鼠类感染沙粒病毒CODEHOP RT-PCR检测方法的建立  

Development of CODEHOP RT-PCR to detect arenaviruses carried by rodents

作　　者：胡群[1] 马思杰[1] 裘炯良[1] 邹春颖[1] 童淑梅[1] 

机构地区：[1]大榭出入境检验检疫局,浙江宁波315812

出　　处：《中国病原生物学杂志》2013年第12期1074-1077,共4页Journal of Pathogen Biology

基　　金：宁波市自然科学基金项目(No.2013A610240);国家质检总局科技支撑项目(No.2012IK233)

摘　　要：目的建立鼠类感染沙粒病毒的CODEHOPRT—PCR检测方法。方法依据沙粒病毒N蛋白氨基酸序列设计1对CODEHOP引物，建立沙粒病毒一步RT—PCR检测方法，分析该引物在对不同种沙粒病毒基因序列上的结合位点及所能获得的产物序列大小；通过检测分离自鼠类的沙粒病毒属淋巴细胞脑膜炎病毒（LCMV）株MS111013，评价方法的特异性和灵敏度；对MS111013扩增产物进行Blast同源性比对。结果从GenBank获得的22种沙粒病毒均存在CODEHOP引物结合位点，扩增产物大小在406～429bp之间。建立的CODEHOPRT—PCR方法可特异性扩增LCMV病毒核酸，目的片段大小与预期结果相符，核酸的最小检出量为11．8Pg。经Blast比对，MS111013与LCMV病毒株Douglas strain（4707）同源性较高，为86％。结论建立的沙粒病毒CODEHOPRT—PCR检测方法敏感、特异，可用于鼠类沙粒病毒感染检测。Objective To develop a method of CODEHOP RT-PCR to detect arenaviruses carried by rodents. Methods Based on the N protein amino acid sequences of arenaviruses, a pair of CODEHOP primers was designed and a one-step RT-PCR technique was developed to detect arenaviruses. Sites accessible to CODEHOP primers in genes from different species of arenaviruses were determined and the size of PCR products was determined. Lymphocyte meningitis virus （LC- MV） strain MS111013 was isolated from rodents to evaluate the specificity and the sensitivity of the method of CODE- HOP RT-PCR. Blast similarity analysis of the nucleocapsid sequences of MS111013 was then performed. Results Twenty-two types of arenaviruses from GenBank had sites accessible to CODEHOP primers, and the size of PCR products ranged from 406-429 bp. CODEHOP RT-PCR specifically detected arenaviruses, the size of the target fragment and the sequence agreed with the anticipated results. The detection limit for arenavirus RNA was 11.8 pg. According to blast, MS111013 had a similarity of 86% to the Douglas strain of LCMV （4707）. Conclusion The proposed method of CODEHOP RT-PCR has a high level of specificity and sensitivity and can be effectively used to detect different arenaviruses carried by rodents.

关 键 词：沙粒病毒 CODEHOP RT—PCR 检测 

分 类 号：R373.9[医药卫生—病原生物学]