EP1276898A1 - Flavivirus detection and quantification assay - Google Patents

Flavivirus detection and quantification assay

Info

Publication number
EP1276898A1
EP1276898A1 EP00973687A EP00973687A EP1276898A1 EP 1276898 A1 EP1276898 A1 EP 1276898A1 EP 00973687 A EP00973687 A EP 00973687A EP 00973687 A EP00973687 A EP 00973687A EP 1276898 A1 EP1276898 A1 EP 1276898A1
Authority
EP
European Patent Office
Prior art keywords
tcc
caa
cat
tct
gct
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00973687A
Other languages
German (de)
French (fr)
Inventor
Huo-Shu H. Houng
Niranjan Kanesa-Thasan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Army Medical Research and Materiel Command USAMRMC
Original Assignee
US Army Medical Research and Materiel Command USAMRMC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/551,161 external-priority patent/US6793488B1/en
Application filed by US Army Medical Research and Materiel Command USAMRMC filed Critical US Army Medical Research and Materiel Command USAMRMC
Publication of EP1276898A1 publication Critical patent/EP1276898A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • the subject invention relates to a nucleic acid based diagnostic system for rapid and specific flavivirus virus identification and quantification and kits which involve a novel set of oligonucleotides which can distinguish and quantitate flavivirus, especially.
  • Dengue viruses belong to the family Flaviviridae, which contains almost 70 viruses, including those causing yellow fever and several encephalitides.
  • Four distinct dengue virus types (dengue 2, 3, and 4) are each capable of causing infection in humans (Henchal and Putnak, 1990).
  • Dengue infections are usually confirmed by serologic detection of dengue-specific antibodies or/and dengue virus isolation through culture in insect cells or mosquito inoculation (Gubler, 1989). These conventional methods for dengue virus detection are very time consuming, labor intensive, and have limited sensitivity for detecting low levels of dengue virus.
  • Rapid molecular diagnostic systems such as RT-PCR, could provide a possible solution for dengue virus identification from clinical samples (Harris et al, 1998).
  • RT-PCR systems for dengue virus detection have been reported, based on different conserved genomic regions, such as genes for nonstructural proteins (Chow et al., 1993; Fulop et al, 1993) and the 3'-noncoding region (Sudiro et ah, 1997). See also U.S. Patent No. 5,939,254.
  • these methods frequently have mismatches corresponding to their amplicons, or even to primer sequences, due to genetic differences among strains of the same dengue serotype.
  • Some of these systems rely upon sequences from the "conserved” regions for probes or primers, such as NS3 and NS5 genes. Alignment of these so-called conserved genes of dengue reveal mismatches among the viruses, in addition to variation among virus strains. These mismatches result in reduced sensitivity of the assay, and as a result, false diagnosis of the specific dengue virus. Further, unpredictable variations in PCR target sequences would yield lower specificity and sensitivity in dengue detection. In addition, these methods are often cumbersome to adapt for routine clinical use and are not quantitative.
  • the 3' noncoding region has been previously studied and sequenced. See, for example, Mandl, C. W., et al, "Spontaneous Engineered Deletions in the 3' noncoding region of Tick-bome Encephalitis Virus: Construction of Highly Attenuated Mutants of a Flavivirus", J. Virology, Vol. 72, (1998) pp. 2132-2140. Proupski, V. et al, "Biological Consequences of Deletions within the 3 '-untranslated Region of Flavivirus May be due to Rearrangements of RNA Secondary Structure", Virus Research (1999) Vol. 64, pp. 107-123.
  • the present invention is directed to designing and utilizing specific genomic region, the conserved 3 - terminal non-coding region, as specific nucleic acid-based diagnostic system for rapid and specific flavivirus, e.g. dengue virus, identification and quantification.
  • the invention focuses on the last 250, more specifically the last 200, even more specifically the last 160 bases of the conserved dengue genome. This would correspond to nucleotides 10558 through 10718 of dengue 1.
  • the invention also utilizes flavivirus specific upstream primers, probes, and downstream primers developed from this region. From the conserved terminal 3' non coding region conserved region, a probe unique to a flavivirus is chosen.
  • Primers can then be generated from conserved areas upstream and downstream of the probe that are unique to the virus of interest, the areas extending to about one kilobase from the 3' terminus of any flavivirus. A preferred region extends to about 200 bases from the 3' terminus. More than one primer set and more than one probe can be chosen, each primer set and probe can be both conserved and specific for a virus.
  • the cDNA is incubated with the primers and probe under condition such that a polymerase is able to synthesize a complementary strand using the primer/template substrate.
  • the polymerase having a 5' ⁇ 3' exonuclease activity, will digest the probe hybridized to its specific complementary virus sequence.
  • the probe may contain a reporter at the 5 ' end which will release a detectable signal when the probe is digested. Quantification and detection of the specific vims for which the probe and primers were designed is possible by detecting' the released probe signal. If the probe does not find its match in the sample, the probe is not digested by the polymerase, and the signal is not released or detected.
  • probes can be highly discriminatory, even single nucleotide substitutions can be reliably detected among different viruses.
  • Oligonucleotide probes and primers described in this invention can be used in a multiplex format to detect and differentiate flaviviruses. It is also possible to formulate individual virus-specific assay mixtures to identify and quantitate one virus at a time. The format of the assay depends on the desired usage. A generic dengue diagnostic system may work well for clinical or epidemiologic use, whereas stereotype-specific dengue assays are required for use in vaccine development. The design of the assay can be refined for single-step multiplex use (processing of a specimen and evaluation in a single reaction mixture), or two step reactions may be used, as described in the material.
  • the flavivirus fluorogenic assays based on the terminal 3' noncoding region offer real-time quantitation of virions (expressed as plaque forming units or as genome copy equivalents). Determination of viral number is extrapolated from reproducible linear standard curves, derived either from titration of viral RNA or from dilutions of flaviviral cDNA (as plasmids).
  • the rapid, sensitive detection of flaviviruses lends itself to several applications. These include: 1. Rapid diagnosis of flaviviral infection: the specific nature of the fluorogenic probes devised for this patent allows confirmation of flaviviral infection.
  • Determination of viral burden the magnitude and duration of viremia during flaviviral infection may be positively associated with flaviviral pathogenicity. This finding may be of prognostic significance in dengue hemorrhagic fever, where increased viral burden (particularly at the time of defervescence) may be a marker of increased risk for severe disease. Studies of viral clearance by different immunologic mechanisms will be made easier. Furthermore, the effectiveness of specific antiviral therapy (such as drugs, immunotherapy, and other approaches) may be gauged by its effect on viral burden in vivo or in vitro.
  • Amplification of flaviviral RNA offers a simplified approach to creation of amplified, near full-length copies of flaviviral genomes.
  • the 3' noncoding region is critical to replication of viral RNA, and the product of the reverse transcription reaction could be manipulated to generate flaviviral replicons.
  • formation of replicative intermediates can be monitored through antisense RNA copies.
  • Flaviviral cDNA products may be used for molecular pharmacologic or therapeutic purposes.
  • Fluorescence accumulation plots derived from PCR with MG ++ concentration of 2.5, 2.0, 1.5, 1.0, and 0.5 mM are shown from left to right.
  • Figure 3 Standard curve of dengue 2 SI 6803. Triplicate Cx values of serially diluted S16803 ranging from 1.3 to 10 6 pfu/ml are shown.
  • Figure 5 A and 5B The basics of the nuclease assay.
  • FIG 8. Copy number dependent West Nile virus (WNV) assay: 1 :5 dilution of WNV cDNA derived from viral sample ranging from 100,000 to 1 pfu per reaction.
  • Figure 9. Linear scale of WNV assay showing background-free results from non-template control (from 1 through 100,000 pfu).
  • Dengue viral RNA was extracted from about 40 ⁇ l of either viral suspension or infected serum and then the total viral RNA was dissolved into buffer, and about 8 ⁇ l of the resulting RNA solution was mixed with about 12 ⁇ l of RT reaction mixture (see below). After reverse transcription, about 2 ⁇ l of the cDNA product was used as DNA template for the fluorogenic PCR assay.
  • each infectious pfu for dengue virus should represent at least 100, or greater of genomic equivalencies.
  • a novel dengue 2 type-specific genomic sequence derived from the 3' noncoding region was use to develop a fluorogenic-based RT-PCR (Taqman, PE Biosystem, Foster City, CA).
  • the assay was formulated to use a uniquely designed internal fluorescence-labeled probe to specifically hybridize a 3'-noncoding target sequence of dengue 2.
  • fluorescent signal is released through 5'-nuclease activity of DNA Taq polymerase that is used to amplify the target sequence (Holland et al. 1991 ; Higgins, et al. 1998). This allows convenient monitoring of a specific PCR product formation over time.
  • the fluorogenic RT-PCR developed in this study is capable of discriminating single nucleotide substitutions in the target sequence.
  • real time detection of dengue PCR product was correlated with input dengue cDNA copy number, to yield a quantitative assay for dengue 2 viruses.
  • Dengue virus strains and other flavivirus isolates shown in Table I were obtained from Departments of Virus Diseases and Biologies Research, Walter Reed Army Institute of Research, Washington, DC. Most virus isolates were routinely obtained from supemates of insect or Vero cell culture. Virus titrations were performed by plaque assay in Vero cells (Eckels et al., 1980). A dengue 2 SI 6803 virus stock suspension (3.8 ⁇ 0.8 x 10 8 , plaque forming units, pfu per ml) was used as a standard for development of the assay.
  • Dengue infected human sera were collected from individuals with confirmed dengue fever in Somalia (Kanesa-thasan et al, 1994) and Haiti (Trofa et al, 1997). These specimens were dengue 2 positive by indirect immunofluorescence after culture in C6/36 insect cells. Monkey sera were obtained from three monkeys following subcutaneous inoculation with 10 4 pfu dengue 2 virus SI 6803. Blood specimens were collected daily for 12 days after infectious challenge from each monkey. For immunocomplexed dengue virus study, pooled human hyperimmune serum with plaque reduction neutralizing antibody titer of 1 :7000 against dengue 2 was kindly provided by the Dept of Virology, AFRIMS, Bangkok, Thailand. AU serum specimens were stored at -80°C until use.
  • Virus RNA used in this study was routinely extracted from virus suspensions or dengue- infected sera (40 VI) according to QiAamp Viral RNA Handbook (Qiagen Inc. Valencia, CA 91355). Total RNA was eluted into 32 ⁇ l of TE- (10 mM Tris- HC1, 1 mM Na 2 EDTA, pH 8.0).
  • oligonucleotide sequences are shown as following: DV2.P1 (nucleotides 10653-10678): 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG- CA-3OV2.L1 (nucleotides 10558-10579): 5'-CAT-TCC-ATT-TTC-TGG-CGT-TCT- 3' DV2.U2 (nucleotides 10680-10700): 5'-AAG-GTG-AGA-TGA-AGC-TGT-AGT- CTC-3'.
  • DV2.P1 consists of oligonucleotide sequence shown as above with a 5 ' - reporter dye (FAM, 6- carboxy-fluorescein) and a down stream 3 '-quencher dye (TAMRA, 6-carboxy-tetramethyl-rhodamine).
  • FAM 5 ' - reporter dye
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • oligonucleotide as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleotides, ribonucleotides, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, or the like.
  • monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units.
  • oligonucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5' — 3' order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted.
  • Analogs of phosphodiester linkages include phosphorothioate, phosphoranilidate, phosphoramidate, and the like.
  • nucleotide includes the natural nucleotides, including 2'- deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
  • "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of specific hybridization.
  • Such analogs include synthetic nucleotides designed to enhance binding properties, reduce degeneracy, increase specificity, reduce activity as enzyme substrates, and the like.
  • Oligonucleotides of the invention can be synthesized by a number of approaches, e.g. Ozaki et al, Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al, Nucleic Acids Research, 18: 5419-5423 (1990); or the like.
  • the oligonucleotide probes of the invention are conveniently synthesized on an automated DNA synthesizer, e.g. a Perkin-Elmer (Foster City, Calif.) Model 392 or 394 DNA/RNA Synthesizer, using standard chemistries, such as phosphoramidite chemistry, e.g.
  • the oligonucleotide probe is in the range of 15-150 nucleotides in length selected from contiguous sequences in the conserved 3'- terminal non-coding region to detect and quantitate specific flaviviruses.
  • the oligonucleotide probe is in the range of 18-30 nucleotides in length.
  • the precise sequence and length of an oligonucleotide probe of the invention depends in part on the nature of the target nucleic acid sequence to which it hybridizes. The binding location and length may be varied to achieve appropriate annealing and melting properties for a particular embodiment. Guidance for making such design choices can be found in many of the above-cited references describing the "Taqman" type of assays. The invention develops these probes from the last 250 nucleotides, more specifically the last 200 nucleotides, even more specifically the last 160 nucleotides of the 3' noncoding region of the flaviviral genome.
