP101_SPET11_3168 656 xpt 3 F 3196 R SP101 SPET11 3511 353 SP101_SPET11 3605 441 5 TMOD F 309 _3629_TMOD_R 664 yqiL 98 SP101 SPET11 3511 353 308 SP101 SPET11_3605 663 yqiL F 3629 R [0122] The primers of Table 7 were used to produce bioagent identifying amplicons from nucleic acid present in the clinical samples. The bioagent identifying amplicons which were subsequently analyzed by mass spectrometry and base compositions corresponding to the molecular masses were calculated.

[0123] Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 8A-C rows all except three samples were found to represent emm3, a Group A Streptococcus genotype previously associated with high respiratory virulence. The three outliers were from samples obtained from healthy individuals and probably represent non-epidemic strains. Archived samples (Tables 8A-C rows 5-13) from historical collections showed a greater heterogeneity of base compositions and emm types as would be expected from different epidemics occurring at different places and dates. The results of the mass spectrometry analysis and emm gene sequencing were found to be concordant for the epidemic and historical samples.

WO 2006/071241 PCT/US2005/006133 60 Table 8A: Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 426 and 430 emm-type by emm-Gene Location murI mutS iof Mass Year (Primer Pair (Primer Pair Instances Spectrometry Sequencing (sample) No. 426) No. 430) 48 3 3ORD San A39 G25 C20 T34 A38 G27 C23 T33 2 6 6 Diego 2002 A40 G24 C20 T34 A38 G27 C23 T33 1 28 28 A39 G25 C20 T34 A38 G27 C23 T33 (Cultured) 3 ND A39 G25 C20 T34 A38 G27 C23 T33 6 3 3 A39 G25 C20 T34 A38 G27 C23 T33 3 5,58 5 A40 G24 020 T34 A38 G27 023 T33 6 6 6 NHRC San A40 G24 C20 T34 A38 G27 C23 T33 1 11 11 Diego- A39 G25 C20 T34 A38 G27 C23 T33 3 12 12 Archive 2003 A40 G24 C20 T34 A38 G26 C24 T33 1 22 22 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 3 25,75 75 A39 G25 C20 T34 A38 G27 C23 T33 4 44/61,82,9 44/61 A40 G24 C20 T34 A38 G26 C24 T33 2 53,91 91 A39 G25 C20 T34 A38 G27 C23 T33 1 2 2 A39 G23 C20 T34 A3G G27 C24 T32 2 3 3 A39 G25 C20 T34 A38 G27 C23 T33 1 4 4 A39 G25 C20 T34 A38 G27 C23 T33 1 6 6 Ft. A40 G24 C20 T34 A38 G27 C23 T33 Leonard 11 25 or 75 75 Wood 2003 A39 G25 C20 T34 A38 G27 C23 T33 25,75, 33, 1 34,4,52,84 75 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 44/61 or 82 1 or 9 44/61 A40 G24 C20 T34 A38 G26 C24 T33 2 5 or 58 5 A40 G24 C20 T34 A38 G27 C23 T33 3 1 1 A40 G24 C20 T34 A38 G27 C23 T33 2 3 3 A39 G25 C20 T34 A38 G27 C23 T33 2003 1 4 4 (Cultured) A39 G25 C20 T34 A38 G27 C23 T33 1 28 28 A39 G25 C20 T34 A38 G27 C23 T33 1 3 3 A39 G25 C20 T34 A38 G27 C23 T33 1 4 4 A39 G25 C20 T34 A38 G27 C23 T33 3 6 6 A40 G24 C20 T34 A38 G27 C23 T33 1 !i 1 Ft. A39 G25 C20 T34 A38 G27 C23 T33 1 13 94** Banning 2003 A40 G24 C20 T34 A38 G27 C23 T33 44/61 or 82 (Cultured) 1 or 9 82 A40 G24 C20 T34 A38 G26 C24 T33 1 5 or 58 58 A40 G24 C20 T34 A38 G27 C23 T33 1 78 or 89 89 A39 G25 C20 T34 A38 G27 C23 T33 2 5 or 58 A40 G24 C20 T34 A38 G27 C23 T33 Lackland 1 2 AFB A39 G25 C20 T34 A38 G27 C24 T32 1 81 or 90 ND 2003 A40 G24 C20 T4 A38 G27 C23 T33 1 78 (Throat A38 G26 C20 T34 A38 G27 C23 T33 1 78 Swabs No detection No detection No detection 7 3 ND A39 G25 C20 T34 A38 G27 C23 T33 1 3 ND MCRD San No detection A38 G27 C23 T33 1 3 ND Diego No detection No detection 1 3 ND (Throat No detection No detection 2 3 ND Swabs) No detection A38 G27 C23 T33 3 No detection ND No detection No detection WO 2006/071241 PCT/US2005/006133 61 Table 8B: Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 elof mm-type by emm-Gene Location xpt yqiL Instanoes Mass Sequencing (enplG) Year (Primer Pair (Primer Pair Spectrometry No. 438) No. 441) 4B 3 3 MCRD San A30 G36 C20 T36 A40 G29 C19 T31 2 16 6 Diego 2002 A30 G36 C20 T36 A40 G29 C19 T31 1 28 28 A30 G36 C20 T36 A41 G28 C18 T32 (Cultured) 3 ND A30 G36 C20 T36 A40 G29 C19 T31 6 3 3 A30 G36 C20 T36 A40 G29 C19 T31 3 5,58 5 A30 G36 C20 T36 A40 G29 C19 T31 6 6 6 A30 G36 C20 T36 A40 G29 C19 T31 NHRC San 1 11 ii Diego- A30 G36 C20 T36 A40 G29 C19 T31 3 12 12 Archive 2003 A30 G36 C19 T37 A40 G29 C19 T31 1 22 22 (Cultured) A30 G36 C20 T36 A40 G29 C19 T31 3 25,75 75 A30 G36 C20 T36 A40 G29 C19 T31 4 44/61,82,9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 53,91 91 A30 G36 C19 T37 A40 G29 C19 T31 1 2 2 A30 G36 C20 T36 A40 G29 C19 T31 2 3 3 A30 G36 C20 T36 A40 G29 C19 T31 1 4 4 A30 G36 C19 T37 A41 G28 C19 T31 1 t 6 Ft. A30 G36 C20 T36 A40 G29 C19 T31 Leonard 11 25 or 75 Wood 2003 A30 G36 C20 T36 A40 G29 C19 T31 25,75, 33, 1 34,4,52,84 75 (Cultured) A30 G36 C19 T37 A40 G29 C19 T31 44/61 or 82 I or 9 44/61 A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 5 A30 G36 C20 T36 A40 G29 C19 T31 3 1 1 A30 G36 C19 T37 A40 G29 C19 T31 2 3 FtA30 G36 C20 T36 A40 G29 C19 T31 2003 1 4 4 (Cultured) A30 G36 C19 T37 A41 G28 C19 T31 1 28 28 A30 G36 C20 T36 A41 G28 C18 T32 1 5 3 A30 G36 C20 T36 A40 G29 C19 T31 1 4 4 A30 G36 C19 T37 A41 