Evaluation of Two Primer Sets Using Newly Developed Internal Amplification Controls for Rapid Human Norovirus Detection by SYBR Green I Based Real-Time RT-PCR

Abstract

Rapid detection of emerging virulent human noroviruses (Genogroups I and II) from clinical and food samples remains an on-going challenge. Development of internal amplification controls (IACs) in real-time RT-PCR assays to eliminate false negatives due to sample inhibition or reaction failure is critical. RNA IACs were constructed for application with two sets of previously described highly reactive degenerate primers (MON and COG) for the detection of human noroviruses (HNoVs) GI and GII. These primer sets were compared for detection sensitivity of HNoVs from outbreak stool samples (6 GI and 9 GII) by SYBR Green I based real-time reverse-transcriptase (RT)-PCR. In order to detect viruses directly from stool samples, heat release was used to expose the viral RNA (95°C, 10 min). PCR conditions were optimized for each primer set before and also after IAC addition to obtain similar detection limits. Both primer sets showed equal detection limits for GII strains (4 log RT-PCR U/sample) and with one-log higher detection for GI strains (10-5 end-point dilution; 5 log RT-PCR U/sample). The melt temperature (Tm) of the COG and MON IAC were 83°C (155 bp) and 83.5°C (150 bp), respectively. Product Tm were 84°C (using MON primers for both genogroups) and 81.5°C (COG for GI) and 84°C (COG for GII). Agarose gel electrophoresis determined product sizes as 85 and 98 bp for GI and GII with COG, respectively, and 213 bp with MON for both genogroups. From our study, COG primers appear to have a broader detection range than the MON primers, using the 15 tested stool samples. This assay can potentially be implemented for routine HNoV detection from clinical and food samples. © 2011 Springer Science + Business Media, LLC.