兔出血症病毒RT-PCR检测方法的建立及应用  

Establishment and Application of RT-PCR Test for Rabbit hemorrhagic disease virus

作　　者：王芳[1] 李超美[2] 杨龙圣[1] 徐为中[1] 张则斌[1] 侯玉峰[2] 陈国强[2] 何孔旺[1] 

机构地区：[1]江苏省农业科学院兽医研究所,南京210014 [2]南京出入境检验检疫局,南京210001

出　　处：《农业生物技术学报》2007年第3期409-413,共5页Journal of Agricultural Biotechnology

基　　金：江苏省南京出入境检验检疫局科研项目(No.JS.99(021))资助

摘　　要：按照GenBank上公布的兔出血症病毒(RHDV)基因序列设计1对引物,以RHDV发病兔的新鲜肝为材料,提取总RNA,进行cDNA的合成和PCR扩增,结果能扩增出与预期的269bp大小一致的片段,该产物序列与公布的RHDV基因序列同源性95.8%～98.5%。该方法可特异性地检测出RHDV,其最小检出RHDV的RNA浓度为1.66ngμL,敏感性为血凝试验(HA)的4×104倍,表明已成功地建立了RHDVRT-PCR检测方法。用该方法和HA对兔出血症发病兔各组织脏器进行检测,结果表明,在检测发病死亡兔各脏器中,HA检测阳性的病料为肝脏、肾脏、脾脏、血液和肺,而RT-PCR检测除粪便外,其它均为阳性;该方法还能够检测出-20℃保存12个月的阳性病料。表明RT-PCR方法检测RHDV特异性强、敏感度高、重复性好,不仅可用于RHD临床诊断和流行病学调查,而且在兔肉等兔类产品的检疫方面具有很好的应用前景。Total RNA was extracted from the flesh liver of rabbit haemorrhagic disease rabbits and a pair of primers were designed according to the sequences of Rabbit haemorrhagic disease virus （RHDV） published in GenBank, and an expect 269 bp segment was amplified by RT-PCR. The sequencing result showed that the homology was 95.8%～98.5% compared with the reference sequence published in GenBank. The least amount of RHDV that could be detected was 1.66 ng/μL, also showed its high specialties, and what＇s more, the sensitivity was 4×10^4 times higher than that of HA （hemagglutination assay） method. The detection of RHDV by RT-PCR was established successfully. Compared to HA, RT-PCR could detect RHDV in all the viscera except for feces, and HA method only in liver, kidney, spleen, blood and lung were positive. Samples kept in -20 ℃ for 12 months could be detected by RT-PCR. All above indicate that this RT-PCR method has strong speciality, high sensitivity and good repetition, and can be used in RHDV clinical diagnosis, epidemiology study and quarantine of rabbit products such as meat.

关 键 词：兔出血症病毒(RHDV) RT-PCR 病毒分布 

分 类 号：S188[农业科学—农业基础科学]