Evaluation and improvement of real-time PCR methods for detecting norovirus

Abstract

Noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time RT-qPCR is currently the gold standard for sensitive and accurate detection for noroviruses and serves as a critical tool for outbreak investigations. However, different surveillance teams may use different assays and the variability of specimen conditions may produce disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCRs robustness in the context of norovirus detection as well as the investigation of practical strategies to enhance assay performance. Four real-time reverse transcription quantitative PCR (RT-qPCR) assays (Assays A-D) were run in parallel to evaluate the performance of different real-time RT-qPCR assays in the detection of norovirus genogroups I and II. Characteristics such as PCR efficiency and limits of quantification and detection were investigated to assess the precision and analytical sensitivities of the assays. RT-PCR assays were also performed to investigate the comparative sensitivity and specificity of the real-time RT-qPCR assays. Overall, Assay D was evaluated to be the most precise and accurate assay in the detection of both norovirus GI and GII. A Zen internal quencher was placed within the probe sequence of Assay D, which further improved the detection range of the assay. This study compared several detection assays for noroviruses and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.