检测肠道病毒71型RT-LAMP技术的建立及其应用

Establishment and application of RT-LAMP technique in detection of enterovirus 71 gene

目的为肠道病毒71型(Enterovirus 71,EV71)感染的快速诊断建立一种简捷、敏感、特异的基因检测方法,即逆转录环介导等温核酸扩增技术(RT-LAMP)。方法针对EV71VP1基因的8个区域设计6条特异性引物,建立检测该靶序列的RT-LAMP方法,对其特异性及敏感性进行了验证;并对手足口病患者标本进行检测,并与RT-PCR及Real-time PCR方法进行了比较。结果利用RT-LAMP方法能成功检测到EV71VP1基因区,等温条件下60min内即可完成检测,该方法简便快捷、敏感性高、特异性好;其阳性率高于RT-PCR方法(P<0.05),与Real-time PCR结果呈现很好的一致性(P>0.05)。结论 RT-LAMP方法具有灵敏度高,特异性强,操作过程简捷快速,无需特复杂仪器等优点。适用于EV71感染的快速检测及分子流行病学的监测,具有很好的开发应用前景。

A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification（RT-LAMP） method was established to provide a new rapid method for detecting Enterovirus 71 gene.A set of six specially primers that recognized eight distinct sequences of VP1 gene of EV71 were designed,RT-LAMP method was established,and the specificity and sensitivity of RT-LAMP were verified.The stool or throat specimens of patients infected with Hand Foot and Mouth Disease（HFMD） were tested by RT-LAMP technique,and the result was compared to that of RT-PCR and Real-time PCR.Results demonstrated that the RT-LAMP technique has been successfully detected VP1 gene of EV71,and showed a good consistency with Real-time PCR results but higher detection rates than RT-PCR method.It＇s suggested that RT-LAMP technique appeared to be high sensitivity and specificity,as well as simple and rapid.It is suitable for the diagnosis of HFMD and for molecular epidemiological surveillance of infection,and with good development prospects.