WO2012009813A1

WO2012009813A1 - Methods of detecting genetic polymorphisms using melting temperature of probe-amplicon complexes

Info

Publication number: WO2012009813A1
Application number: PCT/CA2011/050443
Authority: WO
Inventors: Eric Frost; Sylvie Deslandes; Patrick Dextras Paquette
Original assignee: Socpra Sciences Sante Et Humaines, S.E.C.
Priority date: 2010-07-20
Filing date: 2011-07-20
Publication date: 2012-01-26

Abstract

The present application provides methods and related kits for the detection of a nucleotide difference between a target sequence and standard sequences. The methods include the determination of a melting temperature of a complex between an amplicon of the target sequence and a probe which is substantially identical to one of the standard sequences. The probe comprises a detectable label, linked to its 5' end as well as a quencher, linked to its 3' end. This method can be easily adapted for use with multiple probes and in multiplex PCR.

Description

METHODS OF DETECTING GENETIC POLYMORPHISMS USING MELTING TEMPERATURE OF PROBE-AMPLICON COMPLEXES

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

This application claims priority from U.S. provisional patent application 61/365,921 filed on July

20, 2010, the content of which is incorporated by reference in its entirety.

This application contains a sequence listing submitted herewith electronically. The sequence listing is incorporated by reference in this application.

BACKGROUND

The explosion of genomic research has led to the discovery of single nucleotide polymorphisms (SNP) associated with antibiotic resistance and virulence traits in microorganisms, as well as with cancer, cardiovascular disease, and susceptibility or intolerance to medications in humans and other eukaryotes. Single nucleotide polymorphisms (SNP) in bacterial or viral genes are a common cause of the acquisition of clinically significant resistance to antibiotics or antivirals. They are also at the origin of strain variation. Single nucleotide polymorphisms (SNP) in bacterial genes are the most common cause of the acquisition of clinically significant resistance to the antibiotics usually employed to treat infections due to Campylobacter jejuni (C. jejuni), the most frequent bacterial cause of diarrhea. In addition, the sudden appearance of a pandemic strain of influenza A virus in April 2009 and its rapid dissemination around the globe obliged public health authorities to implement molecular detection strategies to identify this novel virus. This epidemic caused health authorities to realize that antigenic detection strategies were not sufficiently sensitive to detect influenza virus and, as a result, a significant number of hospitals switched from antigenic to molecular detection strategies creating a sudden demand for this kind of test. This epidemic also propelled diagnostic laboratories to sub-type influenza virus so that they could report the pandemic strain, one of the seasonal strains that had circulated earlier in the year or even avian H5N1 virus. Resistance to oseltamivir had become widespread in seasonal H1 N1 influenza and was also occasionally observed in avian H5N1 influenza even though it was very rarely observed in pandemic H1 N1 influenza. The extremely rare appearance of resistant virus following prophylactic use of oseltamivir has also discouraged widespread use. This is somewhat in contradiction with antibacterial usage. Physicians will continue to use normally effective and safe antibacterials until resistance levels exceed 5 to 10% but with influenza, physicians have developed cold feet before influenza resistance reached 1 %.

The detection of SNPs can have multiple diagnostic applications so numerous strategies have been devised for their detection. Some involve temperature specific annealing of a primer or a probe to the target sequence containing the SNP. When both primer and target are derived from wild type sequences or both from the mutant sequence the annealing temperature is usually several degrees higher than when one comes from the mutant and the other from wild type. Small differences in annealing temperature between heterologous and homologous primer and target pairs can be enhanced by using locked nucleic acids at the SNP site or minor groove binders nearby. These differences can be visualized in Invader assays™, or with TaqMan, Molecular Beacon, or fluorescent resonant energy transfer (FRET) probes by performing the detection step at a temperature in the window between the homologous and heterologous annealing temperatures. Allele specific PCR or ligation can be used with primers with the SNP at the 3' end of the primer. When SNPs form part of a naturally occurring or artificially engineered restriction endonuclease site, digestion of the amplicon with such enzymes can reveal the SNP. The melting temperature of FRET probes or the high resolution melting (HRM) profiles of short amplicons can also infer sequence data revealing SNP. When all else fails, amplicons can be sequenced, pyrosequenced, or hybridized to specific probes.

While most of these methods can be performed over several days by high complexity reference laboratories, hospital clinical laboratories will depend on assays run on realtime (RT) PCR apparatus usually using TaqMan™ probes, but occasionally with Molecular Beacon™ or FRET probes or other apparatus that are simple to operate and analyse like those performing Invader™ assays.

TaqMan™ probes are usually designed to be located near one of the PCR primers so that when the Taq polymerase reaches the probe, it will cleave its 5' end. This will separate the fluorophore attached to the 5' end of the probe and notably from a strong quencher attached to the 3' end that would normally absorb all the light emitted from the fluorophore. When this method is applied for the detection of the H1 N1 influenza virus RNA, it is suggested that signal acquisition should be performed at the annealing temperature of 60°C rather than at the extension temperature of 72°C. Of importance, the 5' endonuclease activity is expected to reach its maximum at 72°C, while at 60°C only the fluorophores cleaved during the preceding cycle are expected to be detected. Consequently, it is noted that when the TaqMan™ probe anneals to its target (thereby separating the 5' from the 3' end), even in the presence of a strong quencher, that light might escape and could be detected.

Molecular Beacons™ probes, on the other hand, have complementary sequences at the ends, often referred to as "panhandles", which when hybridized together bring a fluorophore at the 5' end in very close proximity to a quencher at the 3' end. Measurable light is emitted from the fluorophore only when the probe hybridizes to its target and the 5' and 3' ends are spatially separated. Molecular Beacons™ probes are designed so that their complementary ends reanneal near the annealing temperature of the PCR reaction. Thus, melting temperature curves could not usefully be performed below this PCR annealing temperature. A recent modification called "sloppy" Molecular Beacons™, which are characterized by relatively long probe sequences, have been used with melting temperature determinations.

FRET probes involve transfer of light not to a quencher but rather to a second fluorophore that is not excited by the wave length used by the analyzer which excites only the fluorophore fluorescein. The second fluorophore then absorbs the light emitted by the fluorescein moiety to emit at a longer wave length. This occurs when two probes hybridize to adjoining sequences on the target such that the 3' end of one is very near the 5' end of the other. The use of FRET™ probes in melting temperature determinations may fail probably due to the 5' endonuclease activity of the polymerase.

High resolution melting or HRM is a method based on PCR amplification of a short sequence and the use of a double-stranded specific fluorescent dye. Briefly, the sequence is amplified and the dye inserts within the double-stranded amplicon. Following PCR amplification, a very slow temperature ramping induced amplicon melting is performed and fluorescence (or loss thereof) is measured. Since each DNA amplicon possess a unique denaturation pattern and the presence of a polymorphism can modify the amplicon's denaturation pattern, high resolution melting can be used to distinguish between sequences differing in their nucleotide sequence. However, this procedure requires a technically demanding analysis protocol and controls representing each potential target. An adaptation of this method using unlabeled probes representing short specific sequences has also been described, but separate wells must be used for each probe and the presence of the full length amplicon tended to mask the fluorescent peaks obtained with the probe.

It would be highly desirable to be provided with a method of detecting differences in sequences that would be rapid, efficient and cost-effective. It would be also highly desirable to be provided with a method of confirming the detection of sequence mutations.

BRIEF SUMMARY

In accordance with the present invention there is provided a method for detecting a nucleotide different between an unknown sequence (herein referred to as the target sequence) and a known sequence (herein referred to as the standard sequence). In this method, a probe is contacted with the unknown sequence so as to form a complex and the melting of the complex is determined and compared to the melting temperature of a complex between the known sequence and the probe. According to a first aspect, the present application provides a method of detecting a nucleotide difference between a target sequence and a first standard sequence. Broadly, the method comprises: amplifying the target sequence to generate a target amplicon; contacting the target amplicon with a first probe to form a first target amplicon-probe complex, wherein the first probe has a first label covalently linked to the one end of the probe and a first corresponding quencher linked to the other end of the probe; measuring the signal from the first label of the first probe within a range of temperature to determine the melting temperature of the first target amplicon- probe complex; providing the melting temperature of a complex between a corresponding amplicon of the first standard sequence and the first probe; comparing the melting temperature of the first target amplicon-probe complex with the melting temperature of standard sequence amplicon-probe complex; and detecting the presence of the nucleotide difference if the melting temperature of the first target amplicon-probe complex is lower than the melting temperature of the standard sequence amplicon-probe complex, or the absence of the nucleotide difference if the melting temperature of the first target amplicon-probe complex is substantially similar to the melting temperature of the standard sequence amplicon-probe complex. In an embodiment, the sequence of the first probe is substantially identical to a fragment of the corresponding amplicon of the first standard sequence. In an embodiment, the method further comprises contacting the target amplicon with a second probe to form a second target amplicon-probe complex, wherein the second probe has a second label covalently linked to one end of the probe and a second corresponding quencher linked to the other end of the probe and wherein the first and second label are different; measuring the signal from the second label of the second probe within a range of temperature to determine the melting temperature of the second target amplicon-probe complex; providing the melting temperature of a complex between a corresponding amplicon of the first standard sequence and the second probe; determining a first difference in the melting temperature of the first target amplicon-probe complex and the melting temperature of standard amplicon-probe complex; determining a second difference in the melting temperature of the second amplicon-probe complex with the standard amplicon-second probe complex; determining which of the first difference or the second difference is lower; and assessing the identity of the nucleotide difference based on this determination. In this specific embodiment, the sequence of the second probe is substantially identical to a fragment of a second standard sequence and wherein the second standard sequence differs by at least one nucleotide from the first standard sequence. In yet another embodiment, the second standard sequence differs by at least two, at least three, at least four or at least five nucleotides from the first standard sequence. In yet a further embodiment, the first difference in melting temperature and the second difference in melting temperature is equal to or lower than about 25°C, 10°C, 5°C or between about 2°C to 4°C. ln still a further embodiment, the amplification step comprises a polymerase chain reaction. In yet another embodiment, a first primer is used for the polymerase chain reaction and said first primer is substantially identical to a first portion of the target sequence or complement thereof and a first corresponding portion of the first standard sequence or complement thereof and is non-linear to the first and/or second probe. In yet a further embodiment, a second primer is used for the polymerase chain reaction and said second primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is co-linear to the first and/or second probe. In still another embodiment, the concentration of the first primer is higher than the concentration of the second primer in the polymerase chain reaction. In yet another embodiment, the first and/or second probe is present during the polymerase chain reaction and the concentration of the first and/or second probe is higher than the concentration of the second primer. In another embodiment, the range of temperature for determining the melting temperature is between about 30°C to about 95°C or about 45°C to about 70°C. In yet another embodiment, the nucleotide difference is a single nucleotide polymorphism.

In an embodiment, the target sequence and the first standard sequence are derived from Campylobacter jejuni. For example, the target sequence and the first standard sequence can be derived from a 23S ribosomal RNA gene (wherein the first primer comprises the sequence of SEQ ID NO: 1 , SEQ ID NO: 2, or a complement thereof; wherein the second primer comprises the sequence of SEQ ID NO: 1 , SEQ ID NO: 2, or a complement thereof; and/or wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and complementary sequences thereof). In another example, the target sequence and the first standard sequence are derived from a gyrA gene (wherein the first primer comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or a complement thereof; wherein the second primer comprises the sequence of of SEQ ID NO: 6, SEQ ID NO: 7, or a complement thereof; and/or the first probe and/or the second probe comprises is at least one of the following sequences: SEQ ID NO: 8, SEQ ID NO: 9 and complementary sequences thereof)

In another embodiment, the target sequence and the first standard sequence can be derived from Influenza virus. For example, the target sequence and the first standard sequence can be from a matrix gene (wherein the first primer comprises the sequence of SEQ ID NO: 10, SEQ ID NO: 11 , or a complement thereof; wherein the second primer comprises the sequence of (i of SEQ ID NO: 10, SEQ ID NO: 11 , or a complement thereof ; and/or wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and complementary sequences thereof). In another example, the target sequence and the first standard sequence can be from a neuraminidase gene, (wherein the first primer comprises the sequence of SEQ ID NO: 12, SEQ ID NO: 13, or complement thereof; wherein the second primer comprises the sequence of SEQ ID NO: 12, SEQ ID NO: 13, or complement thereof; and/or wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 and complementary sequences thereof).

In a further embodiment, the target sequence and the first standard sequence can be derived from a 16S ribosomal RNA gene of a bacteria. In yet another embodiment, the bacteria can be Dialister pneumosintes (wherein the first primer comprises the sequence of SEQ ID NO: 31 , SEQ ID NO: 32, or a complement thereof; wherein the second primer comprises the sequence of SEQ ID NO: 31 , SEQ ID NO: 32, or a complement thereof; and/or wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 33 or a complementary sequence thereof). In another embodiment, the bacteria can be Anaerococcus sp. (wherein the first primer comprises the sequence of SEQ ID NO: 34, SEQ ID NO: 35, or a complement thereof; wherein the second primer comprises the sequence of of SEQ ID NO: 34, SEQ ID NO: 35, or a complement thereof; and/or wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 36 or a complementary sequence thereof). In another embodiment, the bacteria can be Peptoniphilus sp. other than P. lacrimalis (wherein the first primer comprises the sequence of SEQ ID NO: 37, SEQ ID NO: 38 or a complement thereof; wherein the second primer comprises the sequence of SEQ ID NO: 37, SEQ ID NO: 38 or a complement thereof; and/or wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 39 or a complementary sequence thereof. In another embodiment, the bacteria can be Bifidobacterium sp. (wherein the first primer comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 41 , or a complement thereof, wherein the second primer comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 41 , or a complement thereof; and/or wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 33 or a complementary sequence thereof).

According to a second aspect, the present application provides a method of detecting a plurality of nucleotide differences between a target sequence and a first standard sequence. Broadly, the method comprises: amplifying the target sequence to generate a plurality of target amplicons; contacting the plurality of target amplicons with at least two probes to form at least two target amplicon-probe complexes, wherein the at least two probes each have a label covalently linked to the one end of the probe and a corresponding quencher linked to the other end of the probe and each of the at least two probes have a different label; measuring the signal from each label of the at least two probes within at least one temperature range to determine the melting temperatures of the at least two target amplicon-probe complexes; providing the melting temperature of complexes of at leas two corresponding amplicons of the first standard sequence and the at least two probes; comparing the melting temperature of the at least two of target amplicon-probe complexes with the melting temperatures of the corresponding standard amplicons-probes complexes; and detecting, for each target-amplicon probe complex, the presence or absence of the plurality nucleotide difference based on this comparison. In the method, the sequence of the at least two probes are substantially identical to a fragment of a corresponding amplicon of the first standard sequence. In an embodiment, the amplification comprises a polymerase chain reaction. In yet another embodiment, a first primer is used for the polymerase chain reaction and said first primer is substantially identical to a first portion of the target sequence or a complement thereof and a first corresponding portion of the first standard sequence or a complement and is non-linear to the first of the at least two probes. In another embodiment, a second primer is used for the polymerase chain reaction and said second primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is co-linear to the first of the at least two probes. In still another embodiment, a third primer is used for the polymerase chain reaction and said third primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is non-linear to the second of the at least two probes. In yet another embodiment, a fourth primer is used for the polymerase chain reaction and said fourth primer is substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and is co-linear to the second of the at least two probes. In an embodiment, the concentration of the first primer is higher than the concentration of the second primer in the polymerase chain reaction. In another embodiment, the concentration of the third primer is higher than the concentration of the fourth primer in the polymerase chain reaction. In still another embodiment, the at least two probes are present during the polymerase chain reaction. In yet another embodiment, the concentration of the first probe is higher than the concentration of the second primer. In still another embodiment, the concentration of the second probe is higher than the concentration of the fourth primer. In yet another embodiment, the range of temperature is between about 30°C to about 95°C or between about 45°C to about 70°C.

