Abstract:

Objective To establish a RT-PCR method for detection of Pneumonia virus of mice ( PVM ) in laboratory animals and related samples. Method The primers were designed and synthesized by selecting the conserved G gene sequence of PVM, and the RT-PCR method was established. And the specificity, sensitivity,repeatability and stability of the established method were verified. The established method was used to detect the lung tissue samples of 27 mice,9 rats, 5 gerbils submitted for routine examination and 19 PVM infected mice, and 8 ICLAS international comparison samples. Result The established method had no cross reaction with Sendai virus( SV) , Reovirus type 3 ( Reo3) , ( HV) and Lymphocytic Choriomeningitis Virus ( LCMV) ; The method could detect the minimum template concentration of PVM DNA was 8. 77×102 copies / μL, and could detect the minimum titer of virus was 10- 4. 0 / mL. The primers and PVM plasmids stored at -30 ℃ refrigerator for 12 months were used for PCR detection, and the target fragment of about 249 bp could still be amplified. The lung tissue samples test result of 27 mice, 9 rats and 5 gerbils submitted for routine examination were all negative. The result showed that 7 lung tissue samples of the 19 mice infected with PVM were positive. Among the 8 ICLAS international comparison samples tested, 1 sample was positive for PVM nucleic acid, which was in line with the expected result.Conclusion The established PVM RT-PCR method is good in specificity, sensitivity, repeatability and stability,and can be used for the monitoring of mice, rats, gerbils and other experimental animals and the detection of related samples.

Key words:

pneumonia virus of mice, RT-PCR, experimental animals, application 

摘要：

目的 建立小鼠肺炎病毒 RT-PCR 检测方法,用于实验动物及实验动物相关样本的检测。 方法 选择小鼠肺炎病毒( pneumonia virus of mice,PVM) G 基因保守序列设计合成引物,建立 RT-PCR 方法,并进行方法的特异性、敏感性、重复性和稳定性验证。 应用建立的方法对日常送检的 27 只小鼠、9 只大鼠、5 只沙鼠肺组织样本和 19 只PVM 感染小鼠肺组织样本,及 8 份国际实验动物理事会 ( the International Council for Laboratory Animal Science,ICLAS)国际比对样本进行检测。 结果 建立的方法与仙台病毒、呼肠孤病毒Ⅲ 型、汉坦病毒、淋巴细胞脉络丛脑膜炎病毒无交叉反应。 能够检测 PVM DNA 最小模板浓度为 8. 77× 102 拷贝 / μL,可检测病毒最小滴度为 10- 4. 0 / mL。用放置-30 ℃ 冰箱保存 12 个月的引物和 PVM 质粒进行 PCR 检测,仍能扩增到约 249 bp 的目的条带。 用建立的方法检测日常送检的 27 只小鼠、9 只大鼠、5 只沙鼠肺组织样本,结果均为阴性;检测 19 只 PVM 感染小鼠肺组织样本,有 7 只小鼠肺组织样本结果阳性;检测 8 份 ICLAS 国际比对样本,有 1 份样本小鼠肺炎病毒核酸阳性,均符合预期结果。 结论 建立的 PVM RT-PCR 方法特异、敏感,重复性、稳定性好,能够用于小鼠、大鼠、沙鼠等实验动物的监测及其相关样本的检测。

关键词:

小鼠肺炎病毒, RT-PCR, 实验动物, 应用