肠道病毒 71 型逆转录环介导等温扩增检测方法的建立与评价。

Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71.

机构信息

Department of Clinical Laboratory, The Third Affiliated Hospital of Suzhou University, No, 185 Juqian street, Changzhou, Jiangsu 213003, PR China.

出版信息

BMC Infect Dis. 2011 Jul 18;11:197. doi: 10.1186/1471-2334-11-197.

BACKGROUND

Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China. It is difficult to distinguish between EV71 and coxsackievirus A16 (CVA16) infections in clinical HFMD patients. Routine laboratory diagnosis of EV71 infection is time-consuming and requires expensive instruments. In this study, we have developed a one-step, single tube, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of EV71.

METHODS

Six primers that can recognize 6 distinct regions on the VP2 gene of EV71 were designed for RT-LAMP assay. The amplification was completed by incubating all reagents in a single tube with reverse transcriptase and Bst DNA polymerase under the isothermal condition (60°C) for 60 min, and could be evaluated by using GoldView staining under a handheld ultraviolet torch lamp or electrophoresis analysis.

RESULTS

A total of 123 specimens collected from suspicious patients with HFMD were simultaneously detected by RT-LAMP and PCR fluorescence probing assay. The RT-LAMP amplified products containing EV71 were digested by HinfI and TaqI restriction endonucleases; in contrast, non-specific products with CVA16, coxsackievirus A4 and coxsackievirus B3 could not be detected in RT-LAMP assay. Meanwhile, RT-LAMP assay could amplify EV71 virus with a detection limit of 1 PFU/ml within 60 min. Compared with PCR fluorescence probing assay, RT-LAMP assay exhibited 98.4% identity during the detection of EV71 viral RNA without the missing of positive samples.

CONCLUSION

Our results indicated that RT-LAMP is a rapid, sensitive, specific and accurate method for the detection of EV71 in clinical specimens. Therefore, this developed method has potential application for rapid and comprehensive surveillance for EV71 infection, especially in developing country.

背景

在中国，由肠道病毒 71 型（EV71）引起的手足口病（HFMD）非常常见。在临床 HFMD 患者中，EV71 与柯萨奇病毒 A16（CVA16）感染很难区分。常规实验室诊断 EV71 感染既费时又需要昂贵的仪器。在本研究中，我们开发了一种一步、单管、逆转录环介导等温扩增（RT-LAMP）检测方法，用于快速、灵敏地检测 EV71。

方法

设计了针对 EV71 VP2 基因 6 个不同区域的 6 个引物，用于 RT-LAMP 检测。通过在单个管中孵育所有试剂，在逆转录酶和 Bst DNA 聚合酶的作用下，在等温条件（60°C）下孵育 60 分钟，即可完成扩增，并且可以通过在手持紫外线灯下使用 GoldView 染色或电泳分析进行评估。

结果

共对 123 份疑似 HFMD 患者的标本同时进行了 RT-LAMP 和 PCR 荧光探针检测。用 HinfI 和 TaqI 限制性内切酶消化含有 EV71 的 RT-LAMP 扩增产物；相反，RT-LAMP 检测不到含有 CVA16、柯萨奇病毒 A4 和柯萨奇病毒 B3 的非特异性产物。同时，RT-LAMP 检测法可在 60 分钟内以 1 PFU/ml 的检测限扩增 EV71 病毒。与 PCR 荧光探针检测相比，RT-LAMP 检测法在检测 EV71 病毒 RNA 时具有 98.4%的一致性，没有漏检阳性样本。