G28 C19 T31 3 6 6 A30 G36 C20 T36 A40 G29 C19 T31 1 11 ii Ft. A30 G36 C20 T36 A40 G29 C19 T31 1 13 94** Benning 2003 A30 G36 C20 T36 A41 G28 C19 T31 44/61 or 82 (Cultured) 1 or 9 82 A30 G36 C20 T36 A41 G28 C19 T31 1 5 or 58 58 A30 G35 C20 T36 A40 G29 C19 T31 1 78 or 89 89 A30 G36 C20 T36 A41 G28 C19 T31 2 5 or 58 A30 G36 C20 T36 A40 G29 C19 T31 Lackland 1 2 AFB A30 G36 C20 T36 A40 G29 C19 T31 1 81 or 90 ND 2003 A30 G36 C20 T36 A40 G29 C19 T31 I (Throat A30 G35 C20 T36 A41 G28 C19 '31 Swabs) No detection No detection No detection 7 3 ND A30 G36 C20 T36 A40 G29 C19 T31 1 3 ND MCRD San A30 G36 C20 T36 A40 G29 C19 T31 1 3 ND Diego 20 G36 C20 T36 No detection 1 3 ND (Throat No detection A40 G29 C19 T31 2 3 ND Swabs) A30 G36 C20 T36 A40 G29 C19 T31 3 No detection ND No detection No detection WO 2006/071241 PCT/US2005/006133 62 Table 8C: Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441 emm-type by emm-Gene Location gki gtr of Mass Year (Primer Pair ((Primer Pair instances Spectrometry Sequencing (sample) No. 442) No. 443) 48 3 3 MCRD San A32 G35 C17 T32 A39 G28 C16 T32 2 6 6 Diego 2002 A31 G35 017 T33 A39 G28 C15 T33 1 23 28 A30 G36 C17 T33 A39 G28 C16 T32 (Cultured) 3 ND A32 G35 C17 T32 A39 G28 C16 T32 6 3 3 A32 G35 C17 32 A39 028 016 32 3 5,58 5 A30 G36 C20 T30 A39 G20 C15 T33 6 6 6 NHRC San A31 G35 C17 T33 A39 G28 C15 T33 1 11 11 Dieco- A30 G36 C20 T30 A39 G28 C16 T32 3 12 12 Archive 2003 A31 G35 C17 T33 A39 G28 C15 T33 1 22 22 (Cultured) A31 G35 C17 T33 A38 G29 C15 T33 3 25,75 75 A30 G36 C17 T33 A39 G28 C15 T33 4 44/61,82,9 44/61 A30 G36 C18 T32 A39 G28 C15 T33 2 53,91 91 1 A32 G35 C17 T32 A39 G28 C16 T32 1 2 2 A30 G36 C17 T33 A39 G28 C15 T33 2 3 3 A32 G35 C17 T32 A39 G28 C16 T32 1 4 4 A31 G35 C17 T33 A39 G28 C15 T33 1 6 6 Ft. A31 G35 C17 T33 A39 G28 C15 T33 Leonard 11 25 or 75 75 Wood 2003 A30 G36 C17 T33 A39 G28 C15 T33 25,75, 33, 1 34,4,52,84 75 (Cultured) A30 G36 C17 T33 A39 G28 C15 T33 44/61 or 82 1 or 9 44/61 A30 G36 C18 T32 A39 G28 C15 T33 2 5 or 58 5 A30 G36 C20 T30 A39 G28 C15 T33 3 1 1 SA30 G36 C18 T32 A39 G28 C15 T33 2 3 3 2003 A32 G35 C17 T32 A39 G28 C16 T32 1 4 4 (Cultured) A31 G35 C17 T33 A39 G28 C15 T33 1 28 28 A30 G36 C17 T33 A39 G28 C16 T32 1 3 3 A32 G35 C17 T32 A39 G28 C16 T32 1 4 4 A31 G35 C17 T33 A39 G28 C15 T33 3 6 6 A31 G35 C17 T33 A39 G28 C15 T33 1 11 1i Ft. A30 G36 C20 T30 A39 G28 C16 T32 1 13 94** Benning 2003 A30 G36 C19 T31 A39 G28 C15 T33 44/61 or 82 (Cultured) 1 or 9 82 A30 G36 C18 T32 A39 G28 C15 T33 1 5 or 58 58 A30 G36 C20 T30 A39 G28 C15 T33 1 78 or 89 89 A30 G3518 T32 A39 G28 C15 T33 2 5 or 58 A30 G36 C20 T30 A39 G28 C15 T33 Leckland 1 2 AFB A30 G36 C17 T33 A39 G28 C15 T33 1 81 or 90 ND 2003 A30 G36 C17 T33 A39 G28 C15 T33 1 78 (Throat A30 G36 C18 T32 A39 G28 C15 T33 Swab5) No detection No detection No detection 7 3 ND A32 G35 C17 T32 A39 G28 CL6 T32 1 3 ND MCRD San No detection No detection 1 3 ND Diego A32 G35 C17 T32 A39 G28 C16 T32 A26507 2 13 2 1 3 1 3 ND (Throat A32 G35 C17 T32 No detection 2 3 ND Swabs) A32 G35 C17 T32 No detection 3 No detection ND No detection No detection WO 2006/071241 PCT/US2005/006133 -63- [0124] Example 9: Design of Calibrant Polynucleotides based on Bioagent Identifying Amplicons for Identification of Species of Bacteria (Bacterial Bioagent Identifying Amplicons) [0125] This example describes the design of 19 calibrant polynucleotides based on bacterial bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table 4) and the Bacillus anthracis drill-down set (Table [0126] Calibration sequences were designed to simulate bacterial bioagent identifying amplicons produced by the T modified primer pairs shown in Table 4 (primer names have the designation "TMOD"). The calibration sequences were chosen as a representative member of the section of bacterial genome from specific bacterial species which would be amplified by a given primer pair. The model bacterial species upon which the calibration sequences are based are also shown in Table 9. For example, the calibration sequence chosen to correspond to an amplicon produced by primer pair no. 361 is SEQ ID NO: 722. In Table 9, the forward or reverse primer name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes the forward primer name 16S_EC 713 732 TMOD F indicates that the forward primer hybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence (in this case, the reference sequence is an extraction consisting of residues 4033120-4034661 of the genomic sequence ofE. coli K12 (GenBank gi number 16127994). Additional gene coordinate reference information is shown in Table 10. The designation "TMOD" in the primer names indicates that the 5' end of the primer has been modified with a non-matched template T residue which prevents the PCR polymerase from adding non-templated adenosine residues to the 5' end of the amplification product, an occurrence which may result in miscalculation of base composition from molecular mass data (vide supra).

