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
This work was supported by research grant from the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. program (Grant No. PHD/0085/2554), and the Thai Government Budget through Mahidol University, fiscal year 2015‒2017. The authors thank The Language Center, Faculty of Graduate Studies, Mahidol University for editorial assistance.
KDR performed the experiments and analyzed the data. PCP and PPD participated in the experiments. LRK designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.
Compliance with Ethics Guidelines
All the authors declare that they have no competing interests. The study was approved by the Ethical Review Committee for Human Research, Faculty of Public Health, Mahidol University. The protocol complied with a “Research with Exemption” category.
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