Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories

Mattison, Kirsten; Grudeski, Elsie; Auk, Brian; Brassard, Julie; Charest, Hugues; Dust, Kerry; Gubbay, Jonathan B.; Hatchette, Todd F.; Houde, Alain; Jean, Julie; Jones, T. H.; Lee, Bonita E.; Mamiya, Hiroshi; McDonald, Ryan; Mykytczuk, Oksana; Pang, Xiaoli; Petrich, Astrid; Plante, Daniel; Ritchie, Gordon; Wong, Julie; Booth, Tim F.

doi:10.1016/j.jcv.2010.10.008

Cited by 18 publications

(17 citation statements)

References 38 publications

(50 reference statements)

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“…Fortunately, most reference laboratories use a real-time RT-PCR targeting the most conserved region of the genome (ORF1-ORF2 junction) and use degenerate primers and probes that can tolerate some sequence mismatches [9,10] (Figure 1). In this study, this same real-time RT-PCR was able to detect diverse norovirus genotypes with analytical sensitivities consistent with values previously reported for GII.4 (2006b) (Table 1) [6]. In contrast, EP-JV and EP-SR were far less sensitive and failed to detect certain genotypes (Tables 1, 2 and 3).…”

Section: Discussionsupporting

confidence: 89%

“…Ideally, sequencing would be used to encompass the regions required for genotyping and the primer/probe-binding sites for commonly used RT-PCRs [42,43]. When sequence mismatches are identified in RT-PCR target regions or when new norovirus variants emerges, proficiency panels should be promptly disseminated to clinical laboratories to ensure accurate detection [6]. With the dynamic nature of norovirus epidemiology, this study highlights the importance of routine surveillance and ongoing proficiency testing for circulating norovirus genotypes.…”

Section: Discussionmentioning

confidence: 99%

“…RT-PCR has markedly improved the detection of noroviruses and has become the method of choice for clinical diagnosis [2]. However, the genetic diversity among noroviruses poses a particular challenge for molecular assays [6-11]. Noroviruses are classified into six genogroups, three of which cause human disease (GI, GII, and GIV) [12-14].…”

Section: Introductionmentioning

confidence: 99%

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Detection of circulating norovirus genotypes: hitting a moving target

Rooney

Pettipas

Grudeski³

et al. 2014

Virol J

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BackgroundAlthough national surveillance programs are in place to monitor norovirus epidemiology, the emergence of new strains and the genetic diversity among genotypes can be challenging for clinical laboratories. This study evaluated the analytical and clinical performance characteristics of one real-time RT-PCR and two end-point RT-PCRs commonly used in microbiology laboratories.MethodsLower limit of detection (LoD) was determined using 10-fold dilutions of noroviruses belonging to different genotypes. The clinical performance of the real-time and end-point RT-PCRs was assessed in parallel using nucleic acids extracted from 186 stool specimens.ResultsThe real-time RT-PCR was highly sensitive and specific for the detection of norovirus genotypes that are currently circulating in Canada. In contrast, the two end-point RT-PCRs displayed poor analytical sensitivity or complete failure to detect certain norovirus genotypes, which was correlated to sequence mismatches in the primer-binding sites. In an attempt to improve norovirus detection with the end-point RT-PCRs, both assays were processed concurrently and detection from either assay was considered a positive result. Concurrent testing resulted in only a modest increase in clinical sensitivity (75.0%) compared to each assay alone (62.5% and 71.9%). However, the false positivity rate increased from 1.98% and 3.36% for the assays alone to 5.47% with concurrent testing.ConclusionsThis study emphasizes the benefits of a real-time method and provides support for routine surveillance to monitor norovirus epidemiology and ongoing proficiency testing to ensure detection of circulating norovirus genotypes.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

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Detection of circulating norovirus genotypes: hitting a moving target

Rooney

Pettipas

Grudeski³

et al. 2014

Virol J

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“…In the absence of other assays with detection levels low enough to detect viruses directly, RT-PCR is the only published method that offers the possibility of direct detection of NoVs in environmental samples such as bivalve shellfish or other foods (Lees, 2000;Mattison et al, 2010). Its high sensitivity and accuracy enables the detection of as little as 10 virus copies.…”

Section: Detection Of Nov In Oystersmentioning

confidence: 99%

Scientific Opinion on Norovirus (NoV) in oysters: methods, limits and control options

Publication¹

2012

EFSA Journal

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NoV is highly infectious, and there is no threshold infectivity limit for NoV detected by PCR. The probability of becoming infected increases with the dose but depends also on the characteristics of the organism, the food matrix and the host factors. The relationship between the number of infectious virus particles and the number of virus genome copies detected by quantitative PCR is not a constant, and it is important to realise that the infectious risk associated with low level positive oysters as determined by real‐time PCR may be overestimated. Quantitative data on viral load from areas compliant with current EU legislative requirements (E. coli standards) during January‐March 2010 in 3 selected member states, show that a viral limit of 100, 200, 500, 1000 or 10.000 NoV PCR copies would result in 33.6‐88.9%, 24.4‐83.3%, 10.0‐72.2%, 7.7‐44.4% or 0‐11.1% of non‐compliant batches, respectively. Compliance with any of the above NoV limits would reduce the number of contaminated oysters placed on the market and therefore the risk for consumers to become infected. It is currently not possible to quantify the public health impact of different limits. Microbiological criteria for NoV in oysters are useful for validation and verification of HACCP‐based processes and procedures, and can also be used by competent authorities as an additional control to improve risk management in production areas, during processing and retail. The Panel recommended that risk managers should consider establishing an acceptable limit for NoV in oysters to be harvested and placed on the market. NoV testing of oysters (standardized CEN method) should be used to verify compliance with the acceptable NoV limit established. The most effective public health measure to control human NoV infection from oyster consumption is to produce oysters from areas which are not faecally contaminated, particularly given the ineffectiveness of current depuration and relaying procedures.

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“…Molecular assays for the detection of noroviruses are challenging not only because of the different subtypes but also because stool is the recommended sample for norovirus RNA extraction. [8][9][10] Cytomegalovirus (CMV) has the ability to establish lifelong latent infection following primary infection. Under certain conditions, CMV can reactivate producing severe CMV disease that is generally restricted to the immunocompromised or immunologically immature host.…”

Section: Introductionmentioning

confidence: 99%