EV71、CA16和肠道病毒通用型三重荧光RT-PCR检测技术的建立

A study on the development of a triplex real-time RT-PCR for simultaneous detection of enteroviruses and differentiation of EV 71 and CA 16

目的建立-种可以同时检测EV71、CA16和肠道病毒通用型的三重荧光RT-PCR检测方法,为手足口病监测与应急诊断提供高通量快速检测技术.方法分别在VP1和5’ UTR基因保守区设计EV71、CA16和肠道病毒通用型的特异性检测引物以及Taqman探针,建立并评估三重荧光RT-PCR技术的特异性和敏感性,并对91份疑似手足口病患儿临床标本进行核酸检测与效果验证.结果建立的三重荧光RT-PCR技术可同时对EV71、CA16和肠道病毒通用型进行特异性检测区分,敏感性均可达到0.1TCID50/mL,从核酸提取至完成检测仅需2～3h.91份疑似手足口病患儿临床标本采用三重荧光RT-PCR方法检测,EV71、CA16和其他型别肠道病毒的检出率分别为30.77%、9.89%和5.49%;荧光RT-PCR方法的检出率高于培养分离法.结论建立的三重荧光RT-PCR技术能同时准确分型EV71、CA16和其他型别肠道病毒.

Objective To develop a multiplex real -time RT -PCR assay for simultaneous detection of enteroviruses and differentiation of EV71 and CA16.Methods Specific primers and probes were designed for enteroviruses,EV71 and CA16.The probes labeled with various fluorescent reporter dyes,and a triplex real -time RT -PCR technique was developed to simultaneously detect these viruses.A total of 91 clinical specimens with suspected HFMD were analyzed by this method.Results This assay could simultaneously detect enterovirus and differentiation of EV71 and CA16,and the sensitivity of the assay was up to 0.1 TCID50 /mL,and only need 2 to 3 hours for completing the detection.A total of 91 clinical specimens were detected by this assay in 28 of the 91(30.77%)specimens contained EV71,9 of the 91(9.89%) contained CA16,and 5 of the 91 (5.49%)contained other enteroviruses.Conclusion This assay would be a useful molecular diagnostic tool for large -scale screening of clinical samples,especially at the peroid of HFMD outbreaks.