According to a third aspect, the present application provides a kit for the determination of a nucleotide difference between a target sequence and a standard sequence. The kit comprises a first primer and a second primer for amplifying the target sequence and generate a corresponding target amplicon; and a first probe for forming a first complex with the target amplicon, wherein the first probe has a first label covalently linked to the one end of the probe and a first quencher linked to the other end of the probe and wherein the sequence of the first probe is substantially identical to a portion of the standard sequence, wherein the concentration the concentration of the first probe is higher than the concentration of the second primer. In an embodiment, the kit further comprises a second probe for forming a second complex with the target amplicon, wherein the second probe has a second label covalently linked to the one end of the probe and a second corresponding quencher linked to the other end of the probe, wherein the first label and second label are different and wherein the sequence of the second probe is substantially identical to a portion of a second standard sequence, wherein the second standard sequence differs by at least one nucleotide from the first standard sequence. In still another embodiment, the kit further comprises a thrid primer and a fourth primer for amplifying the target sequence and generate a second corresponding target amplicon; and a second probe for forming a second complex with the second target amplicon, wherein the second probe has a first label covalently linked to the one end of the probe and a first quencher linked to the other end of the probe and wherein the sequence of the second probe is substantially identical to a second portion of the standard sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

Fig. 1 illustrates High Resolution Melting curves (A) without and (B) with an unlabelled probe specific for the resistance sequence. High resolution melting profiles of a 131 bp sequence amplicon from the 23S rRNA gene from erythromycin sensitive (regular line) and erythromycin resistant (dashed line) C. jejuni isolates.

Fig. 2 illustrates asymmetric PCR with a non-fluorescent probe. The 131 bp sequence amplicon from the 23S rRNA gene was amplified in HRM PCR reaction mix with asymmetric primers together with an unlabelled probe specific for the resistant sequence with DNA from erythromycin sensitive (regular line for Ery^s) or erythromycin resistant (dashed line for Ery^R) C. jejuni isolates.

Fig. 3 illustrates amplification profiles generated with different annealing temperatures to distinguish a sequence marker specific for erythromycin sensitivity. Amplification profiles of a 131 bp sequence amplicon from the 23S rRNA gene were performed using a probe specific for the erythromycin sensitive sequence of C. jejuni at an annealing temperature of 55°C (panel A), 58°C (panel B), or 60°C (panel C). The reactions contained DNA from erythromycin sensitive (regular line for Ery^s) or erythromycin resistant (bold line for Ery^R) C. jejuni isolates. A negative control without C. jejuni DNA was also analysed (dashed line).

Fig. 4 illustrates a comparison between different polymerases used for SNP detection using TaqMan™ probes with melting curve analysis. The 131 bp sequence amplicon from the 23S rRNA gene and the 232 bp amplicon of the gyrA gene were simultaneously amplified in the presence of a mix of probes labelled with different fluorophores. Only the probe specific for the erythromycin sensitive sequence of C, jejuni is shown here, but the other three probes gave similar results. In panels A and E, those done with Roche Taq buffer supplied in the 2X Roche LightCycler™ 480 Probe Master Kit, in panels B and F those done with KlenTaq™, in panels C and G done with Taq and in panels D and H those done with VentR™ (exo-). Profiles for DNA from erythromycin sensitive (regular line for Ery^s), erythromycin resistant (dashed line for Ery^R), C. jejuni isolates are shown.

Fig. 5 illustrates a comparison of primer ratios for asymmetric PCR. The 131 bp sequence amplicon from the 23S rRNA gene and the 232 bp amplicon of the gyrA gene were simultaneously amplified with different asymmetric primer concentrations with a mix of probes labeled with different fluorophores. Only amplification profiles (A) and melting temperature profiles (B) detected with the probe specific for erythromycin sensitivitive C. jejuni are shown here. Ratios used for Forward primer/Reverse Primer were: 1 :7 (0.1 μΜ and 0.7 μΜ), 1 :10 (0.07 μΜ and 0.7 μΜ), 1 :14 (0.05 μΜ and 0.7 μΜ), 1 :23 (0.03 μΜ and 0.7 μΜ) and 1 :70 (0.01 μΜ and 0.7 μΜ). Profiles for DNA from erythromycin sensitive (regular line for Ery^s), erythromycin resistant (dashed line for Ery^R), C. jejuni isolates are shown.

Fig. 6 illustrates SNP detection using labelled probes with melting curve analysis. The 131 bp sequence amplicon from the 23S rRNA gene and the 232 bp amplicon of the gyrA gene were simultaneously amplified with asymmetric probe concentrations together with a mix of probes labeled with different fluorophores. Amplification profiles and melting temperature profiles detected with the probe specific for erythromycin sensitivity are shown in panels A and E, those for the erythromycin resistance specific probe in panels B and F, those for the ciprofloxacin sensitive sequence in panels C and G and those for the ciprofloxacin resistance sequences in panels D and H. Profiles for DNA from erythromycin sensitive (regular line for Ery^s), erythromycin resistant (dashed line for Ery^R), ciprofloxacin sensitive (regular line for Cip^s), ciprofloxacin resistant (dashed line for Cip^R) C. jejuni isolates are shown.

Fig. 7 illustrates a distinction of heterozygotes in the 23S rRNA gene by amplification profiles and by melting curves with labeled probes. Artificial mixes of DNA from bacteria sensitive to erythromycin with that from resistant bacteria were made to simulate bacteria with two 23S resistant copies and one 23S sensitive copy; with two 23S sensitive copies and one 23S resistant copy, or with all of three 23S gene copies that are resistant; or sensitive. Amplification curves (panel A) from sensitive bacteria (regular line), for resistant bacteria (dashed line), and mixtures of sensitive and resistant bacteria (bold line) are shown. In the melting curve analysis (panel B), homozygote resistant (regular line) or sensitive (regular line) bacteria presented single peaks typical of sensitive (at around 65°C) or resistant (at around 55°C) bacteria, whereas heterozygotes presented two peaks (bold line) of varying height at temperatures coinciding with those of both sensitive and resistant bacteria. A negative control without C. jejuni DNA was also analysed (bottom line).

Fig. 8 illustrates a sequence alignment between the matrix coding sequence of various representative influenza strains downloaded from GenBank including seasonal H1 N1 isolates A/Maryland/1/2006(H1 N1) (referred to as 2006(H1 N1)), and A/New Jersey/06/2008(H1 N1 ) (referred to as 2008(H1 N1 )), a reference pandemic strain A California/04/2009(H1 N1 ) (referred to as Pandemic 2009(H1 N1 )), a seasonal H3N2 isolate A/California/UR07-0053/2008(H3N2) (referred to as 2008(H3N2)), and an H5N1 isolate A/lndonesia/CDC1046/2007(H5N1) (referred to as 2007(H5N1)). Consensus sequence is shown above the other sequences. Dots indicate the same sequence as the consensus. Primers and the universal probe FluAun209F Tx615 are underlined. The probes used for subtyping purposes are identified as "subtype specific probes" and are underlined.

Fig. 9 illustrates the distinction of Influenza A virus sub-types by amplification and melting curves using labeled probes. Amplification curves (panels A, C, and E) and melting temperatures (panels B, D, and F) were obtained with three probes (A and B, H3N2 probe; C and D, PanH1 N1 probe; E and F, Sea H1 N1 probe; universal probe not shown). Only samples containing the corresponding virus type yielded amplification and melting curves, except for the pandemic H1 1 samples which gave melting temperature peaks at 57°C with the H3N2 probe (panel B), but not amplification curves (panel A).

Fig. 10 illustrates a sequence alignment of a portion of the neuraminidase gene surrounding the H275Y mutation responsible for oseltamivir resistance between various influenza strains (the pandemic probe used in experiments, a seasonal H1 N1 sensitive strain A/New York/UR06- 0253/2007(H1 N1 ) (referred to as Seasonal H1 N1 S), a seasonal H1 N1 resistant strain A/Pennsylvania/09/2008(H1 N1 ) (referred to as Seasonal H1 N1 R), a pandemic sensitive strain A/California/04/2009(H1 N1 ) (referred to as Pandemic H1 N1 S), two H5N1 sensitive strains A/lndonesia/CDC1032T/2007(H5N1 ) and A/Cambodia/R0405050/2007(H5N1) (both referred to as H5N1 S) and an inosine probe. Resistance and sensitivity are in relationship with oseltamivir. The codon for histidine at position 275 is underlined. When a T replaces the initial C in this codon the resulting virus is resistant to oseltamivir.

Fig. 11 illustrates the detection of oseltamivir sensitivity in Influenza A viruses carrying neuraminidase type 1. The amplification curves (panel B) and melting temperatures (panel A) are shown for the neuraminidase probe (universal probe not shown). Melting curves were also determined for the fluorescein labeled neuraminidase probe (panel C) and the unlabeled neuraminidase probe containing inosine residues (panel D) added at 0.1 μΜ to 0.1 μΜ of a synthetic probe representative of the complementary sequences of oseltamivir sensitive and resistant pandemic H1 N1 (PanS and PanR), seasonal H1 N1 (SeaS and SeaR) or avian H5N1 (H5 S, H5 R).

Fig. 12 illustrates a sequence alignment of a portion of the 16S ribosomal gene for Anaerococcus, Dialister, Bifidobacter, Peptinophilus sp., and Peptinophilus lacrimalis as well as the position and sequence identity of various primers and probes used in the subsequent PCR reactions. The numerous differences in the sequences within the primers and probes should assure specificity for each reaction to the desired microorganism.

Fig. 13 illustrates a sequence alignment of a portion of the 16S ribosomal gene for Peptinophilus lacrimalis, Peptinophilus indolicus, Peptinophilus stomatis, Anaerobranca californiensis, Anaerobranca horikoshii, Anaerobranca gottschalkii, Dialister pneumosintes, Dialister invisus and Dialister micraerophilus as well as the position and sequence identity of various primers and probes. There are 4 single nucleotide polymorphisms between the probe sequence for Dialister pneumosintes and the 2 other Dialister species tested. There is one single nucleotide polymorphism between the forward primer (PrimerF) sequences for Dialister pneumosintes and Dialister invisus and again one single nucleotide polymorphism between the reverse primer (PrimerR) sequences for Dialister pneumosintes and Dialister microaerophilus. This variation should not prevent PCR, whereas there are at least 4 single nucleotide polymorphisms in the 8 3^' nucleotides between the forward and reverse primer (PrimerF) sequences for Dialister pneumosintes and the other related micro-organisms in this Table. Thus, 16S ribosomal RNA gene sequences from these organisms should not be amplified. Fig 14 illustrates the classification and identification of bacteria by amplification of 16S ribosomal RNA genes and performing melting curves using TaqMan™ probes. DNA from patient vaginal samples was analysed by TaqTm probing using primers and probes specific for different bacteria. Left panels (A, C and E) show melting curves whereas right panels (B, D and F) show amplification curves. Results for Peptinophilus species other than P. lacrimalis are shown in the top panels (A and B), for Dialister pseumosintes in the middle panels (C and D), for Anaerococcus sp. in the bottom panels (E and F). Positively identified bacteria are presented as regular lines, negative samples as clashed lines, and variant bacteria as bold lines. One variant melting peak at only a slightly lower temperature (about 66°C) than the typical peak (about 67.5°C) can be seen (bold lines) in the upper left panel (A). Again only one variant peak with a lower melting temperature (about 64°C) compared with the typical peak (about 68°C) can be seen in the middle panel (C). A much lower temperature peak (53°C) can be seen in five samples in the lower panel (E) compared with the typical peak (66.5°C).

DETAILED DESCRIPTION

In accordance with the present invention, there is provided a method for the detection of differences between at least two corresponding sequences. This method is based on the amplification of a corresponding portion of the sequence to be analyzed (herein referred to as the target or unknown sequence). The resulting amplicon is then contacted with a probe so as to form a complex with the amplicon. The probe is covalently linked at one of its ends with a detectable label and at its other end with a corresponding quencher. The melting temperature of the resulting complex (between the probe and the amplicon) is then determined, based on the acquisition of the signal from the probe, and compared with a standard melting temperature to characterize the presence or absence of the nucleotide difference. The method can be conveniently modified to accommodate the use of more than one probe, the detection of more than one nucleotide difference as well as to determine the copy number of an allele, on a semiquantitative basis. The method can be particularly convenient to detect mutations associated with antibiotic or antiviral resistance, bacterial and viral subtypes, or human haplotypes.

Under usual PCR conditions, both strands produced by the PCR reaction are present in large enough amounts that they will reanneal in less than 10 seconds and thereby prevent smaller fluorescent probes from reannealing with their complementary strand. In theory, each primer that initiates synthesis of a strand will degrade the probe hybridized to the strand being copied so 0.7 μΜ of primer might consume 0.7 μΜ of probe. Once again, under usual PCR conditions, a fluorescent probe could not be used to determine the melting temperature of the amplicons. On the other hand, the use of a PCR enzyme that does not have a 5' endonuclease activity, like the KlenTaq polymerase (Sigma) or Vent polymerase (New England Biolabs), would not degrade the probe and so could be employed to preserve intact probe. It would not generate a typical PCR curve, however, because it would not degrade the TaqMan™ probe, so signal acquisition must be determined rapidly at the annealing temperature before the polymerase displaced the probe in the course of synthesis of a new DNA strand and even this action did not generate amplification curves with crossing points as low and fluorescent intensities as high as polymerases with a 5' endonuclease activity. However, as shown herein, the methods described use the determination of the melting temperature of a complex between a labeled probe and an amplicon to indicate the presence or absence of base or nucleotide mismatch(es) between the probe and the amplicon.

Definitions

As used herein, the term "base" refers to any nitrogen-containing heterocyclic moiety capable of forming Watson-Crick type hydrogen bonds in pairing with a complementary base or base analog. A large number of natural and synthetic (non-natural, or unnatural) bases, base analogs and base derivatives are known. Examples of bases include purines and pyrimidines, and modified forms thereof. The naturally occurring bases include, but are not limited to, adenine (A), guanine (G), cytosine (C), uracil (U) and thymine (T). As used herein, it is not intended that the invention be limited to naturally occurring bases, as a large number of unnatural (non- naturally occurring) bases and their respective unnatural nucleotides that find use with the invention are known to one of skill in the art. In an embodiment, inosine (I) can be successfully used to increase the melting temperature of an amplicon-probe complex. Examples of such unnatural bases are given below. The term "nucleoside" refers to a compound consisting of a base linked to the C-1' carbon of a sugar, for example, ribose or deoxyribose.

The term "nucleotide" refers to a phosphate ester of a nucleoside, as a monomer unit or within a polynucleotide. "Nucleotide 5'-triphosphate" refers to a nucleotide with a triphosphate ester group attached to the sugar 5 -carbon position, and are sometimes denoted as "NTP", or "dNTP" and "ddNTP". A modified nucleotide is any nucleotide (e.g., ATP, TTP, GTP or CTP) that has been chemically modified, typically by modification of the base moiety. Modified nucleotides include, for example but not limited to, methylcytosine, 6-mercaptopurine, 5- fluorouracil, 5-iodo-2'-deoxyuridine and 6-thioguanine. As used herein, the term "nucleotide analog" refers to any nucleotide that is non-naturally occurring.

