Real-time TaqMan RT-PCR快速检测犬瘟热病毒方法的研究

Study on Rapid Test Method for Canine Distemper Virus by Real-time TaqMan RT-PCR

按照犬瘟热(CDV)N基因序列,设计合成了特异性引物和探针,经各反应条件的优化,建立了Real-time荧光定量RT-PCR技术,对细胞培养物、肝脏、肺脏、脑、脾脏、淋巴结以及鼻腔拭子等组织病料中的CDV进行了特异性检测和敏感性试验。同时,利用建立的Real-time荧光定量RT-PCR方法与常规RT-PCR以及韩国BIOINDIST生产的BIT RAPID CDV检测试剂盒对57份临床样品进行了检测。结果:用20pmol/mL的引物浓度各1uL和20pmol/mL的探针浓度0．3uL,获得的荧光信号最强,曲线平滑。敏感性高,可检测到1．24×10—3ng/uL的病毒RNA;特异性强,与NDV、AIV、NiPV等RNA病毒不发生交叉反应。试验重复性的变异系数(CV)分别为2．3%、2．5%和4．2%;与常规RT-PCR和BIOINDIST生产的BIT RAPID CDV检测试剂盒相比较,该方法具有快速、特异、敏感、可定量,并可同时检测大量样品等优点。

Objective： Develop a Real-time TaqMan RT-PCR to test CDV samples rapidly and accurately. Methods ： Specific primer and probe were designed and synthesized according to CDV N gene. After optimization, a Real-time TaqMan RTPCR was established. Then specificity and sensitivity tests were done by detecting CDV in cell medium, liver, lung, brain, spleen, lymph node and nasal cavity swab. Simultaneously, 57 clinical samples were detected by Real-time RT-PCR method and routine RT-PCR with BIOINDIST BIT RAPID CDV kit. Results： Powerful fluoescence signal and smooth curve were gained with luL primer with a concentration of 20pmol/mL and 0.3uL probe with a concentration of 20pmol/mL. It is of high sensitivity, testing RNA concentration as low as 1.24×10^-3ng/uL; and it is of high specificity, with no cross reaction to NDV, AIV and NiPV. The coefficient variation （CV） of test repeatability was 2.3%, 2.5% and 4.2%, respectively. Conclusion： This method, Real-time TaqMan RT-PCR, is rapid, specific, sensitive, and it can test large quantity of samples in meanwhile.