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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 establish a real-time reverse transcription-polymerase chain reaction (PCR) method for the detection of mitochondrial antiviral signaling protein (MAVS) by H5N1 avian influenza virus (AIV) in guinea pigs, MAVS expression levels. Methods RNA was extracted from the guinea pig lung tissue and cDNA was reverse transcribed. The cDNA template was serially diluted 10 times to 9 concentrations (1.00 E + 10-1.00 E + 02 copies / μl) for PCR amplification. Using β-actin as internal reference, a standard curve was established to compare the amplification efficiency of the two genes, and the primer specificity, repeatability and sensitivity of the method were verified. At the same time, this method was used to detect the expression of MAVS in the lungs of guinea pigs before and after H5N1 AIV challenge. Results The optimal dilution of c DNA template ranged from 1.00 E +09 to 1.00 E + 04. The standard curve slope of the MAVS and β-actin genes in guinea pigs was less than 0.1, R ~ 2 = 0.999, and the amplification efficiencies were 100.2% and 100.3%, respectively. The single peak of the amplification curve of both genes amplified a clear target (CV) were <10%. The sensitivity was 1.00 E + 03 copies / μl. MAVS expression was down-regulated in the lung tissue of guinea pig infected with H5N1 AIV. Conclusion The SYBR Green Ⅰ relative real-time quantitative PCR method for detecting MAVS in H5N1 AIV infected guinea pigs was successfully established. It was found that the infection of H5N1 AIV caused the down-regulation of MAVS expression in guinea pigs.