The terms "polynucleotide", "nucleic acid", "oligonucleotide", "oligomer", "oligo" or equivalent terms, as used herein refer to a polymeric arrangement of monomers that corresponded to a sequence of nucleotide bases, e.g., a DNA, RNA, peptide nucleic acid, or the like. A polynucleotide can be single- or double-stranded, and can be complementary to the sense or antisense strand of a gene sequence, for example. A polynucleotide can hybridize with a complementary portion of a target polynucleotide to form a duplex, which can be a homoduplex or a heteroduplex. A homoduplex being a duplex between two perfectly complementary DNA strands such as those from a single organism, whereas a heteroduplex is a duplex that is not perfectly complementary where one strand of DNA (for example the sense strand) comes from one organism and the other strand (for example the antisense strand) and has a different sequence of bases and thus forms a duplex that is not perfectly complementary. The length of a polynucleotide is not limited in any respect. Linkages between nucleotides can be internucleotide-type phosphodiester linkages, or any other type of linkage. A "polynucleotide sequence" refers to the sequence of nucleotide monomers along the polymer. A "polynucleotide" is not limited to any particular length or range of nucleotide sequence, as the term "polynucleotide" encompasses polymeric forms of nucleotides of any length. A polynucleotide can be produced by biological means (e.g., enzymatically), or synthesized using an enzyme-free system. A polynucleotide can be enzymatically extendable or enzymatically non-extendable.

Polynucleotides that are formed by 3'-5' phosphodiester linkages are said to have 5 -ends and 3 -ends because the nucleotide monomers that are reacted to make the polynucleotide are joined in such a manner that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen (hydroxyl) of its neighbor in one direction via the phosphodiester linkage. Thus, the 5'-end of a polynucleotide molecule has a free phosphate group, a hydroxyl, or other group at the 5' position of the pentose ring of the nucleotide, while the 3' end of the polynucleotide molecule has a free phosphate, hydroxyl, or other group at the 3' position of the pentose ring. Within a polynucleotide molecule, a position or sequence that is oriented 5' relative to another position or sequence is said to be located "upstream", while a position that is 3' to another position is said to be "downstream". This terminology reflects the fact that polymerases proceed and extend a polynucleotide chain in a 5' to 3' fashion along the template strand. Unless denoted otherwise, whenever a polynucleotide sequence is represented, it will be understood that the nucleotides are in 5' to 3' orientation from left to right.

As used herein, it is not intended that the term "polynucleotides" be limited to naturally occurring polynucleotides sequences or polynucleotide structures, naturally occurring backbones or naturally occurring internucleotide linkages. One familiar with the art knows well that in addition to synthetic oligonucleotides identical in sequence and structure to natural oligonucleotides, there is a wide variety of polynucleotide analogues, unnatural nucleotides, non-natural phosphodiester bond linkages and internucleotide analogs that find use with the invention. Non- limiting examples of such unnatural structures include non-ribose sugar backbones, 3'-5' and 2'- 5' phosphodiester linkages, internucleotide inverted linkages (e.g., 3 -3' and 5 -5'), and branched structures. Furthermore, unnatural structures also include unnatural internucleotide analogs, e.g., peptide nucleic acids (PNAs), locked nucleic acids (LNAs), C -C alkylphosphonate linkages such as methylphosphonate, phosphoramidate, Ci-C₆ alkyl-phosphotriester, phosphorothioate and phosphorodithioate internucleotide linkages. Furthermore, a polynucleotide can be composed entirely of a single type of monomeric subunit and one type of linkage, or can be composed of mixtures or combinations of different types of subunits and different types of linkages (a polynucleotide can be a chimeric molecule). As used herein, a polynucleotide analog retains the essential nature of natural polynucleotides in that they hybridize to a single-stranded nucleic acid target in a manner similar to naturally occurring polynucleotides.

As used herein, the term "sequence of a polynucleotide", "nucleic acid sequence", "polynucleotide sequence" and equivalent or similar phrases refer to the order of nucleotides in the polynucleotide. In some cases, a "sequence" refers more specifically to the order and identity of the bases that are each attached to the nucleotides. A sequence is typically read (written or provided) in the 5' to 3' direction. Unless otherwise indicated, a particular polynucleotide sequence of the invention optionally encompasses complementary sequences, in addition to the sequence explicitly indicated.

As used herein, the terms "subsequence", "fragment" or "portion" and the like refer to any portion of a larger sequence (e.g., a polynucleotide or polypeptide sequence), up to and including the complete sequence. The minimum length of a subsequence is generally not limited, except that a minimum length may be useful in view of its intended function. For example, a polynucleotide portion can be amplified to produce an amplicon, which in turn can be used in a hybridization reaction that includes a polynucleotide probe. Thus, in this case, the amplified portion should be long enough to specifically hybridize to a polynucleotide probe. Portions of polynucleotides can be any length, for example, at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150 or 200 nucleotides or more in length.

As used herein, the terms "amplification", "amplifying" and the like refer generally to any process that results in an increase in the copy number of a molecule or set of related molecules. As it applies to polynucleotide molecules, amplification means the production of multiple copies of a polynucleotide molecule, or a portion of a polynucleotide molecule, typically starting from a small amount (undetectable without amplification) of a polynucleotide, until, typically, the amplified material becomes detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other detection. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a template DNA molecule during a polymerase chain reaction (PCR), a strand displacement amplification (SDA) reaction, a transcription mediated amplification (TMA) reaction, a nucleic acid sequence-based amplification (NASBA) reaction, or a ligase chain reaction (LCR) are forms of amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, the generation of multiple cDNA molecules from a limited amount of viral RNA in a sample using RT-PCR is a form of amplification. Furthermore, the generation of multiple RNA molecules from a single DNA molecule during the process of transcription is also a form of amplification.

As used herein, the term "polymerase chain reaction" (PCR) refers to a method for amplification well known in the art for increasing the concentration of a segment of a target polynucleotide in a sample, where the sample can be a single polynucleotide species, or multiple polynucleotides. Generally, the PCR process consists of introducing a molar excess of two or more extendable oligonucleotide primers to a reaction mixture comprising the desired target sequence(s), where the primers are complementary to opposite strands of the double stranded target sequence. The use of the primers enable the production of amplicons that represent a target or standard sequence. The reaction mixture is usually subjected to a program of thermal cycling in the presence of a DNA polymerase, resulting in the amplification of the desired target sequence flanked by the DNA primers. Reverse transcriptase PCR (RT-PCR) is a PCR reaction that uses an RNA template and a reverse transcriptase, or an enzyme having reverse transcriptase activity, to first generate a single stranded DNA molecule prior to the multiple cycles of DNA- dependent DNA polymerase primer elongation. Multiplex PCR refers to PCR reactions that produce multiple copies of more than one product or amplicon in a single reaction, typically by the inclusion of more than two different primers in a single reaction.

For example, the cycles can consist of an initial denaturation step at 94 or 95°C where double stranded DNA is denatured to provide single strands that can be copied; an annealing step usually at 55°C or 60°C allows primers and probes to anneal to the single strands. Primers can initiate synthesis of new DNA copies and this process can be completed in the following step of synthesis at 72°C which is the usual optimal temperature for Taq DNA polymerase activity. Probe fluorescence can be measured for some probes at the annealing temperature while the probes are annealed to the single strand separating the fluorophore from its quencher or bringing together the probes and before the Taq polymerase degrades or displaces the probes to complete synthesis of the new strand. For other probes, such as TaqMan™ probes, fluorescence may be measured during the annealing cycle or at 72°C. Since the melting temperature of most probes is below 72°C, they will have dissociated from the complex and no longer be fluorescent.

As used herein, the expression "asymmetric PCR" refers to the preferential PCR amplification of one strand of a DNA target by adjusting the molar concentration of the primers in a primer pair so that they are unequal. An asymmetric PCR reaction produces a predominantly single- stranded product and a smaller quantity of a double-stranded product as a result of the unequal primer concentrations. As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into a double-stranded DNA amplicon, but the higher concentration primer continues to prime DNA synthesis, resulting in continued accumulation of a single stranded product. Asymmetric PCR also includes the use of a single primer for amplification.

As used herein, the terms "real-time PCR" or "kinetic PCR" refer to real-time detection and/or quantitation of amplicon generated in a PCR, without the need for a detection or quantitation step following the completion of the amplification.

The TaqMan™ PCR reaction uses a thermostable DNA-dependent DNA polymerase that possesses a 5'-3' nuclease activity. During the PCR amplification reaction, the 5 -3' nuclease activity of the DNA polymerase cleaves the labeled probe that is hybridized to the amplicon in a template-dependent manner. The resultant probe fragments dissociate from the primer/template complex, and the reporter fluorophore typically, but not exclusively attached to the 5' end of the probe is then free from the quenching effect of the quencher moiety typically, but not exclusively attached to the 3' end of the probe. Approximately one molecule of reporter fluorophore is liberated for each new amplicon molecule synthesized, and detection of the unquenched reporter fluorophore provides the basis for quantitative interpretation of the data, such that the amount of released fluorescent reporter is directly proportional to the amount of amplicon template.

One measure of the TaqMan™ assay data is typically expressed as the threshold cycle (C_T). Fluorescence levels are recorded during each PCR cycle and are proportional to the amount of product amplified to that point in the amplification reaction. The PCR cycle when the fluorescence signal is first recorded as statistically significant, or where the fluorescence signal is above some other arbitrary level (e.g., the arbitrary fluorescence level, or AFL), is the threshold cycle (C_T).

Variations in methodologies for real-time amplicon detection are also known, and in particular, where the 5'-nuclease probe is replaced by double-stranded DNA intercalating dye resulting in fluorescence that is dependent on the amount of double-stranded amplicon that is present in the amplification reaction.

As used herein, the term "DNA-dependent DNA polymerase" refers to a DNA polymerase enzyme that uses deoxyribonucleic acid (DNA) as a template for the synthesis of a complementary and antiparallel DNA strand. Thermostable DNA-dependent DNA polymerases find use in PCR amplification reactions. Suitable reaction conditions (and reaction buffers) for DNA-dependent DNA polymerase enzymes, and indeed any polymerase enzyme, are widely known in the art. Reaction buffers for DNA-dependent DNA polymerase enzymes can comprise, for example, free deoxyribonucleotide triphosphates, salts and buffering agents. As used herein, the term "RNA-dependent DNA polymerase" refers to a DNA polymerase enzyme that uses ribonucleic acid (RNA) as a template for the synthesis of a complementary and antiparallel DNA strand. The process of generating a DNA copy of an RNA molecule is commonly termed "reverse transcription", or "RT", and the enzyme that accomplishes that is a "reverse transcriptase". Some naturally-occurring and mutated DNA polymerases also possess reverse transcription activity.

As used herein, the term "thermostable", as applied to an enzyme, refers to an enzyme that retains its biological activity at elevated temperatures (e.g., at 55°C or higher), or retains its biological activity following repeated cycles of heating and cooling. Thermostable polynucleotide polymerases find particular use in PCR amplification reactions, and, in an embodiment, they can retain their biological activity even at 94°C or 95°C.

As used herein, the term "primer" refers to an enzymatically extendable oligonucleotide, generally with a defined sequence that is designed to hybridize in an antiparallel manner with a complementary, primer-specific portion of a sequence (e.g. target sequence). Further, a primer can initiate the polymerization of nucleotides in a template-dependent manner to yield a polynucleotide that is complementary to the target polynucleotide. The extension of a primer annealed to a target uses a suitable DNA or RNA polymerase in suitable reaction conditions. One skilled in the art knows well that polymerization reaction conditions and reagents are well established in the art, and are described in a variety of sources.

A primer nucleic acid does not need to be 100% complementary with its template subsequence (such as the target sequence or the standard sequence) for primer elongation or probe binding to occur; primers and probes with less than 100% complementarity can be sufficient for hybridization and polymerase elongation to occur depending on the annealing temperature used. If the melting temperature of the primer, even with less than 100% complementarity, is still above the annealing temperature then elongation will occur. Sequences with less than 100% complementarity can be excluded by choosing an annealing temperature below the standard, but above the target annealing temperature. Optionally, a primer nucleic acid can be labeled, if desired. The label used on a primer can be any suitable label, and can be detected by, for example, by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other detection means.

In an embodiment, a pair of primers (also referred to as a first primer and a second primer or a forward primer and a reverse primer) can be used in the amplification reaction. Such pair of primers are designed to hybridize to complementary strands of the standard sequence and allow the specific extension of a portion of the standard sequence. The pair of primers are also designed to hybridize to corresponding complementary strands of the target sequence and allow the specific extension of a corresponding portion of the target sequence. In a further embodiment, the first primer is non-linear to the probe, i.e. it specifically produces the strand that will hybridize to the probe and the second primer is co-linear to the probe, i.e. it specifically hybridizes to the same strand as the probe and produces the strand that contains the sequence of the probe.

As used herein, the phrase "conditions wherein base-pairing occurs" refers to any hybridization conditions that permit complementary polynucleotides or partially complementary polynucleotides to form a stable hybridization complex.

As used herein, the terms "stringent", "stringent conditions", "high stringency" and the like denote hybridization conditions of generally low ionic strength and high temperature, as is well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Current Protocols in Molecular Biology (Ausubel et al., ed., J. Wiley & Sons Inc., New York, 1997), which are incorporated herein by reference. Generally, stringent conditions for polynucleotides longer than 1000 bases are selected to be about 5-30°C lower than the thermal melting point (T_m) for the hybridization complex comprising the specified sequence at a defined ionic strength and pH. Alternatively, stringent conditions for shorter polynucleotides are selected to be about 5-10°C lower than the T_m for the specified sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the hybridization complexes comprising complementary (or partially complementary) polynucleotides become dissociated. In contrast, the expression "low stringency" denotes hybridization conditions of generally high ionic strength and lower temperature. Under low stringency hybridization conditions, polynucleotides with imperfect complementarity can more readily form hybridization complexes.

As used herein, the terms "complementary" or "complementarity" are used in reference to antiparallel strands of polynucleotides related by the Watson-Crick and Hoogsteen-type base- pairing rules. For example, the sequence 5'-AGTTC-3' is complementary to the sequence 5'- GAACT-3'. The terms "completely complementary" or "100% complementary" and the like refer to complementary sequences that have perfect Watson-Crick pairing of bases between the antiparallel strands (no mismatches in the polynucleotide duplex). However, complementarity need not be perfect; stable duplexes, for example, may contain mismatched base pairs or unmatched bases. The terms "partial complementarity", "partially complementary", "incomplete complementarity" or "incompletely complementary" and the like refer to any alignment of bases between antiparallel polynucleotide strands that is less than 100% perfect (e.g., there exists at least one mismatch or unmatched base in the polynucleotide duplex). For example, the alignment of bases between the antiparallel polynucleotide strands can be at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%, or any value between.

Furthermore, a "complement" of a target polynucleotide refers to a polynucleotide that can combine (e.g., hybridize) in an antiparallel association with at least a portion of the target polynucleotide. The antiparallel association can be intramolecular, e.g., in the form of a hairpin loop within a nucleic acid molecule, or intermolecular, such as when two or more single- stranded nucleic acid molecules hybridize with one another.

The hybridization complex formed as a result of the annealing of a polynucleotide with a probe is termed a "polynucleotide-probe complex". The hybridization complex can form in solution (and is therefore soluble), or one or more component of the hybridization complex can be affixed to a solid phase (e.g., a dot blot, affixed to a bead system to facilitate removal or isolation of the hybridization complexes, or in a microarray). The structure of the probe is not limited, and can be composed of DNA, RNA, analogs thereof, or combinations thereof, and can be single-stranded or double-stranded. A probe can be derived from any source.

As used herein, the term "probe" refers typically to a polynucleotide that is capable of hybridizing to a standard sequence (as well as fragment or amplicon thereof) and a target sequence (as well as fragment or amplicon thereof). The probe is associated (preferably in a covalent manner) with a suitable label(s) or reporter moiety(ies) so that the probe (and the complex it can form with the standard or the target sequence) can be detected, visualized, measured and/or quantitated. In addition, the probe may also be linked at its other end to a quencher. The presence of a quencher is advantageous to suppress the fluorescent signal when the probe is not hybridized to its target or has not been degraded.

