Simultaneous detection of enterovirus 71 and coxsackievirus A16 by multiplex real-time PCR with an internal amplification control

Abstract:

[Objective] To rapidly identify EV71 and CA16 simultaneously. [Methods] A multiplex real-time PCR with an internal control (IC) was developed. The specificity, sensitivity, reproducibility of the real time RT-PCR assay were estimated and more over 400 clinical samples were tested. [Results] The results showed 100% specificity for the selected panel. The assay met the sensitivity of 50% tissue culture infective dose (TCID50) per milliliter samples for CA16 and 0.1 TCID50 for EV71. Analysis with 104-0.1TCID50/mL EV71 or CA16 samples demonstrated high reproducibility with a coefficient of variation (CV) of 0.9-2.0% for EV71 and 0.9-2.3% for CA16. More than 400 clinical samples were detected by real-time PCR, The results showed that the average positive ratio for EV71 and CA16 were 46.1% and 14.2%, higher than common assay (34.5% and 12.8%). Moreover, the result statistics indicated that there were PCR inhibition in stools, rectal swabs and throat swabs specimens with inhibition ratio from 1.8% to 3.4%. The inhibition ratio of these samples showed the importance of IC when PCR was used to detect the RNA of EV71, CA16 or other enterovirus. [Conclusion] As a result of its high specificity, sensitivity and avoiding false negative results by using an internal control , the assay is suitable for rapid clinical diagnosis of EV71 and CA16.