摘要
目的建立长爪沙鼠小鼠肝炎病毒(MHV)RT-PCR检测方法,应用于长爪沙鼠、小鼠等实验动物MHV的检测。方法根据已发表的小鼠肝炎病毒(MHV)S基因序列,设计合成引物。提取MHV细胞毒RNA,以其为模板,进行PCR扩增。优化反应条件,进行特异性、敏感性、稳定性、重复性试验。并对65只长爪沙鼠及12只小鼠进行检测。结果建立的MHV RT-PCR检测方法特异、敏感、稳定。以MHV RNA逆转录产物为模板,所能检测RNA最小模板浓度为3.1 pg/μL,可检测病毒最小滴度为10-3/mL。65只沙鼠经RT-PCR检测,均为阴性,12只小鼠经RT-PCR检测,有3只MHV阳性,测序结果与Genbank中MHV核酸序列同源性均为97%。结论建立的长爪沙鼠小鼠肝炎病毒(MHV)RT-PCR检测方法可用于长爪沙鼠、小鼠等实验动物MHV的检测。
Objective To establish a RT-PCR method for detection of mouse hepatitis virus （MHV） in laboratory mice and Mongolian gerbils. Methods Two pairs of specific primers were designed according to the published S gene sequence of mouse hepatitis virus. With which the MHV RNA was extracted and reversely transcribed to cDNA as a template for PCR amplification. The developed RT-PCR method was optimized, the specificity, sensitivity, stability, and repeatability tests were performed, and the RE-PCR method was used to detect MHV in 65 Mongolian gerbils and 12 mice. Results The developed RT-PCR method is good in specificity, sensitivity, stability, and repeatability. Its minimum detection limit using the recombinant plasmid containing MHV gene as a template was 3. 1 pg/μL, and the lowest virus detection titer was 10-3/mL. The results of RT-PCR detection showed that the 65 Mongolian gerbils were negative, and 3 of the 12 mice were MHV-positive. Compared with the MHV in Genebank, the homologies in nucleotide sequence of all the 3 positive mice were 97%. Conclusions The developed RT-PCR technique can be used to detect mouse hepatitis virus （MHV） in laboratory animals such as mice and Mongolian gerbils.
出处
《中国比较医学杂志》
CAS
2013年第2期58-63,共6页
Chinese Journal of Comparative Medicine
基金
实验动物新品种的种群建立与质量标准化研究(国家科技支撑计划项目:2011BAI15B01)
