尼帕病毒巢式RT-PCR方法的建立和初步应用.pdfVIP

中国人兽共患病学报 2014 , 30 (6) Chinese J ournal of Zoonoses 599 001: 10. 3969/cjz. j. issn. 1002 - 2694.2014.06.011 尼帕病毒巢式 RT-PCR 方法的建立和初步应用 张体银，张志灯，郑 腾，白泉阳，王武军，林素洁 摘 要:目的 建立灵敏、特异的检测尼帕病毒的巢式 RT-PCR 方法。方法 根据 GenBank 公布的尼帕病毒 N 基因序 列，设计 2 对特异性引物(外引物和内引物) ，建立巢式 RT-PCR 方法，优化反应体系，测定特异性和灵敏度，并进行临床样品 的检测。结果 该方法扩增的片段序列与源序列同源性均为 100% 。特异性试验结果表明本试验设计的内外侧引物不能从 新城疫病毒、牛瘟病毒和日本乙型脑炎病毒中扩增出条带。敏感性试验结果表明，该方法最低能检测出的标准模板 RNA 浓 度为 39 fg/μLo 100 份临床样品检测结果全部为阴性。结论 初步建立了快速、灵敏、特异的检测尼帕病毒 N 基因的巢式 RT-PCR 方法，可用于动物疫病监测和检验检疫等领域。 关键词:巢式 RT-PCR; 尼帕病毒 ;N 基因 中图分类号:R373 文献标识码:A 文章编号: 1002 - 2694(2014 )06 - 0599 一 04 Development of nested RT-PCR for detection of Nipah virus ZHANG Ti-yin ,ZHANG Zhi-deng ,ZHENG Teng ,BAI Quan-yang , W ANG W u-jun , LIN Su-jie (Fujian Provincial Key Laboratory 01 lnspection and Quarantine Technology Research /Fujian Entry-E:r it lns户ection and Quarantine Bureau , Fuzhou 350001 , China) ABSTRACT: In the present study , we aimed to establish a specific and sensitive nested reverse transcription polymerase chain reaction (RT-PCR) assay for detection of Nipah virus. Two pairs of specific-primers were designed according to the se- quence of N gene of Nipah virus. and the RNA positive controls of synthetic was used for the nested RT• PCR reaction system. The system was optimized and the specificity and sensitivity of this system were evaluated and used to detect the 100 clinical samples. The results showed that the homology of the outer primers and the inner primers were all 100 %. comparing with the origin gene. As demonstrated by the specificity test. the outer and inner primers designed in this study could not be amplified from the Newcastle disease v