[0127] The 49 calibration sequences described in Tables 9 and 10 were combined into a single calibration polynucleotide sequence (SEQ ID NO: 741 which is herein designated a "combination calibration polynucleotide") which was then cloned into a pCR®-Blunt vector (Invitrogen, Carlsbad, CA). This combination calibration polynucleotide can be used in conjunction with the primers of Table 9 as an internal standard to produce calibration amplicons for use in determination of the quantity of any bacterial bioagent. Thus, for example, when the combination calibration polynucleotide vector is present in an amplification reaction mixture, a calibration amplicon based on primer pair 346 (16S rRNA) will be produced in an amplification WO 2006/071241 PCT/US2005/006133 -64reaction with primer pair 346 and a calibration amplicon based on primer pair 363 (rpoC) will be produced with primer pair 363. Coordinates of each of the 19 calibration sequences within the calibration polynucleotide (SEQ ID NO: 783) are indicated in Table Table 9: Bacterial Primer Pairs for Production of Bacterial Bioagent Identifying Amplicons and Corresponding Representative Calibration Sequences Primer Forward Primer Name Forward Reverse Primer Name Reverse Calibration Calibration Pair No. Primer Primer Sequence Model Sequence (SEQ ID (SEQ ID Species (SEQ ID NO:) NO:) NO:) 361 16S EC 1090 1111 2 T 5 16S EC 1175 1196 TMODB R 370 Bacillus 764 MOD F anthracis 346 16S EC 713 732 TMOD 27 16 EC 789 809 TMOD R 389 Bacillus 765 F anthracis 347 169 EC 785 806 TMOD 30 16S F.C_880 897 TMOD R 392 Bacillus 766 F anthracis 348 16S EC 960 981 TMOD 38 16S EC 1054 1073 TMOD R 363 Bacillus 767 F anthracis 349 23S EC 1826 1843 TMO 49 23S EC 1906 1924 TMOD R 405 Bacillus 768 D F anthracis 360 23S EC 2646 2667 TMO 60 238_EC2745_2765_TMOD_. 416 Dacillus 769 D F anthracis 350 CAPC BA 274 303 TMOD 98 CAPC 3A 349 376 TMOD R 452 Bacillus 770 F anthracis 351 CYA BA 1353 1379 TMO 128 CYA BA 1448 1467 TMOD R 483 Bacillus 771 D F Ianthracis 352 INFB EC1365 1393 TM 161 INFB EC 1439 1467 TMOD 516 Bacillus 772 OD F R anthracis 353 LEF BA 756 781 TMOD 175 LEF BA 843 872 TMOD R 531 Bacillus 773 F anthracis 356 RPLB EC 650_79 TMOD 232 RPL EC 739 762 TMOD R 592 Clostridium 774 F hotulinum 449 RPLB EC_690 710_F 237 RPLB EC 737 758_ R 5B9 Clostridium 775 botalinum 359 RPOB EC 1845 1866 TM 241 RPOB EC 1909_1929 TMOD 597 Yersinia 776 OD F R Pestis 362 RPOB EC 3799 3821 TM 245 RPOB EC 3862 3888 TMOD 603 Burkholderia 777 OD F R mallei 363 RPOC EC 2146 2174 TM 257 RPOC EC 2227 2245 TMOD_ 621 Burkholderia 778 OD F R mallei 354 RPOC EC 2218 2241 TM 262 RP0C EC 2313_2337 TMOD_ 625 Bacillus 779 OD F R anthracis 355 SSPE BA 115 137 TMOD 321 SSPE BA 197 222 TMOD R 687 Bacillus 780 F anthracis 367 TUFB EC 957 979 TMOD 345 TUFB EC 1034 1058 TMOD 701 Burkholderia 781 F R mallei 358 VALS EC 1105 1124TM 350 VALS EC 1195 1218_TMOD 712 Yersinia 782 OD F R Pestis Table 10: Primer Pair Gene Coordinate References and Calibration Polynucleotide Sequence Coordinates within the Combination Calibration Polynucleotide Bacterial Gene Gene Extraction Coordinates Reference GenBank GI No. of Primer Pair Coordinates of Calibration and Species of Genomic or Plasmid Sequence Genomic or Plasmid No. Sequence in Combination Sequence Calibration Polynucleotide (SEQ IDNO:783 16S E, coli 4033120..4034661 16127994 346 16..109 16S E. coli 4033120..4034661 16127994 347 83..190 165 E. coli 4033120..4034661 16127994 348 246..353 16S E. coli 4033120..4034661 16127094 361 368..469 23S E. coli 4166220..4169123 16127994 349 743..837 23S E. coli 4166220..4169123 16127994 360 865..981 rpo8 E. 4178823..4182851 16127994 359 1591..1672 coli. (complement strand) rpoB E. coli 4178823..4182851 16127994 362 2081..2167 (complement strand) cpoC E. coli 4182928..4187151 16127994 354 1810..1926 rpoC E. coli 4182928..4187151 16127994 363 2183..2279 infB E. coli 3313655..33109B3 16127994 352 1692..1791 (complerent strand) tufB E. coli 4173523..4174707 16127994 367 2400..2498 rplB E. coli 3449001..3448180 16127994 356 1945..2060 rplB E. coli 3449001..3440180 16127994 449 1986..2055 valS E. coli 4481405..4470550 16127994 358 1462..1572 (complement strand) WO 2006/071241 PCT/US2005/006133 capC 56074..55628 (complement 6470151 350 2517..2616 B. anthracis strand) cya 156626..154288 4894216 351 1338..1449 B, anthracis (complement strand) lef 127442..129921 4894216 353 1121..1234 e. anthracis sspE 226496..226783 30253828 355 1007-1104 B. anthracis [0128] Example 10: Use of a Calibration Polynucleotide for Determining the Quantity of Bacillus Anthracis in a Sample Containing a Mixture of Microbes [0129] The process described in this example is shown in Figure 7. The capC gene is a gene involved in capsule synthesis which resides on the pX02 plasmid of Bacillus anthracis. Primer pair number 350 (see Tables 9 and 10) was designed to identify Bacillus anthracis via production of a bacterial bioagent identifying amplicon. Known quantities of the combination calibration polynucleotide vector described in Example 3 were added to amplification mixtures containing bacterial bioagent nucleic acid from a mixture of microbes which included the Ames strain of Bacillus anthracis. Upon amplification of the bacterial bioagent nucleic acid and the combination calibration polynucleotide vector with primer pair no. 350, bacterial bioagent identifying amplicons and calibration amplicons were obtained and characterized by mass spectrometry. A mass spectrum measured for the amplification reaction is shown in Figure 8).