  • the oligonucleotide probes of the present invention include fluorescer and quencher molecules attached to the oligonucleotide.
  • quenching or “fluorescence energy transfer” refer to the process whereby when a fluorescer molecule and a quencher molecule are in close proximity, whenever the fluorescer molecule is excited, a substantial portion of the energy of the excited state nonradiatively transfers to the quencher where it either dissipates nonradiatively or is emitted at a different emission wavelength than that of the fluorescer.
  • Fluorescer-quencher pairs may be xanthene dyes, including fluoresceins, and rhodamine dyes.
  • Another group of fluorescent compounds are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1- dimethylaminonaphthyl-5-sulfonate, l-anilino-8-naphthalene sulfonate and 2-p- toluidinyl-6-naphthalene sulfonate.
  • dyes include 3-phenyl-7- isocyanatocoumarin, acridines, such as 9-isothiocyanatoacridine acridine orange; N- (p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, pyrenes, and the like.
  • RT Reverse Transcription
  • PCR Polymerase Chain Reaction
  • RNA extract was transcribed into cDNA using Taqman RT kit (PE ABI, Foster City, CA).
  • the RT reactions were performed in 20 W volume containing 8 ⁇ l extracted RNA, 241 1 0 X RT buffer (PE ABI), 4 ⁇ l 2.5 mM dNTPs, 4.4 ⁇ l 25 mM MgCl 2 , 0.4 ⁇ l RNAase inhibitor, 0.5 ⁇ l MuLV reverse transcriptase (50 units/ ⁇ l), and 1 ⁇ l DV2.L1 primer (20 pmole/ ⁇ l).
  • RT reaction mixtures were incubated at 25°C for 10 minutes followed at 48°C for 30 minutes, then 95°C for 5 minutes.
  • the resulting cDNA was used as template in the subsequent PCR reaction.
  • PCR reactions were conducted in 50 VI volume containing 2.0 VI cDNA template, Taqman buffer (PE ABI, Foster City, CA), 0.1 mM deoxyribonucleotide triphosphates x (dNTPs), 2.0 MM MgCl 2 , 200 nM DV2.U2, 20 nM DV2.P 1 , 60 nM DV2.L1, and 25 units AmpliTaq Gold DNA polymerase (PE ABI, Foster City, CA).
  • Gene detection system 7700 and sequence detection system software version, 6.3 were employed for PCR cycling reaction, real time data collection and analysis. 6 PCR mixtures were pre-incubated at 50°C for 2 minutes, then 95°C for 10 minutes followed by 40 cycles of two-step incubations at 95°C for 15 seconds and 60°C for 1 minute. Results
  • the cycle number to yield detectable fluorescence level, Cj usually was automatically selected by using the average of samples' fluorescences from cycle 3 through 15 as the baseline. Parallel curves were derived from different concentrations of dengue 2 viral cDNA templates. Greater initial viral cDNA copy numbers required fewer amplification cycles (smaller C T value) to reach a detectable fluorescence level. As the number of viral copies was reduced by serial I dilutions, the curves corresponding to each individual dilution were shifted to the night, with greater Cj values. The furthermost right plot, showing C T value of approximately 37, represents non- template (water) controls.
  • the dengue 2- specific primers and the probe uniquely designed for the fluorogenic RT-PCR system are capable of detecting dengue 2 viral cDNA in a dose-dependent fashion over 7 serial 5-fold dilutions (more than 3.5 logs dilution).
  • the assay is capable of discriminating specific dengue 2 viral stock cDNA from background levels (non- template controls).
  • the sensitivity of the assay can be improved by enhancing the relationship between dengue viral cDNA copy number and C T value.
  • DV2. LI primer was with universal poly-oligo (T)l 6 primer (PE Applied Biosystem Inc. . Foster City, CA) for reverse transcription of dengue RNA.
  • Universal poly-oligo (T)16 primer is frequently used as generic RT primer to generate dengue cDNA during reverse transcription process.
  • the quantities of cDNA generated by RT reactions of the same dengue viral RNA (from dengue 2 virus suspension of 3.8 X 10 6 pfu/ml) were examined using DV2.L1 and poly-oligo (T) 16 primers, respectively.
  • Figure 2 shows that use of DV2.L1 primer in the RT reaction yielded much greater number of cDNA copies than the poly-oligo (T)16 primer by dengue 2 fluorogenic PCR, i.e. C T of 15.4 vs. 22.5.
  • C T 15.4 vs. 22.5.
  • DV2.L1 was used for all RT reactions in this study.
  • the effects of varying the concentrations of Mg** on the PCR reaction were examined. It was shown in Fig 3 that Mg' concentration from 1.5 to 2.5 mM had very little effect on the C T value of the assay. A midrange Mg concentration of 2.0 mM was selected as optimal for the assay.
  • the background (non-template control) was reduced by examining the effect of different concentrations and ratios of the flanking primers, DV2.L1 and DV2.U2, respectively.
  • Preliminary observations showed that the dengue 2 fluorogenic RT-PCR would yield saturating, high fluorescence levels when DV2.U2 and DV2.L1 primers were used at equimolar concentrations of 200 nM (as shown in Figure 1).
  • non- template/water controls would yield detectable background fluorescent signal after 36 to 37 cycles of amplification. Little or zero background fluorescence level after 40 amplification cycles (defined as C T of 40 for non-template control wells) is desirable for dengue 2 detection from samples containing low virus concentrations.
  • the optimized primer ratio was empirically determined as 200 nM for DV2.U2 primer and 60 nM for DV2.L1 primer.
  • Table 2 shows results of triplicate assays using cDNA derived from serially diluted dengue 2 virus standard, Consistent triplicate Cj values were obtained for nearly the entire range of dengue concentrations, from 6.4 - 10 6 pfu/mi. However only 2/3 and 1/3 of the assays for 1.3 and 0.3 pfu/ml samples, respectively, had C T values less than background level of 40. Thus, the sensitivity of the dengue 2 fluorogenic PCR was established as approximately 6.4-10 pfu per ml. Note that the lowest virus samples are clearly distinguishable from normal sera alone, or from non-template (water) controls, indicating that background-free dengue 2 RT-PCR was achieved.
  • Figure 3 shows a standard curve by plotting threshold cycle (C T ) VS the known quantity of original virus concentrations in the reaction (pfu/ml).
  • C T threshold cycle
  • dengue 2 virus genotypes were universally detected and identified by the assay. Furthermore, the assay could not detect all other flaviviruses fisted in Table I, such as dengue 1, 3 and 4 viruses, as well as other flaviviruses, including Japanese encephalitis, St. Louis encephalitis, yellow fever, and Kunjin viruses. All viruses other than dengue 2 viruses were indistinguishable from background (defined as C T of 40), indicating that the fluorogenic PCR assay is dengue 2-specific.
  • dengue specific antisera can neutralize dengue virus and consequently prevent viral replication in cell lines (Johnson, 1976). Furthermore, sera specimens from Individuals with secondary dengue may contain variable amounts of non-neutralizing antibody bound to vims. Serially diluted dengue 2 virus suspensions used for previous normal sera spiking experiment (sensitivity study) were also extracted after incubating with dengue 2-specific antisera (1 :500 virus reduction titer of human hyperimmune sera described in Materials and methods) at 37°C for one hour. The resulting neutralized dengue virus was efficiently recognized and detected by fluorogenic PCR: there was no difference in C T values from assays using either native virus or immunocomplexed virus (data not shown). Thus, it was demonstrated that the fluorogenic RT- PCR could detect immunocomplexed dengue viruses that may be undetectable by conventional mosquito cell isolation.
  • dengue 2 fluorogenic RT-PCR assay was used to quantitatively identify dengue virus from infected sera.
  • Dengue 2 viruses were identified in clinical sera specimens collected from patients with acute febrile illness in Somalia (1993) and Haiti (1997) that yielded positive dengue 2 vims isolations
  • the fluorogenic assay was able to identify and confirm most of dengue 2 infections with calculated viral concentrations ranging from 10 to 6.2X10 5 pfu/ml of sera.
  • the fluorogenic RT-PCR assay was applied to non-human primates following experimental dengue infection with wild-type S 16803 virus.
  • the C T value of dengue 2 specific RT-PCR is established as reproducibly dependent on viral titer. Furthermore, zero background conditions for the assay, i.e., negative (non- template) control wells yield C of 40 after 40 amplification cycles is systematically defined and achieved.
  • the optimized RT-PCR protocol developed in this study allows the detection of low level viremia in infected monkey and human sera, with dengue 2 viral titers ranging from 10-10 6 pfu per ml.
  • the method of the invention provides an early diagnostic capability well before infected hosts develop antibodies. In addition, virus detection can be made in hours in contrast to traditional viral isolation in mosquito cells requiring days.
  • the reagents employed in the present invention can be provided in a kit packaged combination with predetermined amounts of reagents for use in determining and/or quantitating flavivirus.
  • a kit can comprise in packaged combination with other reagents any or all of primers or probes described herein.
  • the oligonucleotide probes can be packaged to permit the assay to be performed in a hetero- or homogeneous format.
  • the oligonucleotide probes can be labeled or bound to a support or can be provided with groups that permit the probe or primer to be subsequently labeled or bound to a support.
  • the kit can further include in the packaged combination buffers, developing systems, if needed, nucleoside triphosphates and the like.
  • the kit may optionally contain a denaturation solution, a hybridization buffer, a wash solution and an assay device, e.g. test strip, microwell plate, etc. It is also envisioned that the kit could contain an internal calibration standard.
  • a standard curve of dengue 2 cDNA derived from known viral concentrations was demonstrated to have a nearly linear relationship over 5 logs, from pfu per ml, of viral concentrations and could be an internal calibration standard.
  • This standard curve would be valid only if the reverse transcription efficiency of different dengue 2 RNA concentrations is not the rate-limiting step. This assumption was preliminarily proven by examining C T values derived from viral RNA extract of spiked normal sera. However, the reverse transcription reaction does become a rate- limiting step when using RNA extracts derived from samples containing dengue virus concentrations greater than 10 7 pfu per ml.
  • DVl-lU 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3' (nucleotides 10543-10566, DV1 genome size 10718 nucleotides)
  • DV2-2U 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3* (nucleotides 10558-10579, DV2 genome size 10724 nucleotides)
  • DV3-1U 5'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3'
  • Dengue Viruses Dengue serotype 1, 2, 3, and 4 virus isolates and sera collected from dengue- infected humans and monkeys were Obtained from collections maintained at the Department of Virus Diseases.
  • step 5 by applying the rest of "4" mixture. 7. Add 500 ⁇ l AW buffer (QiAamp kit) and spin column and centrifuge at 20,000 X 9 for 3 minutes.
  • step 7 Repeat “step 7 " and add 50 ⁇ l preheated (80°C) molecular grade water, Centrifuge at 6000X g for 1 minute,
  • RNAase inhibitors 20 units/ ⁇ l 0.4 ⁇ l
  • composition of primers and probe to be used for each dengue serotype-specific Taqman assay is shown in the table below.
  • Example 2 The assay is performed according to the methodology of Example 1 using the materials below. Materials for JE Taqman assay:
  • the listing shows that all viral cDNA can be distinctly identified as positive by only one of the four dengue type specific master mixture, i.e., type 1, 2, 3 and 4. Only those strains yielded positive PCR product before the end of 40 cycle amplification, i.e., C t value smaller than 40, were identified as positive. This indicates that each serotype specific master can specifically detect only its own corresponding dengue serotype. There is no cross reactivity among different dengue serotypes detected by serotype-specific RT-PCR. See table below. Detection and identification of dengue strains by serotype specific 3'-based fluorogenic RT- PCR assays. Total of 40 cycles of amplification was carried out using fluorogenic PCR for dengue virus detection and identification. Positive identification was made for those samples yielded C t value of less than 40 cycles.
  • Example 4 The assay is performed according to the methodology of Example 1 using the materials below. Formulation of JE Taqman assay:
  • the new F214' primer can detect genotype 3 JE strains, but barely can detect other genotypes' JE. Both F214 and F214' were used as upper primers for the test and results were shown as below:
  • Example 5 The assay is performed according to the methodology of Example 1 using the materials below.
  • WNV-Ul 5'-CCTGGGATAGACTAGGAGATCTTCTG-3* and WNV-Pl probe: 6FAM-TCTGCACAACCAGCCACACGGC-3*

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Fluorescent DNA probes specific and flanking primer pairs are designed based on the sequence information found in the conserved terminal 3'-noncoding region of flavivirus, e.g. nucleotides 10653-10678 of dengue virus. Fluorogenic polymerase chain reaction employing these primers and probes produce results that permit specific flavivirus identification. The assays can be both quantitative and qualitative. Optimal assay conditions with zero background are disclosed which permit the detection of low levels of flavivirus from clinical specimens. Specifically, Dengue virus isolates from different geographic regions can be universally detected and identified by the disclosed fluorogenic RT-PCR assay. The fluorogenic RT-PCR assay readily detected viremia in sera collected from individuals ill with dengue fever.