The probe is preferentially substantially identical or even identical over its entire length to a portion of a standard sequence. In an embodiment, the probe is free (or devoid) of complementary, self-annealing 3' and 5' ends that are that are not present in the standard sequence.

Detection systems for labeled probes include, but are not limited to, the detection of fluorescence, fluorescence quenching, fluorescence resonant energy transfer (FRET), enzymatic activity, absorbance, molecular mass, radioactivity, luminescence or binding properties that permit specific binding of the reporter (e.g., where the reporter is an antibody, antigen, or small molecule with high affinity binding properties such as biotin). In some embodiments, a probe can be an antibody or DNA binding protein, rather than a polynucleotide, that has binding specificity for a nucleic acid nucleotide sequence of interest. It is not intended that the present invention be limited to any particular probe label or probe detection system. The source of the polynucleotide used in the probe is not limited, and can be produced synthetically in a non-enzymatic system, or can be a polynucleotide (or a portion of a polynucleotide) that is produced using a biological (e.g., enzymatic) system (e.g., in a bacterial cell).

Typically, a probe is sufficiently complementary (e.g. identical) to the standard sequence (or fragment or amplicon thereof) to form a stable hybridization complex with the standard sequence under a selected hybridization condition, such as, but not limited to, a stringent hybridization condition. In an embodiment, the probe is sufficiently complementary to the target sequence (or fragment or amplicon thereof) to form a stable hybridization complex with the target sequence under a selected hybridization condition, such as, but not limited to, a stringent hybridization condition. In an embodiment, the length of the probe smaller than the length of the amplicon.

As used herein, the terms "label" or "reporter", in their broadest sense, refer to any moiety or property that is detectable, or allows the detection of, that which is associated with it. For example, a polynucleotide that comprises a label is detectable (and in some aspects is referred to as a probe). Ideally, a labeled polynucleotide permits the detection of a hybridization complex that comprises the polynucleotide. In some aspects, e.g., a label is attached (preferably covalently) to a probe. In various aspects, a label can, alternatively or in combination: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the second label, e.g., FRET; (iii) stabilize hybridization, e.g., duplex formation; (iv) confer a capture function, e.g., hydrophobic affinity, antibody/antigen, ionic complexation, or (v) change a physical property, such as electrophoretic mobility, hydrophobicity, hydrophilicity, solubility, or chromatographic behavior. Labels vary widely in their structures and their mechanisms of action.

Examples of labels include, but are not limited to, fluorescent labels (including, e.g., quenchers or absorbers), non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels, mass-modifying groups, antibodies, antigens, biotin, haptens, enzymes (including, e.g., peroxidase, phosphatase, etc.), and the like. To further illustrate, fluorescent labels may include dyes that are negatively charged, such as dyes of the fluorescein family, or dyes that are neutral in charge, such as dyes of the rhodamine family, or dyes that are positively charged, such as dyes of the cyanine family. Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NAN and ZOE. Dyes of the rhodamine family include, e.g., ROX, R110, R6G, Carboxytetramethylrhodamine (TAMRA), tetramethylrhodamine (TMR) and its isothiocyanate derivative (TRITC), sulforhodamine 101 (and its sulfonyl chloride form Texas Red) and Rhodamine Red. Alternatives to rhodamine dyes include, but are not limited to, Alexa 546™, Alexa 555™, Alexa 633™, DyLight 549™ and DyLight 633™. Dyes of the cyanine family include, e.g., Cy2, Cy3, Cy5, Cy 5.5 and Cy7. Alternatives to cyanine dyes include, but are not limited to, Freedom Dyes TEX™, and TYE™, Alexa Fluor™ dyes, Dylight™, IRIS™ Dyes, Seta™ dyes, SeTau™ dyes, SRfluor™ dyes and Square™ dyes.

When a fluorescent label is used, a quencher can be matched to limit signal emission from the fluorescent label when the probe is not hybridized with a corresponding sequence. Examples of quenchers that can be linked to the probe include, but are not limited to DDQ-I A, Dabcyl, Eclipse B, Iowa Black FQ C, BHQ-1 D, QSY-7 E, BHQ-2 D, DDQ-II A, Iowa Black RQ C, QSY- 21 E and BHQ-3 D.

Methods for detecting nucleotide differences

The first step of the method described herein comprises the amplification of a target sequence. As used herein, a "target sequence" is an unknown sequence suspected of different from the standard sequence (e.g. being difference in at least one nucleotide), similar or identical to the probe and/or the standard (known) sequence or sequences. In opposition to the target unknown sequence, the nucleotide identity of a "standard sequence" (or a fragment thereof) is known and is similar (preferably identical) to the probe used. When the probe is present during the amplification reaction, the method must be realized so that a fraction of the initial probe population remains intact (e.g. label remains attached to one of its ends) and is available to form a complex with the amplification product or amplicon. In an embodiment, the amplification can be performed in the presence of the probe, in conditions allowing that a fraction of the initial population of probes remains intact after the amplification reaction. In another embodiment, the amplification step can be performed in the absence of the probe and the probe can be added after the amplification step. The amplification step can also be preceded by a reverse- transcription step when the staring material is RNA.

The nucleic acid identity and length of the amplification primer pair must be adjusted in order that, during the amplification step, a single fragment of the target sequence is amplified. When more than one amplification primer pairs are used, for example in a multiplex application, the primers must be designed to limit the interference between the primer pairs and allow the specific amplification of the various fragments of the target sequence. The amplification primers used in the amplification step can, in an embodiment, have a length between 10 and 30 nucleotides. Modifications known to those skilled in the art, for example the used of locked nucleic acid bases or inosine, can help reducing the length of the primer without altering the specificity. Under the appropriate conditions, the amplification reaction results in the formation of amplicons of the target sequence (also referred to as target amplicon). As used herein, the term "amplicon" refers to a polynucleotide molecule (or collectively the plurality of molecules) produced following the amplification of a particular nucleic acid. The amplification method used to generate the amplicon can be any suitable method, most typically, for example, by using a PCR methodology. An amplicon is typically, but not exclusively, a DNA amplicon. An amplicon can be single-stranded or double-stranded, or in a mixture thereof in any concentration ratio. In an embodiment, the amplicon is generated using real-time qPCR. In another embodiment, the amplicon has a length between about 50 bp to 500 bp or even between 100 bp and 300 bp.

Once an amplicon is generated, it is contacted with a probe to form a complex. The probe can be present in the initial amplification mixture or can be added once amplification is terminated. The contact between the amplicon and the probe should be made under conditions favoring the hybridization or annealing of the amplicon to the probe. As used herein, the terms "hybridization" and "annealing" and the like are used interchangeably and refer to the base- pairing interaction of one polynucleotide with another polynucleotide (typically an antiparallel polynucleotide) that results in formation of a duplex or other higher-ordered structure, typically termed a hybridization complex or complex. The primary interaction between the antiparallel polynucleotide molecules is typically base specific, e.g., A/T and G/C, by Watson/Crick and/or Hoogsteen-type hydrogen bonding. It is not a requirement that two polynucleotides have 100% complementarity over their full length to achieve hybridization. The present method used the ability of the mismatch probe and amplicon to anneal together to determine the presence of possible nucleotide mismatch.

The probe is intended to specifically hybridize (e.g. being substantially identical) with the standard sequence or a portion of the standard sequence. In an embodiment, the probe is identical to the standard sequence or a complement thereof. The probe possesses an increased specificity towards the standard sequence in comparison with its specificity to the target sequence, when the standard sequence and the target sequence differ in their nucleotide sequence. As used herein, the phrases "specifically hybridize", "specific hybridization" and the like refer to hybridization resulting in a complex where the annealing pair show complementarity, and preferentially bind to each other to the exclusion of other potential binding partners in the hybridization reaction. It is noted that the term "specifically hybridize" does not require that a resulting hybridization complex have 100% complementarity; hybridization complexes that have mismatches can also specifically hybridize and form a hybridization complex. The degree of specificity of the hybridization can be measured using a distinguishing hybridization property, e.g., the melting temperature of the hybridization complex (T_m). The nucleic acid identity and length of the probe must be adjusted in order that, during the annealing step, the probe will specifically associate with its corresponding amplicon. When more than one probes are used, they must be designed to limit the interference with other amplicons. The probes can, in an embodiment, have a length between 15 and 40 nucleotides, preferably between 20 and 40 nucleotides. Modifications known to those skilled in the art, for example the used of locked nucleic acid bases, minor groove binders, or inosine, can help reducing the length of the probe without altering its specificity.

When two probes are used for determining the identity of the nucleotide difference, the number of nucleotides mismatch between the two probes is, in an embodiment, five, less than five nucleotides, less than four nucleotides, less than three nucleotides, less than two nucleotides or one nucleotides. In a further embodiment, the number of nucleotide mismatches between the two probes is between one and five, preferably between two and four. In yet another embodiment, the difference in melting temperature between the standard amplicon-first probe complex and the standard amplicon-second probe complex is 25°C, less than 25°C, less than 20°C, less than 15°C, less than 10°C, less than 5°C, less than 4°C, less than 3°C or less than 2°C. In still a further embodiment, the difference in melting temperature between the standard amplicon-first probe complex and the standard amplicon-second probe complex is between about 2°C to about 4°C.

When the probe is not associated to the target sequence (fragment or amplicon thereof), it adopts a random coil structure and the quencher at one end captures the signal emitted by the fluorescent dye at the other end. However, once the probe associates with the target sequence (fragment or amplicon thereof) and a complex between the probe and the target is formed, the quencher is too far from the fluorescent dye for it to efficiently capture the fluorescence emitted. This fluorescent emission can then be measured, for examplem by a fluorometer incorporated in the real-time PCR cycler or by an independent fluorometer generating a signal. Consequently, the presence of the probe in the complex is associated with a signal from the label of the probe and the dissociation of the probe from the complex is associate with a loss of signal from the label of the probe. This very specific property enables the determination of the melting temperature of the amplicon-probe complex. As used herein, the melting temperature (T_m) is a temperature-dependent distinguishing hybridization property of the complex.

The melting temperature of an amplicon-probe complex is dependant on the degree of identity between the probe. The higher the degree of identity between the amplicon and the probe, the higher the melting temperature. The melting temperature is the temperature at which one half of a population of double-stranded polynucleotides or nucleobase oligomers (e.g., hybridization complexes), in homoduplexes or heteroduplexes, become dissociated into single strands. The prediction of a T_m of a duplex polynucleotide takes into account the base sequence as well as other factors including structural and sequence characteristics and nature of the oligomeric linkages. Methods for predicting and experimentally determining T_m are known in the art. For example, a T_m is traditionally determined using a melting curve, wherein a duplex nucleic acid molecule is heated in a controlled temperature program, and the state of association/dissociation of the two single strands in the duplex is monitored (in the present method with by assessing the signal from the probe) and plotted until reaching a temperature where the two strands are completely dissociated. The T_m is read from this melting curve.

In the present method, when a melting curve is used to determine the melting temperature, a signal from the probe is measured within a specified temperature range. At temperatures below the melting temperature, the complexes are in majority annealed and the signal measured from the probe is high. As the temperature increases, the complex begins its dissociation and the probe is progressively released from the complex, thereby decreasing the measured value of the signal. At temperatures above the melting temperature, the majority of the complexes are dissociated. As the temperatures continue to increase, the probe has been completely released from the complex and the value of the signal is at a minimum. Fluctuation in signal may still occur for the probes as the random coil assumed by the free probe may fold differently depending on temperature, thus creating minor signal variation. Melting temperature curves are usually presented as second derivatives of fluorescence and represent the change in fluorescent signal so they appear as peaks with a maximum at the melting temperature.

Alternatively, a T_m can be determined by an annealing curve, wherein a duplex nucleic acid molecule is heated to a temperature where the two strands are completely dissociated. The temperature is then lowered in a controlled temperature program, and the state of association/dissociation of the two single strands in the duplex is monitored and plotted until reaching a temperature where the two strands are completely annealed. The T_m is read from this annealing curve.

In the present method, when a melting curve is used to determine the melting temperature, a signal from the probe is measured within a specified temperature range. At temperatures below the melting temperature, the complexes are in majority associated and the signal measured from the probe is high. As the temperature increases, the complex begins to melt and the probe is progressively separated from the complex, thereby decreasing the measured value of the signal. At temperatures above the melting temperature, the majority of the probes is dissociated from the amplicon and the signal is low. The melting curve is a first derivative of the temperature change where the midpoint of the transition is considered the melting temperature. This should reflect the temperature where exactly half of the DNA strands have separated.

In an analogous manner, an annealing curve (or its first or second derivative) can be used to determine the melting temperature.

However, the melting temperature is not the sole temperature-dependent hybridization property that can be used in the method to provide useful information on the presence (or absence) of a mismatch between the probe and the target sequence. For example, the temperature at which 25% of a population of double-stranded polynucleotides or nucleobase oligomers (e.g., hybridization complexes), in homoduplexes or heteroduplexes, become dissociated into single strands (T₂₅) is also a defining characteristic of the hybridization complex. Similarly, the temperature at which 75% of a population of double-stranded polynucleotides or nucleobase oligomers (e.g., hybridization complexes), in homoduplexes or heteroduplexes, become dissociated into single strands (T₇₅) is also a defining property of the hybridization complex. Alternatively, the percentage of dissociation of a hybridization complex at any series of defined temperatures is a quantitative temperature dependent hybridization property and this usually forms the basis for High Resolution Melting (HRM).

Once the melting temperature of the complex has been determined, it is compared to the melting temperature of a complex between a corresponding amplicon of a standard sequence and the probe. The amplicon of the standard sequence "corresponds" to the amplicon of the target sequence, in the sense that both amplicons are derived from the same corresponding region and were generated using similar methods (e.g. same primer oligonucleotide pair). Both amplicons are approximately the same length and also possess a certain degree of identity. This similarity in length and sequence is necessary to generate melting temperature that can be compared to detect nucleotide differences between the target and the standard sequences.

If a nucleotide difference exists between the amplicon of the target sequence and of the standard sequence and that this nucleotide difference is also present between the target sequence and the probe, it is then assumed that the melting temperature of the complex between the amplicon of the target sequence and the probe will be different from (in an embodiment, it will be lower than) the melting temperature of the complex between the amplicon of the standard sequence and the probe. This difference in melting temperature is attributed to the presence of a mismatch or difference between the target sequence and the probe and/or between the standard sequence and the probe. If there is no nucleotide difference between the amplicon of the target sequence and of the standard sequence, it is then assumed that the melting temperature of the complex between the amplicon of the target sequence and the probe will be substantially similar (i.e. indistinguishable). This lack of difference in melting temperature is attributed to the absence of a mismatch or difference between the target sequence and the probe. For example, when a 20 to 30 bp probe is used, a single base mismatch between the probe and the target sequence will typically lower the melting temperature of the probe-amplicon complex by at least 2°C (with respect to the standard amplicon-probe complex). As such, a difference of less than 1°C is usually not considered to be a substantially dissimilar temperature. For a difference in temperature to be substantially similar, this difference must be less than about 1°C. For a difference in temperature to be considered substantially dissimilar and indicative of the presence of a nucleotide difference, a difference of at least about 2°C is required. Differences between 1 ° and 2°C are unusual and require further investigation because they indicate the possible presence of a mismatch.

The method described herein can be used to detect a specific known nucleotide difference between the target sequence and standard sequences. However, in some instances, some additional unknown nucleotide differences can be present between the target and the standard sequences. In those instances, the unknown additional nucleotide differences can modify the melting temperature of the probe-amplicon complex and the detection of the "known" nucleotide difference can be difficult to perform. As such, it may be advantageous to use a further probe (e.g. second probe) in the method. This further probe is linked to a detectable label (different from the label of the first probe so as to provide a clear distinct signal from the first probe) and, optionally, a corresponding quencher. The method is not limited to the addition of a single further probe, multiple probes can be used, as long as they can be labeled with different labels that will generate distinct signals.

