An RT-qPCR Assay for Rapid Detection of Coxsackievirus A4

Coxsackievirus A4 (CV-A4) is a major cause of hand, foot, and mouth disease and herpangina in children, and has been linked to severe complications. However, current detection methods, including commercial RT-qPCR kits, suffer from false-negative and false-positive results. This study aimed to develop a sensitive and specific TaqMan-based RT-qPCR assay for the rapid detection of CV-A4, targeting the conserved VP1 region covering genotypes A-D and subgenotypes C1-C5, with emphasis on the predominant C2 subgenotype in China. The assay was evaluated using a panel of enteroviruses, influenza virus, adenovirus, and rhinovirus, and compared with two commercial kits using 82 CV-A4-positive and 100 negative clinical specimens. The established assay demonstrated high specificity with no cross-reactivity, a detection limit of 2.1 TCID₅₀ equivalents/mL, and excellent reproducibility (CV ≤ 3.03%). In clinical testing, it achieved a 100% detection rate (82/82) with no false positives within 1.5 h, outperforming the two commercial kits, which showed positive rates of 91.5% and 89.0%, and false-positive rates of 0% and 1%, respectively. These findings indicate that our RT-qPCR assay offers superior sensitivity and specificity for CV-A4 detection, particularly for the C2 subgenotype, and represents a promising tool for laboratory diagnosis and epidemiological surveillance.