The molecular masses of the bioagent identifying amplicons provided the means for identification of the bioagent from which they were obtained (Ames strain of Bacillus anthracis) and the molecular masses of the calibration amplicons provided the means for their identification as well. The relationship between the abundance (peak height) of the calibration amplicon signals and the bacterial bioagent identifying amplicon signals provides the means of calculation of the copies of the pX02 plasmid of the Ames strain of Bacillus anthracis. Methods of calculating quantities of molecules based on internal calibration procedures are well known to those of ordinary skill in the art.

[0130] Averaging the results of 10 repetitions of the experiment described above, enabled a calculation that indicated that the quantity of Ames strain of Bacillus anthracis present in the sample corresponds to approximately 10 copies of pX02 plasmid.

[0131] Example 11: Drill-down Genotyping of Campylobacter Species [0132] A series of drill-down primers were designed as described in Example 1 with the objective of identification of different strains of Campylobacterjejuni. The primers are listed in Table 11 with the designation "CJST_CJ." Housekeeping genes to which the primers hybridize and produce bioagent identifying amplicons include: tkt (transketolase), glyA (serine WO 2006/071241 PCT/US2005!006133 66 hydroxymethyltransferase), gitA (citrate synthase), aspA (aspartate ammonia lyase), ginA (glutamnine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).

Table 11: Campylobacter Drill-down Primer Pairs Primer Forward Primer Name Forward Primer Reverse Primer Name Reverse Primer Target Gene Pair (SEQ ED NO:) (SEQ ED NO:) 1053 CJST CJY 1080 1110 F 102 COST CJ 1166 1198 R 456 gltA 1064 CJTST CT 1680 1713 F 107 CJST Ca 1795 1822 R 461 glyA 1054 CJST CJT 2060 2090 F 109 CJST CJ 2148 2174 R 463 PgJM 1049 OJST Cj 2636 2668F 1 113 CJST CJ 2753 2777 R 467 tkt 1048 OJST WJ 360 394 F 119 CJST CJ 442 476 R 472 aspA 1047 CS J54616_F 121 CJST CJ 663 692 R 474 glnA [0133] The primers were used to amplify nucleic acid from 50 food product samples provided by the USDA, 25 of which contained Camipylobacterjejuni and 25 of which contained Campylobacter coli. Primers used in this study were developed primarily for the discrimination of Camipylobacterjejuni clonal complexes and for distinguishing Campylobacterjejuni from Cainpylobacter coli. Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates demonstrate polymorphisms between strains. The conclusions of the comparison of base composition analysis with sequence analysis are shown in Tables 12A-C.