Description

FLAVIVIRUS DETECTION AND QUANTIFICATION ASSAY
Cross-Reference to Related Applications
This application is a continuation-in-part of application No. 09/551,161, filed April 14, 2000, to which priority is claimed and which application is based on provisional application No. 60/153,685, filed on September 14, 1999 and on provisional application No. 60/129,713, filed on April 16, 1999. The content of these documents is expressly incorporated herein.
Technical Field
The subject invention relates to a nucleic acid based diagnostic system for rapid and specific flavivirus virus identification and quantification and kits which involve a novel set of oligonucleotides which can distinguish and quantitate flavivirus, especially.
Background Art
Dengue viruses belong to the family Flaviviridae, which contains almost 70 viruses, including those causing yellow fever and several encephalitides. Four distinct dengue virus types (dengue 2, 3, and 4) are each capable of causing infection in humans (Henchal and Putnak, 1990). Dengue infections are usually confirmed by serologic detection of dengue-specific antibodies or/and dengue virus isolation through culture in insect cells or mosquito inoculation (Gubler, 1989). These conventional methods for dengue virus detection are very time consuming, labor intensive, and have limited sensitivity for detecting low levels of dengue virus.
Development of sensitive, type-specific dengue diagnostic systems is crucial because of explosive growth in dengue virus transmission and the increasing incidence of dengue hemorrhagic fever (Monath, 1994).
Rapid molecular diagnostic systems, such as RT-PCR, could provide a possible solution for dengue virus identification from clinical samples (Harris et al, 1998). Several RT-PCR systems for dengue virus detection have been reported, based on different conserved genomic regions, such as genes for nonstructural proteins (Chow et al., 1993; Fulop et al, 1993) and the 3'-noncoding region (Sudiro et ah, 1997). See also U.S. Patent No. 5,939,254. However, these methods frequently have mismatches corresponding to their amplicons, or even to primer sequences, due to genetic differences among strains of the same dengue serotype. Some of these systems rely upon sequences from the "conserved" regions for probes or primers, such as NS3 and NS5 genes. Alignment of these so-called conserved genes of dengue reveal mismatches among the viruses, in addition to variation among virus strains. These mismatches result in reduced sensitivity of the assay, and as a result, false diagnosis of the specific dengue virus. Further, unpredictable variations in PCR target sequences would yield lower specificity and sensitivity in dengue detection. In addition, these methods are often cumbersome to adapt for routine clinical use and are not quantitative.
The 3' noncoding region has been previously studied and sequenced. See, for example, Mandl, C. W., et al, "Spontaneous Engineered Deletions in the 3' noncoding region of Tick-bome Encephalitis Virus: Construction of Highly Attenuated Mutants of a Flavivirus", J. Virology, Vol. 72, (1998) pp. 2132-2140. Proupski, V. et al, "Biological Consequences of Deletions within the 3 '-untranslated Region of Flavivirus May be due to Rearrangements of RNA Secondary Structure", Virus Research (1999) Vol. 64, pp. 107-123.
The invention herein disclosed addresses these and other associated problems.
Disclosure of the Invention
The present invention is directed to designing and utilizing specific genomic region, the conserved 3 - terminal non-coding region, as specific nucleic acid-based diagnostic system for rapid and specific flavivirus, e.g. dengue virus, identification and quantification. In this regard, the invention focuses on the last 250, more specifically the last 200, even more specifically the last 160 bases of the conserved dengue genome. This would correspond to nucleotides 10558 through 10718 of dengue 1. The invention also utilizes flavivirus specific upstream primers, probes, and downstream primers developed from this region. From the conserved terminal 3' non coding region conserved region, a probe unique to a flavivirus is chosen. Primers can then be generated from conserved areas upstream and downstream of the probe that are unique to the virus of interest, the areas extending to about one kilobase from the 3' terminus of any flavivirus. A preferred region extends to about 200 bases from the 3' terminus. More than one primer set and more than one probe can be chosen, each primer set and probe can be both conserved and specific for a virus.
Once the cDNA is isolated from the samples containing the viruses to be identified, the cDNA is incubated with the primers and probe under condition such that a polymerase is able to synthesize a complementary strand using the primer/template substrate. The polymerase, having a 5' → 3' exonuclease activity, will digest the probe hybridized to its specific complementary virus sequence. The probe may contain a reporter at the 5 ' end which will release a detectable signal when the probe is digested. Quantification and detection of the specific vims for which the probe and primers were designed is possible by detecting' the released probe signal. If the probe does not find its match in the sample, the probe is not digested by the polymerase, and the signal is not released or detected.
The advantages of this invention are that probes can be highly discriminatory, even single nucleotide substitutions can be reliably detected among different viruses. Oligonucleotide probes and primers described in this invention can be used in a multiplex format to detect and differentiate flaviviruses. It is also possible to formulate individual virus-specific assay mixtures to identify and quantitate one virus at a time. The format of the assay depends on the desired usage. A generic dengue diagnostic system may work well for clinical or epidemiologic use, whereas stereotype-specific dengue assays are required for use in vaccine development. The design of the assay can be refined for single-step multiplex use (processing of a specimen and evaluation in a single reaction mixture), or two step reactions may be used, as described in the material.
Applications of the flavivirus fluorogenic RT-PCR assay
The flavivirus fluorogenic assays based on the terminal 3' noncoding region offer real-time quantitation of virions (expressed as plaque forming units or as genome copy equivalents). Determination of viral number is extrapolated from reproducible linear standard curves, derived either from titration of viral RNA or from dilutions of flaviviral cDNA (as plasmids). The rapid, sensitive detection of flaviviruses lends itself to several applications. These include: 1. Rapid diagnosis of flaviviral infection: the specific nature of the fluorogenic probes devised for this patent allows confirmation of flaviviral infection. Use of selected flaviviral probes (for example, for a particular geographic region) would support clinical/epidemiologic efforts to quickly and accurately establish the cause of specific illnesses such as febrile illness. Typically, the diagnostic apparatus required to confirm specific flaviviral infection is cumbersome and restricted to reference laboratories. The new methods allow rapid, bedside diagnosis and confirmation of flavivirus infection. This use will be greatly facilitated by the availability of portable field-tested diagnostic platforms during future deployments to endemic regions.
2. Determination of viral burden: the magnitude and duration of viremia during flaviviral infection may be positively associated with flaviviral pathogenicity. This finding may be of prognostic significance in dengue hemorrhagic fever, where increased viral burden (particularly at the time of defervescence) may be a marker of increased risk for severe disease. Studies of viral clearance by different immunologic mechanisms will be made easier. Furthermore, the effectiveness of specific antiviral therapy (such as drugs, immunotherapy, and other approaches) may be gauged by its effect on viral burden in vivo or in vitro.
3. Quality control of flaviviral biologies: the precise and specific determination of viral titer in flaviviral vaccines and other biologies is a difficult and time-consuming effort, often requiring three separate bioassays. The availability of a nucleic acid-based assay that is both specific and sensitive provides a significant advance in analysis (identification and characterization) of biologies, central to the quality assurance components of the manufacturing documentation required for any vaccine. Moreover, this methodology may be used with both live, recombinant, and killed flaviviral vaccines; it is also applicable to complex combinations of flaviviruses, such as multivalent dengue vaccines.
4. Amplification of flaviviral RNA: the described method offers a simplified approach to creation of amplified, near full-length copies of flaviviral genomes. The 3' noncoding region is critical to replication of viral RNA, and the product of the reverse transcription reaction could be manipulated to generate flaviviral replicons. In addition, formation of replicative intermediates can be monitored through antisense RNA copies. Flaviviral cDNA products may be used for molecular pharmacologic or therapeutic purposes.
Brief Description of the Drawings Figure 1. Dengue 2 detection by fluorogenic RT-PCR. Seven 5-fold serially diluted cDNA derived from RNA extract of dengue 2 S16803 (3.8 X 107 pfu/ml) were used as DNA template. Accumulation in fluorescence (ΔRn) as amplification progresses for each diluted cDNA are shown by different color of sigmoid plots. Two non-template controls are represented by two plots locating on the far right. Figure 2. Effects of Mg^ concentrations on fluorogenic RT-PCR.
Fluorescence accumulation plots derived from PCR with MG++ concentration of 2.5, 2.0, 1.5, 1.0, and 0.5 mM are shown from left to right.
Figure 3. Standard curve of dengue 2 SI 6803. Triplicate Cx values of serially diluted S16803 ranging from 1.3 to 106 pfu/ml are shown. Figure 4. Infection courses of dengue 2 SI 6803 in Rhesus monkey. Viral concentrations were determined from three monkeys' sera (JEH, GWP, an GKB) following subcutaneous inoculation with 104 pfu dengue 2 SI 6803.
Figure 5 A and 5B. The basics of the nuclease assay.
Figure 6. Nucleic acid dot matrix analysis of the 3'-end regions for the dengue 3 and dengue 4.
Figure 7. Nucleic acid dot matrix analysis of the 3 '-end regions for representative dengue 3 and dengue 4.
Figure 8. Copy number dependent West Nile virus (WNV) assay: 1 :5 dilution of WNV cDNA derived from viral sample ranging from 100,000 to 1 pfu per reaction. Figure 9. Linear scale of WNV assay showing background-free results from non-template control (from 1 through 100,000 pfu).
Figure 10. Standard Curve of WNV assay.
Figure 11. Quantitative detection of WNV isolate from NY99.
BEST MODE FOR CARRYING OUT THE INVENTION
Dengue viral RNA was extracted from about 40 μl of either viral suspension or infected serum and then the total viral RNA was dissolved into buffer, and about 8 μl of the resulting RNA solution was mixed with about 12 μl of RT reaction mixture (see below). After reverse transcription, about 2 μl of the cDNA product was used as DNA template for the fluorogenic PCR assay. The total sample input for each fluorogenic PCR reaction was calculated as follows: 40 Iii specimen used for RNA extraction X 8 μl RNA used for RT/32 μl total RNA X 2 μl cDNA/20 μl total cDNA = 1 μl of dengue 2 virus specimen.
For practical purpose, the expressed viral concentration is stated in terms of pfu-per-ml sample. The actual virus detected per PCR reaction should be only 1/1000 of dengue sample concentration, pfu/n-J. Based on the sensitivity defined in this study, the lower limit of dengue detection is only 0.01 pfu per PCR assay. It is not possible for any known PCR assay to detect fraction of integral genomic sequence. Thus, each infectious pfu for dengue virus should represent at least 100, or greater of genomic equivalencies.
According to the above general method, a novel dengue 2 type-specific genomic sequence derived from the 3' noncoding region (NCR) was use to develop a fluorogenic-based RT-PCR (Taqman, PE Biosystem, Foster City, CA). The assay was formulated to use a uniquely designed internal fluorescence-labeled probe to specifically hybridize a 3'-noncoding target sequence of dengue 2. After hybridizing to the target PCR product, fluorescent signal is released through 5'-nuclease activity of DNA Taq polymerase that is used to amplify the target sequence (Holland et al. 1991 ; Higgins, et al. 1998). This allows convenient monitoring of a specific PCR product formation over time. In contrast to traditional dengue RT-PCR, the fluorogenic RT-PCR developed in this study is capable of discriminating single nucleotide substitutions in the target sequence. In this study, real time detection of dengue PCR product was correlated with input dengue cDNA copy number, to yield a quantitative assay for dengue 2 viruses.
