Simultaneous Detection of Selected Enteric Viruses in Water Samples by Multiplex Quantitative PCR

Human rotavirus (HRV), adenovirus (HAdV), and norovirus (HNV) are the most common causes of viral gastroenteritis in humans worldwide. Application of quantitative PCR (qPCR) coupled with reverse transcription (RT), known as RT-qPCR, has greatly improved detection sensitivity for enteric viruses, but it has been mostly used in monoplex mode where only a single virus type can be quantified per assay. Here, we report the development of a simple and specific multiplex RT-qPCR assay for simultaneous quantification of rotavirus, adenovirus, and norovirus in ground water, river water, and wastewater samples. For all three virus groups, the monoplex and multiplex RT-qPCR yielded virtually identical PCR efficiency, dynamic quantification range, and detection sensitivity, indicating the reliability of the multiplex RT-qPCR assay for simultaneous quantification of three viruses. The multiplex assay also accurately quantified the target genes of a small number (40 copies per PCR) in the presence of competing viral genes of up to 10(4)-fold more abundant. Specificity of the assay was estimated to be 100 % for all three viruses when tested against 19 common waterborne microorganisms. The results showed that our multiplex RT-qPCR assay holds considerable promise in quantifying the three enteric viruses in environmental water samples.