Table 12A Results of Base Composition Analysis of 50 Campylobacter Samples with Drilldown MLST Primer Pair Nos: 1048 and 1047 Base Base SILST type or Composition of Composition of Clonal S4LST Type fliomant Bioagent Isle Cmpe b rCoalb Strain Identifying Identifying origin Base A b uplinon Asaplicon Compositysis Obtained with Obtained with anlssanalysis Primer Pair No: Primer Pair 1048 (aspA) No: 1047 (ginA) 9T-1 Goose ST 690ST91 R37 A3G2C1T4 A7G1C6T5 jeu' 692/707/991 S 9 937 3 2 1 4 4 2 1 2 J-2 0* Hmn Complex ST 356, Human 26/8/5 complex 01M4192 A30 G25 016 T46 A48 G21 017 T23 J-3 Human Complex ST 4138 RM44194 A30 G25 015 T47 A48 G21 018 T22 j ej UOn.L 354/179 J-4 C..ST 257, .24Human Complex 257 complex 0344197 A30 G25 016 T48 A48 G21 018 T22 jejunj.257 je-un Human Complex 52 ST.5,e 014R4277 A30 925 016 T46 A48 921 C17 T23 ST 51, 2344275 A30 G25 C15 T47 A48 G21 017 T23 J- C Human Complex 443 complex jeui443 0344279 A30 925 018 T47 A48 G21 017 T23 2- Human Complex 42 ST 804, 0416 A39205T7 A8G20812 J- 3.ej ona complex 42 R16 3 2 1 4 4 2 1 2 J8Human Complex ST 362, J -8 o 42/49/362 complex 0343193 A30 G25 015 T47 A48 921 018 122 .29 C. Human Complex ST 147, 0343203 A30 925 015 T47 A47 921 018 T23 j ej Ln J 45/283 Complex 45 Human Consistent ST 828 0944183 A31 927 020 T29 A48 921 016 T24 WO 2006/071241 PCT/US2005/006133 -67with 74 closely related sequence types (none belong to a clonal complex) I ST 832 RM1169 A31 G27 C20 T39 A48 G21 C16 T24 Poultry C. coli ST 1056 RM1857 A31 G27 C20 T39 A48 G21 C16 T24 ST 889 6M1166 A31 G27 C20 T39 A48 G21 C16 T24 ST 829 RM1182 A31 G27 C20 T39 A48 G21 C16 T24 ST 1050 RM1518 A31 G27 C20 T39 A48 G21 C16 T24 ST 1051 RM1521 A31 627 C20 T39 A48 G21 C16 T24 ST 1053 RM1523 A31 G27 C20 T39 A48 G21 C16 T24 ST 1055 RM1527 A31 G27 C20 T39 A48 G21 C16 T24 ST 1017 RM1529 A31 G27 C20 T39 A48 G21 C16 T24 ST 860 RM1840 A31 G27 C20 T39 A48 G21 C16 T24 ST 1063 142219 A31 G27 C20 T39 A48 G21 C16 T24 ST 1066 RM2241 A31 G27 C20 139 A48 G21 C16 T24 ST 1067 RM2243 A31 G27 C20 T39 A48 G21 C16 T24 ST 1068 RM2439 A31 G27 C20 T39 A48 G21 C16 T24 ST 1016 RM3230 A31 G27 C20 T39 A48 G21 C16 '124 ST 1069 RM3231 A31 G27 C20 T39 A48 G21 C16 T24 ST 1061 RM1904 A31 G27 C20 T39 A48 G21 C16 T24 ST 825 RM1534 A31 G27 C20 T39 A48 G21 C16 T24 Swine Unknown ST 901 RM1505 A31 G27 C20 T39 A4B G21 C16 T24 C-2 C. coli Human ST 895 ST 895 RM1532 A31 G27 C19 T40 A4B G21 C16 T24 Consistent ST 1064 RM2223 A31 G27 C20 T39 A48 G21 C16 T24 with 63 closely ST 1082 RM1178 A31 G27 C20 T39 A48 G21 C16 T24 Poultry related C-3 C. coli saquence ST 1054 RM1525 A31 G27 C20 T39 A48 G21 C16 T24 types (none belong to a ST 1049 RM1517 A31 G27 C20 T39 A48 G21 C16 T24 clonal Marmoset complex) ST 891 RM1531 A31 G27 C20 T39 A48 G21 C16 T24 Table 12B Results of Base Composition Analysis of 50 Campylobacter Samples with Drilldown MLST Primer Pair Nos: 1053 and 1064 Group Species Isolate origin MLST type or Clonal Complex by Base Composition analysis MLST Type or Clonal Complex by Sequence analysis Strain Base Composition of Bioagent Identifying Amplicon Obtained with Primer Pair No: 1053 (gcltA) Base Composition of Bioagent Identifying Amplicon Obtained with Primer Pair No: 1064 (glyA) A24 G25 C23 T47 A40 G29 C29

J-

1 .n Goose ST9 9 07/991 ST991 RM3673 jeJuni /692/707/991 Complex ST 356, R24 G25 C23 T47 A40 G29 C29 -2 C. Human omple206/48/353 complex RM4192 jejuni 206/48/353 353 J-3 C. a Complex ST 436 RM4194 A24 G25 C23 T47 A40 G29 C29 jejuni Human 354/179 ST 257, A24 G25 C23 T47 A40 G29 C29 J-4 Human Complex 257 complex RM4197 jejuni 257 C. ST 52, RM4277 A24 G25 C23 T47 A39 G30 C26 T48 J- .ejuni Human Complex 52 complex 52 o p e 2 ST 51, RM4275 A24 G25 C23 T47 A39 G30 C28 T46 J-6 i Human Complex 443 complex jenj 443 RM4279 A24 G25 C23 T47 A39 G30 C28 T46 A24 G25 C23 T47 A39 G30 C26 T48 en ST 604, 601864 J-7 1 e Human Complex 42 T 64,e RM1864 iejuni I- complex 42 WO 2006/071241 PCT/US2005/006133 68 C. Complex ST 362, A24 G25 C23 T47 A38 G31 C28 T46 jejuni 42/49/362 complex RM3193 362 A24 G25 C23 T47 A38 G31 C28 T46 C. Complex ST 147, 9043203 J ejuni Human 45/283 Complex 45 C. ST 828 904483 A23 G24 C26 T46 A39 G30 C27 T47 jejuni Human ST 832 RM1169 A23 G24 C26 T46 A39 G30 C27 T47 ST 1056 90M1857 A23 G24 C26 T46 A39 G30 C27 T47 ST 889 R1166 A23 924 C26 T46 A39 G30 C27 T47 ST 829 R41182 A23 G24 C26 T46 A39 G30 C27 T47 ST 1050 M1518 A23 G24 C26 T46 A39 G30 C27 T47 ST 1051 R01521 A23 G24 C26 T46 A39 G30 C27 T47 ST 1053 RM1523 A23 G24 C26 T46 A39 G30 C27 T47 Consistent with 74 ST 1055 R41527 A23 G24 C26 T46 A39 G30 C27 T47 Poultry closely related ST 1017 RM1529 A23 G24 C26 T46 A39 G30 C27 T47 sequence C-I C. coll types (none ST 860 RM1840 A23 G24 926 T46 A39 930 927 T47 belong to a clonal ST 1063 RM2219 A23 924 926 T46 A39 930 927 T47 complex ST 106 2241 A23 G24 C26 T46 A39 G30 C27 T47 ST 1067 RM2243 A23 924 926 T46 A39 930 927 T47 ST 1060 903439 A23 G24 C26 T46 A39 G30 C27 T47 ST 1016 903230 A23 G24 C26 T46 A39 G30 C27 T47 swine ST 1068 RM3231 A23 924 926 T46 HO DATA ST 1061 901904 A23 G24 C26 T46 A39 G30 C27 T47 ST 825 RM90534 A23 G24 C26 T46 A39 G30 C27 T47 Unknown ST 901 RM1505 A23 G24 C26 T46 A39 G30 C27 T47 C-2 C. coll Human ST 895 ST 895 RM1532 A23 914 926 T46 A39 930 927 T47 Consistent ST 1064 R42223 A23 G24 C26 T46 A39 G30 C27 T47 with 63 closely ST 1082 941178 A23 G24 C26 T46 A39 G30 C27 T47 Poultry related 9-3 C. C01 sequence ST 1054 9041525 A23 G24 C25 T47 A39 930 C27 T47 types (none belong to a ST 1049 RM1517 A23 924 926 T46 A39 930 927 T47 clonal Marmoset complex) ST 891 RMI531 A23 924 926 T46 A39 930 927 T47 Table 12C Results of Base Composition Analysis of 50 Campylobacter Samples with Drilldown MLST Primer Pair Nos: 1054 and 1049 Base Base LST type or TComposition of Composition of Clonal or Conal Bioagent Bioagent Isolate Complex by Coal Identifying Identifying Group Species origin Base Ctran Amplicon Amplioon Composition Sequence Obtained with Obtained with analysis analysis Primer Pair No: Primer Pair 1054 (pgm) No: 1049 (tkt) J-i Goose ST 690 S" 991 RM3673 A26 G33 C16 T38 A41 G28 C35 T38 jejalLL /692/707/991 C. Cumplex ST 356, J-2 C Human 2064835 complex RM4192 A26 G33 C19 T37 A41 028 C36 T37 2 jejun 206/48/353 353 J-3 C Human Complex ST 436 9044194 jejuni 354/179 A27 G32 C19 T37 A42 G28 C36 T36 WO 2006/071241 PCT/US2005/006133 -69c.

