Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II
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Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three TaqMan real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit (assay A: Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays (assay B: LightCycler RNA Master Hybprobe and assay C: RealTime ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity (103 DNA copies/mL) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups. No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean quantification cycle (Cq) value of assay B for GII was lower than assays A and C with statistical significance (P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17, and GII.21. This study suggests assay B as a suitable assay for the detection and quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.
Keywordsnorovirus genogroup real-time RT-PCR quantification
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- Green KY. 2013. Caliciviridae: the noroviruses. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE. (Eds.), Fields Virology, 6th ed. Philadelphia: Lippincott Williams & Wilkins, pp. 582–608.Google Scholar
- Mattison K, Grudeski E, Auk B, Brassard J, Charest H, Dust K, Gubbay J, Hatchette TF, Houde A, Jean J, Jones T, Lee BE, Mamiya H, McDonald R, Mykytczuk O, Pang X, Petrich A, Plante D, Ritchie G, Wong J, Booth TF. 2011. Analytical performance of norovirus real-time RT-PCR detection protocols in Canadian laboratories. J Clin Virol 50: 109–113.CrossRefPubMedGoogle Scholar
- Neesanant P, Sirinarumitr T, Chantakru S, Boonyaprakob U, Chuwongkomon K, Bodhidatta L, Sethabutr O, Abente EJ, Supawat K, Mason CJ. 2013. Optimization of one-step real-time reverse transcription-polymerase chain reaction assays for norovirus detection and molecular epidemiology of noroviruses in Thailand. J Virol Methods, 194: 317–325.CrossRefPubMedGoogle Scholar
- Siebenga JJ, Vennema H, Zheng DP, Vinjé J, Lee BE, Pang XL, Ho EC, Lim W, Choudekar A, Broor S, Halperin T, Rasool NB, Hewitt J, Greening GE, Jin M, Duan ZJ, Lucero Y, O’Ryan M, Hoehne M, Schreier E, Ratcliff RM, White PA, Iritani N, Reuter G, Koopmans M. 2009. Norovirus illness is a global problem: emergence and spread of norovirus GII.4 variants, 2001-2007. J Infect Dis, 200: 802–812.CrossRefPubMedGoogle Scholar
- Wenzel JJ, Panning M, Kaul KL, Mangold KA, Revell PA, Luna RA, Zepeda H, Perea L, Vazquez-Perez JA, Young S, Rodic-Polic B, Eickmann M, Drosten C, Jilg W, Reischl U. 2010. Analytical performance determination and clinical validation of the novel Roche RealTime Ready Influenza A/H1N1 Detection Set. J Clin Microbiol, 48: 3088–3094.CrossRefPubMedPubMedCentralGoogle Scholar