Evaluation of real-time RT-PCR assays for detection and quantification of norovirus genogroups I and II.

ABSTRACT

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.