荧光定量RT-PCR检测柯萨奇病毒A组6和10型的方法建立及应用

Construction and application of the real time RT-PCR methods for detecting Coxsackievirus A6 and A10

目的 建立柯萨奇病毒A组6（Coxsackievirus A6,CVA6）和10型（CVA10）的SYBRGreen Ⅰ荧光定量RT-PCR方法并进行评价及初步应用.方法 根据GenBank上的CVA6和CVA10序列,分别设计特异性引物,建立SYBR Green Ⅰ荧光定量RT-PCR方法体系,并分别进行灵敏度,重复性和特异性检测,然后利用该方法对649份2014年天津市未明确分型的肠道病毒阳性标本进行检测.结果 成功建立CVA6和CVA10的SYBR GreenⅠ荧光定量RT-PCR方法及定量标准曲线,其检出限均为101 copies/μL,重复性实验的变异系数均小于2％.该方法特异性强,与柯萨奇病毒（A组2、4、5、16型）、肠道病毒EV71型、诺如病毒、轮状病毒、麻疹病毒和乙型流感病毒均无交叉反应.利用该方法检测649份未分型标本,发现136份为CVA6阳性和174份为CVA10阳性,分别占未分型阳性标本的20.96％和26.81％.结论 本研究建立的检测CVA6和CVA10荧光定量RT-PCR方法灵敏度高、重复性好、特异性强,应用于2014年天津市手足口病监测,发现C VA6和CVA10已成为优势型病原体.

Objective To construct two SYBR Green Ⅰ real time RT-PCR methods to detect Coxsackievirus A6（CVA6） and A10 （CVA10）, and make the assessments and initial applications.Methods Specific primers were designed for CVA6 and CVA10 respectively, according to the CVA6 and CVA10 sequences published in GenBank.The SYBR Green Ⅰ real time RT-PCR methods for detecting CVA6 and CVA10 were constructed, respectively.The sensitivity, repeatability and specificity of the methods were assessed respectively.The newly developed methods were used to detect 649 unidentified enterovirus-positive specimens from Tianjin.Results The two SYBR Green Ⅰ real time RT-PCR methods for detecting CVA6 and CVA10, and their standard curves were all constructed successfully.Both detection limits of the two methods were all 101 copies/μL.The coefficients of variation of the repeatability tests were both less than 2%.Both methods were excellent in specificity.There was no nonspecific reactivity with Coxsackievirus A2,A4, A5, A16, enterovirus 71, norovirus, rotavirus, measles and inflluenza b virus.The new methods were used to detect 649 specimens.136 CVA6-postive and 174 CVA10-postive specimens were found, and accounted for 20.96% and 26.81% of total specimens, respectively.Conclusions The two real time RT-PCR methods were confirmed to be excellent in specificity, repeatability and sensitivity.CVA6 and CVA10 were found to be the dominant pathogens by using the two methods in the surveillance of hand-foot-mouth disease in Tianjin, 2014.