内标多重荧光RT-PCR同时检测肠道病毒71型和柯萨奇病毒A16型

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

摘要:

【目的】为对当前爆发的手足口病进行快速准确的检测， 【方法】本研究建立了含内标的同时检测EV71和CA16的多重荧光RT-PCR方法，对该方法的特异性、灵敏度等进行评估，并对400多份临床样品进行了检测。【结果】实验结果表明，该检测方法特异性强，对10株EV71病毒、8株CA16病毒和25株其他人类病毒进行了检测，特异性为100%；该检测方法对EV71和CA16的检测灵敏度分别达到0.1 TCID50和1 TCID50；将0.1-104TCID50/ml EV71和CA16样本进行重复性实验，其变异系数分别为0.9-2.0%和0.9-2.3%。对400多份临床样品分别进行荧光RT-PCR检测和传统方法检测，结果显示，荧光RT-PCR对EV71和CA16的阳性检出率平均为46.1%和14.2%，比传统方法（34.5%和12.8%）的阳性检测率高。另外，实验数据显示，在粪便、直肠拭子、咽喉拭子样本中，PCR抑制物存在的比例为1.8%-3.4%，表明内标对监控PCR抑制物的存在具有重要作用。【结论】本方法能同时对EV71和CA16进行快速检测，并且灵敏度高，特异性好，由于加入了内标，能有效地监控假阴性的出现，适合于手足口病的临床检测。

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.