H5N1禽流感病毒感染豚鼠体内线粒体抗病毒信号蛋白SYBR GreenⅠ相对荧光定量PCR检测方法的建立及应用  

Development and application of relative fluorescent quantitative PCR for mitochondrial antiviral signaling protein SYBR Green Ⅰ in guinea pigs infected with avian influenza H5N1 virus

作　　者：孙丽娟[1,2] 许微微[2,3] 张坤[2] 郭骐源[1,2] 刘旭光[1] 杨松涛[2] 王铁成[2] 黄耕[2] 高玉伟[2] 夏咸柱[2] 张雪梅[1] 

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130062 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [3]吉林农业大学动物科技学院,吉林长春130122

出　　处：《中国生物制品学杂志》2016年第1期79-83,共5页Chinese Journal of Biologicals

基　　金：国家重点基础研究发展计划(973计划;2011CB50502)

摘　　要：目的建立H5N1禽流感病毒(avian influenza virus,AIV)感染豚鼠体内线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)SYBR GreenⅠ相对荧光定量PCR检测方法,并检测H5N1 AIV感染前、后豚鼠肺脏组织中MAVS的表达水平。方法提取豚鼠肺脏组织RNA,反转录合成cDNA,将cDNA模板按10倍系列稀释为9个浓度(1.00 E+10~1.00 E+02 copies/μl),进行PCR扩增。以β-actin为内参,建立标准曲线,比较两基因的扩增效率,验证该方法的引物特异性、重复性及灵敏度。同时用该方法检测H5N1 AIV攻毒前、后豚鼠肺脏组织中MAVS的表达水平。结果 c DNA模板最佳稀释度范围为1.00 E+09~1.00 E+04。豚鼠MAVS和β-actin基因的标准曲线斜率差值<0.1,R^2=0.999,扩增效率分别为100.2%和100.3%;两基因扩增溶解曲线峰单一,可扩增出清晰的目的条带,且无非特异性扩增;两基因Ct值的变异系数(CV)均<10%;灵敏度为1.00 E+03 copies/μl。感染H5N1 AIV的豚鼠肺脏组织中MAVS的表达水平下调。结论成功建立了H5N1 AIV感染豚鼠体内MAVS的SYBR GreenⅠ相对荧光定量PCR检测方法,并发现H5N1 AIV感染可导致豚鼠体内MAVS表达水平下调。Objective To develop a relative fluorescent quantitative PCR method for mitochondrial antiviral signaling protein（MAVS） SYBR GreenⅠ in guinea pigs infected with avian influenza virus（AIV） subtype H5N1, and determine the expression levels of MAVS in lung tissue of guinea pigs before and after infection. Methods RNA was extracted from the lung tissue of guinea pigs and reversely transcribed into c DNA, which was 10-fold serially diluted to 9 concentrations（1. 00 E ＋ 10 ~ 1. 00 E ＋ 02 copies / μl） for PCR amplification. A standard curve was plotted using β-actin as an internal reference. The amplification efficiencies of two genes were compared, and the method was verified for specificity,reproducibility and sensitivity. The expression levels of MAVS in lung tissue of guinea pigs before and after challenge with H5N1 AIV. Results The dilution range of c DNA template was 1. 00 E ＋ 09 ~ 1. 00 E ＋ 04. The difference between slopes of standard curves of MAVS and β-actin genes was less than 0. 1, with a R2 value of 0. 999, while the amplification efficiencies were 100. 2% and 100. 3% respectively. Either of the melting curves of the two genes showed a single peak,and clear target fragments were amplified, no non-specific amplification was observed. Both the coefficients of variation（CV）of Ct values of the two genes were less than 10%. The sensitivity of the developed method was 1. 00 E ＋ 03 copies / μl.The expression level of MAVS in lung tissue of guinea pigs infected with H5N1 AIV was down-regulated. Conclusion A relative fluorescent quantitative PCR method for MAVS SYBR Green Ⅰ in guinea pigs infected with AIV subtype H5N1 was successfully developed, and infection with H5N1 AIV decreased the expression level of MAVS in guinea pigs.

关 键 词：H5N1禽流感病毒 豚鼠 线粒体抗病毒信号蛋白 SYBR GreenⅠ相对荧光定量PCR 

分 类 号：R373.13[医药卫生—病原生物学] Q789[医药卫生—基础医学]