人3型副流感病毒滴度TaqMan实时荧光定量RT-PCR检测方法的建立  

Development of a TaqMan real-time RT-PCR for the rapid titer detection of human parainfluenza virus type 3

作　　者：关文竹 王云瑾 火文 王名强 马超 李雄雄 包红 寇桂英 白慕群 GUAN Wen-zhu;WANG Yun-jin;HUO Wen;WANG Min-qiang;MA Chao;LI Xiong-xiong;BAO Hong;KOU Gui-ying;BAI Mu-qun(Second Department of Research,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司第二研究室,甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2021年第4期30-37,共8页Progress In Microbiology and Immunology

摘　　要：目的利用TaqMan实时荧光RT-PCR(real-time RT-PCR)技术建立测定人3型副流感病毒培养物滴度的方法。方法根据人3型副流感病毒HN基因的保守序列设计引物及探针,采用10倍系列稀释的体外转录HN RNA为模板,建立定量标准曲线。验证方法的专属性、敏感性、重复性,并对人3型副流感病毒的病毒培养液滴度进行检测。结果建立的方法对HN RNA标准品的检出灵敏度为4.7 copies/μL,标准曲线相关系数为0.998,扩增效率为105%。标准品的实验内重复性CV均<0.50%,实验间重复性CV<3.60%。系列稀释HNRNA标准品在低温(-80℃)下保存3年后,所测标准曲线的斜率和截距差异均无统计学意义(P=0.673)。用该方法对人3型副流感病毒分离株LZ17、LZ1501和LZ1728进行检测,结果均为阳性,3个病毒分离株滴度检测重复性CV均<10.00%;对7种呼吸道非人3型副流感病毒病原体检测均为阴性,表明该方法具有很高的专属性、敏感性和重复性。结论建立的TaqMan探针实时荧光定量RT-PCR技术可以快速、准确地对人3型副流感病毒滴度进行检测。Objective To develop a rapid method for titer detection of HPIV-3. Methods Primers and probe specific for HN gene of HPIV-3 were designed, and in vitro transcripted HN RNA was serially diluted as the standard RNA, a TaqMan probe fluorescence real-time quantitative RT-PCR was established. The specificity, sensitivity and stability of the assay were assessed. Results The correlation coefficient of the standard curve generated with in vitro transcripted HN RNA was 0.998, the PCR efficiency was 105% and the minimum detection limit of this method was 4.7 copies/μL. This method was used to detect three HPIV-3 isolates and seven other respiratory pathogens, the results were all positive for HPIV-3 isolates and were all negative for others. There was no significant difference in the slope and intercept of the standard curve after the serial diluted HN RNA standardwas stored at low temperature(-80 ℃) for 3 years. Conclusion This method for titer detection of HPIV-3 had higher specificity, sensitivity and repeatability.

关 键 词：人3型副流感病毒 实时荧光PT-PCT 滴度检测 标准曲线 方法建立 

分 类 号：R373.19[医药卫生—病原生物学]