Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis

Pang, Xiaoli; Preiksaitis, Jutta K.; Lee, Bonita

doi:10.1016/j.jcv.2004.12.014

Cited by 98 publications

(78 citation statements)

References 13 publications

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“…In the present study, we found that the median viral load of the positive samples was 1.02 ϫ 10 4 copies per ml of stool (range, 1.67 ϫ 10 2 to 4.27 ϫ 10 9 copies per ml), which was far below the viral loads in other enteric-virus-associated gastroenteritis. For example, the median viral load of norovirus was at least 8.4 ϫ 10 5 copies/g or ml of stool specimen in previous studies (5,16). Based on the hypothesis that high viral loads are an indication of pathogenic relevance, the relatively low virus loads of HBoV2 in stool specimens do not seem to support its clinical significance.…”

Section: Discussionmentioning

confidence: 91%

Development of a Real-Time PCR Assay for Detecting and Quantifying Human Bocavirus 2

Cheng

et al. 2011

J Clin Microbiol

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Human bocavirus 2 (HBoV2) is a parvovirus that has been recently identified in stool samples from children. Any association between the virus and clinical disease is unclear. A rapid, reliable diagnostic method is necessary to address this issue. In this study, we developed a sensitive and specific HBoV2 quantitative real-time PCR assay that targets the HBoV2 NP-1 gene, based on the TaqMan method. The assay could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV2 genome, with a dynamic range of 8 log units (10

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Section: Discussionmentioning

confidence: 91%

Development of a Real-Time PCR Assay for Detecting and Quantifying Human Bocavirus 2

Cheng

et al. 2011

J Clin Microbiol

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“…These assays were demonstrated to have better sensitivities and specificities than electron microscopy or antigen-detection assays (4,21,22,26,27,29). Of these studies, two have described the use of multiplex assays for the detection of viral gastroenteritis (2,31).…”

Section: Discussionmentioning

confidence: 99%

Evaluation and Verification of the Seeplex Diarrhea-V ACE Assay for Simultaneous Detection of Adenovirus, Rotavirus, and Norovirus Genogroups I and II in Clinical Stool Specimens

Higgins¹,

Beniprashad²,

Cardona³

et al. 2011

J Clin Microbiol

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Acute viral gastroenteritis is an intestinal infection that can be caused by several different viruses. Here we describe the evaluation and verification of Seeplex Diarrhea-V ACE (Seeplex DV), a novel commercial multiplex reverse transcription-PCR (RT-PCR) assay that detects 5 diarrheal pathogens, including adenovirus, rotavirus, norovirus genogroup I (GI) and GII, and astrovirus. We describe a retrospective study of 200 clinical specimens of which 177 were stool specimens previously tested for the presence of gastrointestinal viruses by electron microscopy (EM) and/or real-time RT-PCR (rRT-PCR). The remaining 23 specimens comprised other human pathogens of viral or bacterial origin. Discordant norovirus GI and GII results were resolved using a commercial kit; discordant adenovirus and rotavirus results were resolved using a home brew multiplex rRT-PCR assay. Diagnostic sensitivities and specificities were calculated before and after discordant analysis. After discordant analysis, estimated diagnostic sensitivities were 100% for adenovirus, rotavirus, and norovirus GI and 97% for norovirus GII. Diagnostic specificities after discordant analysis were 100% for adenovirus, rotavirus, and norovirus GI and 99.4% for norovirus GII. The 95% limits of detection were 31, 10, 2, and 1 genome equivalent per reaction for adenovirus, rotavirus, and norovirus GI and GII, respectively. The results demonstrate that the Seeplex DV assay is sensitive, specific, convenient, and reliable for the simultaneous detection of several viral pathogens directly in specimens from patients with gastroenteritis. Importantly, this novel multiplex PCR assay enabled the identification of viral coinfections in 12 (6.8%) stool specimens.Acute viral gastroenteritis is the second most common infectious disease worldwide (20, 5), affecting humans of all age groups (23, 17) but mostly the young, the elderly, and people in enclosed communities, such as hospitals, nursing homes, military bases, and cruise ships. Known enteric viral pathogens include adenovirus group F serotypes 40 and 41, rotavirus, norovirus, and astrovirus, with rotavirus and norovirus being the predominant causative agents of gastroenteritis (3,7,30). In recent years the number of reported gastroenteritis outbreaks of suspected viral etiology has increased, hence the need for fast, sensitive, and reliable diagnostic assays.The established method for detection of enteric viruses at the Ontario Agency for Health Protection and Promotion (OAHPP) Public Health Laboratories (PHL) relies on the conventional and labor-intensive electron microscopy (EM). In recent years, home brew real-time reverse transcription-PCR (rRT-PCR) assays were implemented for the detection of norovirus GI and GII (11). Similar home brew rRT-PCR assays for the detection of adenovirus (6) and rotavirus (35) have been described. These rRT-PCR assays enable the detection of a specific virus by the amplification of a unique genomic sequence within its RNA or DNA. However, the simultaneous detection of several viruses c...

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“…TaqMan real-time RT-PCR assays for GI and GII NoVs were conducted in each laboratory according to published procedures (see Table S1 through S3 in the supplemental material) (12,13,22,26). Laboratory A used a GI and GII multiplex format with QuantiTect probe RT-PCR kits (Qiagen).…”

Section: Norovirusmentioning

confidence: 99%

“…In addition, they support identification of newly emerging strains and of common-source outbreaks so that transmission routes can be interrupted (5,15,18). Because NoVs cannot be routinely grown in cell culture, most clinical laboratories use either conventional or real-time reverse transcription (RT)-PCR for detection of the virus (4,7,13,22,25,26,29,30). Currently, classification of NoV genotypes is based on genetic diversity within the entire capsid gene (2,16,33).…”

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confidence: 99%

Multicenter Comparison of Two Norovirus ORF2-Based Genotyping Protocols

et al. 2009

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Point source norovirus outbreaks can be difficult to track due to high background levels of the virus in the environment and the limited strain variation in some genotyping regions. However, rapid and accurate source identification can limit the spread of a foodborne outbreak and reduce the number of cases. Harmonization of genotyping assays is critical for enabling the rapid exchange of sequence data nationally and internationally. Several regions of the genome have been proposed for this purpose, but no consensus has been reached. In the present study, two standardized genotyping protocols (region C and region D) were evaluated by nine laboratories in Canada and the United States, using a coded panel of 96 fecal specimens representing 22 different norovirus genotypes. Overall, region C typing had a success rate of 78% compared to 52% for region D; however, region D provides greater nucleotide sequence diversity for identifying new GII.4 variant strains. Significant differences in the genotyping success rate were observed among the nine participating laboratories (10% to 100%) and among the different genotypes (6% to 100%). For several genogroup II strains, reduced region D amplification correlated directly with mismatches between primer sequences and the template. Based on overall performance, we recommend the region C protocol for routine genotyping of noroviruses, while the region D protocol may be useful for identifying new GII.4 variants. Standardized genotyping protocols will enable rapid exchange of outbreak and sequence data through electronic norovirus surveillance networks.

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Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis

Cited by 98 publications

References 13 publications

Development of a Real-Time PCR Assay for Detecting and Quantifying Human Bocavirus 2

Development of a Real-Time PCR Assay for Detecting and Quantifying Human Bocavirus 2

Evaluation and Verification of the Seeplex Diarrhea-V ACE Assay for Simultaneous Detection of Adenovirus, Rotavirus, and Norovirus Genogroups I and II in Clinical Stool Specimens

Multicenter Comparison of Two Norovirus ORF2-Based Genotyping Protocols

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