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Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard

Inactive Publication Date: 2006-05-25
HEALTH & HUMAN SERVICES GOVERNMENT OF THE US SEC DEPT OF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[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.

Problems solved by technology

Noroviruses can not be propagated by cell culture techniques.
SYBR® 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).

Method used

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  • Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard
  • Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard
  • Quantitative real-time assay for noroviruses and enteroviruses with built in quality control standard

Examples

Experimental program
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Effect test

example 1

Development of a Quantitative Multiplex RT-PCR Assay for the Detection of Noroviruses and Enteroviruses

[0078] The use of RT-PCR based assays has increased detection sensitivity of Enteroviruses and reduced analysis time. The predominant human Noroviruses, which are placed into two genogroups, GI and GII, have not been cultured. Thus, Norovirus detection is based primarily upon non-quantitative RT-PCR assays. To date, no single assay has been capable of simultaneous detection and enumeration of GI genogroup Norovirus and GII genogroup Norovirus.

[0079] In the present study, a multiplex qRT-PCR assay using the Cepheid SmartCycler® (Cepheid, Sunnyvale, Calif.) system has been developed for the simultaneous detection and quantification of viruses from the Enterovirus genus. The following example demonstrated that the multiplex qRT-PCR can simultaneously detect Enterovirus, Norovirus genogroup I, and Norovirus genogroup II. The assay also incorporated a novel quantitative universal inte...

example 2

Quantitative Multiplex RT-PCR Assay for the Detection of Noroviruses and Enteroviruses

[0093] RNA template was serially diluted 1:10 until a dilution of 10−9. Using the different concentrations of RNA template tested the effect of template overload and the assay's threshold of detectable levels of RNA.

[0094] Methods

[0095] The cDNAs from the RT-PCR of Example I were purified using a NucleoSpin® Extraction Kit (BD Biosciences Clontech, Palo Alto, Calif.) and cloned using a TOPO TA Cloning® Kit (Invitrogen Corp., Carlsbad, Calif.) by known methods. Clones with full-length inserts and proper orientation were subjected to a QIAprep® Miniprep Kit (Qiagen) to purify their plasmids. Plasmids were purified and sequenced in both the forward and reverse direction to determine and / or verify their sequences. Each plasmid was linearized by restriction digest with BamHI (New England BioLabs®, Inc., Beverly, Mass.) and run on a 1% agarose gel to confirm linearization. The linearized plasmid was ...

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Abstract

A method is provided for reverse transcription-polymerase chain reaction (RT-PCR) comprising a) amplifying a reverse transcribed cDNA in a mixture comprising Norovirus Genogroup I and Norovirus Genogroup II primers and probes, wherein said Norovirus primers and probes distinguish between Genogroup I and Genogroup II viruses; b) quantifying virus; and c) normalizing data based on a universal internal RNA control. Optionally, the method may also include primers and probes for Enteroviruses. A reaction mixture comprising Norovirus Genogroup I and Norovirus Genogroup II primers and probes, wherein said Norovirus primers and probes distinguish between Genogroup I and Genogroup II viruses and universal internal RNA control primers and probes are also included.

Description

GOVERNMENTAL INTERESTS [0001] This invention was developed employing support from the United States Food and Drug Administration. The Government of the United States of America may have certain rights in this invention.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 eve...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12P19/34
CPCC12Q1/701
Inventor BURKHARDT, WILLIAMVICKERY, MICHAELNORDSTROM, JESSICA
Owner HEALTH & HUMAN SERVICES GOVERNMENT OF THE US SEC DEPT OF
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