Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

Abstract

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities.

Norovirus

“…These limitations were addressed with the development of real-time RT-PCR for norovirus diagnostics. These assays used numerous detection methods, including SYBR green (190)(191)(192)(193), hydrolysis (TaqMan) (194)(195)(196)(197)(198)(199)(200)(201)(202), and hybridization probes (203,204). While many of these assays were directed at the viral polymerase gene, further sequence analysis revealed that a conserved region at the ORF1-ORF2 polymerase-capsid junction could also be used as an effective target for detection.…”

Detección de norovirus en niños con diarrea adquirida en la comunidad o nosocomial en el Hospital Guillermo Grant Benavente de Concepción, Chile

“…La introducción del diagnóstico empleando RPC en tiempo real ha sido de gran importancia para mejorar el diagnóstico y establecer el rol relevante de NoV en brotes epidémicos agudos y en casos esporádicos de diarrea. Este método diagnóstico ha permitido demostrar la amplia heterogeneidad y rápida evolución de las variantes genéticas de NoV 8,9,[11][12][13][14][15] ; La diversidad antigénica y la falta de sistema de cultivo in vitro del virus mantendrá al diagnóstico molecular como el mejor método de detección de NoV. La RPC a tiempo real ha emergido como el estándar para el diagnóstico y detección de NoV, ya que combina un alto rendimiento, reproducibilidad, sensibilidad y especificidad 11,27,[42][43][44][45][46] . El 36,6% de los pacientes en este estudio fueron ingresados al hospital con un diagnóstico diferente al de una gastroenteritis, adquiriendo la infección probablemente en forma nosocomial (plazo de latencia para los síntomas de 3 o más días), siendo mayor para NoV (32,1%) que para RV (5,7%).…”

Nucleic Acid Detection of Major Foodborne Viral Pathogens Human: Noroviruses and Hepatitis A Virus

“…There are two major groups of sequence-specific oligonucleotide probes: hydrolysis (e.g., TaqMan) probes and hybridization probes (e.g., molecular beacons and fluorescence resonance energy transfer probes); both groups are homologous to the internal region of amplified products. TaqMan probes have been frequently used in RT-qPCR for detecting hNOVs [67][68][69][70][71][72][73]. While RNA polymerase and capsid genes are the primary targets for amplification, within the NoV genomes, a junction of ORF1-ORF2-polymerase-capsid has been demonstrated to be the most highly conserved region that can serve as an effective target for amplification.…”