In an embodiment, the first probe is substantially identical to a first standard sequence (or amplicon or fragment thereof) and the second probe is substantially identical to a second standard sequence (or amplicon or fragment thereof). The first and second standard sequence differ in nucleotide identity and this difference is known. The first and second standard sequence may be overlapping and, in an embodiment, may only differ at a single nucleotide. For example, the first standard sequence can correspond to a wild-type sequence, whereas the second standard sequence can correspond to a known mutant sequence. The melting temperature of the target amplicon-probe complexes with both of the probes is then determined and compared to the melting temperature derived from the standard amplicons-probes complexes with both of the probes. Dissimilarity in melting temperature between the target amplicon-first probe complex and the first standard amplicon-first probe complex indicates the presence of a nucleotide difference between the target sequence and the standard sequence (or first probe). This result can be optionally confirmed (and the identity of this difference be reasonably inferred) by the similarity in melting temperature between the target amplicon- second probe complex and the second standard amplicon-second probe complex. If a dissimilarity still exists between the target amplicon-second probe complex and the second standard amplicon-second probe complex, it can be assumed that the difference in the nucleotide sequence of the target is different than the one(s) known between the first and second standard sequence. Even in this case where the nucleotide sequence varies at a nucleotide other than the one known between the first and second standard sequences, it is still possible to distinguish the presence of the wild type or known mutant sequence as either the first or second probe will have a higher melting temperature then the other probe, because this probe will have only one nucleotide mismatch whereas the other probe will have this mismatch in addition to the mismatch at the wild type/mutant site. However, if both a standard and the target sequence do not contain a nucleotide difference at the level of the probe, even though they contain differences elsewhere, then the melting temperature of all their amplicon-probe complexes derived from this same probe, would be similar.

The method presented herewith can also be adapted for multiplex PCR. Amplicons from more than one portion of the standard and/or target sequence can be generated. These different amplicons can be contacted with a single or a combination of probes, under conditions favoring the formation of a complex between the plurality of amplicons and the probe(s). The melting temperatures of these complexes are determined and compared to the melting temperatures of complexes between the plurality of amplicons of the standard sequences and the probe(s). If one of the amplicons of the target sequence is dissimilar to the probe (and consequently to the standard sequence), then its melting temperature will be different from the corresponding amplicon from the standard sequence. If more than one amplicon of the target sequence is dissimilar to the probes (and consequently to the standard sequence), then their melting temperatures will be different from the corresponding amplicons from the standard sequence. In a multiplex situation where all probes are labeled with the same fluorophore or where dyes are used, extreme care must be taken to avoid, when possible, overlap of the melting temperatures between the different complexes between the amplicons from the standard sequence and the probes to provide more accurate data. When each probe is labeled with a distinct fluorophore, then such care is not necessary.

For example, the first standard sequence can correspond to a sequence in one gene, whereas the second standard sequence can correspond to a sequence in another gene (and even in another organism). The melting temperature of the target amplicon-probe complexes with both of the probes is then determined and compared to the melting temperature derived from the standard amplicons-probes complexes with both of the probes. Absence of a melting temperature between an amplicon and a probe indicates the absence of the corresponding standard sequence (or gene) in the target sequence. Alternatively, the presence of a melting temperature between an amplicon and a probe indicates the presence of the corresponding standard sequence (or gene) in the target sequence. Similarity/dissimilarity in melting temperature between the target amplicon-probe complexes indicates the absence/presence of a nucleotide difference between the target sequence and the standard sequence. This result can be optionally confirmed (and the identity of this difference be reasonably inferred) by the similarity/dissimilarity in melting temperature between the target amplicon and a further probe complex, as discussed above. As such, the method presented herewith can be used in a sample for detecting various target sequences (overlapping or not) as well as the presence of a nucleotide difference between the target and the standard sequence.

When more than one probe is used, it is possible to add them in the original amplification reaction mixture or, alternatively, add them to the mixture once amplification of the target amplicon(s) has been done.

The methods presented herewith can be applied to any target sequence derived from any sample, provided that the target sequence can be amplified and can form a complex with a probe. As used herein, the term "sample" is used in its broadest sense, and refers to any material subject to analysis. The term "sample" refers typically to any type of material of biological origin, for example, any type of material obtained from animals or plants. A sample can be, for example, any fluid or tissue such as blood or serum, and furthermore, can be human blood or human serum. A sample can be cultured cells or tissues, cultures of microorganisms (prokaryotic or eukaryotic), viruses or any fraction or products produced from or derived from biological materials (living or once living). Optionally, a sample can be purified, partially purified, unpurified, enriched or amplified. Where a sample is purified or enriched, the sample can comprise principally one component, e.g., nucleic acid. More specifically, for example, a purified or amplified sample can comprise total cellular RNA, total cellular mRNA, DNA, cDNA, cRNA, or an amplified product derived there from.

The sample used in the methods of the invention can be from any source, and is not limited. Such sample can be an amount of tissue or fluid isolated from an individual or individuals, including, but not limited to, for example, skin, plasma, serum, whole blood, blood products, spinal fluid, saliva, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid, urine, feces, tears, blood cells, blood products, semen, seminal fluid, nasal secretions, vaginal or cervical secretions, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, paraffin embedded tissues, lesion scrapings, etc. Samples also can include constituents and components of in vitro cell cultures, including, but not limited to, conditioned medium resulting from the growth of cells in the cell culture medium, recombinant cells, cell components, etc.

As indicated above, the target and standard sequences can be derived from various organisms. As used herein, the expression "derived from" refers to a component that is isolated from or made using a specified sample, molecule, organism or information from the specified molecule or organism. For example, a nucleic acid molecule that is derived from an influenza virus can be a molecule of the influenza genome, or alternatively, a transcript from the influenza genome.

Commercial packages

The method provided herewith can be easily applied in the clinical use. Kits approved for clinical use are often based on fluorescent resonant energy transfer (FRET) where melting temperatures between the target and a probe distinguish mutant from wild type sequences, which is a more robust methodology (simpler to interpret) for clinical laboratories.

The present application does provide a kit for determining the presence or absence as well as the identity of a nucleotide difference between a target sequence and a standard sequence. The kit, for use in the methods described herewith, comprises at least two components: a pair of primers for amplifying a fragment of the target sequence and a probe that is able to bind specifically to the amplified target sequence.

If the identity of the nucleotide difference is to be determined, then the kit may also comprise a second probe that is able to specifically bind to the amplified target sequence. The second is similar to the first probe and its nucleic acid identity is known. In an embodiment, the second is identical to the first probe except in at least one nucleotide.

If the kit is to be used in a multiplex PCR, it also comprise a second pair of primers for amplifying a fragment in the target sequence as well as an additional probe that is able to bind specifically to the amplified target sequence.

Computer generated methods, associated medium and systems

While it is recognized that the amplification and measuring steps are conducted using standard laboratory equipment, the comparison, assessment and detection steps may be assisted with a computer for ease of use. Providing melting temperature. Since the sequence of the probe is known and the sequence of the corresponding amplicon of the standard sequence is known, it is possible to store the properties of the melting temperature and/or melting curve of the standard amplicon-probe in a memory card and retrieve such information when the comparison is performed.

Comparing the melting temperature. The comparison can be made in a comparison module. Such comparison module may comprise a processor and a memory card to perform an application. The processor may access the memory to retrieve data (such as the stored standard amplicon-probe melting temperature). The processor may be any device that can perform operations on data. Examples are a central processing unit (CPU), a front-end processor, a microprocessor, a graphics processing unit (PPU/VPU), a physics processing unit (PPU), a digital signal processor and a network processor. The application is coupled to the processor and configured to determine if a difference exists between the target amplicon-probe melting temperature and the standard amplicon-probe melting temperature. An output of this comparison may be transmitted to a display device. The memory, accessible by the processor, receives and stores data, such as measured melting temperatures or any other information generated or used. The memory may be a main memory (such as a high speed Random Access Memory or RAM) or an auxiliary storage unit (such as a hard disk, a floppy disk or a magnetic tape drive). The memory may be any other type of memory (such as a Read-Only Memory or ROM) or optical storage media (such as a videodisc or a compact disc).

Detection of a presence of a nucleotide difference and/or nucleotide identity. Once the comparison between the melting temperature is made, then it is possible to detect the presence or absence and, implicitly the identity of the nucleotide difference. This characterization is possible because, as shown herein, the presence of a nucleotide difference on the target sequence (with respect to the standard sequence) modifies (e.g. lowers) the resulting melting temperature of the target amplicon-probe with respect to the standard amplicon-probe. As such, the presence or absence of the nucleotide difference is based on that premise. If a nucleotide difference is detected, its identity can be determined by comparing the melting temperature of additional amplicon-probe complexes as indicated above. The determination can be made with a processor and a memory card to perform an application. The processor may access the memory to retrieve data. The processor may be any device that can perform operations on data. Examples are a central processing unit (CPU), a front-end processor, a microprocessor, a graphics processing unit (PPU/VPU), a physics processing unit (PPU), a digital signal processor and a network processor. The application is coupled to the processor and configured to determine the presence (or absence) of a nucleotide difference in the target sequence. The memory, accessible by the processor, receives and stores data, such as measured parameters or any other information generated or used. The memory may be a main memory (such as a high speed Random Access Memory or RAM) or an auxiliary storage unit (such as a hard disk, a floppy disk or a magnetic tape drive). The memory may be any other type of memory (such as a Read-Only Memory or ROM) or optical storage media (such as a videodisc or a compact disc).

Screening system. The present application also provides a screening system for determining the presence or absence of a nucleotide difference between the target sequence and the standard sequence. This screening system comprises a reaction vessel for combining the target amplicon and the probe, a processor in a computer system, a memory accessible by the processor and an application coupled to the processor. The application or group of applications is(are) configured for receiving a value of a melting temperature of the target amplicon-probe; comparing the value of the melting temperature of the target amplicon-probe to the melting temperature of the standard amplicon-probe and/or determining the presence of a nucleotide difference in the target sequence with the compared melting temperatures are different. Further assessment can be made with respect to the identity of the nucleotide difference when more than one probe is used.

Software product. The present application also provides a software product embodied on a computer readable medium. This software product comprises instructions for determining the presence of a nucleotide sequence in a target sequence. The software product comprises a receiving module for receiving a value of a melting temperature of a target amplicon-probe complex; a comparison module receiving input from the receiving module for determining if the melting temperature of the target amplicon-probe complex different from the melting temperature of the standard amplicon-probe complex; a determination module receiving input from the comparison module for determining the presence of a nucleotide sequence in a target sequence. The nucleotide difference is considered present when a difference in the melting temperatures is observed. On the other hand, the nucleotide difference is considered absent when no (or a statistically insignificant) difference in the melting temperatures is observed. The comparison module and determination module may each comprise a processor, a memory accessible by the processor to perform an application.

Applications

The methods described herein can be useful for determining any nucleotide differences (single or multiple) in any organism (viral, prokaryotic and eukaryotic). Specific examples are presented below for multiplex detection of mutations associated with antibiotic resistance in the bacteria Campylobacter jejuni, subtyping of influenza A virus with simultaneous detection of the mutation associated oseltamivir resistance across three highly divergent neuraminidase genes, and bacterial classification and typing by identifying nucleotide differences in the 16S ribosomal RNA gene.

Multiplex detection of mutations associated with antibiotic resistance in the bacteria Campylobacter jejuni. Campylobacter jejuni is the leading reported cause of bacterial enteritis in developed countries. In 2004 in Canada, it was the leading notifiable enteric food- and waterborne disease, with 9345 reported cases. In Quebec province alone, nearly 3000 cases of diarrheal illness are attributed annually to Campylobacter enteritis, more than the combined total caused by Salmonella and Shigella species, E. coli 0157:1-17 and Yersinia enterocolitica. It was recently concluded that even these numbers appear to represent a substantial underestimate of the public health burden of this enteric pathogen and that for every case of Campylobacter infection reported in Canada each year, there are an additional unreported 23 to 49 cases.

These infections usually resolve spontaneously without treatment, but certain patients require antibiotic treatment, especially immuno-compromised patients, who may develop severe complications. Antibiotic sensitivity determination for C. jejuni using culture methods is more complex than for other common bacteria and is rarely performed in clinical laboratories. It would thus be convenient to have molecular methods to detect antibiotic resistance to the two most commonly employed antibiotics erythromycin and ciprofloxacin. Resistance to erythromycin in C. jejuni is usually provoked by a specific mutation in the 23S ribosomal RNA gene, whereas resistance to ciprofloxacin is usually associated with a particular mutation in the DNA gyrase gene, gyrA. A multiplex PCR procedure using melting curve determination of probe/amplicon complexes is described herein to detect the most common mutations associated with C. jejuni resistance to erythromycin and ciprofloxacin.

Subtyping of influenza A virus with simultaneous detection of the mutation associated oseltamivir resistance across three highly divergent neuraminidase genes. Influenza is a major public health problem because of its considerable mortality in elderly people and morbidity in all. It appears as an epidemic during the winter months usually lasting 6 to 8 weeks in each particular location. Spread of the virus is by contact with respiratory secretions on infected hands and aerosols created by sneezing. As the much less severe common cold virus has similar symptoms, it is important to rapidly and accurately diagnosis influenza infections, especially in hospitalized patients as well as in nursing and retirement home residents and staff in order to prevent spread. Appropriate treatment should be given hospitalized patients and prophylaxis used to curtail spread. An improved method is presented here to rapidly diagnose influenza virus, to identify whether it is a pandemic strain or not, and to identify whether it is susceptible or not to Oseltamivir (Tamiflu™) the most common influenza antiviral agent.

Bacterial classification and typing by identifying nucleotide differences in the 16S rRNA gene. Bacterial classification and identification initially relied on phenotypic characterization. The pioneering work of Carl Woese since the 1970s identified the gene sequence of 16S ribosomal RNA as a highly discriminate marker. Despite the shift made by reference texts, clinical microbiology laboratories still rely almost exclusively on phenotypic characteristics to identify bacteria. This is no doubt largely due to the difficulty to sequence bacterial genomes and interpret the results in a clinical laboratory context. Indeed, only recently has it been possible to sequence, economically, with greater than 99% accuracy. It is still challenging for hospital technicians to identify microorganisms by differences gleaned from mostly identical sequences. Detecting these differences by primers or probes using conventional or even real-time PCR is not a technologically simple procedure as separate reactions would be necessary for each nucleotide difference and thus would be unmanageable.

Sequence differences in the 16S rRNA gene, usually single nucleotide polymorphisms (SNP), can distinguish bacteria. The ability to detect these SNPs simply, rapidly and with high confidence in clinical laboratories could have a considerable impact on health care.

Numerous strategies have been devised to detect SNPs. Most involve annealing a primer with the SNP at its 3' end or a probe with a target sequence containing the SNP at temperatures near the maximal annealing temperature. When both primer and target are derived from wild type sequences or both from the mutant sequence the annealing temperature in PCR is usually several degrees higher than when one comes from the mutant and the other from wild type. These differences can be visualized by performing the PCR detection step at a temperature in the "window" between the homologous and heterologous annealing temperatures. However, existing technologies are associated with several drawbacks. This window may vary between different apparatus or reaction mixes yielding weak amplification curves that the PCR apparatus software may call positive or negative making interpretation uncertain for clinical laboratory personnel. Additional SNPs in the target sequence prevent these methods from yielding accurate results. Such additional SNPs may appear in bacterial genes, especially in 16S rRNA genes due to the immense variety of bacteria.

