Identification of enterovirus 71 hand, foot and mouth disease from other enteroviruses by microfluidic digital PCR

Enterovirus71 (EV71) is a major etiological agent of severe hand, foot, and mouth disease (HFMD), which can lead to central nervous system damage in children. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) often produces false-negative results during the early stage of EV71 infection, attributable to the low viral load. To address this limitation, we developed a rapid, precise, and highly sensitive multiplex reverse transcription digital polymerase chain reaction (RT-dPCR) assay for differential diagnosis of EV71-induced HFMD and non-EV71 pathogens. The EV71 RT-dPCR assay targeted the viral structural protein VP2 using a TaqMan minor groove binder (MGB) probe. This method achieved a detection sensitivity of 7.406 copies per microliter. Specificity analysis confirmed no cross-reactivity with non-EV71 enteroviruses. When validated against 60 clinical samples, the assay demonstrated 100% overall agreement (60/60) with a kappa value of 1.0 (p < 0.001). The developed EV71 RT-dPCR assay may facilitate early diagnosis of severe EV71-associated HFMD, enabling timely intervention for affected children.