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An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping. 2007

K J Rolfe, and S Parmar, and D Mururi, and T G Wreghitt, and H Jalal, and H Zhang, and M D Curran
Health Protection Agency, East of England Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, United Kingdom. kathryn.rolfe@addenbrookes.nhs.uk

BACKGROUND Reverse transcription (RT)-PCR for norovirus detection is prone to false-negative results due to inhibitory substances in faeces. An internal control is needed to monitor extraction efficiency and to detect inhibition. OBJECTIVE To further develop a one-step RT-PCR assay for norovirus detection/genogrouping by addition of MS2 bacteriophage as an internal control. METHODS Our norovirus RT-PCR assay was modified by addition of MS2 phage to the extraction tray and primers/probe for MS2 detection to the reaction mix. The effect of addition of MS2 phage and MS2 primers/probe on the sensitivity/specificity of the PCR assay was examined. RESULTS The addition of MS2 as an internal control showed no loss of sensitivity or specificity for norovirus detection. CONCLUSIONS A triplex, one-step, type-specific, real-time RT-PCR with MS2 internal control has been developed for use in routine laboratory diagnosis of norovirus infection.

UI MeSH Term Description Entries
D005243 Feces Excrement from the INTESTINES, containing unabsorbed solids, waste products, secretions, and BACTERIA of the DIGESTIVE SYSTEM.
D005759 Gastroenteritis INFLAMMATION of any segment of the GASTROINTESTINAL TRACT from ESOPHAGUS to RECTUM. Causes of gastroenteritis are many including genetic, infection, HYPERSENSITIVITY, drug effects, and CANCER. Gastroenteritides
D005838 Genotype The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS. Genogroup,Genogroups,Genotypes
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D017250 Caliciviridae Infections Virus diseases caused by CALICIVIRIDAE. They include HEPATITIS E; VESICULAR EXANTHEMA OF SWINE; acute respiratory infections in felines, rabbit hemorrhagic disease, and some cases of gastroenteritis in humans. Calicivirus Infections,Infections, Caliciviridae,Infections, Calicivirus,Norovirus Infections,Caliciviridae Infection,Calicivirus Infection,Infection, Caliciviridae,Infection, Calicivirus,Infection, Norovirus,Infections, Norovirus,Norovirus Infection
D017909 Levivirus A bacteriophage genus of the family LEVIVIRIDAE, whose viruses contain the short version of the genome and have a separate gene for cell lysis. Bacteriophage MS2,Coliphage MS2,Enterobacteria phage MS2,Enterobacteria phage R17,Enterobacteria phage f2,Enterobacteria phage fr,MS2 Phage,Phage MS2,Pseudomonas phage PP7,Leviviruses,MS2 Phages,MS2, Bacteriophage,MS2, Coliphage,MS2, Phage,Phage, MS2,Phages, MS2
D017931 DNA Primers Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques. DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide
D020133 Reverse Transcriptase Polymerase Chain Reaction A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols. Polymerase Chain Reaction, Reverse Transcriptase,Reverse Transcriptase PCR,PCR, Reverse Transcriptase,Transcriptase PCR, Reverse

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