鼠肺炎病毒实时荧光定量聚合酶链反应方法的建立及其在新型冠状病毒感染模型用小鼠等动物检测中的应用  

Establishment of Q-PCR method for pneumonia virus of mice and its application in detection of mouse and other animal models of COVID-19 infection

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] 梁春南[1] WANG Ji;WANG Shasha;WANG Shujing;LI Wei;QIN Xiao;LI Xiaobo;FU Rui;YUE Bingfei;LIANG Chunnan(National Institutes for Food and Drug Control(NIFDC),National Rodent Laboratory Animal Resources Center,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实验动物资源研究所(国家啮齿类实验动物资源库)国家实验动物微生物遗传检测中心,北京102629

出　　处：《中国病毒病杂志》2023年第4期264-271,共8页Chinese Journal of Viral Diseases

基　　金：国家重点研发计划(2022YFF0711002)。

摘　　要：目的建立鼠肺炎病毒(pneumonia virus of mice,PVM)实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,Q-PCR)检测方法,用于新型冠状病毒感染模型用小鼠等实验动物及其动物相关样本的检测。方法选择PVM G基因保守序列设计合成引物和探针,建立Q-PCR方法。并进行方法的线性、特异性、敏感性、重复性、稳定性和可信度验证。应用建立的方法对包括用于构建新型冠状病毒感染模型用hACE2等8个品系142只小鼠,及15只大鼠、5只沙鼠、4只黄鼠、63只裸鼹鼠和19只PVM感染小鼠的肺组织样本进行PVM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。方法的线性范围为1×10^(1)-1×10^(8)拷贝/μl,检测病毒质粒标准品灵敏度达101拷贝/μl;重复性、稳定性结果显示实验内和实验间平均变异系数均<5%。用建立的方法检测用于构建新型冠状病毒感染模型用hACE2等8个品系142只小鼠,及15只大鼠、5只沙鼠、4只黄鼠和63只裸鼹鼠,结果均为阴性;检测19只PVM感染小鼠肺组织样本,PVM阳性率为36.84%(7/19),与RT-PCR检测结果符合率为100%。结论建立的PVM Q-PCR方法特异性、敏感性,重复性、稳定性好,检测结果可靠,能够用于构建新型冠状病毒感染模型常用品系小鼠等实验动物的监测及其相关样本的检测。未发现用于构建新型冠状病毒感染模型常用品系小鼠携带PVM。Objective To establish a real-time fluorescent quantitative polymerase chain reaction(Q-PCR)method for detection of pneumonia virus in mice(PVM)to detect mouse model of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection and animal-related samples.Methods The specific primers and probe were designed and synthesized based on selected conserved regions of PVM G gene sequence to establish the Q-PCR method.The linearity,specificity,sensitivity,repeatability,stability,and reliability of the method were validated.The established method was used to detect PVM in lung tissue samples from 142 mice of 8 strains,including the hACE2 strain used to construct SARS-CoV-2 infection model,15 rats,5 gerbils,4 ground squirrels,63 naked mole rats and 19 PVM-infected mice.Results The established Q-PCR method for PVM had no cross reaction with Sendai virus(SV),mammalian orthoreovirus type 3(Reo3),hantavirus(HV)and lymphocytic choriomeningitis virus(LCMV).The linear range of the method was 101-108 copies/μl.The detection sensitivity reached 101 copies/μl.The results of repeatability and stability showed that the intra-and inter-assay average coefficient of variation were both under 5%.The detection results of the established method in 142 mice from 8 strains including the hACE2 strain used to construct SARS-CoV-2 infection model,15 rats,5 gerbils,4 ground squirrels and 63 naked mole rats were all negative;and PVM positive rate in lung tissue samples from 19 PVMinfected mice was 36.84%(7/19).The consistency rate between the established method and RT-PCR was 100%.Conclusions The established Q-PCR method for PVM detection has satisfactory specificity,sensitivity,repeatability,stability,and reliability.It can be used for the monitoring of laboratory animals such as the mice used to construct COVID-19 infection models and the detection of related samples.PVM is not found in the mice used to construct COVID-19 infection models.

关 键 词：鼠肺炎病毒 实时荧光定量聚合酶链反应 新型冠状病毒 实验动物 检测方法 动物模型 

分 类 号：R33[医药卫生—人体生理学]