Material and methods
Viruses
Dengue virus strains and other flavivirus isolates shown in Table I were obtained from Departments of Virus Diseases and Biologies Research, Walter Reed Army Institute of Research, Washington, DC. Most virus isolates were routinely obtained from supemates of insect or Vero cell culture. Virus titrations were performed by plaque assay in Vero cells (Eckels et al., 1980). A dengue 2 SI 6803 virus stock suspension (3.8 ± 0.8 x 108, plaque forming units, pfu per ml) was used as a standard for development of the assay.
Table I. List of Flavivirus strains
Virus Strains Origins Comments
Dengue 1 WP74 West Pacific WRAIR vaccine strain
Dengue 1 Hawaii Hawaii
Dengue 1 Haiti 059 Haiti
Dengue 2 S16803 Thailand WRAIR vaccine strain
Dengue 2 NGC New Guinea
Dengue 2 SOM 13 Somalia Kanesa-thasan et al, 1998
Dengue 2 SOM 58 Somalia
Dengue 2 Haiti 103 Haiti
Dengue 2 Haiti 120 Haiti
Dengue 2 Haiti 121 Haiti
Dengue 2 16681 Thailand
Dengue 2 PRS-1 Puerto Rico
Dengue 2 21868 Thailand
Dengue 2 ALI 088 Brazil
Dengue 2 ALI 072 Brazil
Dengue 2 ALI 013 Brazil
Dengue 3 CH53489 Thailand WRAIR vaccine strain
Dengue 3 H87 Philippines
Dengue 3 SOM 79 Somalia
Dengue 4 341750 Caribbean WRAIR vaccine strain
Dengue 4 H241 Philippines
Dengue 4 Haiti 119 Haiti
Japanese encephalitis SA14-14-2 China
Yellow fever 17D Nigeria
Kunjin - -
St. Louis encephalitis -
The last four hundred nucleotides of the 3 '-noncoding regions of all 4 dengue serotypes (Genbank accession number M87512;Denl, M20558;Den2, M93130;Den3, M17255;Den4 see Fig 1) were used for alignment comparison by the ClustalW algorithm program from MacVector 6.5 DNA analysis program (Oxford Molecular Inc., 575 Science Drive, Madison, WI 53711, USA). Sequences for oligonucleotide primers and fluorogenic oligonucleotide probes are shown in Figure 2b. Both labeled and non-labeled oligonucleotides were synthesized by the oligonucleotide factory of PE Applied Biosystem (Foster City, CA, USA).
Sera
Dengue infected human sera were collected from individuals with confirmed dengue fever in Somalia (Kanesa-thasan et al, 1994) and Haiti (Trofa et al, 1997). These specimens were dengue 2 positive by indirect immunofluorescence after culture in C6/36 insect cells. Monkey sera were obtained from three monkeys following subcutaneous inoculation with 104 pfu dengue 2 virus SI 6803. Blood specimens were collected daily for 12 days after infectious challenge from each monkey. For immunocomplexed dengue virus study, pooled human hyperimmune serum with plaque reduction neutralizing antibody titer of 1 :7000 against dengue 2 was kindly provided by the Dept of Virology, AFRIMS, Bangkok, Thailand. AU serum specimens were stored at -80°C until use.
Extraction of Viral RNA
Virus RNA used in this study was routinely extracted from virus suspensions or dengue- infected sera (40 VI) according to QiAamp Viral RNA Handbook (Qiagen Inc. Valencia, CA 91355). Total RNA was eluted into 32 μl of TE- (10 mM Tris- HC1, 1 mM Na2EDTA, pH 8.0).
Design and Synthesis of Primers and Fluorogenic Hybridization Probe
An unique design of dengue 2 fluorogenic probe (DV2.PI) and its flanking primers (DV2.U2 and DV2.L1) based on its 3'-end genomic sequence (Genbank
Accession # N120558) were selected by using Primer Express software (PE Applied Biosystems Inc., Foster City These oligonucleotide sequences are shown as following: DV2.P1 (nucleotides 10653-10678): 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG- CA-3OV2.L1 (nucleotides 10558-10579): 5'-CAT-TCC-ATT-TTC-TGG-CGT-TCT- 3' DV2.U2 (nucleotides 10680-10700): 5'-AAG-GTG-AGA-TGA-AGC-TGT-AGT- CTC-3'. DV2.P1 consists of oligonucleotide sequence shown as above with a 5 ' - reporter dye (FAM, 6- carboxy-fluorescein) and a down stream 3 '-quencher dye (TAMRA, 6-carboxy-tetramethyl-rhodamine). (See Figure 6A and 6B for an overview of primers, upper and lower suitable for the detection and/or quantitation of Dengue types 1-4.) Both labeled and non-labeled oligonucleotides were synthesized by Oligo-factory of PE Applied Biosystem Inc., Foster City, CA.
Oligonucleotide Probes
The term "oligonucleotide" as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleotides, ribonucleotides, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5' — 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphoranilidate, phosphoramidate, and the like.
As used herein, "nucleotide" includes the natural nucleotides, including 2'- deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in reference to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso that they are capable of specific hybridization. Such analogs include synthetic nucleotides designed to enhance binding properties, reduce degeneracy, increase specificity, reduce activity as enzyme substrates, and the like.
Oligonucleotides of the invention can be synthesized by a number of approaches, e.g. Ozaki et al, Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al, Nucleic Acids Research, 18: 5419-5423 (1990); or the like. The oligonucleotide probes of the invention are conveniently synthesized on an automated DNA synthesizer, e.g. a Perkin-Elmer (Foster City, Calif.) Model 392 or 394 DNA/RNA Synthesizer, using standard chemistries, such as phosphoramidite chemistry, e.g. disclosed in the following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. Pat. No. 4,980,460; Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679; and the like. Preferably, the oligonucleotide probe is in the range of 15-150 nucleotides in length selected from contiguous sequences in the conserved 3'- terminal non-coding region to detect and quantitate specific flaviviruses. More preferably, the oligonucleotide probe is in the range of 18-30 nucleotides in length. The precise sequence and length of an oligonucleotide probe of the invention depends in part on the nature of the target nucleic acid sequence to which it hybridizes. The binding location and length may be varied to achieve appropriate annealing and melting properties for a particular embodiment. Guidance for making such design choices can be found in many of the above-cited references describing the "Taqman" type of assays. The invention develops these probes from the last 250 nucleotides, more specifically the last 200 nucleotides, even more specifically the last 160 nucleotides of the 3' noncoding region of the flaviviral genome. The oligonucleotide probes of the present invention include fluorescer and quencher molecules attached to the oligonucleotide. As used herein, the terms "quenching" or "fluorescence energy transfer" refer to the process whereby when a fluorescer molecule and a quencher molecule are in close proximity, whenever the fluorescer molecule is excited, a substantial portion of the energy of the excited state nonradiatively transfers to the quencher where it either dissipates nonradiatively or is emitted at a different emission wavelength than that of the fluorescer. It is well known that the efficiency of quenching is a strong function of the proximity of the fluorescer and the quencher, i.e., as the two molecules get closer, the quenching efficiency increases. As quenching is strongly dependent on the physical proximity of the reporter molecule and quencher molecule, it has been assumed that the quencher and reporter molecules must be attached to the probe within a few nucleotides of one another, usually with a separation of about 6-16 nucleotides, e.g. Lee et al Nucleic Acids Research, 21 : 3761-3766 (1993); Mergny et al, Nucleic Acids Research, 22: 920-928 (1994); Cardullo et al, Proc. Natl. Acad. Sci., 85: 8790-8794 (1988); Clegg et al, Proc. Natl. Acad. Sci., 90: 2994-2998 (1993); Ozaki et al, Nucleic Acids
Research, 20: 5205-5214 (1992); and the like. Typically, this separation is achieved by attaching one member of a reporter-quencher pair to the 5' end of the probe and the other member to a base 6- 16 nucleotides away. . The selection of appropriate fluorescer-quencher pairs for particular probes is well known in the literature. See, for e example Wu et al., Anal. Biochem., 218: 1-13 (1994). Pesce et al, editors, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al, Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); and the like. Fluorescer-quencher pairs may be xanthene dyes, including fluoresceins, and rhodamine dyes. Another group of fluorescent compounds are the naphthylamines, having an amino group in the alpha or beta position. Included among such naphthylamino compounds are 1- dimethylaminonaphthyl-5-sulfonate, l-anilino-8-naphthalene sulfonate and 2-p- toluidinyl-6-naphthalene sulfonate. Other dyes include 3-phenyl-7- isocyanatocoumarin, acridines, such as 9-isothiocyanatoacridine acridine orange; N- (p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, pyrenes, and the like.
Reverse Transcription (RT) and Polymerase Chain Reaction (PCR)
Each viral RNA extract was transcribed into cDNA using Taqman RT kit (PE ABI, Foster City, CA). The RT reactions were performed in 20 W volume containing 8 μl extracted RNA, 241 1 0 X RT buffer (PE ABI), 4 μl 2.5 mM dNTPs, 4.4 μl 25 mM MgCl2, 0.4 μl RNAase inhibitor, 0.5 μl MuLV reverse transcriptase (50 units/μl), and 1 μl DV2.L1 primer (20 pmole/μl). RT reaction mixtures were incubated at 25°C for 10 minutes followed at 48°C for 30 minutes, then 95°C for 5 minutes. The resulting cDNA was used as template in the subsequent PCR reaction.
PCR reactions were conducted in 50 VI volume containing 2.0 VI cDNA template, Taqman buffer (PE ABI, Foster City, CA), 0.1 mM deoxyribonucleotide triphosphates x (dNTPs), 2.0 MM MgCl2, 200 nM DV2.U2, 20 nM DV2.P 1 , 60 nM DV2.L1, and 25 units AmpliTaq Gold DNA polymerase (PE ABI, Foster City, CA). Gene detection system 7700 and sequence detection system software version, 6.3 (PE ABI, Foster City, CA) were employed for PCR cycling reaction, real time data collection and analysis. 6 PCR mixtures were pre-incubated at 50°C for 2 minutes, then 95°C for 10 minutes followed by 40 cycles of two-step incubations at 95°C for 15 seconds and 60°C for 1 minute. Results
Preliminary observations
Real time amplification assays based on 3 '-noncoding sequence of dengue 2 virus were initially demonstrated using serially diluted cDNA derived from RNA extract of 10 diluted dengue 2 S 16803 stock (3.8 X 107 pfu/ml). Seven 5-fold serial dilutions of viral cDNA were used as DNA templates for the dengue 2 fluorogenic RT-PCR assay. Figure shows typical sigmoid plots representing formation of the dengue 2 specific PCR product by accumulation in fluorescence (ΔR) as amplification progresses (cycle number). Threshold Cycle (C*r) for each cDNA concentration is defined as cycle number where the sequence detection application software detects the increase of fluorescence associated with exponential growth of PCR product. The cycle number to yield detectable fluorescence level, Cj usually was automatically selected by using the average of samples' fluorescences from cycle 3 through 15 as the baseline. Parallel curves were derived from different concentrations of dengue 2 viral cDNA templates. Greater initial viral cDNA copy numbers required fewer amplification cycles (smaller CT value) to reach a detectable fluorescence level. As the number of viral copies was reduced by serial I dilutions, the curves corresponding to each individual dilution were shifted to the night, with greater Cj values. The furthermost right plot, showing CT value of approximately 37, represents non- template (water) controls. These preliminary results established that the dengue 2- specific primers and the probe uniquely designed for the fluorogenic RT-PCR system are capable of detecting dengue 2 viral cDNA in a dose-dependent fashion over 7 serial 5-fold dilutions (more than 3.5 logs dilution). In addition, the assay is capable of discriminating specific dengue 2 viral stock cDNA from background levels (non- template controls).
Optimization of dengue 2 fluorogenic RT-PCR assay
