Development of a Multiplex TaqMan Real-Time RT-PCR Assay for the Rapid Differential Detection of Classic, MLB-Clade and VA-Clade Human Astroviruses

The classical human astrovirus (HAstV) causes acute or severe gastroenteritis mainly among children and the elderly populations. The novel divergent HAstV-VA and HAstV-MLB clades related to encephalitis in immunocompromised patients, are genetically more similar to certain animal astroviruses, and have the potential for cross-species transmission. To identify all the three HAstV clades in a single reaction, we developed a quantitative reverse transcription PCR (RT-qPCR) method. Primers and probes were designed based on the conserved regions of the HAstV genomes that exhibited inter-clade divergence. This method was highly sensitive, as the detection limits were 95, 12.5 and 25 copies/μl for the classical HAstV, HAstV-MLB and HAstV-VA strains, respectively. Intra- and inter-assay variability revealed excellent reproducibility. Furthermore, this multiplex assay showed no cross-reactivity with other human pathogens. A total of 326 anal swabs from outpatients with AGE were examined: 5.83% were positive for the classical HAstV, 1.53% for the HAstV-MLB, and 0.31% for the HAstV-VA, while 0.31% for co-infections of HAstV-MLB/HAstV-VA. Conclusively, the developed multiplex RT-qPCR assay represents a tool with potential for laboratory and clinical diagnoses, epidemiological surveillance, prevention and control of the HAstV infection.