多重荧光RT-PCR方法检测手足口肠道病毒方法的研究

    周冬根，孙大为，王燕，倪敏君，张升，翟敏

    宁波国际旅行卫生保健中心，浙江 宁波 315012

    摘要：目的 为了解手足口病的流行和感染情况，并进行快速准确的检测。方法本研究建立了含非竞争性内标的同时检测肠道病毒通用型、肠道病毒EV71型及柯萨奇病毒CA16型的四重荧光RT-PCR方法,对该方法的特异性、灵敏度等进行评价，并对多份临床样本进行了应用检测。结果实验结果表明，该检测方法特异性强，对肠道病毒及其他人类非肠道病毒进行了检测，显示了良好的特异性；该检测方法对EV71型和CA16型的检测灵敏度分别达到31.25TCID50和1.25×102 TCID50；将浓度为1×104 TCID50及5×102TCID50的EV71样本进行重复性实验，其变异系数均小于1.5%；将浓度为5×102~5×105TCID50的EV71和CA16样本进行定量范围实验，其相关系数R2值在0.982~0.998之间。采用本研究建立的方法检测40份疑似临床样本，最后检出31份肠道病毒阳性样本，检出率为77.5%，其中8例为EV71型阳性，13例为CA16型阳性。另外，实验数据表明内标对监控PCR抑制物的存在具有重要作用。结论本方法能同时快速检测所有肠道病毒并进行EV71型及CA16型的分型，并且灵敏度高、特异性好、扩增效率高，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。

    关键词：四重荧光RT-PCR；非竞争性内标；手足口病；肠道病毒；检测

    中图分类号：R183.4　文献标识码：B

    Research on multiplex real-time PCR detectionof enteroviruses of

    hand, foot and mouth disease

    ZHOU Dong-gen， SUN Da-wei， WANG Yan， NIMin-jun， ZHANG Sheng， ZHAI Min

    Ningbo International Travel HealthcareCenter, Ningbo, Zhejiang 315012,China

    Abstract:   Objective  To understand the status of hand, foot and mouth disease prevalenceand infection, and to detect HFMD enterovirus simultaneously. Methods   An up to 4-plex real-time PCR with anon-competent internal control (IC) was developed to detect genevaltype EV71 and CA16. The specificity, sensitivity, reproducibilityand linearity of the real time RT-PCR assay were estimated andabout 40 clinical samples were tested.  Results  The results showed good specificity for the selected virus. Thesensitivity of samples for EV71 and CA16 were 31.25 TCID50 and 1.25TCID50, respectively. Analysis with 1×104 TCID50 and 5×102 TCID50EV71 samples demonstrated high reproducibility with a coefficientof variation (CV) below 1.5%. The linearity analysis with5×102~5×105 TCID50 for EV71 and CA16 showed a good correlation andamplification efficiency. R2 value of the correlation coefficientwas in 0.982~0.998. A total of 40 suspected clinical samples weredetected by real-time PCR, the results showed that the positiveratio was 77.5% (31/40). Moreover, the result indicated that therewas PCR inhibition in some samples. The inhibition in these samplesshowed the importance of IC when PCR was used to detect the RNA ofEV71, CA16 or other enterovirus.  Conclusion   As aresult of its high specificity, sensitivity, linearity and avoidingfalse negative results by using an internal control, the assay issuitable for rapid clinical diagnosis of human enterovirus andgenotyping of EV71 and CA16.

    Key words:   Multiplex real-timeRT-PCR; Non-competent internal control; Hand, foot and mouthdisease; Enterovirus; Identify

    《中国国境卫生检疫杂志》2012年第6期