The sensitivity of the assay can be improved by enhancing the relationship between dengue viral cDNA copy number and CT value. DV2. LI primer was with universal poly-oligo (T)l 6 primer (PE Applied Biosystem Inc. . Foster City, CA) for reverse transcription of dengue RNA. Universal poly-oligo (T)16 primer is frequently used as generic RT primer to generate dengue cDNA during reverse transcription process. The quantities of cDNA generated by RT reactions of the same dengue viral RNA (from dengue 2 virus suspension of 3.8 X 106 pfu/ml) were examined using DV2.L1 and poly-oligo (T)16 primers, respectively. Figure 2 shows that use of DV2.L1 primer in the RT reaction yielded much greater number of cDNA copies than the poly-oligo (T)16 primer by dengue 2 fluorogenic PCR, i.e. CT of 15.4 vs. 22.5. Thus, DV2.L1 was used for all RT reactions in this study. The effects of varying the concentrations of Mg** on the PCR reaction were examined. It was shown in Fig 3 that Mg' concentration from 1.5 to 2.5 mM had very little effect on the CT value of the assay. A midrange Mg concentration of 2.0 mM was selected as optimal for the assay. The background (non-template control) was reduced by examining the effect of different concentrations and ratios of the flanking primers, DV2.L1 and DV2.U2, respectively. Preliminary observations showed that the dengue 2 fluorogenic RT-PCR would yield saturating, high fluorescence levels when DV2.U2 and DV2.L1 primers were used at equimolar concentrations of 200 nM (as shown in Figure 1). However, under such conditions, non- template/water controls would yield detectable background fluorescent signal after 36 to 37 cycles of amplification. Little or zero background fluorescence level after 40 amplification cycles (defined as CT of 40 for non-template control wells) is desirable for dengue 2 detection from samples containing low virus concentrations. The optimized primer ratio was empirically determined as 200 nM for DV2.U2 primer and 60 nM for DV2.L1 primer. Using dengue 2- specific RT primer with optimal Mg" concentration and primer ratios, we demonstrated zero background RT-PCR assays (shown in sensitivity study, Table 2).
Table 2. C values of spiked sera containing serially diluted dengue 2 S 16803 stock.
Sensitivity and specificity of dengue 2 fluorogenic RT-PCR The upper and lower detection limits of the fluorogenic PCR assay were determined using optimized assay conditions identified in the previous section. Normal human sera were spiked with serial 1.5 dilutions of 1 : 100 diluted stock dengue 2 S16803 virus suspension (3.8X106 pfu ml). The resulting ten spiked sera contained dengue 2 virus concentrations ranging from 0.3 - 106 pfu/ml. Viral RNA extracted from each serum was used to generate cDNA as described in Materials and methods. Table 2 shows results of triplicate assays using cDNA derived from serially diluted dengue 2 virus standard, Consistent triplicate Cj values were obtained for nearly the entire range of dengue concentrations, from 6.4 - 106 pfu/mi. However only 2/3 and 1/3 of the assays for 1.3 and 0.3 pfu/ml samples, respectively, had CT values less than background level of 40. Thus, the sensitivity of the dengue 2 fluorogenic PCR was established as approximately 6.4-10 pfu per ml. Note that the lowest virus samples are clearly distinguishable from normal sera alone, or from non-template (water) controls, indicating that background-free dengue 2 RT-PCR was achieved. Figure 3 shows a standard curve by plotting threshold cycle (CT) VS the known quantity of original virus concentrations in the reaction (pfu/ml). For practical purposes, we expressed viral concentration as pfu per ml sample rather than pfu per RT-PCR reaction. Threshold cycle decreases as the concentration of virus sample is increased to 106 pfu/ml. A nearly perfect linear relationship was established between cDNA concentration and corresponding CT value over 5 logs of cDNA (r = .973). To demonstrate specificity of the fluorogenic PCR assay, all dengue 2 isolates from different geographic origins fisted in Table I, such as strains from Thailand, Puerto Rico, Haiti, Brazil, and Somalia were examined by the fluorogenic RT-PCR. These different dengue 2 virus genotypes were universally detected and identified by the assay. Furthermore, the assay could not detect all other flaviviruses fisted in Table I, such as dengue 1, 3 and 4 viruses, as well as other flaviviruses, including Japanese encephalitis, St. Louis encephalitis, yellow fever, and Kunjin viruses. All viruses other than dengue 2 viruses were indistinguishable from background (defined as CT of 40), indicating that the fluorogenic PCR assay is dengue 2-specific.
Detection of immunocomplexed dengue 2 virus by fluorogenic RT-PCR
It is known that dengue specific antisera can neutralize dengue virus and consequently prevent viral replication in cell lines (Johnson, 1976). Furthermore, sera specimens from Individuals with secondary dengue may contain variable amounts of non-neutralizing antibody bound to vims. Serially diluted dengue 2 virus suspensions used for previous normal sera spiking experiment (sensitivity study) were also extracted after incubating with dengue 2-specific antisera (1 :500 virus reduction titer of human hyperimmune sera described in Materials and methods) at 37°C for one hour. The resulting neutralized dengue virus was efficiently recognized and detected by fluorogenic PCR: there was no difference in CT values from assays using either native virus or immunocomplexed virus (data not shown). Thus, it was demonstrated that the fluorogenic RT- PCR could detect immunocomplexed dengue viruses that may be undetectable by conventional mosquito cell isolation.
Illustration of dengue 2 viremia from natural infection using fluorogenic RT-PCR. In order to demonstrate the utility of this newly developed dengue assay, dengue 2 fluorogenic RT-PCR assay was used to quantitatively identify dengue virus from infected sera. Dengue 2 viruses were identified in clinical sera specimens collected from patients with acute febrile illness in Somalia (1993) and Haiti (1997) that yielded positive dengue 2 vims isolations The fluorogenic assay was able to identify and confirm most of dengue 2 infections with calculated viral concentrations ranging from 10 to 6.2X105 pfu/ml of sera. The fluorogenic RT-PCR assay was applied to non-human primates following experimental dengue infection with wild-type S 16803 virus. Sera systematically collected daily from three rhesus monkeys from 2 through 12 days after infectious challenge (see Materials and methods) were subjected to fluorogenic RT-PCR assays. Using the dengue 2 standard curve established in this study, calculated viremia counts (expressed as pfu per ml) were determined for monkey sera as shown in Figure 4. It was shown that two out of three monkeys had detectable low level viremia, 10 pfu/ml from day 3 after viral challenge. All three monkeys showed significant viral concentration of 40 pfu/ml or higher in sera from day 5 through day 8. Reduction of viremia to 0 pfu/ml was observed for all three monkeys after day 10.
The CT value of dengue 2 specific RT-PCR is established as reproducibly dependent on viral titer. Furthermore, zero background conditions for the assay, i.e., negative (non- template) control wells yield C of 40 after 40 amplification cycles is systematically defined and achieved. The optimized RT-PCR protocol developed in this study allows the detection of low level viremia in infected monkey and human sera, with dengue 2 viral titers ranging from 10-106 pfu per ml. The method of the invention provides an early diagnostic capability well before infected hosts develop antibodies. In addition, virus detection can be made in hours in contrast to traditional viral isolation in mosquito cells requiring days.
Kit Packaging
As a matter of convenience, the reagents employed in the present invention can be provided in a kit packaged combination with predetermined amounts of reagents for use in determining and/or quantitating flavivirus. For example, a kit can comprise in packaged combination with other reagents any or all of primers or probes described herein.
Generally, it is desirable to include the requisite number of probes and/or primers to afford identification of all of the flavivirus of interest. The oligonucleotide probes can be packaged to permit the assay to be performed in a hetero- or homogeneous format. The oligonucleotide probes can be labeled or bound to a support or can be provided with groups that permit the probe or primer to be subsequently labeled or bound to a support. The kit can further include in the packaged combination buffers, developing systems, if needed, nucleoside triphosphates and the like.
Additionally, the kit may optionally contain a denaturation solution, a hybridization buffer, a wash solution and an assay device, e.g. test strip, microwell plate, etc. It is also envisioned that the kit could contain an internal calibration standard.
A standard curve of dengue 2 cDNA derived from known viral concentrations was demonstrated to have a nearly linear relationship over 5 logs, from pfu per ml, of viral concentrations and could be an internal calibration standard. In addition to using extracted RNA from various concentrations of dengue 2, we also used serially diluted cDNA suspension to establish dengue 2 standard curve (data not shown). This standard curve would be valid only if the reverse transcription efficiency of different dengue 2 RNA concentrations is not the rate-limiting step. This assumption was preliminarily proven by examining CT values derived from viral RNA extract of spiked normal sera. However, the reverse transcription reaction does become a rate- limiting step when using RNA extracts derived from samples containing dengue virus concentrations greater than 107 pfu per ml.
The following examples describe the application of the present invention for detecting and quantifying the four different dengue viruses in a sample.
EXAMPLES Example 1 MATERIALS 1. QiAamp viral RNA kit (Part number 29504, Qiagen Inc. 28159 Avenue Stanford, Valencia, California 91355) 2. Dengue Serotype-Specific Oligonucleotide primers and Taqman probes: Serotype-speciflc Upstream Primers (4):
DVl-lU: 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3' (nucleotides 10543-10566, DV1 genome size 10718 nucleotides)
DV2-2U: 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3* (nucleotides 10558-10579, DV2 genome size 10724 nucleotides) DV3-1U: 5'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3'
(nucleotides 10523-10546, DV3 genome size 10697 nucleotides) DV4-lU: 5*-GAT-CAA-GCT-TAAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3' (nucleotides 10488-10502, DV4 genome size 10645 nucleotides) Serotype-speciflc Fluorescent Probes (3):
DV1-P1 : 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3*
(nucleotides 10647-10672) DV4-P1 : 5'-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3'
(nucleotides 10574-10599) DV2-P1: 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3'
(used for both D2 and D3 vimses: nucleotides 10653-10678, DV2 genome; nucleotides 10626-1065 1, DV3 genome) Serotype-specific Downstream Primers (2): DV4-lL: 5'-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3* (nucleotides 10601-10621, DV4 genome size 10645 nucleotides)
DV2-lL: 5'-GAT-CGA-ATT-CCAT-TCC-ATT-TTC-TGG-CGT-TCT-3'
(used for Dl, D2, and D3 viruses: nucleotides 10674-10694, DVl genome; nucleotides 10680-10700, DV2 genome; 10653-10673, DV3 genome)
3. Dengue Viruses: Dengue serotype 1, 2, 3, and 4 virus isolates and sera collected from dengue- infected humans and monkeys were Obtained from collections maintained at the Department of Virus Diseases.
4. PE Applied Biosystem 7700 Gene Detection System (Perkin Elmer, Foster City, CA)
5. PE Applied Biosystem 2400 cycler (Perkin Elmer, Foster City, CA).
METHODS
A. Dengue RNA extraction (as per Qiagen kit handbook):
1. Pipette 140 μl dengue suspension (from dengue-infected Vero cell culture supemate) or serum, from dengue-infected animals into a 1.5 ml microfuge tube.
2. Add 560 μl of AVL buffer containing carrier RNA (QiAamp kit) to sample and mix well.
3. Incubate at room temperature for 10 minutes. 4. Add 560 μl of 95% ethanol to the mixture and mix thoroughly.
5. Apply 630 μl of "4" mixture to the QiAmp spin column and centrifuge at 6000X g for I minute.
6. Repeat step 5 by applying the rest of "4" mixture. 7. Add 500 μl AW buffer (QiAamp kit) and spin column and centrifuge at 20,000 X 9 for 3 minutes.
8. Repeat "step 7 " and add 50 μl preheated (80°C) molecular grade water, Centrifuge at 6000X g for 1 minute,
9. Store the obtained RNA at -20°C fill usage for reverse transcription. B. Reverse Transcription (RT):
1. Prepare RT master mixture as follows:
1 OX Taqman RT buffer 2 μl dNTPs 2. 5 mM 4 μl
25 MM MgCl2 4.4 μl RNAase inhibitors 20 units/μl 0.4 μl
Rtase, superscript 200 units/μl, 0.5 μl
RT primer (DV2.1L & DV4.11), 20 pmole/μl 1.0 μl
2. Mix 8 μl RNA extract with 12 μl PT master mixture.
3. Incubate at room temperature 10 minutes, followed by 30 minutes at 48°C, and inactivate the Rtase at 95°C, 5 minutes.
C. Taqman assay:
1. Prepare Dengue Taqman master mixture as follows:
10 X Taqman Buffer 5 μl dNTPs mix, 1.25 mM 4 μl MgCl2, 25 mM 4 μl
Dengue upper primer, 100 pmole/μl 0.1 μl
Dengue lower primer, 100 pmole/μl 0.1 μl
Dengue Taqman probe, 1 pmole/μl 1.0 μl
Molecular grade water 35 μl AmpliTaq polymerase Gold, 5 units/μl 0.25 μl
The composition of primers and probe to be used for each dengue serotype- specific Taqman assay is shown in the table below.
2. Mix 2 μl of cDNA from RT reaction with 48 μl Taqman master mixture in optical tube and capped with optical cap.
3. Place the tube into PE 7700 gene detection system, heat activate the AmpliTaq Gold 95°C for 10 minutes followed by 40 cycles of following conditions: 95°C, 15 seconds; 60°C, minutes.
