【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。更多还原

【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 10⁴～0.1 TCID₅₀/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.更多还原