小鼠脑脊髓炎病毒RT-PCR方法的建立及初步应用

Development and application of RT-PCR for detection of TMEV

目的建立小鼠脑脊髓炎病毒RT-PCR方法并对方法进行初步应用。方法根据NCBI发表的TMEV GD VII株(GI:62039)基因组序列设计特异引物,建立RT-PCR方法,验证方法的敏感性和特异性;脑腔接种方式感染9只BALB/c小鼠,感染后第6天采集动物的脑、心、肝、脾、肺、肾、盲肠内容物和血清等样本,用建立的方法进行检测;同时检测100份小鼠盲肠内容物样本,对方法进行初步应用。结果以GD VII RNA为模板,建立的RT-PCR方法能够扩增出约371bp的单一目的条带,敏感性验证显示能够检测到的最低GD VII c DNA量为0.69pg/μL;以脑心肌炎病毒、淋巴细胞脉络丛脑膜炎病毒、日本乙型脑炎病毒、小鼠诺如病毒及正常小鼠脑组织为对照进行方法特异性验证,均未出现目的条带。脑腔接种感染小鼠,所有小鼠在接种第3天均产生不同程度的精神萎靡后肢麻痹等症状,其中2只小鼠于第5天死亡;采集心、肝、脾、肺、肾、脑、盲肠内容物及血清等样本进行检测,所有小鼠的脑组织均检测到GD VII RNA,而其他组织均未检测到;对100份小鼠盲肠内容物进行检测,结果均为阴性。结论建立的TMEV GDVII株RT-PCR方法能够高效的检测小鼠组织中的病毒感染,可作为实验动物国家标准的有力补充。

Objective To develop RT-PCR for detection of TMEV and apply the method. Methods To design specific primers on the basis of GD VII（ GI： 62039） genome sequences published in NCBI and establish RT-PCR. To verify the sensitivity and specificity of method after optimizing PCR. We infected 9 BALB / c mice intracerebrally and collected brain,heart,liver,spleen,lung,kidnet,cecal contents and serum samples the 6thday postinfection. The samples were tested by the TMEV RT-PCR. 100 mouse cecal contents samples were also detected to apply the established method.Results The 371 bp single band was amplified using GDVII as template. Sensitivity test showed that the RT-PCR method can detect as low as 0. 69 pg / μL GDVII c DNA. There were no objective band amplified when encephalomyocarditis virus,lymphocytic choriomeningitis virus,Japanese B encephalitis virus,murine norovirus and normal mouse brain tissue were used as case-control. All infected mice showed symptom of different degrees such as depression and hind limb paralysis the3 th day postinoculation and two of infected mice died the 5th day postinoculation. Tissues such as heart,liver,spleen,lung,kidney,brain,cecal contents and serum were collected and tested for TMEV. All the brain samples were detected positive for GDVII and other tissues were all negative; The 100 cecal contents samples were tested and all were negative.Conclusions RT-PCR for TMEV GDVII strain can detect virus infection in mouse tissues efficiently and can be used as a powerful supplement for the national standard of lab animal.