Example 2 The assay is performed according to the methodology of Example 1 using the materials below. Materials for JE Taqman assay:
10X Taqman buffer 10 μl
10 mM dNTPs 8 μl
25 mM MgCl2 8 μl
JE.P1(1 pmole/μl) l μl
JE-F214(100 pmole/μl) 0.2 μl
JE-R382(20 pmole/μl) 0.35 μl dH2O 75 μl
Amplitaq-Gold 0.25 μl
JE.P1 (JE specific fluorescent probe):
TCTGCTCTATCTCAACATCAGCTACTAGGCACAGA
JE.F214 (JE RT-PCR, upstream primer): CAAGCCCCCTCGAAGCTGT
JE.R382 (JE RT-PCR, downstream primer): CACCAGCTACATACTTCGGCG
Example 3
Two panels of 4 different dengue serotypes (Dengue 1- WP74 and Den 1 Hawaii; Dengue 2- SI 6803 and Den 2 N6C; Dengue 3- CH53489 and Den 3 H87; Dengue 4- were used to test the specificity of fluorogenic dengue RT-PCR assays developed in this study. Viral RNA from these samples were extracted through silica dioxide method as described above. A generic dengue RT primer set comprising of two anti-sense primers (DV.L1 and DV.L2) was used to transcribe dengue viral RNA of all four possible dengue serotypes into cDNA The resultant cDNA were employed as templates for PCR amplifications using different dengue fluorogenic PCR master mixtures specially formulated for dengue serotyping.
Listing of Oligonucleotide sequences for primers and probes:
DVl .P 1 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3'
DV4.1L 5*-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3'
DV2. IL 5'-CAT-TCC-ATT-TTC-TGG-CGT-TCT-3' DV4.P1 5'-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3'
DV2.P1 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3'
DVl .1U 5'-ACA-CCA-GGG-GAA-GCT-GTA-TCC-TGG-3'
DV2.2U 5'-AAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3'
DV3.1U 5'-AGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3* DV4.1U 5--AAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3'
The listing shows that all viral cDNA can be distinctly identified as positive by only one of the four dengue type specific master mixture, i.e., type 1, 2, 3 and 4. Only those strains yielded positive PCR product before the end of 40 cycle amplification, i.e., Ct value smaller than 40, were identified as positive. This indicates that each serotype specific master can specifically detect only its own corresponding dengue serotype. There is no cross reactivity among different dengue serotypes detected by serotype-specific RT-PCR. See table below. Detection and identification of dengue strains by serotype specific 3'-based fluorogenic RT- PCR assays. Total of 40 cycles of amplification was carried out using fluorogenic PCR for dengue virus detection and identification. Positive identification was made for those samples yielded Ct value of less than 40 cycles.
Sample Den 1 Den 1 Den 2 Den 2 Den 3 Den 3 Den 4 Den 4 ID Ct pfu/ml Ct pfu/ml Ct pfu/ml Ct pfu/ml
1 (D1) 30.82 5559.04 40.00 0.00 40.00 0.00 40.00 0.00
1 (D1) 31.00 5011.87 40.00 0.00 40.00 0.00 40.00 0.00
2 (D1) 35.23 439.04 40.00 0.00 40.00 0.00 40.00 0.00
2 (D1) 34.46 683.91 40.00 0.00 40.00 0.00 40.00 0.00
3 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
3 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
4 (D1) 35.10 473.15 40.00 0.00 40.00 0.00 40.00 0.00
4 (D1) 34.26 767.36 40.00 0.00 40.00 0.00 40.00 0.00
5 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
5 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
6 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
6 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
7 (D1) 34.88 537.03 40.00 0.00 40.00 0.00 40.00 0.00
7 (D1) 34.22 785.24 40.00 0.00 40.00 0.00 40.00 0.00 (D1&3) 28.59 20067.81 40.00 0.00 33.73 1041.12 40.00 0.00 (D1&3) 27.78 31988.95 40.00 0.00 34.06 860.99 40.00 0.00
9 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
9 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
10 (D2) 40.00 0.00 27.23 43903.59 40.00 0.00 40.00 0.00
10 (D2) 40.00 0.00 26.18 80352.61 40.00 0.00 40.00 0.00
11 ((D2) 40.00 0.00 31.28 4265.80 40.00 0.00 40.00 0.00
11 ((D2) 40.00 0.00 31.29 4241.31 40.00 0.00 40.00 0.00
12 ((D2) 40.00 0.00 28.05 27384.20 40.00 0.00 40.00 0.00
12 ((D2) 40.00 0.00 29.31 13258.67 40.00 0.00 40.00 0.00
15 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
15 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
16 (D2) 40.00 0.00 28.14 26001.60 40.00 0.00 40.00 0.00
16 (D2) 40.00 0.00 28.34 23173.95 40.00 0.00 40.00 0.00
17 (D2) 40.00 0.00 28.28 23988.33 40.00 0.00 40.00 0.00
17 (D2) 40.00 0.00 28.65 19386.53 40.00 0.00 40.00 0.00
18 (-) 40.00 0.00 38.40 70.79 40.00 0.00 40.00 0.00
18 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
19 (D3) 40.00 0.00 39.79 31.81 24.76 181970.09 40.00 0.00
19 (D3) 40.00 0.00 40.00 0.00 24.60 199526.23 40.00 0.00
20 (D3) 40.00 0.00 40.00 0.00 29.60 11220.18 40.00 0.00
20 (D3) 40.00 0.00 40.00 0.00 29.87 9605.06 40.00 0.00
22 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 29.43 12373.71
22 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 29.26 13645.83
23 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 28.69 18945.24
23 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 29.41 12516.99
24 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
24 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
25 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
25 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
26 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 29.66 10839.27
26 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 29.78 10115.79
27 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
27 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00 Sample Den 1 Den 1 Den 2 Den 2 Den 3 Den 3 Den 4 Den 4
ID Ct pfu/ml ct pfu/ml ct pfu/ml Ct pfu/ml
28 (D2&4) 40.00 0.00 33.10 1496.24 40.00 0.00 29.57 11415.63
28 (D2&4) 40.00 0.00 33.03 1557.76 40.00 0.00 29.47 12092.05
29 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 32.73 1851.40
29 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 33.62 1109.17
30 (D2) 40.00 0.00 37.87 96.05 40.00 0.00 40.00 0.00
30 (D2) 40.00 0.00 37.59 112.85 40.00 0.00 40.00 0.00
33 (D2) 40.00 0.00 34.18 803.53 40.00 0.00 40.00 0.00
33 (D2) 40.00 0.00 35.78 319.89 40.00 0.00 40.00 0.00
34 (D2) 40.00 0.00 36.08 269.15 40.00 0.00 40.00 0.00
34 (D2) 40.00 0.00 35.96 288.40 37.12 147.91 40.00 0.00
35 (D2&4) 40.00 0.00 35.71 333.04 40.00 0.00 27.28 42657.95
35 (D2&4) 40.00 0.00 36.19 252.64 40.00 0.00 26.59 63459.99
38 (-) 40.00 0.00 39.77 32.17 40.00 0.00 40.00 0.00
38 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
39 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
39 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
40 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
40 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
43 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
43 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
44 (D2&3) 40.00 0.00 25.71 105317.37 38.48 67.61 40.00 0.00
44 (D2&3) 40.00 0.00 25.57 114156.33 37.23 138.84 40.00 0.00
45 (D2) 40.00 0.00 34.13 826.99 40.00 0.00 40.00 0.00
45 (D2) 40.00 0.00 31.80 3162.28 40.00 0.00 40.00 0.00
46 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 33.08 1513.56
46 (D4) 40.00 0.00 40.00 0.00 40.00 0.00 33.12 1479.11
47 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
47 (-) 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
48 (D2) 40.00 0.00 23.15 459726.99 40.00 0.00 40.00 0.00
48 (D2) 40.00 0.00 22.41 703882.23 40.00 0.00 40.00 0.00
49 (D2) 40.00 0.00 35.01 498.31 40.00 0.00 40.00 0.00
49 (D2) 40.00 0.00 35.27 429.04 40.00 0.00 40.00 0.00
50 (D2) 40.00 0.00 35.47 382.38 40.00 0.00 40.00 0.00
50 (D2) 40.00 0.00 35.72 331.13 40.00 0.00 40.00 0.00
NTC control 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
NTC control 40.00 0.00 40.00 0.00 39.95 0.00 40.00 0.00
NTC control 40.00 0.00 40.00 0.00 40.00 0.00 40.00 0.00
Example 4 The assay is performed according to the methodology of Example 1 using the materials below. Formulation of JE Taqman assay:
1 OX Taqman buffer 10 μl
10 mM dNTPs 8 μl
25 mM MgCl2 8 μl
JE-P1(1 pmole/μl) l μl
JE-F214(100 pmole/μl) 0.2 μl
JE-R382(20 pmole/μl) 0.35 μl dH2O 75 μl
Amplitaq-Gold 0.50 μl
It was found that several strain of genotype 3 JE can not be detected by JE Taqman assay mixture shown here.
Degenerate JE-F214 primer was incorporate into the mixture as shown below:
JE-U1(F214): 5'-CAAGCCCCCTCGAAGCTGT-3*
JE-U2(F214'): 5'-CAAGCTTCCTCAAAGCTGT-3'
The new F214' primer can detect genotype 3 JE strains, but barely can detect other genotypes' JE. Both F214 and F214' were used as upper primers for the test and results were shown as below:
Table 9. Detection of different JE isolates by Taqman assay:
Example 5 The assay is performed according to the methodology of Example 1 using the materials below.
Fluorogenic RT-PCR for West Nile virus (WNV) detection:
A. Primers and probes sequences for West Nile virus detection: WNV-L1: 5'-CCATTGTCGGCGCACTG-3'
WNV-Ul : 5'-CCTGGGATAGACTAGGAGATCTTCTG-3* and WNV-Pl probe: 6FAM-TCTGCACAACCAGCCACACGGC-3*
B . Formulation of WNV specific fluorogenic RT-PCR:
10X Taqman buffer 10 μl 10 mM dNTPs 8 μl 25 mM MgCl2 8 μl WNV-Pl (1 pmole/μl) l μl WNV-Ul (100 pmole/μl) 0.2 μl JE-L1 (20 pmole/μl) 0.35 μl dH2O 75 μl Amplitaq-Gold 0.50 μl The results are shown in Figures 8 and 9. A standard curve is shown in Figure 10. Quantitative results are shown in Figure 11.
References:
Chow, V.T.K., Seah, C.L.K. and Chan, Y.C. (1993) Use of NS-3 Consensus Primers for the Polymerase Chain Reaction Amplification and Sequencing of Dengue Viruses and Other Flaviviruses. Arch. Virol. 133, 157-170. Eckels, K.H., Harrison, V.R., Summers, P.L. and Russell, P.K. (1980) Dengue-2 vaccine: preparation from a small-plaque virus clone. Infect. Immun. 27(1), 175- 180.
Figueiredo, L.T., Batista, W.C, Kashima, S. andNassar, E.S. (1998) Identification of Brazilian flaviviruses by a simplified reverse transcription-polymerase chain reaction method using Flavivirus universal primers. Am J Trop Med Hyg 59(3),
357-62.
Fulop, L., Barrett, A.D.T., Phillpotts, R., Martin, K., Leslie, D. and Titball, R.W. (1993) Rapid Identification of Flaviviruses Based on Conserved NS5-Gene Sequences. J. Virol. Methods 44, 79-188. Gubler, D.J. (1989) Surveillance for dengue and dengue hemorrhagic fever. Bull. Pan. Am. Health Organ. 23, 397-404.
Harris, E., Roberts, T.G., Smith, L., Selle, J., Kramer, L.D., Valle, S., Sandoval, E. and Balmaseda, A. (1998) Typing of dengue viruses in clinical specimens and mosquitoes by single- tube multiplex reverse transcriptase PCR. J Clin Microbiol 36(9), 2634-9.
Henchal, E.A. and Putnak, J.R. (1990) The dengue viruses. Clin. Microbiol. Rev. 3(4), 376-396.
Higgins, J. A., J. Ezzell, B. J. Hinnebusch, M. Shipley, E. A. Henchal and M. S. Ibrahim. 1998. A 5 ' -nuclease assay for the detection of Yersinia pestis . J. Clin. Microbiol. 36(8)-.2284- 2288.
Holland P. M., R. D. Abramson, R. Watson, and D. H. Gelfand. 1991. Detection of specific polymerase chain reaction product by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. 88: 7276- 7280. Johnson, B.K.aV., M.G.R. (1976) Infection of an Aedes aegypti cell fine with infectious arbovirus-antibody complexes. Trans Royal Soc Trop Med Hyg 70(3), 230-34.
Leitmeyer KC, V.D., Watts DM Salas R, Villalobos de Chacon I, Ramosa C, Rico- Hesse R. (1999) Dengue virus structural differences that correlate with pathogenesis. J Virol 73 (6), 4738-4747.
Lewis, J.A., Chang, G.J., Lanciotti, R.S., Kinney, R.M., Mayer, L.W. and Trent, D.W. (1993) Phylogenetic Relationships of Dengue-2 Viruses. Virology 197, 216-224. Kanesa-thasan, N., Iacono Connors, L., Magill, A., Smoak, B., Vaughn, D.W., Dubois, D., Burrous, J. and Hoke, C.H., Jr. (1994) Dengue Serotypes 2 and 3 in Us Forces in Somalia, Lancet 343, 678.
Monath, T.P. (I 994) Dengue: The risk to developed and developing countries Proc. Natl. Acad. Sci. USA 91 , 2395-2400.
Pierre, V., Drouet, M.T. and Deubel, V. (1994) Identification of Mosquito-Bome Flavivirus Sequences Using Universal Primers and Reverse Transcription Polymerase Chain Reaction. Res. Virol. 145, 93-104.
Sudiro, T.M., Ishiko, H., Green, S., Vaughn, D.W., Nisalak, A., Kalayanarooj, S., Rothman, A.L. Raengsakulrach, B., Janus, J., Kurane, I. and Ennis, F.A. (1997)
Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3'-noncoding region universal primers. Am. J. Trop. Med. Hyg. 56(4), 424-9.
Sudiro, T.M., Ishiko, H., Rothman, A.L., Kershaw, D.E., Green, S., Vaughn, D.W., Nisalak, A., Kalayanarooj, S. and Ennis, F. A. (1998) Microplate-reverse hybridization method to determine dengue virus serotype. J Virol Methods 73(2), 229-35.
Tanaka, M. (1993) Rapid Identification of Flavivirus Using the Polymerase Chain Reaction. Virol. Methods 41, 311-322. Trofa, A.F., DeFraites, R.F., Smoak, B.L., Kanesa-thasan, N., King, A.D., Burrous, J.M., MacArthy, P.O., Rossi, C. and Hoke, C.H., Jr. (I 997) Dengue fever in US military personnel in Haiti. Jama 277(19), 1546-8.
INCORPORATION BY REFERENCE To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.

