[采用含内对照的多重实时荧光定量PCR法同时检测肠道病毒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 control].

作者信息

Xiao Xinglong, He Yaqing, Yu Yigang, Yang Hong, Li Huifang, Yang Xiaoquan, Zhang Jingwei, Chen Gu, Liu Dongmei, Li Xiaofeng, Wu Hui

机构信息

Institut of Food Safety, Department of Food Science, College of Light Industry & Food Science, South China University of Technology, Guangzhou, China.

出版信息

Wei Sheng Wu Xue Bao. 2009 Jan;49(1):98-104.

PMID:19388271

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 10(4) - 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.

摘要

目的

同时快速鉴定肠道病毒71型（EV71）和柯萨奇病毒A16型（CA16）。

方法

建立了一种带有内对照（IC）的多重实时荧光定量PCR方法。对实时荧光定量逆转录PCR检测方法的特异性、敏感性、重复性进行评估，并对400余份临床样本进行检测。

结果

结果显示该方法对所选检测组的特异性为100%。该检测方法对CA16每毫升样本的敏感性达到50%组织培养感染剂量（TCID50），对EV71为0.1 TCID50。对10⁴ - 0.1 TCID50/mL的EV71或CA16样本进行分析，显示出高重复性，EV71的变异系数（CV）为0.9% - 2.0%，CA16为0.9% - 2.3%。通过实时荧光定量PCR检测了400余份临床样本，结果显示EV71和CA16的平均阳性率分别为46.1%和14.2%，高于常规检测方法（34.5%和12.8%）。此外，结果统计表明粪便、直肠拭子和咽拭子标本中存在PCR抑制，抑制率为1.8%至3.4%。这些样本的抑制率表明，在使用PCR检测EV71、CA16或其他肠道病毒RNA时，内对照的重要性。