jejuni Human Complex 257 ST 257, complex 257 RM4197 A27 G32 C19 T37 A41 G29 C35 T37 C. Hman Complex 52 ST 52, 277 jejuni Han Complex 52 complex 52 RM4277 A26 G33 C18 T38 A41 G28 C36 T37 C. ST 51, RM4275 A27 31 C19 T38 A41 G2 C36 T37 J-6 jej un Human Complex 443 complex A27 G31 019 T38 A41 G2 C36 T37 4443 KM4279 443 R47 A27 G31 C19 T38 A41 G28 C36 T37 J-7 C. Human Complex 42 ST 604, RM1864 A27

G

32 C19 T37 A42 G2 8 C35 T37 jejuni complex 42 J-8 C H4m36 ComplexST 362 J- jejni Human C42/49/362 complex RM3193 A26 033 C19 T37 A42 G28 C35 T37 362 C. Human Complex ST 147, RM3203 A28 31 C19 T3 A43 28 C36 9 jejuni Hun 45/283 Complex 45 20 28 31 19 T37 43 G2 36

C.

jej uni C. coli ST 828 P44183 A27 G30 C19 T39 A46 G28 C32 T36 Human Poultry Consistent with 74 closely related sequence types (none belong to a clonal complex) ST 832 RM1169 ST M A27 G30 C19 T39 A46 G28 C32 T36 ST 1056 RM1357 A27 G30 C19 T9 A46 G28 C32 T36 ST 889 RM166 A27 G30 C19 TO9 A46 G28 C32 T36 ST 1050 RM1518 A2 G3 C1 TI- A4 G2 C3 T36 ST 829 RMI4182 ST 105 RM15 A27 G30 C19 T39 A46 G28 C32 T36 ST 1050 RM152318 A27 G30 C19 T9 A46 G28 C32 T36 ST 1051 RM1521 A27 G30 C19 T39 A46 G28 C32 T36 ST 8 6 0 RMB40 G 1523 S 1063A27 230 C19 T39 A46 228 C32 T36 ST 1055 R041527 S 1 0 6 6 R M 2 2 4 1 A27 230 C19 T39 A46 228 C32 T36 ST 1017 RM41529 A27 230 C19 T39 A46 G28 C32 T36 ST 860 2041840 S T 1 0 1 6 27 30 C19 T39 A46 G28 C32 T36 ST 1063 RM22219 A27 230 C19 T39 A46 G28 C32 T36 ST 1066 RM1904 2241 S T 8 2 RM 1 5 3 4 A27 G30 C19 T39 A46 G28 C32 T36 ST 1067 2042243 227 00 019 TOO A46 228 032 TOO ST 1068 2142409 227 230 019 T39 246 228 002 106 ST 1018 2143230 227 00 019 TOO A46 228 002 TO6 ST 1069 2140201 227 00 018 T39 246 228 002 T36 ST 1061 2041904 A27 G00 019 T39 246 228 032 TO6 ST 825 2041504 227 230 019 T39 246 228 002 T36 Swine Unknown ST 901 RM1505 A27 G30 C19 T39 A46 G28 C32 T36 C-2 C. coli Human ST 895 ST 895 BM1532 G 227 00 019 TOO A45 229 002 T06 ST 1004 2042220 Consistent STA27 G30 C19 T39 A45 29 C32 T36 with 63 closely ST 1082 RM1178 A27 30 C19 T39 A45 29 C32 T36 Poultry related C-3 C. culi sequence ST 1054 RM1525 types (none A27 G30 C19 T39 A45 G29 C32 T36 belong to a ST 1049 RM1517 clonal 27 G30 C19 T39 A45 G29 C32 T36 complex) Marmoset ST 991 RM1531 complex)__ ST_ R__1531_ 227 030 C19 T39 A45 29 C32 T36 [0134] The base composition analysis method was successful in identification of 12 different strain groups. Campylobacterjejuni and Campylobacter coli are generally differentiated by all WO 2006/071241 PCT/US2005/006133 loci. Ten clearly differentiated Campylobacterjejuni isolates and 2 major Campylobacter coli groups were identified even though the primers were designed for strain typing of Campylobacterjejuni. One isolate (RM4183) which was designated as Campylobacterjejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli by full MLST sequencing.

