脑心肌炎病毒(EMCV)RT-PCR检测方法的建立及初步应用

Development and preliminary application of RT-PCR method for determination of encephalomyocarditis virus

目的建立脑心肌炎病毒(EMCV)RT-PCR检测方法,应用于长爪沙鼠、小鼠等实验动物EMCV的检测。方法根据已发表的EMCV VP1基因序列设计合成引物。建立RT-PCR方法,并对方法的特异性、敏感性、稳定性等进行验证。用该方法检测62只长爪沙鼠、12只小鼠。结果建立的EMCV RT-PCR检测方法特异、敏感、稳定。以EMCV RNA逆转录产物为模板,所能检测RNA最小模板浓度为4.1 pg/μL,可检测病毒最小滴度为10-7mL-1。经RT-PCR检测,62只沙鼠均为阴性;12只小鼠中有1只EMCV核酸阳性,测序结果与GenBank中EMCV标准株核苷酸序列进行比对,其同源性为85%。结论建立的脑心肌炎病毒(EMCV)RT-PCR检测方法特异、敏感、稳定,可用于长爪沙鼠、小鼠等实验动物EMCV的检测。

Objective To develop a RT-PCR method for determination of Eneephalomyocarditis virus （EMCV） in Mongolian gerbils and Laboratory mice. Methods The primers were designed and synthesised according to the published EMCV specific sequences of VPI gene. RT - PCR method is established, carries on the specificity, sensitivity, stability test. The method is used to detected 62 Mongolian gerbils and 12 mice. Results The developed RT-PCR method is good in specificity, ensitivity, stability; and its minimum detection limit using the recombinant plasmid containing EMCV gene as atemplate was 4. lpg/pLL, and the lowest detection virus titer is 10 -~ ml-l. The 62 Mongolian gerbils after RT - PCR detection were negative; The 12 mice after RT - PCR detection, there were one EMCV positive, compared with the EMCV in Genebank, The homologies in nucleotide sequence of one positive mice is 85% ; there were 5 mice can be detected EMCV in the body of the 6 mice by artificial infection EMCV. Conclusion The developed RT-PCR method is good in specificity, ensitivity, stability, can be used in detecting the EMCV in laboratory animal, such as Mongolian gerbils and mice.