Claims

WHAT IS CLAIMED IS:
1. Isolated DNA segments having any one of the following sequences or sequences complimentary thereto: Serotype-speciflc Upstream Primers (8):
DVl-lU: 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3' DV2-2U: 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3' DV3-lU: 5'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3* DV4-lU: 5'-GAT-CAA-GCT-TAAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3' JE.F214: 5'-CAAGCCCCCTCGAAGCTGT
JE-U1 (F214): 5'-CAAGCCCCCTCGAAGCTGT-3' JE-U2(F214'): 5'-CAAGCTTCCTCAAAGCTGT-3* WNV-Ul : 5'-CCTGGGATAGACTAGGAGATCTTCTG-3' Serotype-specific Fluorescent Probes (5): DV1-P1: 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3* DV4-P1 : 5'-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3' DV2-P1 : 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3' JE.P1 : 5'-TCTGCTCTATCTCAACATCAGCTACTAGGCACAGA-3' WNV-Pl : 5'-TCTGCACAACCAGCCACACGGC-3' Serotype-specific Down-stream Primer (4):
DV4-lL: 5'-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3' DV2-lL: 5'-GAT-CGA-ATT-CCAT-TCC-ATT-TTC-TGG-CGT-TCT-3' JE.R382: 5'-CACCAGCTACATACTTCGGCG-3' WNV-L1: 5*-CCATTGTCGGCGCACTG-3'
2. The isolated DNA segments of claim 1 wherein said segments are probes and are labeled.
3. The probe of claim 2 wherein the label is fluorescent.
4. The probe of claim 2 wherein the label is a quencher.
5. The probe of claim 2 wherein the segment is labeled at both the 3' and 5' end, respectively, where one label is a quencher and the other is a fluorescent.
6. A PCR-based diagnostic kit for detecting or quantitating flavivirus comprising isolated DNA segments having any one of the following sequences or sequences complimentary thereto:
Serotype-specific Upstream Primers (8):
DVl-lU: 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3'
DV2-2U: 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3* DV3-1U: 5'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3'
DV4-lU: 5'-GAT-CAA-GCT-TAAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3'
JE.F214: 5'-CAAGCCCCCTCGAAGCTGT
JE-U1(F214): 5'-CAAGCCCCCTCGAAGCTGT-3'
JE-U2(F214'): 5'-CAAGCTTCCTCAAAGCTGT-3' WNV-Ul: 5'-CCTGGGATAGACTAGGAGATCTTCTG-3'
Serotype-specific Fluorescent Probes (5):
DV1-P1 : 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3'
DV4-P1 : 5'-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3*
DV2-P1: 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3' JE.P1: 5'-TCTGCTCTATCTCAACATCAGCTACTAGGCACAGA-3'
WNV-Pl : 5'-TCTGCACAACCAGCCACACGGC-3*
Serotype-specific Down-stream Primer (4):
DV4-lL: 5'-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3'
DV2-lL: 5'-GAT-CGA-ATT-CCAT-TCC-ATT-TTC-TGG-CGT-TCT-3' JE.R382: 5'-CACCAGCTACATACTTCGGCG-3'
WNV-Ll: 5'-CCATTGTCGGCGCACTG-3'.
7. The kit of claim 6 wherein said segments are labeled probes.
8. The kit of claim 7 wherein the label is fluorescent.
9. The kit of claim 7 wherein the label is a quencher.
10. The kit of claim 6 wherein the segment is labeled at both the 3' and 5' end, respectively, where one label is a quencher and the other is a fluorescent.
11. A method for detecting or quantifying one or more species of species of flavivirus contained in a sample comprising the steps of: i) collecting a sample suspected of containing a flavivirus; ii) preparing said sample for PCR amplification; iii) adding to said prepared sample PCR reagents wherein the primer pairs are selected from the following groups: Serotype-specific Upstream Primers (8):
DVl-lU: 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3' DV2-2U: 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3' DV3-lU: 5'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3' DV4-lU: 5*-GAT-CAA-GCT-TAAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3' JE.F214: 5'-CAAGCCCCCTCGAAGCTGT
JE-U1(F214): 5'-CAAGCCCCCTCGAAGCTGT-3' JE-U2(F214'): 5'-CAAGCTTCCTCAAAGCTGT-3' WNV-Ul : 5'-CCTGGGATAGACTAGGAGATCTTCTG-3' Serotype-specific Fluorescent Probes (5): DV1-P1: 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3' DV4-P1 : 5'-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3* DV2-P1 : 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3* JE.P1 : 5'-TCTGCTCTATCTCAACATCAGCTACTAGGCACAGA-3' WNV-Pl : 5'-TCTGCACAACCAGCCACACGGC-3' Serotype-specific Down-stream Primer (4):
DV4-lL: 5'-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3' DV2-lL: 5'-GAT-CGA-ATT-CCAT-TCC-ATT-TTC-TGG-CGT-TCT-3' JE.R382: 5*-CACCAGCTACATACTTCGGCG-3' WNV-L1: 5'-CCATTGTCGGCGCACTG-3' iv) maintaining the sample under conditions suitable for amplification; v) detecting or quantifying one or more of the flavivirus species.
12. The method of claim 11 wherein said species-specific probes of step (v) are labeled with a fluorescent label.
13. The method of claim 11 wherein said species-specific probes of step (v) are labeled with a quencher.
14. The method of claim 11 wherein the segment is labeled at both the 3' and 5' end, respectively, where one label is a quencher and the other is a fluorescent.
15. The method of claim 10 wherein said flavivirus is Dengue.
16. The method of claim 15 wherein said Dengue virus is Dengue 1, 2, 3, or 4.
17. A method for detecting or quantifying dengue virus comprising the steps of contacting a sample suspected of containing a flavivirus with PCR reagents, including at least two PCR primers selected from the following groups:
Serotype-specific Upstream Primers (8):
DVl-lU: 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3' DV2-2U: 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3'
DV3-lU: 5'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3*
DV4-lU: 5'-GAT-CAA-GCT-TAAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3'
JE.F214: 5'-CAAGCCCCCTCGAAGCTGT
JE-U1(F214): 5'-CAAGCCCCCTCGAAGCTGT-3* JE-U2(F214'): 5*-CAAGCTTCCTCAAAGCTGT-3'
WNV-Ul : 5'-CCTGGGATAGACTAGGAGATCTTCTG-3'
Serotype-specific Down-stream Primer (4):
DV4-lL: 5'-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3'
DV2-lL:5'-GAT-CGA-ATT-CCAT-TCC-ATT-TTC-TGG-CGT-TCT-3* JE.R382: 5'-CACCAGCTACATACTTCGGCG-3'
WNV-L1: 5*-CCATTGTCGGCGCACTG-3' and a polymerase enzyme, and an oligonucleotide probe selected from the following group: Serotype-specific Fluorescent Probes (5): DV1-P1 : 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3' DV4-P1 : 5*-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3' DV2-P1 : 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3' JE.P1: 5*-TCTGCTCTATCTCAACATCAGCTACTAGGCACAGA-3' WNV-Pl : 5'-TCTGCACAACCAGCCACACGGC-3* and wherein a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to a second end of the oligonucleotide such that the quencher molecule substantially quenches the fluorescer molecule whenever the oligonucleotide probe is in a single-stranded state and such that the fluorescer is substantially unquenched whenever the oligonucleotide probe is hybridized to the target nucleic acid; a 5' end which is rendered impervious to digestion by the 5' — 3' exonuclease activity of a polymerase; and a 3' end which is rendered impervious to the 5' → 3' extension activity of the polymerase; and subjecting the sample, the oligonucleotide probe, and the PCR reagents to thermal cycling, including a polymerization step, the thermal cycling being sufficient to amplify the target nucleic acid specified by the PCR reagents.
18. The method of claim 17 further comprising the step of measuring the extent of fluorescence quenching of the oligonucleotide probe, such measurement being performed subsequent to thermocycling and at a probe hybridization temperature.
19. The method of claim 17 further comprising the step of measuring the extent of fluorescence quenching of the oligonucleotide probe at a probe hybridization temperature in a manner which locates the probe within the individual cells originally containing the target nucleic acid sequence.
20. The method of claim 17 wherein the sample, the PCR reagents, and the oligonucleotide probe are located in a containment assembly.
21. The method of claim 17 wherein the probe hybridization temperature is less than or equal to the temperature of the polymerization step of the thermocycling.
22. In a nucleic acid amplification assay for detecting flavivirus wherein the improvement comprises a probe or primer having at least 15 contiguous nucleotides selected from the last 250 nucleotides of the 3' noncoding region of the flaviviral genome.
23. The nucleic acid amplification assay of claim 22 wherein the primer and probe are selected from the last 200 nucleotides of the 3' noncoding region of the flaviviral genome.
24. The nucleic acid amplification assay of claim 22 wherein the primer and probe are selected from the last 160 nucleotides of the 3' noncoding region of the flaviviral genome.
25. The nucleic acid amplification assay of claim 22 wherein the amplification assay is fluorogenic RT-PCR assay.
26. The amplification assay of claim 22 wherein the primer and probe are selected from the following groups respectively:
Serotype-specific Upstream Primers (8):
DVl -1U: 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3*
DV2-2U: 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3' DV3-1U: 5'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-3'
DV4-lU: 5'-GAT-CAA-GCT-TAAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3'
JE.F214: 5'-CAAGCCCCCTCGAAGCTGT
JE-U1(F214): 5'-CAAGCCCCCTCGAAGCTGT-3'
JE-U2(F214'): 5'-CAAGCTTCCTCAAAGCTGT-3* WNV-Ul: 5*-CCTGGGATAGACTAGGAGATCTTCTG-3' Serotype-specific Fluorescent Probes (5): DV1-P1 : 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3* DV4-P1 : 5*-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3' DV2-P1 : 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3' JE.P 1 : 5'-TCTGCTCTATCTCAACATCAGCTACTAGGCACAGA-3' WNV-Pl : 5'-TCTGCACAACCAGCCACACGGC-3' Serotype-speciflc Down-stream Primer (4): DV4-lL: 5'-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3' DV2-lL: 5'-GAT-CGA-ATT-CCAT-TCC-ATT-TTC-TGG-CGT-TCT-3' JE.R382: 5'-CACCAGCTACATACTTCGGCG-3'. WNV-L1: 5'-CCATTGTCGGCGCACTG-3'
27. In a nucleic acid amplification kit for detecting flavivirus wherein the improvement comprises a probe or primer having at least 15 contiguous nucleotides selected from the last 250 nucleotides of the 3' noncoding region of the flaviviral genome.
28. The nucleic acid amplification assay of claim 27 wherein the primer and probe are selected from the last 200 nucleotides of the 3' noncoding region of the flaviviral genome.
29. The nucleic acid amplification assay of claim 27 wherein the primer and probe are selected from the last 160 nucleotides of the 3' noncoding region of the flaviviral genome.
30. The nucleic acid amplification assay of claim 27 wherein the amplification assay is fluorogenic RT-PCR assay.
31. The amplification assay of claim 27 wherein the primer and probe are selected from the following groups respectively:
Serotype-specific Upstream Primers (8):
DVl-lU: 5'-GAT-CAA-GCT-TACA-CCA-GGG-GAA,GCT-GTA-TCC-TGG-3' DV2-2U: 5'-GAT-CAA-GCT-TAAG-GTG-AGA-TGA-AGC-TGT-AGT-CTC-3'
DVS-IU^'-GAT-CAA-GCT-TAGC-ACT-GAG-GGA-AGC-TGT-ACC-TCC-S*
DV4-lU: 5'-GAT-CAA-GCT-TAAG-CCA-GGA-GGA-AGC-TGT-ACT-CCT-3'
JE.F214: 5'-CAAGCCCCCTCGAAGCTGT
JE-U1(F214): 5'-CAAGCCCCCTCGAAGCTGT-3' JE-U2(F214'): 5'-CAAGCTTCCTCAAAGCTGT-3'
WNV-Ul : 5'-CCTGGGATAGACTAGGAGATCTTCTG-3'
Serotype-specific Fluorescent Probes (5):
DV1-P1 : 5'-CTG-TCT-CTA-CAG-CAT-CAT-TCC-AGG-CA-3'
DV4-P1 : 5*-CTG-TCT-CTG-CAA-CAT-CAA-TCC-AGG-CA-3' DV2-P1: 5'-CTG-TCT-CCT-CAG-CAT-CAT-TCC-AGG-CA-3'
JE.P1 : 5'-TCTGCTCTATCTCAACATCAGCTACTAGGCACAGA-3'
WNV-Pl : 5'-TCTGCACAACCAGCCACACGGC-3'
Serotype-specific Down-stream Primer (4):
DV4-lL: 5'-CAA-TCC-ATC-TTG-CGG-CGC-TCT-3' DN2-1L: 5'-GAT-CGA-ATT-CCAT-TCC-ATT-TTC-TGG-CGT-TCT-3'
JE.R382: 5'-CACCAGCTACATACTTCGGCG-3'
WΝN-L1: 5'-CCATTGTCGGCGCACTG-3'.
EP00973687A 2000-04-14 2000-10-19 Flavivirus detection and quantification assay Withdrawn EP1276898A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US09/551,161 US6793488B1 (en) 1999-04-16 2000-04-14 Flavivirus detection and quantification assay
PCT/US2000/028961 WO2001079546A2 (en) 2000-04-14 2000-10-19 Flavivirus detection and quantification assay
US551161 2009-08-31

