| Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard -> Monitor Keywords |
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Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standardRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or BacteriophageQuantitative real-time assay for noroviruses and enteroviruses with built in quality control standard description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060110724, Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0002] This invention relates to methods and reagents for detecting and quantifying viruses including Norovirus or Enterovirus by, for example, detecting or quantifying nucleic acids. BACKGROUND OF THE INVENTION [0003] Noroviruses are estimated to be responsible for two-thirds of the non-bacterial food-borne illness and nearly all (96%) of the non-bacterial gastrointestinal illnesses each year in the United States. Norovirus infection occurs via the fecal-oral route from contaminated food such as oysters (Berg et al., 2000, J. Infect. Dis., 181:S381-S386; Shieh et al., 2000, J. Infect. Dis., 181: S360-S366), water (Yoder et al., 2004, Morb. Mortal. Wkly. Rep., 53(SS-8): 1-15; Blackburn et al., 2004, Morb Mortal Wkly Rep, 53(SS-8): 23-39) and even bakery products (Deneen et al., 2000, J. Infect. Dis., 181: S281 -S283). Infections also occur by droplet transmission, contact with contaminated fomites, or person-to-person transmission in contained or semi-contained areas such as cruise ships (Center for Disease Control and Prevention, 2002, Morb. Mortal. Wkly. Rep., 51(49): 1112-1115), hospitals, nursing homes, restaurants, and schools (Gallimore et al., 2004, J. Clin. Microbiol., 42: 1396-1401). [0004] Noroviruses can not be propagated by cell culture techniques. However, Noroviruses are now detectable by various Reverse Transcription Polymerase Chain Reaction (RT-PCR) techniques. Real-time RT-PCR assays have been developed using either SYBR.RTM. Green and TaqMan.RTM. style assays for the detection and subsequent quantification of these viruses (Donaldson et al., 2002, Water Res., 36: 2505-2514; Kageyama et al., 2003, J. Clin. Microbiol., 41: 1548-1557; Kojimaetal., 2002,J. Virol. Methods, 100: 107-114; Richards et al., 2004, J. Virol. Methods, 116: 63-70). SYBR.RTM. green assays, while useful for detection, are not reliable in quantification of template concentration due to its non-specific intercalation into any double stranded DNA (primer-dimer and products of non-specific amplification). There remains a need for methods and reagents that can detect or quantify more than one type of Norovirus and/or Enterovirus in a reaction mixture. SUMMARY OF THE INVENTION [0005] The present invention includes methods and reagents for detecting and quantifying viruses including Norovirus and Enterovirus by, for example, detecting or quantifying nucleic acids. The methods can employ and the reagents can include primers and oligonucleotide probes configured for a multiplex, real-time quantitative RT-PCR (qRT-PCR) assay. The present method can employ a universal internal RNA control. This internal control nucleic acid molecule can provide more efficient RT-PCR, allow normalization of results, and/or can detect inhibitors of RT-PCR. [0006] In an embodiment, the present method can detect and quantify Norovirus Genogroup I and Norovirus Genogroup II in a single reaction. Detection and quantification of the two viruses from the two genogroups can be simultaneous. The reaction mixture can include an universal internal RNA control. The method can employ primers and oligonucleotide probes that distinguish between Norovirus Genogroups I and II. Optionally, the method of invention can employ primers and oligonucleotide probes that hybridize to Enterovirus nucleic acid. [0007] In an embodiment, the present invention includes a reaction mixture including Norovirus Genogroup I and Norovirus Genogroup II primers and oligonucleotide probes, an internal control nucleic acid molecule, and internal RNA control primers and oligonucleotide probe. Said Norovirus primers are capable of distinguishing Genogroup I and Genogroup II. Optionally, a reaction mixture can also include primers and oligonucleotide probes for Enterovirus nucleic acids. An embodiment includes Norovirus Genogroups I and II primers and probes, an internal control nucleic acid molecule, and internal control primers and