[0135] Example 12: Identification ofAcinetobacter baumannii Using Broad Range Survey and Division-Wide Primers in Epidemiological Surveillance [0136] To test the capability of the broad range survey and division-wide primer sets of Table 4 in identification ofAcinetobacter species, 183 clinical samples were obtained from individuals participating in, or in contact with individuals participating in Operation Iraqi Freedom (including US service personnel, US civilian patients at the Walter Reed Army Institute of Research (WRAIR), medical staff, Iraqi civilians and enemy prisoners). In addition, 34 environmental samples were obtained from hospitals in Iraq, Kuwait, Germany, the United States and the USNS Comfort, a hospital ship.

[0137] Upon amplification of nucleic acid obtained from the clinical samples, primer pairs 346- 349, 360, 361, 354, 362 and 363 (Table 4) all produced bacterial bioagent amplicons which identified Acinetobacter baumannii in 215 of 217 samples. The organism Klebsiella pneumoniae was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain of Acinetobacter baumannii) were identified and assigned arbitrary numbers from 1 to 14. Strain type 1 was found in 134 of the sample isolates and strains 3 and 7 were found in 46 and 9 of the isolates respectively.

[0138] The epidemiology of strain type 7 of Acinetobacter baumannii was investigated. Strain 7 was found in 4 patients and 5 environmental samples (from field hospitals in Iraq and Kuwait).

The index patient infected with strain 7 was a pre-war patient who had a traumatic amputation in March of 2003 and was treated at a Kuwaiti hospital. The patient was subsequently transferred to a hospital in Germany and then to WRAIR. Two other patients from Kuwait infected with strain 7 were found to be non-infectious and were not further monitored. The fourth patient was diagnosed with a strain 7 infection in September of 2003 at WRAIR. Since the fourth patient was not related involved in Operation Iraqi Freedom, it was inferred that the fourth patient was the subject of a nosocomial infection acquired at WRAIR as a result of the spread of strain 7 from the index patient.

WO 2006/071241 PCT/US2005/006133 -71- [0139] The epidemiology of strain type 3 ofAcinetobacter baumannii was also investigated.

Strain type 3 was found in 46 samples, all of which were from patients (US service members, Iraqi civilians and enemy prisoners) who were treated on the USNS Comfort hospital ship and subsequently returned to Iraq or Kuwait. The occurrence of strain type 3 in a single locale may provide evidence that at least some of the infections at that locale were a result of a nosocomial infections.

[0140] This example thus illustrates an embodiment of the present invention wherein the methods of analysis of bacterial bioagent identifying amplicons provide the means for epidemiological surveillance.

[0141] Example 13: Selection and Use of MLST Acinetobacter baumanii Drill-down Primers [0142] To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by multilocus sequence typing (MLST) such as the MLST methods of the MLST Databases at the Max- Planck Institute for Infectious Biology (web.mpiib-berlin.mpg.de/mlst/dbs/Mcatarrhalis/ documents/primersCatarrhalis_html), an additional 21 primer pairs were selected based on analysis of housekeeping genes of the genus Acinetobacter. Genes to which the drill-down MLST analogue primers hybridize for production of bacterial bioagent identifying amplicons include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutY), fumarate hydratase (fumC), and pyrophosphate phospho-hydratase (ppa). These 21 primer pairs are indicated with reference to sequence listings in Table 13. Primer pair numbers 1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers 1155-1157 hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers 1165-1170 hybridize to and amplify segments of fumC. Primer pair number 1171 hybridizes to and amplifies a segment of ppa. The primer names given in Table 13 indicates the coordinates to which the primers hybridize to a reference sequence which comprises a concatenation of the genes TrpE, efp (elongation factor adk, mutT, fumC, and ppa. For example, the forward primer of primer pair 1151 is named AB_MLST-11-OIF007_62 91_F because it hybridizes to the Acinetobacter MLST primer reference sequence of strain type 11 in sample 007 of Operation Iraqi Freedom (OIF) at positions 62 to 91.

WO 2006/071241 PCT/US2005/006133 -72- Table 13: MLST Drill-Down Primers for Identification of Sub-species characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter Primer Forward Primer Name Forward Reverse Primer Name Reverse Pair Primer Primer No. (SEQ ID NO:) (SEQ ID NO:) 1151 AB MLST-11-OIF007 62 91 F 83 AB MLST-11-OIF007 169 203 R 426 1152 AB MLST-11-01F007 165 214 F 76 AB MLST-11-OIF007 291 324 R 432 1153 AB MLST-11-OIF007 260 289 F 79 AB MLST-11-OIF007 354 393 R 434 1154 AB MLST-11-01F007 206 239 F 78 AB MLST-11-OIF007 318 344 R 433 1155 AB MLST-11-01F007 522 552 F 80 AB MLST-11-OIF007 557 610 R 435 1156 AB MLST-11-OIF007 547 571 F 81 AB MLST-11-OIF007 636 686 R 436 1157 AB MLST-11-OIF007 601 627 F 82 AB MLST-11-OIF007 710 736 R 437 1158 AB MLST-11- OIF007 1202 1225 F AB MLST-11-OIF007 1266 1296 R 420 1159 AB MLST-11- DIF007 1202 1225 F AB MLST-11-OIF007 1299 1316 R 421 1160 AB MLST-11- 66 0IF007 1234 1264 F AB MLST-11-OIF007 1335 1362 R 422 1161 AB MLST-11- 67 01F007 1327 1356 F AB MLST-11-OIF007 1422 1448 R 423 1162 AB MLST-11- 68 OIF007 1345 1369 F AE MST-11-OIF007 1470 1494 R 424 1163 AB MLST-11- 69 OIF007 1351 1375 F AB MLST-11-OIF007 1470 1494 R 424 1164 AB MST-11- 01F007 1387 1412 F AB MLST-11-OIF007 1470 1494 R 424 1165 AB MLST-11- 71 OIF007 1542 1569 F AB MLST-11-OIF007 1656 1660 R 425 1166 AB MLST-11- 72 OIF007 1566 1593 F AB MLST-11-OIF007 1656 1680 R 425 1167 AB MLST-11- 73 01F027 1611 1638 F AB MLST-11-OIF007 1731 1757 R 427 1168 AB MLST-11- 74 OIF007 1726 1752 F AB MLST-11-0IF007 1790 1821 R 428 1169 AB MLST-11- 018007 1792 1826 F AB MLST-11-01F007 1876 1909 R 429 1170 AB MLST-11- 01F007 1792 1826 F AB MLST-11-OIF007 19895 1927 R 430 1171 AB MLST-11- 77 AB MLST-11-01F00 7 2097 2118 R 431 WO 2006/071241 PCT/US2005/006133 -73- 01F007 1970 2002 F [0143] Analysis of bioagent identifying amplicons obtained using the primers of Table 13 for over 200 samples from Operation Iraqi Freedom resulted in the identification of 50 distinct strain type clusters. The largest cluster, designated strain type 11 (ST11) includes 42 sample isolates, all of which were obtained from US service personnel and Iraqi civilians treated at the 2 8 th Combat Support Hospital in Baghdad. Several of these individuals were also treated on the hospital ship USNS Comfort. These observations are indicative of significant epidemiological correlation/linkage.