As shown below in Example III, an application of TaqTm probing to identify bacteria by their signature sequences in 16S ribosomal RNA genes. Infections are usually caused by a single bacterial species and only a limited number of different bacteria cause over 95% of infections at particular sites. In example III, the identity of bacteria present in vaginosis was determined based on the assumption that probes will react with highest melting temperatures for their exact intentional target, but will give lower melting temperatures for other organisms, allowing us to detect their presence. A large number of samples was analysed to explore the frequency of variation and its possible impact on this technology. TaqTm probing strategies as shown herein could be employed to identify bacteria by their signature sequences in 16S ribosomal RNA genes. Alternatively, cpn60 genes could also have been employed. This procedure could be used for urinary tract infections as they are the most frequently tested in clinical laboratories.

Urinary tract infection. A variety of possible primer pairs were analyzed for their ability to efficiently amplify enterobacteria and staphylococci and should amplify most other bacteria as well. For example, five different colored probes can be designed, one each for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, group B streptococci and the fifth for a positive control. These probes are expected to react with highest melting temperatures for their intentional target, but give lower melting temperatures for related organisms, allowing us to detect their presence. Traditionally, confirmation of these other organisms and antibiotic sensitivity determination would require culture or subsequent PCR reactions. However, in the method described herein, rapid results for most infections (including antibiotic sensistivity) could be obtained. Slight adaptation of these sequences could be applied to other infection types like wounds, blood cultures, pneumonia or meningitis.

Commercial packages or kits

The methods described herein can be implemented with a commercial package or a kit. As used herein, the term "kit" is in reference to a combination of articles that facilitate a process, method, assay, analysis or manipulation of a sample. Kits can contain written instructions describing how to use the kit (e.g., instructions describing the methods), chemical reagents or enzymes required for the method, primers and probes, as well as any other components. These kits can include, for example but not limited to, reagents for sample collection, reagents for the collection and purification of RNA from blood, a reverse transcriptase, primers suitable for reverse transcription and first strand and second strand cDNA synthesis to produce an amplicon, probes, a thermostable DNA-dependent DNA polymerase and buffers containing deoxyribonucleotide triphosphates. In some embodiments, the enzyme comprising reverse transcriptase activity and thermostable DNA-dependent DNA polymerase activity are the same enzyme.

In some embodiments, the kits are diagnostic kits, where the information obtained from performing the methods enabled by the kits is used to identify the presence or absence of a microorganism or virus including drug resistance in or the subtype of the microorganism or virus. The kits can provide any or all of the synthetic oligonucleotides used in methods described herein. For example, the kits can provide oligonucleotide primer(s) suitable for priming reverse transcription from a viral RNA molecule to produce a viral cDNA. The kits can provide amplification primers suitable for amplification of any suitable portion of a genome. The invention provides suitable amplification primers that can be included in kits of the invention. It is understood that the invention is not limited to the primers recited herein, as any other suitable amplification primers also find use with the invention.

The kits of the invention can include oligonucleotide probes suitable for the determination of the melting temperature as well as amplification curves. It is understood, however, that the kits of the invention are not limited to the primers and probes described in the experimental section, as the invention also provides guidance for the identification and synthesis of additional suitable primers and probes. The probes provided in kits of the invention are preferably labeled (e.g. via a covalent link). In addition, kits of the present invention can also include, for example but not limited to, apparatus and reagents for sample collection and/or sample purification (e.g., isolation of RNA from a blood sample), sample tubes, holders, trays, racks, dishes, plates, instructions to the kit user, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples (e.g., positive controls, negative controls or calibration controls). Kits of the present invention can also be packaged for convenient storage and shipping, for example, in a container having a lid. The components of the kits may be provided in one or more containers within the kit, and the components may be packaged in separate containers or may be combined in any fashion. In some embodiments, kits of the invention can provide materials to facilitate high-throughput analysis of multiple samples, such as multiwell plates that can be read in a suitable real time PCR thermal cycler or fluorescence spectrophotometer.

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I - DETECTION OF MARKERS FOR RESISTANCE TO ERYTHROMYCIN AND CIPROFLOXACIN IN CAMPYLOBACTER JEJUNI USING POST PCR MELTING CURVE ANALYSIS WITH FLUORESCENT PROBES

Bacterial DNA preparation. C. jejuni was isolated from humans, bovines, birds and water. Crude bacterial DNA was extracted with a hot NaOH lysis procedure from bacterial colonies. Normally 1 μΙ of bacterial sample DNA was added to 14 μΙ of PCR reaction mix. Primers and probes preparation. The sequence of the primers and probes used are presented in Tables 1 and 2. The nucleic acids were synthesized by IDT-DNA, Coralville, IA. The nucleic acids were dissolved in water to make 50 μΜ stock solutions. Stock solutions were then diluted to 5 μΜ working solutions.

Table 1 A. 23S Ribosomal RNA primers for erythromycin resistance detection

Table 1 B. Erythromycin resistance (Ery^R) and sensitivity (Ery^s) probes

Table 1C. Melting temperatures observed with the probes of Table 1 B

Table 2A. gyrA primers for ciprofloxacine resistance detection

Table 2B. Ciprofloxacine resistance (Cip^R) and sensitivity (Cip^s) probes

Table 2C. Melting temperatures observed with the probes of Table 2B

Probe name Probe type Cip^R C. jejuni Cip^s C. jejuni

CampGyrMut665 Cip^R 62.3°C 58.4°C

CampGyrWtHex Cip^s 54.3°C 63.0^UC PCR protocol and apparatus. PCR consisted of an initial denaturation step at 95°C [10 min], followed by 40 cycles (95°C [10 s] for denaturation, 60°C [30 s] for annealing and 72°C [15 s] for elongation). Fluorescent levels were acquired at 72°C after each cycle. Real-time PCR reactions were carried out in a 96-well plate in a Roche LightCycler™ II 480 real-time PCR system apparatus.

Melting temperature determinations. Melting temperature determinations were measured theoretically using the DINAmelt™ program (http://dinamelt.bioinfo.rpi.edu/). A temperature melting curve (Tm) was generated by denaturing products at 95°C [1 min] and followed by a renaturation step at 40°C [1 min], then temperature was ramped from 45°C to 95°C at 0.03°C/sec, 5 acquisitions/°C. Fluorescence data generated by Tm curves were plotted and analyzed by the LightCycler™ 480 Tm calling Software.

For High Resolution Melting (HRM), the reaction mix contained 0.2 μΜ of the primers, 7.5 μΙ of 2X Roche LightCycler™ 480 High Resolution Melting Master Kit that containing Resolight™ fluorescent dye and 3 mM of MgCI₂. PCR reactions were followed by an HRM program consisting of a denaturation step at 95°C [1 min] and renaturation step at 40°C [1 min]. Melting curves were obtained by gradually heating from 65°C to 95°C at 0.02°C/sec, with 25 acquisitions/°C. Fluorescence data generated by HRM curves were normalized, plotted and analyzed by the LightCycler™ 480 Gene Scanning Software.

For HRM with an unlabelled probe, 0.7 μΜ of unlabelled probe was added and a modified primer concentration of 0.7 μΜ for the primers producing the DNA strand complementary to the probe and 0.07 μΜ of the primer co-linear with the probe were used.

For symmetric PCR with fluorescent probes, the reaction mix contained 0.2 μΜ of forward and reverse primers, 0.1 μΜ of fluorescent probes and 7.5 μΙ of 2X Roche LightCycler™ 480 Probe Master Kit.

For asymmetric PCR with unlabelled probe, the mix contained 0.1 μΜ of unlabelled probe. Primer concentrations were modified to 0.7 μΜ for the forward primers and to 0.07 μΜ for the reverse primers. The HRM reaction mix and conditions were used except that the melting temperature determination curve rather than an HRM analysis was performed.

SNP detection using TaqMan probes with melting curve analysis. The mix contained 0.2 μΜ of fluorescent probes with the asymmetric primer concentrations described above.

Polymerases for PCR. VentR™ (exo-) (New England Biolab, Pickering ON), KlenTaqDV ReadyMix™ (Sigma Aldrich, Oakville ON), Taq (Qiagen, Mississauga ON), KlenTaq™, Pfu Turbo™, Taq and Roche Taq supplied in the 2X Roche LightCycler™ 480 Probe Master Kit (Roche Diagnostics, Laval, QC).

HRM profiles (Fig. 1) for a 131 bp sequence DNA amplicon from the 23S rRNA gene were produced without (Fig. 1A) and with an unlabelled probe specific for the resistance sequence (Fig. 1 B) using bacterial isolates where a single mutated nucleotide (A2075G) conferred resistance to erythromycin. It was found however that HRM profiles were very similar with or without the probe. When a melting temperature of the amplicon with a fluorescent probe was performed, it was much easier to distinguish the resistant from sensitive isolates (Fig. 2).

The standard TaqMan amplification curves with different annealing temperatures was performed to distinguish a sequence marker specific for erythromycin sensitivity using differently-labeled probes identical to the sensitive and resistant sequences (Fig. 3). With an annealing temperature of 60°C, there was an excellent discrimination between the resistant and sensitive bacterial isolates, but at 55°C the distinction was not as good as there was some erroneous amplification (Fig. 3). It is hypothesized that, if silent mutations had also been present in the sensitive isolates they would probably have been miss-classified as resistant, even at 60°C. It was also observed that, using an HRM reaction mix, a melting temperature for an added probe could be obtained and that these melting temperatures would provide a more precise distinction between "sensitive" and "resistant" bacteria (see Fig. 2).

Different polymerases were also evaluated (Fig. 3) to determine which one produces the more suited amplification profiles and melting temperature profiles. The Roche Taq polymerase in the Roche LightCycler™ 480 Probe Master Kit (Fig. 3A and 3E), KlenTaq (Fig. 3B and 3F), Taq (Fig. 3C and 3G) and Vent (exo-) (Fig. 3D and 3H) were tested. The KlenTaqDV ReadyMix™, PFU Turbo and Taq provided by Qiagen were also tested. Among the polymerases, Roche Taq and Taq provided the two highest fluorescent intensities (Fig. 3). The Roche enzyme in kit form proved to be the most robust for a large number of C. jejuni isolates. Although these polymerases improved slightly the melting curves at the expense of the PCR fluorescent acquisition over cycles curve, as shown herein, the use of different concentrations of the primers (e.g. asymmetric PCR) in order to produce more of one strand (the strand that is complementary to the probe) than the other seemed to be a key contributor to the method.

To perform an asymmetric PCR, the primer concentration ratio that provided a large quantity of the DNA strand complementary to the probe and minimized production of the DNA strand co- linear with the probe was determined. Five different ratios were compared (Fig. 5) for forward and reverse primers (1 :7, 1 :10, 1 :14, 1 :23 and 1 :70). These ratios were applied to a PCR producing a 131 bp amplicon from the 23S rRNA gene with a mix of probes labelled with different fluorophores. Only amplification profiles (Fig. 5A) and melting temperature profiles (Fig. 5B) detected with the probe specific for erythromycin sensitive C. jejuni are shown but the others gave similar results. Amplification profiles and melting temperature profiles were higher with ratios 1 :7, 1 :10 and 1 :14. A primer ratio of 1 :10 (0.7 μΜ of the reverse primer with 0.07 μΜ forward primer) was selected for further optimization.

Amplification profiles (Fig. 6A to 6D) followed by a melting curve analysis (Fig. 6.E to H) for SNP detection using asymmetric PCR with fluorescent probes, were obtained after simultaneous amplification of a 131 bp amplicon from the 23S rRNA gene and a 232 bp amplicon from the gyrA gene using erythromycin sensitive and a resistant C. jejuni isolates and ciprofloxacine sensitive and resistant C. jejuni isolates (Ery^R/Cip^s or Ery^s/Cip^R). Erythromycin and ciprofloxacine sensitive or resistant strains were detected by four different fluorescent probes specific for the sensitive or resistant sequence (refer to table 1 B and 2B). The mutated nucleotide conferring erythromycin resistance (A2075G) or ciprofloxacin resistance (C258T) produced a mismatch between probes specific for the sensitive sequence and resistant DNA strand (Fig. 6E, Ery^R or Fig. 6G, Cip^R) or between probes specific for the resistance sequence and sensitive DNA strand (Fig. 6F, Ery^s or Fig. 6H, Cip^s). Mismatches decreased the annealing temperature. Perfect matches between sensitive specific probes and sensitive DNA strand (Fig. 6E, Ery^s or Fig. 6G, Cip^s), or perfect match between resistance specific probes and resistance DNA strand (Fig. 6.F, Ery^R or Fig. 6H, Cip^R), showed higher annealing temperatures. Observation of these differences in annealing temperature, improves our ability to distinguish between sensitive and resistant C. jejuni isolates.

The 23S rRNA gene in C. jejuni is present in 3 copies, which may be identical, or occasionally, different. Correctly identifying bacteria with heterologous allelic sequences can be challenging. To simulate bacteria with two 23S resistant copies and one 23S sensitive copy (Ery^R/Ery^R/Ery^s) or bacteria with two 23S sensitive copies and one 23S resistant copy, DNA from bacteria sensitive to erythromycin were artificially mixed with resistant bacteria. PCR was performed on these two artificial 23S bacteria mixes and on natural bacteria with all of three 23S gene copies that are resistant (Ery^R/Ery^R/Ery^R); or sensitive (Ery^s/Ery^s/Ery^s). Mixes produced ambiguous amplification curves where both sensitive and resistant specific probes gave partial reactions, but it was clear form the melting temperature analysis that both sensitive and resistant sequences were present and could be detected (Fig. 7).

EXAMPLE II - DETECTION OF INFLUENZA VIRUS VARIANTS AND OSELTAMIVIR

RESISTANCE Viral RNA. Nasopharyngeal aspirates containing either pandemic influenza A/H1 N1 , seasonal influenza A/H1 N1 , or influenza A H3N2 were obtained in 0.25 ml or 0.5 ml aliquots. Nucleic acids were extracted using either the MagnaPure Compact™ (Roche Diagnostics, Laval, Quebec) or the EasyMag™ apparatus (BioMerieux Canada, Montreal, Quebec). Using the total nucleic acid extraction kit and protocol on the MagnaPure Compact™, 0.25 ml of clinical sample was added to 465 μΙ of Roche lysis buffer, containing 0.001 % DTT, 20 ng polyA/ml, and internal control (RNA bacteriophage P7) to give a crossing point between 25 and 27 in RT-PCR. Samples were eluted in 50 μΙ with the EasyMag™ apparatus which delivers lysis buffer into 8 chamber reaction vessels. Briefly, patient samples (usually 0.5 ml) were added to each vessel, mixed, and incubated 10 min. Silica beads together with internal control (RNA bacteriophage P7) were then added to each vessel. Samples were eluted in 50 pi.

RT-PCR. One μΙ aliquot of viral RNA or dilutions in pooled extracts of viral RNA samples was added to 14 μΙ of reaction mix containing 0.7 μΜ of both primers or, for asymmetric PCR, usually 1 μΜ of the primer producing the DNA strand complementary to the probe and 0.07 μΜ of the primer co-linear with the probe, usually 0.2 μΜ probes (see Table 3A and 3B for primer and probe sequences synthesized by IDT-DNA), 3 μΙ of QuantiTec™ Virus Master 5X (Qiagen), and 0.15 μΙ QuantiTec™ RT mix (Qiagen). Amplification started with 20 min at 50°C for reverse transcription, 5 minutes at 95°C for denaturation of DNA/RNA products and activation of the enzyme, followed by 45 cycles of 15 sec at 95°C, 30 sec at 60°C with a single acquisition, and 15 sec at 72°C. A melting curve was generated by denaturing products for 10 sec at 95°C, renaturing at 30°C or 40°C for 30 sec, then gradually heating to 80°C or 95°C and measuring fluorescence at each temperature.

