MIRA/PfAgo-Mediated Biosensor for Multiplex Human Enteroviruses Virus Typing Detection on HFMD

Hand, foot, and mouth disease (HFMD), caused by enteroviruses, mostly including EV71, CVA6, CVA10, and CVA16, is an acute infectious disease commonly found in children. Due to no approved antiviral therapies and available vaccines, except for EV71, developing accurate diagnostic methods of HFMD is essential for controlling its spread and mitigating its impact on public health. Here, we create a MIRA-HEV-PAND multiple nucleic acid typing method that utilizes PfAgo to identify enterovirus type A pathogens (EV71, CVA6, CVA10, and CVA16) and universal type EVU. The MDC (minimum detection concentration) level of MIRA-HEV-PAND is within the range of 1.66 aM (1.0 copy/microL), which was matched to that of qPCR assays and even more sensitive up to 10%. Importantly, the MIRA-HEV-PAND method exhibits higher sensitivity and less time-consuming efficiency compared to the approach that combines PCR amplification instead of MIRA amplification. Meanwhile, though the quintuple and single-tube multiple MIRA-HEV-PAND detection system can be used for one viral target or multiple viral target detection, the single-tube detection system detects more efficiently and rapidly than the quintuple-tube multiple detection system. Moreover, the diagnostic results obtained by evaluating clinical samples using MIRA-HEV-PAND show a complete consistency of 100% with qPCR assays. The MIRA-HEV-PAND method can screen a wider range of target regions using low-cost guide DNA without being limited to PAM sequences, compared to the MARPLES based on the CRISPR-Cas12a. The utilization of this correlation can be beneficial for the application of molecular testing for clinical diagnoses and the study of human enteroviruses A infection and virus typing on an epidemiological scale.