Simultaneous detection of enterovirus 70 and coxsackievirus A24 variant by multiplex real-time RT-PCR using an internal control.

Epidemics of acute hemorrhagic conjunctivitis are always explosive and extensive, and have been recognized as a serious international public health problem. Enterovirus 70 and coxsackievirus A24 variant have been identified as the major etiological agents in acute hemorrhagic conjunctivitis outbreaks worldwide. A novel multiplex real-time RT-PCR assay was developed for simultaneous detection, identification and quantitation of enterovirus 70 and coxsackievirus A24 variant. The specificity, sensitivity and reproducibility of the method were analyzed and 125 clinical samples were tested using this method. No cross-reactivity with other enteroviruses strains was detected. The detection limits achieved were 10 copies/tube of enterovirus 70 and 100 copies/tube of coxsackievirus A24 variant respectively, and the addition of the internal control does not compromise the sensitivity or specificity. One hundred and twenty five clinical samples were tested and the results were consistent with the results obtained by using virus isolation followed by neutralization and sequencing of VP1 region. Due to its high specificity, sensitivity and elimination of false negative results by the internal control, this assay is suitable for both research applications and rapid clinical diagnosis of enterovirus 70 and coxsackievirus A24 variant.