Establishment and Application of a Digital PCR Method for Coxsackievirus A6

Background The emergence of Coxsackievirus A6 recombinant strains may result in a significant public health burden due to hand, foot, and mouth disease. Traditional quantitative Polymerase Chain Reaction (qPCR) methods are widely used for laboratory diagnosis but have low sensitivity and cannot provide accurate quantification. This study established and evaluated a droplet digital Polymerase Chain Reaction (dPCR) method for the detection of Coxsackievirus A6, aiming to achieve precise quantitative detection of pathogen detection. Methods Specific primers and probes for Coxsackievirus A6 were synthesized, and a plasmid standard was used. The dPCR method was optimized by adjusting primer concentration, probe concentration, and cycle number. The evaluation of method specificity, sensitivity, reproducibility, and 47 clinical sample testing was conducted, and the performance was compared with the results of real-time fluorescent quantitative Polymerase Chain Reaction. Results A droplet dPCR method for Coxsackievirus A6 was successfully established, with a lower limit of detection ranging from 5.23 to 10.55 copies/µL. There was no cross-reactivity with other enteroviruses besides Coxsackievirus A6. Repeated measurements of samples above the minimum detection concentration showed a relative standard deviation value of less than 25%, demonstrating excellent specificity and reproducibility. Testing 47 clinical samples using the optimized dPCR method, with qPCR as the gold standard, showed that the true positive rate was 92.86%, the true negative rate was 100%, and the Kappa coefficient was 0.91. Conclusions This study successfully developed an absolute quantification method for Coxsackievirus A6 nucleic acid based on droplet dPCR technology. The method enables accurate identification of Coxsackievirus A6 with a low detection limit, making it suitable for detecting low-concentration samples. It is stable, reliable, and exhibits excellent reproducibility. This method holds significant potential for nucleic acid detection and quantitative analysis of Coxsackievirus A6 and provides technical support for the epidemiological study of Coxsackievirus A6 in community sewage. It shows promising applications in clinical diagnosis and viral monitoring.