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 (10 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.

Age, primary symptoms, and genotype characteristics of norovirus outbreaks in Shanghai schools in 2017

“…In the present study, the viral genogroup and genotype were determined by partial VP1 sequences; thus, we could not fully study the recombinant noroviruses. Although this method is commonly used 26 , 27 , increasing evidence has supported the proposal to adopt a dual nomenclature using both ORF1 and VP1 sequences 28 because recombination is common and the recognition of recombinant viruses may be relevant to their epidemiological characteristics 23 , 28 – 30 .…”

Diagnostic Accuracy of Immunochromatographic Tests for the Detection of Norovirus in Stool Specimens: a Systematic Review and Meta-Analysis

“…Generally, rRT-PCR can detect and quantify norovirus genomes, which provides rapid results and reduces the risk of carryover contamination ( 38 , 39 ). However, the sensitivity and specificity of rRT-PCR can vary, since noroviruses have very highly diverse genomes and different rRT-PCR protocols utilize different primers or probes and reagents and have different reaction conditions ( 65 – 68 ).…”

Clinical significance and intestinal microbiota composition in immunocompromised children with norovirus gastroenteritis

“…The reactions used 10-fold serial dilutions of norovirus GII.4 DNA as positive controls at starting concentrations of 10 8 DNA copies/mL. To evaluate the amplification efficiency of the real-time RT-PCR assays, standard curves were generated for NoV GII.4 DNA copy numbers versus Cq values [ 12 ]. The coefficient of determination (R2) in the linear regression analysis was 0.99, indicating a strong correlation between the copy number and Cq value.…”