柯萨奇病毒A组5型实时荧光RT-PCR检测方法的建立与评价  

Establishment and evaluation of a real-time fluorescent RT-PCR method for detection of Coxsackievirus A5 RNA

作　　者：罗娟 田维霞 李永文 宋光敏 姚淼 陈佳丽 胡雨萌 刘娇阳 罗南宁 艾远航 吴凯峰 LUO Juan;TIAN Weixia;LI Yongwen;SONG Guangmin;YAO Miao;CHEN Jiali;HU Yumeng;LIU Jiaoyang;LUO Nanning;AI Yuanhang;WU Kaifeng(Department of Clinical Laboratory/Scientific Research Laboratory,Zunyi Medical University Third Affiliated Hospital(the First People′s Hospital of Zunyi),Zunyi,Guizhou 563000,China)

机构地区：[1]遵义医科大学第三附属医院(遵义市第一人民医院)检验科/中心实验室,贵州遵义563000

出　　处：《国际检验医学杂志》2023年第7期803-807,共5页International Journal of Laboratory Medicine

基　　金：贵州省科学技术厅省市科技合作项目(省市科合[2015]38号);贵州省教育厅创新群体研究项目(黔教合KY字[2021]019项目)。

摘　　要：目的建立柯萨奇病毒A组5型(CVA5)特异的一步法实时荧光反转录聚合酶链反应(RT-PCR)核酸检测方法。方法以流行于遵义地区的CVA5病毒株和NCBI基因库中随机下载的CVA5 VP1基因序列作为参考序列,分析这些序列的保守区域,在其保守区设计特异性的引物与Taqman探针,建立检测CVA5的一步法实时荧光RT-PCR。通过构建含CVA5 VP1保守区域的重组质粒,绘制标准曲线,并对检测方法的灵敏度、特异性、重复性及临床样本检出限进行评价。利用该方法对遵义地区273份未明确分型的肠道病毒阳性样本进行检测,将检出的部分CVA5阳性产物进行测序。结果成功建立一种基于实时荧光RT-PCR的CVA5检测方法,该方法在10^(4)~10^(9)copy/mL范围内具有良好的线性关系(R 2>0.999),平均扩增效率为102.26%。灵敏度高,对临床样本的检出限为103copy/mL。该方法的特异性强,与柯萨奇病毒A组2型(CVA2)、柯萨奇病毒A组4型(CVA4)、柯萨奇病毒A组6型(CVA6)、丙型肝炎病毒、呼吸道合胞病毒和鼻病毒等均不发生交叉反应。重复性实验的变异系数低于1.5%。273份未明确分型肠道病毒阳性样本中共检出25份CVA5,占未明确分型阳性样本的9.16%。结论该研究建立的一步法CVA5RT-PCR检测方法灵敏度高、特异性强、重复性好,可用于CVA5的检测。Objective To establish a specific one-step real-time fluorescent reverse transcription pdymeease chain reaction(RT-PCR)method for the detection of Coxsackieviruses A5(CVA5)RNA.Methods The CVA5 virus strain prevalent in Zunyi and the CVA5 VP1 gene sequence randomly downloaded from the NCBI gene library were taken as the reference sequence,the conserved regions of these sequences were analyzed,and specific primers and Taqman probes were designed in the conserved regions to establish a one-step real-time fluorescent RT-PCR for detecting CVA5.By constructing the recombinant plasmid containing the conserved region of CVA5 VP1,the standard curve was drawn,and the sensitivity,specificity,repeatability and detection limit of clinical samples were evaluated.Then 273 enterovirus positive samples without clear typing in Zunyi were detected by this method,and the detected CVA5 positive products were sequenced.Results A new method for CVA5 detection based on real-time fluorescence RT-PCR was successfully established.The method had good linearity in the range of 10^(4)~10^(9) copies/mL(R 2>0.999),and the average amplification efficiency of the method was 102.26%.The sensitivity was high,the detection limit was 103 copies/mL.This method had strong specificity and did not cross react with CVA2,CVA4,CVA6,hepatitis C virus,respiratory syncytial virus and rhinovirus.The coefficient of variation of repeatability experiments was lower than 1.5%.A total of 25 cases of CVA5 were detected in 273 positive samples of without clear typing,accounting for 9.16%of the positive samples of without clear typing.Conclusion The one-step CVA5 real-time fluorescence RT-PCR method established in this study has high sensitivity,specificity,strong repeatability and could be used for the detection of CVA5.

关 键 词：柯萨奇病毒A5 实时荧光反转录聚合酶链反应 肠道病毒 手足口病 疱疹性咽峡炎 

分 类 号：R446.6[医药卫生—诊断学]