Melting temperature determinations. Melting temperature determinations were measured theoretically using the DINAmelt™ program (http://dinamelt.bioinfo.rpi.edu/) for 0.1 μΜ DNA strands in 50 ttiM NaCI and 3 mM MgCI₂. Melting curves were also performed using fluorescent or unlabelled probes together with synthetic unlabelled DNA with the sequence of pandemic H1 N1 , seasonal H1 N1 , seasonal H3N2 or avian H5N1 from the genome portion complementary to the probes (see Table 3C). Both the probe and the synthetic DNA were used at 0.1 μΜ in RT- PCR hybridization buffer (50 mM NaCI, 10 mM Tris-HCI, pH 8.8, 3 mM MgCI₂) or in 1X QuantiTec™ Virus Master. SyBr™ green (Molecular Probes) was added to 1X when unlabelled probes were used.

Table 3A. Primers for the detection of the Influenza A virus matrix gene and neuraminidase gene

Target Name Sequence

Matrix FluAM52C CTTCTAACCGAGGTCGAAACG (SEQ ID NO: 10) Matrix FluAM253R AGGGCATTYTGGACAAAGCGTCTA (SEQ ID NO: 11)

Neuraminidase FluNA664F ATATTGAGAACACAAGAGTCTGAATG (SEQ ID NO: 12)

Neuraminidase FluNA896R2 GARCCATGCCAGTTRTCCCTG (SEQ ID NO: 13)

Table 3B. Probes for the detection of sequences in the Influenza A matrix gene identifying subtypes and for the influenza H1 neuraminidase gene with oseltamivir sensitivity

Table 3C. Synthetic unlabeled DNA representing sequences that distinguish Influenza A subtypes and oseltamivir resistant and sensitive strains

Target Name Sequence

Seasonal H3N2 FluMH3h140R GATCTGTGTTTTTCCCAGCAAAGACATCTT

Matrix CAAGT (SEQ ID NO: 20)

Seasonal H1 N1 FluMH1h140R GATCGGTATTCTTTCCAGCAAATACATCTT

Matrix CAAGT (SEQ ID NO: 21)

Pandemic FluMH1s140R GATCTGTGTTCTTTCCTGCAAAGACACTTT

H1 N1 Matrix CCAGT (SEQ ID NO: 22)

Avian H5N1A FluMH5a140R GATCGGTGTTCTTTCCTGCAAAGACATCTT

Matrix CAAGT (SEQ ID NO: 23)

Avian H5N1C FluMH5c140R GATCGGCGTTCTTTCCTGCAAAGACATCTT

Matrix CAAGT (SEQ ID NO: 24)

Pandemic FluN1swWT890R GCATTCCTCATAGTGATAATTAGGGGCATT

H1 N1 ACCACA (SEQ ID NO: 25)

Neuraminidase

Sensitive

Pandemic FluN1swRes890R GCATTCCTCATAGTAATAATTAGGGGCATT

H1 N1 ACCACA (SEQ ID NO: 26)

Neuraminidase

Resistant

Seasonal H1 N1 FluN1saiWT890R GCATTCCTCATAATGAAAATTGGGTGCATT

Neuraminidase ACCACA (SEQ ID NO: 27)

Sensitive

Seasonal H1 N1 FluN1saiRes890R GCATTCCTCATAATAAAAATTGGGTGCATT

Neuraminidase ACCACA (SEQ ID NO: 28)

Resistant

Seasonal H5N1 FiuN1 H5WT890R GCATTCCTCATAGTGATAATTAGGAGCATC

Neuraminidase ACCACA (SEQ ID NO: 29)

Sensitive

Seasonal H5N1 FluN1 H5Res890R GCATTCCTCATAGTAATAATTAGGAGCATC

Resistant

Matrix gene for detection and subtyping. Most influenza genes are highly variable between subtypes with the gene coding for the Matrix protein being the most highly conserved. It was reported that primers in this gene would amplify all influenza sub-types. Within this amplicon, there are regions conserved among most influenza subtypes where probes to allow RT-PCR detection have been designed (see Figure 8). Within the amplified sequence used for detection, there is also a region where many influenza subtypes differ (see Figure 8, subtype specific probes are identified) that is not necessarily amplified by other techniques. Using the 3 sub-type specific probes, it was determined that, with an annealing temperature of 60°C, each probe would be specific for its sub-type within the 3 most common human sub-types. It was also noted that the pandemic probe might react with the avian H5N1 influenza amplicon. RT-PCR using these probes with pandemic H1 N1 , seasonal H1 N1 , seasonal H3N2 was performed. Briefly, purified RNA was extracted from patient samples containing different sub-types of influenza A virus. Two dilutions of pandemic H1 N1 , seasonal H1 N1 , and H3N2 were amplified in reaction mixes containing matrix primers (Forward primer at 0.07 μΜ and reverse primer at 0.7 μΜ) that amplify all subtypes as well as four probes (all in Forward orientation at 0.2 μΜ) with different fluorophores: one specific for each of pandemic H1 N1 , seasonal H1 N1 , H3N2 and a universal probe that should detect all subtypes. Representative amplification curves are shown in Fig 9A, 9C, and 9E and confirm the specificity of the method.

The melting temperature of the various amplicons was determined. Briefly, "observed" values were obtained by mixing synthetic targets (Table 3C) with the individual probe (Table 3B) separately and performing a melting curve. The "predicted" value was obtained using the DINAmelt™ software as described above. Hybridizing the sub-type specific probes with synthetic unlabelled DNA with the sequence of pandemic H1 N1 , seasonal H1 N1 , seasonal H3N2 or avian H5N1 from the genome portion complementary to the probes indicated that avian H5N1 would cross-react with the pandemic probe (see Table 4), but with a melting temperature lower than that obtained for the pandemic target. The pandemic H1 N1 complementary sequence hybridized with the H3N2 probe with a melting temperature of 55°C, but did not produce an amplification curve when RT-PCR was performed with an annealing temperature of 60°C (see Fig. 9A).

Table 4. Melting temperatures (Observed/Predicted)

Probe

FluApand107F FluH1 h111 F FluH3-111 F

Synthetic Target

Pandemic H1 N1 Matrix 70.6/69.1 46.7/42.2 55.0/48.7 Seasonal H1 1 Matrix 40.6/42.9 67.9/66.0 42.0/42.8

Seasonal H3N2 Matrix 52.0/43.5 45.6/44.2 70.4/68.6

Avian H5N1A Matrix 64.1/61.7 59.4/56.9 55.8/48.9

Virus amplified by PCR

Pandemic H1 N1 70.6/69.1 <40/42.2 56.5/48.7

Seasonal H1 N1 <40/42.9 67.6/66.0 <40/42.8

Seasonal H3N2 <40/43.5 <40/44.2 70.9/68.6

Melting curves for probes and asymmetric PCR. Since it was observed that melting curves could be observed with probes when they were mixed with their complementary sequence, it was realized that a curve could be obtained if undegraded probe and its complementary strand remained at the end of the PCR reaction. Under normal PCR conditions, both strands produced by the PCR reaction are present in large enough amounts that they will reanneal in less than 10 seconds and prevent smaller probes from reannealing with their complementary strand. The PCR reaction normally consumes probes, so they are no longer available. In theory, each primer that initiates synthesis of a strand will degrade the probe hybridized to the strand being copied so 0.7 μΜ of primer might consume 0.7 μΜ of probe. Thus, by using 0.1 μΜ primer and 0.2 μΜ probe might leave intact probe at the end of the PCR reaction in order for a melting curve to be established. Alternatively, a PCR enzyme that does not have a 5' endonuclease activity and would not degrade the probe could be employed to preserve intact probe. It would not generate a typical PCR curve however because it would not degrade the probe, so signal acquisition would have to be determined rapidly at the annealing temperature before the polymerase displaced the probe in the course of synthesis of a new DNA strand.

Fluorescence intensity and crossing points (cycle when fluorescence becomes first visible) in the fluorescent acquisition over cycles curve as well as the fluorescence intensity of melting temperature peaks for a fixed amount of the primer producing the strand complementary to the probe (0.7 μΜ) and progressively lower amounts of the other primer from 0.2 to 0.01 μΜ was evaluated (see Table 5). With the exception of the lowest concentration of the reverse primer where no amplification curve was observed, the crossing points (Cp) did not vary with different amounts of the reverse primer. The maximum fluorescence intensity of the amplification curve decreased only slightly between 0.2 and 0.03 μΜ. At all of the reverse primer concentrations tested, a melting temperature peak at the appropriate temperature was observed. The maximum fluorescence intensity of this melting temperature peak increased when the concentration of the reverse primer was decreased from 0.2 to 0.1 μΜ and then the intensity decreased or remained similar to 0.03 μΜ of reverse primer. Similar results were observed for all of the probe-virus combinations, each at two dilutions, including the mismatched melting peak observed for the pandemic H1N1 virus with the H3N2 probe. Doubling of the probe concentration significantly increased the fluorescence intensity except for the mismatched H1 N1 virus with the H3N2 probe (Table 5).

Table 5. Fluorescent intensity of amplification and melting temperature curves with varying ratios of primers and probes.

The melting temperatures observed for each of the available viruses in a PCR reaction closely paralleled the values obtained from the probes mixed with synthetic unlabeled complementary probes (see Table 3 and Fig 9), indicating that values obtained for H5N1 virus synthetic probes should mimic the values for this virus. For most combinations except the pandemic H1 N1 virus with the H3N2 probe (Fig. 1 B), the heterologous probes did not form a melting temperature peak with the other virus sub-types (Fig. 1 D, 1 F) as they did in experiments with synthetic targets reported in Table 4. This was no doubt due to the presence of the homologous probe, also present in the mix, which bound to its target excluding heterologous probes. Even though an amplification curve was not observed when H1 N1 virus was amplified in the presence of the H3N2 probe (Fig 9A), a peak with a melting temperature of 56°C was observed in the melting temperature curve (Fig 9B). This result suggests the possibility that other probes with melting temperatures below the annealing temperature of RT-PCR necessary to maintain a specific PCR product might yield melting temperature curves that would allow them to be used with other non-identical targets.

Neuraminidase probes to distinguish oseltamivir sensitivity. A single FAM labeled probe with a sequence corresponding to that of the region surrounding the mutation causing resistance to oseltamivir in pandemic H1 N1 virus yielded melting temperatures with synthetic probes corresponding to sensitive and resistant pandemic H1 N1 , seasonal H1 N1 and H5N1 virus (see Figure 10 and Figure 11C). Using this probe in a multiplex RT-PCR reaction with primers that amplify the matrix gene target as well as primers that amplify the neuraminidase gene, the matrix and neuraminidase targets can be efficiently amplified and probes used to detect them. In Figure 11 A & 11 B, it is shown that an osteltamivir sensitive pandemic H1 N1 and oseltamivir sensitive and resistant seasonal H1 N1 virus amplified with matrix and neuraminidase primers with the universal probe for Influenza A matrix gene labeled with the fluorophore TEX615 and the neuraminidase probe labeled with FAM. Amplification curves are detected for both subtypes by fluorescence at 615 nM (not shown), but only the pandemic H1 N1 virus yields an amplification curve with fluorescence at 510 (FAM). Both influenza sub-types give the same melting temperature peak at 615 nM (not shown). The pandemic H1 N1 isolate gives a melting temperature of 65°C typical of oseltamivir sensitivity whereas the seasonal H1 N1 isolates give melting temperature peaks at 42°C typical of oseltamivir sensitivity and at 37°C typical of oseltamivir resistance in this sub-type (Fig. 11 A), even though amplification curves were not observed (Fig. 11 B).

Neuraminidase probes with inosine bases. The melting temperatures of the oseltamivir sensitive and resistant seasonal H1 N1 virus are near the limits of detection with most thermal cyclers. The inclusion of inosine bases at some of the bases of mismatch would slightly reduce melting temperatures for the pandemic and H5N1 sub-types, but would significantly increase melting temperatures for the seasonal H1 N1 (see Figure 10 and Fig 11 D) making detection more straightforward.

EXAMPLE III - BACTERIAL CLASSIFICATION AND IDENTIFICATION: ANALYSIS OF 16S

RIBOSOMAL RNA GENES

Patients and bacterial DNA preparation. Between January 2004 and April 2005, women complaining of vaginal discharge were recruited in nine healthcare facilities in West Africa. Specimens of vaginal fluid were collected in transport medium (Roche, Laval, Quebec) and transported to Sherbrooke, Quebec. They were then treated with an equal volume of CT/NG specimen diluent (Roche) in preparation for PCR analysis.

Primers and probes preparation. Primers and probes (see Table 6) were synthesized by IDT- DNA, Coraiville, IA and were dissolved in water to make 50 μΜ stock solutions. Stock solutions were then diluted to 5 μΜ working solutions.

Table 6A. Primers used in Example III

Target Name Sequence

Dialister pneumosintes (16S rRNA Dial476F TGACGGTACCGGAAAAGC (SEQ ID gene) - 196 bp amplicon NO: 31 )

Dialister pneumosintes (16S rRNA Dial662R CTCTCCGATACTCCAGCTTC (SEQ ID gene) - 196 bp amplicon NO: 32)

Anaerococcus sp. (16S rRNA gene) AnCoc318F ATTGGGACTGAGACACGGC (SEQ ID - 334 bp amplicon NO: 34)

Anaerococcus sp. (16S rRNA gene) AnCoc642R CACTAGGAATTCCACTTTCCCT (SEQ - 334 bp amplicon ID NO: 35)

Peptoniphilus other than lacrimalis Pepton1003F GACCGGTATAGAGATATACCCT (SEQ (16S rRNA gene) - 182 bp amplicon ID NO: 37)

Peptoniphilus other than lacrimalis Pepton1184R CACCTTCCTCCGATTTATCATC (SEQ (16S rRNA gene) - 182 bp amplicon ID NO: 38)

Bifidobacterium sp. (16S rRNA Bifi592F CTCGTCGCGTCYGGTGTGA (SEQ ID gene) - 245 bp amplicon NO: 40)

Bifidobacterium sp. (16S rRNA Bifi836R CCACATCCAGCRTCCAC (SEQ ID NO: gene) - 245 bp amplicon 41)

Table 6B. Probes used in Example III

PCR protocol. Reaction mixes in a final volume of 15 μΙ contained Roche Fast Start RT-PCR master mix, were analysed on a Roche LC480™ thermal cycler. Two (2) μΙ of a mixture of equal volumes of specimen and specimen diluent were added to a final volume of 12 μΙ containing the LC480 Probe Master kit™ (Roche) and 0.2 μΜ of labelled probe, 0.7 μΜ of primers for the strand complementary to the probe, and 0.07 μΜ of primers for the strand collinear to the probe. After 10 minutes at 95°C, 50 cycles of 10 sec at 93°C, 15 sec at 55°C and 15 sec at 72°C were performed. A melting temperature curve (Tm) was then generated by denaturing products at 95°C [1 min], followed by a renaturation step at 40°C [1 min], the temperature was then ramped from 45°C to 95°C at 0.03°C/sec, 5 acquisitions/°C. Fluorescence data generated by Tm curves were plotted and analyzed by the LightCycler™ 480 Tm calling Software. Real-time PCR reactions were carried out in a 96-well plate in a Roche LightCycler™ II 480 real-time PCR system apparatus.

Detection of nucleic acids of Trichomonas vaginalis, Gardnerella vaginalis, Atopobium vaginae, Prevotella spp., Mobiluncus spp., Mycoplasma hominis, Lactobacillus spp, Leptotrichia spp., Eggerthella spp., Megasphaera elsdenii, Dialister spp., Bifidobacterium spp., Peptoniphilus spp. with primers that excluded P. lacrimalis, and Anaerococcus spp. in all vaginal samples was performed. Most of these pathogens have already been identified as potential causes of vaginosis and PCR conditions have been described elsewhere. In order to confirm the presence of new agents Dialister pneumosintes, Bifidobacterium spp., Peptoniphilus spp. other than P. lacrimalis, and Anaerococcus spp., the specificity of the PCR reactions was assessed by performing melting temperature curves.

