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