Publications (1)

Publication Number Publication Date
EP1276898A1 true EP1276898A1 (en) 2003-01-22

Family

ID=24200105

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00973687A Withdrawn EP1276898A1 (en) 2000-04-14 2000-10-19 Flavivirus detection and quantification assay

Country Status (5)

Country Link
EP (1) EP1276898A1 (en)
AU (1) AU2001212172A1 (en)
BR (1) BR0017215A (en)
CA (1) CA2405960A1 (en)
WO (1) WO2001079546A2 (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2890882T3 (en) * 2002-10-16 2022-01-24 Gen Probe Inc Compositions and methods for detecting West Nile virus
AU2008200221B2 (en) * 2002-10-16 2012-04-05 Gen-Probe Incorporated Compositions and methods for detecting west nile virus
US7927840B2 (en) 2006-09-11 2011-04-19 Gen Probe Incorporated Method for detecting West Nile Virus nucleic acids in the 3′ non-coding region
WO2013080307A1 (en) * 2011-11-29 2013-06-06 株式会社 東芝 Primer set for amplifying mosquito-borne virus, assay kit for detecting mosquito-borne virus, and detection method using said primer set and said assay kit
CN104342498B (en) * 2013-07-30 2019-04-16 中国疾病预防控制中心病毒病预防控制所 Culex flavivirus real-time fluorescence quantitative RT-PCR detection method and kit
CN104357582B (en) * 2014-10-31 2017-02-15 中国检验检疫科学研究院 Real-time fluorescence RT-PCR detection kit and method of Kunjin virus
CN107893128A (en) * 2017-11-09 2018-04-10 贵州医科大学 The primer pair combination, probe and application of 10 kinds of arboviruses of detection simultaneously
CN110846441B (en) * 2019-12-20 2023-04-11 浙江省淡水水产研究所 Specific primer, probe and rapid detection kit for detecting macrobrachium flaviviridae-1

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999034020A1 (en) * 1997-12-31 1999-07-08 Akzo Nobel N.V. Isothermal transcription based assay for the detection and genotyping of dengue virus
US5939254A (en) * 1997-04-28 1999-08-17 University Of Massachusetts Methods and reagents for rapid diagnosis of dengue virus infection

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5939254A (en) * 1997-04-28 1999-08-17 University Of Massachusetts Methods and reagents for rapid diagnosis of dengue virus infection
WO1999034020A1 (en) * 1997-12-31 1999-07-08 Akzo Nobel N.V. Isothermal transcription based assay for the detection and genotyping of dengue virus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1994, PIERRE V ET AL: "Identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction.", XP002222878, Database accession no. PREV199497336271 *
RESEARCH IN VIROLOGY, vol. 145, no. 2, 1994, pages 93 - 104, ISSN: 0923-2516 *
See also references of WO0179546A3 *

Also Published As

Publication number Publication date
CA2405960A1 (en) 2001-10-25
WO2001079546A2 (en) 2001-10-25
WO2001079546A3 (en) 2003-06-05
BR0017215A (en) 2004-02-10
AU2001212172A1 (en) 2001-10-30

Similar Documents

Publication Publication Date Title
Houng et al. Development of a fluorogenic RT-PCR system for quantitative identification of dengue virus serotypes 1–4 using conserved and serotype-specific 3′ noncoding sequences
Houng et al. Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on 3′-noncoding sequence
Bae et al. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay
Lanciotti Molecular amplification assays for the detection of flaviviruses
CN108330210B (en) Zika virus, dengue virus and chikungunya virus nucleic acid detection kit and application thereof
JP2004533805A (en) Multiplex quantification of nucleic acids in diseased cells
US6855521B2 (en) Serotype and dengue group specific flurogenic probe based PCR (TaqMan) assays against the respective C and NS5 genomic and 3′ non-coding regions of dengue virus
TW202200788A (en) Assays for the detection of sars-cov-2
CA3017265C (en) Compositions and methods for detection of zika virus
US20230151444A1 (en) Pcr based diagnostic kit, compositions and methods for amplification and detection of sars-cov-2
CA2539703A1 (en) Detection of human papilloma virus (hpv) utilizing invasive cleavage structure assays
JP5754100B2 (en) Detection method and detection reagent for enterovirus 71 RNA
WO2012153153A1 (en) Procedure for rapid determination of viruses using nucleic acid-based molecular diagnostics, and a kit for this purpose
Garin et al. Highly sensitive Taqman® PCR detection of Puumala hantavirus
WO2001079546A2 (en) Flavivirus detection and quantification assay
CA2536335A1 (en) Multi-allelic molecular detection of sars-associated coronavirus
US6793488B1 (en) Flavivirus detection and quantification assay
JP4898210B2 (en) How to detect sapovirus
JP6592697B2 (en) Serotype 2 dengue virus detection kit, serotype 3 dengue virus detection kit, and dengue virus detection kit
Houng et al. Development and evaluation of an efficient 3′-noncoding region based SARS coronavirus (SARS-CoV) RT-PCR assay for detection of SARS-CoV infections
US8048630B2 (en) Methods and agents for detecting Parechovirus
KR101755037B1 (en) Method for detection of HCV at the real time PCR with intercalating dye
KR102516022B1 (en) Detection and Differentiation of African Swine Fever Virus and Classical Swine Fever Virus by One Step Duplex Reverse Transcriptase Quantitative PCR Assay
KR20210073221A (en) Primer and probe sets for diagnosing zika virus
WO2005106045A1 (en) System and method for detecting west nile virus

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20021120

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

D17D Deferred search report published (deleted)
R17P Request for examination filed (corrected)

Effective date: 20021114

D17D Deferred search report published (deleted)
17Q First examination report despatched

Effective date: 20050603

17Q First examination report despatched

Effective date: 20050603

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080826