Use of TaqMan Real-Time Reverse Transcription-PCR for Rapid Detection, Quantification, and Typing of Norovirus

Abstract

Noroviruses (NoVs) are the most commonly identified cause of outbreaks and sporadic cases of acute gastroenteritis. We evaluated and optimized NoV-specific TaqMan real-time reverse transcription (RT)-PCR assays for the rapid detection and typing of NoV strains belonging to genogroups GI and GII and adapted them to the LightCycler platform. We expanded the detection ability of the assays by developing an assay that detects the GIV NoV strain. The assays were validated with 92 clinical samples and 33 water samples from confirmed NoV outbreaks and suspected NoV contamination cases. The assays detected NoV RNA in all of the clinical specimens previously confirmed positive by conventional RT-PCR and sequencing. Additionally, the TaqMan assays successfully detected NoV RNA in water samples containing low viral concentrations and inhibitors of RT and/or PCR, whereas the conventional method with region B primers required dilution of the inhibitors. By means of serially diluted NoV T7 RNA transcripts, a potential detection limit of <10 transcript copies per reaction mixture was observed with the GII assay and a potential detection limit of <100 transcript copies per reaction mixture was observed with the GI assay. These results and the ability to detect virus in water that was negative by RT-PCR demonstrate the higher sensitivity of the TaqMan assay compared with that of a conventional RT-PCR assay. The TaqMan methods dramatically decrease the turnaround time by eliminating post-PCR processing. These assays have proven useful in assisting scientists in public health and diagnostic laboratories report findings quickly to outbreak management teams.

High prevalence of noroviruses among hospitalized diarrheal patients in Bangladesh, 2011

“…The detection rate in this study (28%) was much higher than observed in previous studies in Bangladesh [7,8] and is comparable to recent published reports from other countries [9,10]. Designing of appropriate primer set to detect the full range of NoV types has been difficult due to extensive genomic diversity.…”

“…Realtime RT-PCR assay with NoV type specific primers and probes to detect NoV GI, GII and GIV targeting ORF1-ORF2 junction region of NoV genome [9] was performed. In brief, a final reaction mixture (25µl) consisting of 5µl RNA, 1µl of Ag-Path enzyme mix, 12.5µl Ag-Path buffer (Ambion Inc. Austin, USA), 0.8 and 0.2 picomol of each primer and probe was prepared.…”

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

“…Since the diversity of the contemporary NoV strains has been increasing during previous years, the success rate will depend on the mismatches of primers in region C for the specimens being tested. The assay is useful for routine diagnostic as it eliminates post-amplification product processing, thus shortening the turn around time 7 . Apart from diagnosis, this protocol can also be used for clarifying the epidemiology of NoV infections, thus being useful for public health control of this disease.…”

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

“…Se utilizaron 5 ml de ARN de la muestra para la síntesis de cDNA mediante el kit Qiagen One-Step RT-PCR seguida de RPC a tiempo real con sondas Taqman específicas para cada genogrupo de NoV. Utilizamos 1µM de cada partidor COG2-F y COG2-R para amplificación de NoV GII y los partidores COG-1F y COG-1R para la amplificación de NoV GI, que tienen secuencias conservadas del genoma de NoV localizadas en la unión de la región ORF1 (región polimerasa) y ORF2 (región cápside viral) 27,29 . La hibridación del producto de RPC se realizó con 0,1 µM de cada sonda Taqman específica, Ring-2TP y Ring-1A/Ring-1B para los GII y GI, respectivamente 27,29 .…”

“…Para cumplir este objetivo se utilizó el método Taqman de RPC-TR a tiempo real recomendado por el Centers for Disease Control and Prevention (CDC) de Atlanta, E.U.A. 27 y que es el método actualmente utilizado por el ISP para la detección de NoV en Chile.…”