probes packaged together in a kit. BRIEF DESCRIPTION OF THE FIGURES [0008] FIG. 1 is the standard curve established for the quantification of Norovirus Genogroup I using FAM-labeled oligonucleotide probes. [0009] FIG. 2 is the standard curve established for the quantification of Norovirus Genogroup II using a TET-labeled oligonucleotide probe. [0010] FIG. 3 is the standard curve established for the quantification of viruses from the Enterovirus group using a CyS-labeled oligonucleotide probe. FIG. 4 is the standard curve established for the internal RNA control using a TxR-labeled oligonucleotide probe. [0011] FIG. 4 is the standard curve established for the internal RNA control using a TxR-labeled oligonucleotide probe. [0012] FIG. 5 is a 4% TAE agarose gel to determine the effect of template overload. Lane 1-10.sup.0 dilution; Lane 2-10.sup.-1 dilution; Lane 3-10.sup.-2 dilution; Lane 4-10.sup.-3 dilution; Lane 5-10.sup.-4 dilution; Lane 6-10.sup.-5 dilution; Lane 7-10.sup.-6 dilution; Lane 8-10.sup.-7 dilution; Lane 9-10.sup.-8 dilution; Also shown is a negative control lacking viral template RNA, a 100 bp ladder (Invitrogen, Inc., Carlsbad, Calif.), and a 25 bp ladder (Invitrogen). [0013] FIG. 6 is determination of the dynamic range for Noroviruses and Enteroviruses. Norovirus Genogroup I, y=-3.71x +10.78, r.sup.2=0.983, efficiency=86%; Norovirus Genogroup II, y=-3.68x +8.56, r.sup.2=0.998, efficiency=87%; Enterovirus, y=-3.26x +14.92, r.sup.2=0.970, Efficiency=103%; Universal internal RNA Control, r.sup.2=0.11 DETAILED DESCRIPTION Definitions [0014] The term "biological sample" refers to a body sample from any animal, but preferably is from a mammal, more preferably from a human. Such samples include biological fluids such as serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, and tissue culture medium, as well as tissue extracts such as homogenized tissue, and cellular extracts. [0015] As used herein, "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. Buffers may optionally comprise a salt such as MgCl.sub.2, MnCl.sub.2, or the like. Buffers may also optionally comprise other constituents to improve the efficiency of reverse transcription or amplification, including, but not limited to, betaine, bovine serum albumin, etc. [0016] The term "cDNA" refers to a complementary DNA molecule synthesized using a ribonucleic acid strand (RNA) as a template. The RNA may be mRNA, tRNA, rRNA, or another form of RNA, such as viral RNA. The cDNA may be single-stranded, double-stranded or may be hydrogen-bonded to a complementary RNA molecule as in an RNA/cDNA hybrid. [0017] The term "Enterovirus group" refers to icosahedral, nonenveloped, single-stranded, positive-sense RNA viruses of the family Picornaviridae. The Enterovirus group is subdivided into poliovirus, coxsackievirus (Groups A and B), echovirus, and enterovirus. Enteroviruses cause a myriad of clinical pathologies in humans, including gastroenteritis. As used herein, "Enterovirus group" refers to the genus, and "enterovirus" refers to the enterovirus species that is part of the Enterovirus group. [0018] The term "food sample" refers to a substance that is ingested by an animal, preferably a human. Food sample includes, but is not limited to, water, shellfish (e.g., bivalve molluscan shellfish such as oysters, mussels, and clams), and bakery products. [0019] The term "Hepatitis A Virus" or "HAV" refers to a single stranded, positive-sense RNA virus of the family Picornaviridae. Hepatitis A or type A viral hepatitis was also formerly known as any of the following: infectious hepatitis, epidemic hepatitis, epidemic hepatitis, epidemic jaundice, catarrhal jaundice, infectious icterus, Botkins disease, and MS-1 hepatitis. Hepatitis A is usually self-limiting and produces the acute symptoms of fever, malaise, nausea, and abdominal discomfort followed by an extended period of jaundice. The minimum infectious dose is unknown but hypothesized to be between 10 and 100 virions. Hepatitis A is primarily transmitted via the fecal-oral route. Usually, persons infected in outbreaks acquire the disease through the ingestion of contaminated food, mostly water, shellfish, and salads. Continue reading about Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard... Full patent description for Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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