小鼠肺炎病毒RT-PCR方法的建立及在实验动物感染和国际比对样本检测中的应用  

Establishment and Application of RT-PCR Method for Detection Pneumonia Virus of Mice in Infection Laboratory Animals and International Comparison Samples

作　　者：王吉[1] 王莎莎[1] 王淑菁[1] 李威[1] 秦骁 李晓波[1] 付瑞[1] 岳秉飞[1] 贺争鸣[1] WANG Ji;WANG Shasha;WANG Shujing;LI Wei;QIN Xiao;LI Xiaobo;FU Rui;YUE Bingfei;HE Zhengming(National Institutes for Food and Drug Control,National Center for Quality of Laboratory Animal,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院,国家实验动物微生物遗传检测中心,北京102629

出　　处：《实验动物科学》2022年第2期1-10,共10页Laboratory Animal Science

基　　金：国家科技支撑计划(2015BAI09B02);普通级封闭群裸鼹鼠种群的建立既乃低氧机制的初步研究。

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8.77×10拷贝/μL,可检测病毒最小滴度为10/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。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 8.77×10copies/μL, and could detect the minimum titer of virus was 10/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.

关 键 词：小鼠肺炎病毒 RT-PCR 实验动物 应用 

分 类 号：Q95-3[生物学—动物学]