Optimization of one-step real-time reverse transcription-polymerase chain reaction assays for norovirus detection and molecular epidemiology of noroviruses in Thailand

Abstract

Noroviruses (NoVs) are an important human pathogen associated with acute viral gastroenteritis worldwide. NoVs display a significant amount of genetic heterogeneity, making it difficult to develop comprehensive detection assays. In this study, primer sets and probes were designed for a TaqMan®-based real-time reverse transcription-polymerase chain reaction (RT-PCR) for norovirus detection purposes. The assay was optimized and utilized as a multiplex real-time RT-PCR assay for genogroup I (GI) detection, and a singleplex real-time RT-PCR assay for genogroup II (GII) detection. The assays showed high specificity for NoV detection and no cross-reactivity was observed between GI and GII. The detection limit of the assay was as low as 10 and 50 RNA copies per reaction for GI and GII, respectively. The optimized protocol was employed to assess the presence of NoV strains in clinical samples collected throughout Thailand during December 2005 to November 2006. The percentage of NoV infections among children with acute gastroenteritis (case) was 23.8% (119/500) and for children without acute gastroenteritis (control) it was 6.8% (30/441). The frequency of NoV infections varied geographically, with the highest frequency observed in the central region and the lowest frequency in the northern region (P>0.0001). Of the 149 positive case and control specimens, GII was found to be the predominant genogroup (98.6%). Partial capsid sequences were successfully obtained from 67 NoV-positive specimens and a phylogenetic analysis was performed to genotype the viral strains. GII.4 was the most common genotype detected.