IAV绝对定量PCR构建及其在体外感染巨噬细胞中的实验研究  

Construction of IAV absolute quantitative PCR and its experimental study on macrophage infection in vitro

作　　者：阙婷 蓝利 张潇剑 袁江浪 孔星星 樊晓晖[1] 张增峰[1] 唐深[1,2] 罗小玲 Que Ting;Lan Li;Zhang Xiaojian;Yuan Jianglang;Kong Xingxing;Fan Xiaohui;Zhang Zengfeng;Tang Shen;Luo Xiaoling(School of Basic Medical Sciences,Guangxi Medical University,Nanning 530021,China;Key Laboratory of Basic Medical Research in Guangxi Colleges and Universities,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学基础医学院,南宁530021 [2]广西医科大学广西高校基础医学研究重点实验室,南宁530021

出　　处：《广西医科大学学报》2022年第4期637-642,共6页Journal of Guangxi Medical University

基　　金：广西自然科学基金资助项目(No.2021GXNSFAA196019)。

摘　　要：目的:构建实时荧光定量PCR(RT-qPCR)绝对定量法检测甲型流感病毒(IAV),研究其在检测IAV体外感染巨噬细胞(Mφ)中病毒复制动态变化的应用效能。方法:佛波酯(PMA)诱导THP-1细胞刺激活化为Mφ,分为Mφ组和Mφ感染组[IAV的感染复数(MOI)为0.1、0.5、1、1.5、2、5、10、15、20,共9个感染剂量组]。构建RT-qPCR绝对定量检测法检测IAV体外感染Mφ模型中细胞内和培养上清病毒核酸拷贝数;CCK-8法检测24 h内不同MOI的流感病毒对Mφ细胞活性的影响,选取10^(5)~10^(6) TCID50的最佳MOI作为感染模型剂量;采用红细胞凝集实验(HA)检测感染模型细胞内和培养上清的病毒生物合成水平。结果:构建RT-qPCR的标准曲线为Y=-3.345X+38.454,具有良好的线性关系,检测下限为10^(2) copies/μL,10倍稀释梯度在10^(3)~10^(8)区分度和重复性良好;与Mφ组相比,随着MOI增加,Mφ活性下降(P<0.05),感染后贴壁Mφ形态出现变圆悬浮、胞质内大量颗粒、胞体模糊和细胞破裂等变化,且MOI越大,病变程度越严重;以105~106TCID50为依据,选取MOI=0.5、1、2、5、10作为Mφ体外感染的剂量;体外感染的细胞内和培养上清病毒核酸浓度随MOI增大而升高;Mφ感染组细胞内病毒标本HA效价均为1∶64,培养上清的HA效价在1∶32~1∶64。结论:构建RT-qPCR绝对定量法成功检测到体外感染Mφ细胞内和培养上清病毒核酸拷贝数呈逐渐增加趋势,并与细胞病变程度呈正相关关系;HA结果无法准确区分病毒复制趋势,RT-qPCR绝对定量法的敏感度和区分度均较HA高。Objective:To establish a RT-qPCR absolute quantitative method for detection of influenza A virus(IAV),and to study its efficacy in detecting the dynamic changes of virus replication in macrophages(Mφ)infected by IAV in vitro.Methods:THP-1 cells stimulated by phorbol ester(PMA)were divided into Mφgroup and Mφinfection group[multiple of infection(MOI)of IAV was 0.1,0.5,1,2,5,10,15,20,totally 9 infection groups].RT-qPCR absolute quantitative method was established to detect the copy number of viral nucleic acid in the intracellular and culture supernatant of IAV infected Mφmodel in vitro.CCK-8 method was used to detect the effect of influenza viruses of different MOI on Mφcell activity within 24 hours,and the best MOI of 10^(5)-10^(6) TCID50 was selected as the infection model dose.Hemagglutination(HA)was used to detect the level of virus biosynthesis in infected model cells and culture supernatant.Results:The standard curve Y=-3.345X+38.454 of RT-qPCR had a good linear relationship,the detection limit was 10^(2) copies/μL,and the 10 times dilution gradient had good differentiation and repeatability at 10^(3)-10^(8).Compared with Mφgroup,the activity of Mφdecreased with the increase of MOI(P<0.05).The morphology of adherent Mφbecame round and suspended,and a large number of particles in the cytoplasm,blurring of cell body and cell rupture appeared after infection.The larger the MOI was,the more serious the lesion was.On the basis of 10^(5)-10^(6) TCID50,MOI=0.5,1,2,5 and 10 were selected as the doses of Mφinfection in vitro,and the concentration of viral nucleic acid in the intracellular and culture supernatant increased with the increase of MOI.The HA titer of intracellular virus samples in Mφinfection group was 1:64,and the HA titer of culture supernatant was 1:32-1:64。Conclusion:Construction of RT-qPCR absolute quantitative method successfully detect that the copy number of viral nucleic acid in Mφcells and culture supernatants infected in vitro shows a gradual increasing trend,which was positively

关 键 词：荧光定量PCR IAV 巨噬细胞 体外模型 

分 类 号：R374.13[医药卫生—病原生物学]