双重实时荧光RT-PCR法检测肠道病毒方法的建立及应用

Establishment and application of dual real-time fluorescent RT-PCR method for detection of Enterovirus

目的建立检测肠道病毒(EV)和肠道病毒71型(EV71)的双重实时荧光RT-PCR法。方法设计特异性引物和探针,建立双重实时荧光RT-PCR反应体系,绘制标准曲线,对该方法的灵敏度、精密度等进行评价。收集109例临床手足口病患者粪便和咽拭子标本用该法进行检测,并与EV71商品化试剂盒检测结果进行比较。结果双重荧光PCR法建立的EV和EV71标准曲线相关系数(r)均为0.998;该法检测EV和EV71的灵敏度分别达到0.5TCID50/mL和0.05TCID50/mL;检测EV和EV71的批内精密度均小于3%,总精密度均小于或等于4%;对肠道病毒及其他人类非肠道病毒进行检测,显示了良好的特异性。109例临床样本用该法检测出84例EV病毒阳性样本,其中EV71阳性56例,EV71总阳性率为51.4%;与单重荧光PCR商品化试剂盒的检测结果完全一致(P=1.000)。结论建立的双重实时荧光RT-PCR法灵敏度高、稳定性好,对手足口病的早期高通量快速诊断具有重要意义。

Objective To develop a dual real-time fluorescent RT-PCR method for rapid detection of enterovirus（EV）and enterovirus type 71（EV71）.Methods Specific primers and probes were designed and the dual real-time fluorescent RT-PCR reaction system was established.The quantitative standard curve was drawn;its sensitivity and precision were evaluated.Feces and throat swab specimens of 109 clinical patients with hand foot and mouth disease were collected and tested by using this method.Then the obtained results were compared with those detected by commercial EV71 PCR kit.Results The relative coefficient（2）of EV and EV71 standard curve established by the dual real-time fluorescent RT-PCR method were both 0.998.Its sensitivity reached 0.5TCID50/mL for detecting EV and 0.05TCID50/mL for detecting EV71.The within-run precision for detecting EV and EV71was3% and total precision≤4%.The results showed good specificity for the detection of enterovirus and non-enterovirus.In 109 detected clinical samples,84 cases of EV positive samples were detected,in which 56 cases were EV71 positive with the total positive rate of 51.4%,which was consistent with the result of simple fluorescent RT-PCR commercialization kit（P=1.000）.Conclusion The established dual real-time fluorescent RT-PCR method has high sensitivity and good stability,which has an important significance for early high throughput rapid diagnosis of hand foot and mouth disease.