[01441 All of the sample isolates were tested against a broad panel of antibiotics to characterize their antibiotic resistance profiles. As an example of a representative result from antibiotic susceptibility testing, ST11 was found to consist of four different clusters of isolates, each with a varying degree of sensitivity/resistance to the various antibiotics tested which included penicillins, extended spectrum penicillins, cephalosporins, carbipenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics.

Thus, the genotyping power of bacterial bioagent identifying amplicons, particularly drill-down bacterial bioagent identifying amplicons, has the potential to increase the understanding of the transmission of infections in combat casualties, to identify the source of infection in the environment, to track hospital transmission ofnosocomial infections, and to rapidly characterize drug-resistance profiles which enable development of effective infection control measures on a time-scale previously not achievable.

[0145] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety.

Claims (15)

1. A composition comprising an oligonucleotide primer 21 to 35 nucleobases in a length comprising 70% to 100% sequence identity with SEQ ID NO:183.

2. The composition of claim 1, wherein said oligonucleotide primer comprises at c 5 least one modified nucleobase.

3. The composition of claim 1 or 2, further comprising a second oligonucleotide Oprimer 21 to 35 nucleobases in length comprising 70% to 100% sequence identity with SSEQ ID NO:538.

4. The composition of claim 3 wherein either or both of said oligonucleotide to primers comprises at least one modified nucleobase. 1292052 I:JIN WO 2006/071241 PCT/US2005/006133 The composition of claim 3 wherein either or both of said oligonucleotide primers comprises a non-templated T residue on the

6. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises at least one non-template tag.

7. The composition of claim 3 wherein either or both of said oligonucleotide primers comprises at least one molecular mass modifying tag.

8. A kit comprising the composition of claim 3.

9. The kit of claim 8 further comprising at least one calibration polynucleotide. The kit of claim 8 further comprising at least one ion exchange resin linked to magnetic beads.

11. A method for identification of an unknown bacterium comprising: amplifying nucleic acid from said bacterium using the composition of claim 3 to obtain an amplification product; determining the molecular mass of said amplification product; optionally determining the base composition of said amplification product from said molecular mass; and comparing said molecular mass or base composition of said amplification product with a plurality of molecular masses or base compositions of known bacterial bioagent identifying amplicons, wherein a match between said molecular mass or base composition of said amplification product and the molecular mass or base composition of a member of said plurality of molecular masses or base compositions identifies said unknown bacterium.

12. The method of claim 11 wherein said molecular mass is determined by mass spectrometry.

13. A method of determining the presence or absence of a bacterium of a particular clade, genus, species, or sub-species in a sample comprising: WO 2006/071241 PCT/US2005/006133 76 amplifying nucleic acid from said sample using the composition of claim 3 to obtain an amplification product; determining the molecular mass of said amplification product; optionally determining the base composition of said amplification product from said molecular mass; and comparing said molecular mass or base composition of said amplification product with the known molecular masses or base compositions of one or more known clade, genus, species, or sub-species bioagent identifying amplicons, wherein a match between said molecular mass or base composition of said amplification product and the molecular mass or base composition of one or more known clade, genus, species, or sub-species bioagent identifying amplicons indicates the presence of said clade, genus, species, or sub-species in said sample.

14. The method of claim 13 wherein said molecular mass is determined by mass spectrometry. A method for determination of the quantity of an unknown bacterium in a sample comprising: contacting said sample with the composition of claim 3 and a known quantity of a calibration polynucleotide comprising a calibration sequence; concurrently amplifying nucleic acid from said bacterium in said sample with the composition of claim 3 and amplifying nucleic acid from said calibration polynucleotide in said sample with the composition of claim 3 to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon; determining the molecular mass and abundance for said bacterial bioagent identifying amplicon and said calibration amplicon; and distinguishing said bacterial bioagent identifying amplicon from said calibration amplicon based on molecular mass, wherein comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in said sample.

16. The method of claim 15 further comprising determining the base composition of said bacterial bioagent identifying amplicon. 00 17. A method for identification of an unknown bacterium substantially as O Ohereinbefore described with reference to any one of the examples.

18. A method of determining the presence or absence of a bacterium substantially )as hereinbefore described with reference to any one of the examples.

19. A method for determination of the quantity of an unknown bacterium substantially as hereinbefore described with reference to any one of the examples. Dated 1 September 2008 ci Isis Pharmaceuticals, Inc. O 1o Patent Attorneys for the Applicant/Nominated Person C- SPRUSON FERGUSON 1292052 I:JIN