A TaqMan probe-based multiplex real-time quantitative pcr for simultaneous detection of kobuvirus, parechovirus B, rosavirus B, and hunnivirus carried by murine rodents and shrews

Abstract

Background: Picornaviruses, common infectious agents in humans and various animal species, pose significant health threats. Conventional monoplex PCR is widely employed in laboratory diagnostics but is relatively time-intensive and laborious. Results: In this study, we developed a multiplex TaqMan probe-based real-time quantitative PCR (qPCR) assay for the rapid and simultaneous detection of kobuvirus, parechovirus B, rosavirus B and hunnivirus in murine rodent and shrew samples. The approach demonstrated high sensitivity and specificity, with detection limits of 1 × 102 copies/µL for kobuvirus, parechovirus B, and rosavirus B, and 50 copies/µL for hunnivirus. Evaluation using 149 clinical samples showed strong concordance with conventional PCR methods. Conclusions: This work developed an effective multiplex qPCR method for the simultaneous detection of emerging picornaviruses particularly in rodents, including kobuvirus, parechovirus B, rosavirus B, and hunnivirus. Our findings contribute valuable insights into the monitoring and prevention of zoonotic diseases associated with these pathogens.