Highly sensitive and rapid determination of Coxsackievirus A16 using restriction endonuclease-mediated reverse transcription multiple cross displacement amplification.

BACKGROUND: As a major etiological agent of hand, foot, and mouth disease in humans, Coxsackievirus A16 (CVA16) exerts a detrimental impact on the health of infants and toddlers. In the absence of effective antiviral drugs and vaccines targeting CVA16, a diagnostic strategy with sensitivity, specificity and rapid response is indispensable for the prevention and management of CVA16 infection.

RESULTS: In this study, a novel molecular diagnostic approach, coupling real-time fluorescence technique with reverse transcription multiple cross-displacement amplification (RT-MCDA), was developed for the sensitive, rapid, and specific identification of CVA16 (termed CVA16-E-RT-MCDA). To construct the system, a unique set of CVA16-E-RT-MCDA primers was successfully designed targeting the VP1 gene of CVA16, and the optimal conditions for CVA16-E-RT-MCDA were determined to be 63 ℃ for 35 min. The limit of detection for the CVA16-E-RT-MCDA assay was 14 copies/µL (approximately 70 copies per reaction) for the RNA standard template of CVA16. The CVA16-E-RT-MCDA assay exhibited high specificity and anti-interference. The findings demonstrated that the applicability of the CVA16-E-RT-MCDA method was promptly and successfully confirmed using clinical samples from patients with suspected CVA16 infections.

CONCLUSIONS: The CVA16-E-RT-MCDA assay established in the present study exhibits sensitivity, specificity and rapidity for CVA16 detection, rendering it a promising diagnostic tool for clinical applications.