Sequences of the target organisms were compared with other bacteria of similar genera in order to identify primers and probes that would be specific. Examples of the location of some of the primers and probes used are shown in Figure 12. Figure 13 shows an example of the comparison of primers and probes within very similar genera. Differences within the sequence of the probes and primers for the genera Dialister are highlighted. The differences within the probe sequence between D. pseumosintes, D. invisus, and D. microaerophilus should yield maximal melting temperatures by TaqTm probing for the homologous D. pseumosintes, but much lower temperatures for D. invisus, and D. microaerophilus. With the PCR protocol presented herewith, it is not expect that species other than D. pseumosintes would register a positive result. Other species not shown, or indeed unknown, might have fewer mismatches and yield a positive result by conventional PCR with a re-association temperature of 55°C, but if they had even one mismatch would give a renaturation temperature lower than the temperature expected for D. pseumosintes and could thus be detected. A variant D. pseumosintes strain with a slightly different 16S rRNA gene sequence would also give a lower melting temperature, but we would see this and be able to further characterize such very similar isolates.

In order to validate the method, without reference strains, 100 samples were analyzed with our primers (Table 6A) using probes (Table 6B) as well as with only the primers with SyBr green and melting temperature analysis in real-time PCR or with conventional PCR and agarose gels in order to verify the molecular weight of the product. Identical results were obtained for each of the four micro-organisms tested. Real time PCR in multiplex was attempted with all four genes, but found interference between the reactions and thus performed analysis with duplexes of Dialister with Bifidobacterium and Anaerococcus together with Peptoniphilus. Unclear results were repeated separately for each target.

It was also attempted to detect slight variation in the probe/am plicon hybrids using TaqTm probing to identify slight differences that would not have been detected by the other methods used and that would have raised the possibility that other microbes had been responsible. This analysis of 1555 samples of vaginal fluid from different West African patients revealed 387 positive for D. pseumosintes, 309 positive for Anaerococcus spp. with a melting temperature of 53°C and 257 with a melting temperature of 66.5°C, 308 positive for Peptoniphilus other than P. lacrimalis, and 974 positive for Bifidobacterium sp.. Melting temperatures of 68°C, 67.5°C and 67,5°C were observed for D. pseumosintes, Peptoniphilus other than P. lacrimalis, and for Bifidobacterium sp. respectively. All Bifidobacterium samples had the exact melting temperature, whereas 50 Dialister positive samples and 6 Peptoniphilus positive specimens had lower melting temperatures (64°C and 66°C, respectively). Some examples are shown in Figure 14.

It has been shown herein that melting temperatures can be obtained readily for rRNA genes amplified from a variety of bacteria found in vaginal fluid using TaqTm probing. Maximal melting temperatures probably indicate the precise bacteria or species whereas lower temperatures probably reveal related or variant organisms. It was presented that most of the positive reactions observed had an exact melting temperature match with the probe, except for Anaerococcus where two different targets were detected with quite different probe:amplicon melting temperatures. It will be important to sequence amplified DNA from some of these samples in order to identify more precisely the microorganism(s) present and the possible variations they carry.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting a nucleotide difference between a target sequence and a first standard sequence, said method comprising:

(a) amplifying the target sequence to generate a target amplicon;

(b) contacting the target amplicon with a first probe to form a first target amplicon-probe complex, wherein the first probe has a first label covalently linked to the one end of the probe and a first corresponding quencher linked to the other end of the probe;

(c) measuring the signal from the first label of the first probe within a range of temperature to determine the melting temperature of the first target amplicon-probe complex;

(d) providing the melting temperature of a complex between a corresponding amplicon of the first standard sequence and the first probe;

(e) comparing the melting temperature of the first target amplicon-probe complex with the melting temperature of step (d); and

(f) detecting the presence of the nucleotide difference if the melting temperature of the first target amplicon-probe complex is lower than the melting temperature of step (d), or the absence of the nucleotide difference if the melting temperature of the first target amplicon-probe complex is substantially similar to the melting temperature of step (d); wherein the sequence of the first probe is substantially identical to a fragment of the corresponding amplicon of the first standard sequence.

2. The method of claim 1 , further comprising:

(g) contacting the target amplicon with a second probe to form a second target amplicon-probe complex, wherein the second probe has a second label covalently linked to one end of the probe and a second corresponding quencher linked to the other end of the probe and wherein the first and second label are different; measuring the signal from the second label of the second probe within a range of temperature to determine the melting temperature of the second target amplicon-probe complex;

(i) providing the melting temperature of a complex between a corresponding amplicon of the first standard sequence and the second probe;

(j) determining a first difference in the melting temperature of the first target amplicon-probe complex and the melting temperature of step (d);

(k) determining a second difference in the melting temperature of the second amplicon-probe complex with the melting temperature of step (i);

(I) determining which of the first difference of step (j) or the second difference of step (k) is lower; and

(m) assessing the identity of the nucleotide difference based on the determination of step (I); wherein the sequence of the second probe is substantially identical to a fragment of a second standard sequence and wherein the second standard sequence differs by at least one nucleotide from the first standard sequence.

3. The method of claim 2, wherein the second standard sequence differs by at least two nucleotides from the first standard sequence.

4. The method of claim 2, wherein the second standard sequence differs by at least three nucleotides from the first standard sequence.

5. The method of claim 2, wherein the second standard sequence differs by at least four nucleotides from the first standard sequence.

6. The method of claim 2, wherein the second standard sequence differs by at least five nucleotides from the first standard sequence.

7. The method of any one of claims 1 to 6, wherein the difference between the melting temperature of step (d) and the melting temperature of step (i) is equal to or lower than about 25°C.

8. The method of any one of claims 1 to 6, wherein the difference between the melting temperature of step (d) and the melting temperature of step (i) is equal to or lower than about 10°C.

9. The method of any one of claims 1 to 6, wherein the difference between the melting temperature of step (d) and the melting temperature of step (i) is equal to or lower than about 5°C.

10. The method of any one of claims 1 to 6, wherein the difference between the melting temperature of step (d) and the melting temperature of step (i) is between about 2°C to 4°C.

11. The method of any one of claims 1 to 10, wherein the amplification step (a) comprises a polymerase chain reaction.

12. The method of claim 11 , wherein a first primer is used for the polymerase chain reaction and said first primer is (i) substantially identical to a first portion of the target sequence or a complement thereof and a first corresponding portion of the first standard sequence or a complement thereof and (ii) non-linear to the first and/or second probe.

13. The method of claim 11 or 12, wherein a second primer is used for the polymerase chain reaction and said second primer is (i) substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or complement thereof and (ii) co-linear to the first and/or second probe.

14. The method of claim 13, wherein the concentration of the first primer is higher than the concentration of the second primer in the polymerase chain reaction.

15. The method of claim 14, wherein the first and/or second probe is present during the polymerase chain reaction and the concentration of the first and/or second probe is higher than the concentration of the second primer.

16. The method of any one of claims 1 to 15, wherein the range of temperature of step (c) and/or (i) is between about 30°C to about 95°C.

17. The method of any one of claims 1 to 15, wherein the range of temperature of step (c) and/or (i) is between about 45°C to about 70°C.

18. The method of any one of claims 1 to 17, wherein the nucleotide difference is a single nucleotide polymorphism.

19. The method of any one of claims 1 to 18, wherein the target sequence and the first standard sequence are derived from Campylobacter jejuni.

20. The method of claim 19, wherein the target sequence and the first standard sequence are derived from a 23S ribosomal RNA gene.

21. The method of claim 20, wherein the first primer comprises the sequence of SEQ ID NO: 1 , SEQ ID NO: 2 or a complementary sequence thereof.

22. The method of claim 20 or 21 , wherein the second primer comprises the sequence of SEQ ID NO: 1 , SEQ ID NO: 2 or a complementary sequence thereof.

23. The method of any one of claims 20 to 22, wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and complementary sequences thereof.

24. The method of claim 19, wherein the target sequence and the first standard sequence are derived from a gyrA gene.

25. The method of claim 24, wherein the first primer comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or a complementary sequence thereof.

26. The method of claim 24 or 25, wherein the second primer comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or a complementary sequence thereof.

27. The method of any one of claims 24 to 26, wherein the first probe and/or the second probe comprises is at least one of the following sequences: SEQ ID NO: 8, SEQ ID NO: 9 and complementary sequences thereof.

28. The method of any one of claims 1 to 18, wherein the target sequence and the first standard sequence are derived from Influenza virus.

29. The method of claim 28, wherein the target sequence and the first standard sequence are from a matrix gene.

30. The method of claim 29, wherein the first primer comprises the sequence of SEQ ID NO: 10, SEQ ID NO: 11 , or a complementary sequence thereof.

31. The method of claim 29 or 30, wherein the second primer comprises the sequence of SEQ ID NO: 10, SEQ ID NO: 11 , or a complementary sequence thereof.

32. The method of any one of claims 29 to 31 , wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and complementary sequences thereof.

33. The method of claim 28, wherein the target sequence and the first standard sequence are from a neuraminidase gene.

34. The method of claim 33, wherein the first primer comprises the sequence of SEQ ID NO: 12, SEQ ID NO: 13, or a complementary sequence thereof.

35. The method of claim 33 or 34, wherein the second primer comprises the sequence of SEQ ID NO: 12, SEQ ID NO: 13, or a complementary sequence thereof.

36. The method of any one of claims 33 to 35, wherein the first probe and/or the second probe comprises at least one of the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 and complementary sequences thereof.

37. The method of any one of claims 1 to 18, wherein the target sequence and the first standard sequence are derived from a 16S ribosomal RNA gene of a bacteria.

38. The method of claim 37, wherein the bacteria is Dialister pneumosintes.

39. The method of claim 38, wherein the first primer comprises the sequence of SEQ ID NO: 31 , SEQ ID NO: 32, or a complementary sequence thereof.

40. The method of claim 38 or 39, wherein the second primer comprises the sequence of SEQ ID NO: 31 , SEQ ID NO: 32, or a complementary sequence thereof.

41. The method of any one of claims 38 to 40, wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 33 or a complementary sequence thereof.

42. The method of claim 37, wherein the bacteria is Anaerococcus sp..

43. The method of claim 42, wherein the first primer comprises the sequence of SEQ ID NO: 34, SEQ ID NO: 35, or a complementary sequence thereof.

44. The method of claim 42 or 43, wherein the second primer comprises the sequence of SEQ ID NO: 34, SEQ ID NO: 35, or a complementary sequence thereof.

45. The method of any one of claims 42 to 44, wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 36 or a complementary sequence thereof.

46. The method of claim 37, wherein the bacteria is Peptoniphilus sp. other than P. lacrimalis.

47. The method of claim 46, wherein the first primer comprises the sequence of SEQ ID NO: 37, SEQ ID NO: 38, or a complementary sequence thereof.

48. The method of claim 46 or 47, wherein the second primer comprises the sequence of SEQ ID NO: 37, SEQ ID NO: 38, or a complementary sequence thereof.

49. The method of any one of claims 46 to 48, wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 39 or a complementary sequence thereof.

50. The method of claim 37, wherein the bacteria is Bifidobacterium sp..

51. The method of claim 50, wherein the first primer comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 41 , or a complementary sequence thereof.

52. The method of claim 50 or 51 , wherein the second primer comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 41 , or a complementary sequence thereof.

53. The method of any one of claims 50 to 52, wherein the first probe and/or the second probe comprises the sequence of SEQ ID NO: 33 or a complementary sequence thereof.

54. A method of detecting a plurality of nucleotide differences between a target sequence and a first standard sequence, said method comprising:

(a) amplifying the target sequence to generate a plurality of target amplicons;

(b) contacting the plurality of target amplicons with at least two probes to form at least two target amplicon-probe complexes, wherein the at least two probes each have a label covalently linked to the one end of the probe and a corresponding quencher linked to the other end of the probe and each of the at least two probes have a different label;

(c) measuring the signal from each label of the at least two probes within at least one temperature range to determine the melting temperatures of the at least two target amplicon-probe complexes;

(d) providing the melting temperature of complexes of at leas two corresponding amplicons of the first standard sequence and the at least two probes;

(e) comparing the melting temperature of the at least two of target amplicon-probe complexes with the melting temperatures of step (d); and

(f) detecting, for each target-amplicon probe complex, the presence or absence of the plurality nucleotide difference based on the comparison of step (e); wherein the sequence of the at least two probes are substantially identical to a fragment of a corresponding amplicon of the first standard sequence.

55. The method of claim 54, wherein the amplification step (a) comprises a polymerase chain reaction.

56. The method of claim 55, wherein a first primer is used for the polymerase chain reaction and said first primer is (i) substantially identical to a first portion of the target sequence or a complement thereof and a first corresponding portion of the first standard sequence or a complement thereof and (ii) non-linear to the first of the at least two probes.

57. The method of claim 55 or 56, wherein a second primer is used for the polymerase chain reaction and said second primer is (i) substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and (ii) co-linear to the first of the at least two probes.

58. The method of any one of claims 55 to 57, wherein a third primer is used for the polymerase chain reaction and said third primer is (i) substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and (ii) non-linear to the second of the at least two probes.

59. The method of any one of claims 55 to 58, wherein a fourth primer is used for the polymerase chain reaction and said fourth primer is (i) substantially identical to a second portion of the target sequence or a complement thereof and a second corresponding portion of the first standard sequence or a complement thereof and (ii) co-linear to the second of the at least two probes.

60. The method of any one of claims 56 to 59, wherein the concentration of the first primer is higher than the concentration of the second primer in the polymerase chain reaction.

61. The method of claim 58 or 59, wherein the concentration of the third primer is higher than the concentration of the fourth primer.

62. The method of claim 60 or 61 , wherein the at least two probes are present during the polymerase chain reaction.

63. The method of claim 62, wherein the concentration of the first probe is higher than the concentration of the second primer.

64. The method of claim 62 or 63, wherein the concentration of the second probe is higher than the concentration of the fourth primer.

65. The method of any one of claims 54 to 64, wherein the range of temperature of step (c) and/or (i) is between about 30°C to about 95°C.

66. The method of any one of claims 54 to 64, wherein the range of temperature of step (c) and/or (i) is between about 45°C to about 70°C.

67. A kit for the determination of a nucleotide difference between a target sequence and a standard sequence, said kit comprising:

• a first primer and a second primer for amplifying the target sequence and generate a corresponding target amplicon; and

• a first probe for forming a first complex with the target amplicon, wherein the first probe has a first label covalently linked to the one end of the probe and a first quencher linked to the other end of the probe and wherein the sequence of the first probe is substantially identical to a portion of the standard sequence

wherein the concentration the concentration of the first probe is higher than the concentration of the second primer.

68. The kit of claim 67, further comprising a second probe for forming a second complex with the target amplicon, wherein the second probe has a second label covalently linked to the one end of the probe and a second corresponding quencher linked to the other end of the probe, wherein the first label and second label are different and wherein the sequence of the second probe is substantially identical to a portion of a second standard sequence, wherein the second standard sequence differs by at least one nucleotide from the first standard sequence.

69. The kit of claim 67, further comprising:

• a thrid primer and a fourth primer for amplifying the target sequence and generate a second corresponding target amplicon; and

• a second probe for forming a second complex with the second target amplicon, wherein the second probe has a first label covalently linked to the one end of the probe and a first quencher linked to the other end of the probe and wherein the sequence of the second probe is substantially identical to a second portion of the standard sequence.