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。
[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 TCIDs0 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.
出处
《微生物学报》
CAS
CSCD
北大核心
2009年第1期98-104,共7页
Acta Microbiologica Sinica
基金
国家自然科学基金(20676042)~~
