Preliminary Establishment Of The Real-time PCR For Detection Of OPA

OPA is a d-type pulmonary adenomas in JSRV cause is a chronic, progressive, contagiousdiseases of lung tumors.Sheep pulmonary Adenomatosis occurs in many countries and regions ofthe world there, sheep pulmonary Adenomatosis morbidity after passing sensitive sheep up-50%for the first time. Adult sheep susceptibility of secondary infection and pathogenesis of respiratorydiseases, growth slow, dramatic weight loss, mortality100%, seriously affecting the developmentof sheep industry in the context of the world.In this study, according to the gag gene of JSRV, select the conservative sequence fragments asdetection target fragment, primers were designed and synthesized in the corresponding genomicDNA extraction, the lung tumor tissue by natural infection in cases as template, was obtained byPCR gene fragment, were compared and analyzed with the published GenBank gag gene sequence,determine its conservative sequence the design of primers, and TaqMan fluorescence probe.Connect the conventional PCR products into the pMD18-T vector, plasmid DNA was extracted,transformed, quantitative, to10times than the serial dilution of recombinant plasmid pMD-gag,made of101-107copies/μ l positive recombinant plasmid standard template, positive Ct value atabout23of the recombinant plasmid standard established for detecting system, gradientoptimization the experiment to gradually change the reaction system of PCR concentrations of eachcomponent of each single factor density fractions, in order to establish the system of compositionconcentration.Objective gene of conventional PCR results showed, the objective gene gene length isconsistent with the length of1843bp and Genebank released, similarity of99%.The other sheep are susceptible to viruses, bacteria and parasites disease nucleic acid detectionresults, only positive template amplified specific "S" curve, all other samples of nucleic acids andnegative controls showed no fluorescence signal that increases, primer and probe is highly specificfor sheep lung tumor virus; detection the continuous dilution of the positive plasmid standard, theCt value by logarithmic fitting standard curve, amplification equation is Y=-0.304X+38.4,correlation coefficient R2=0.999, the amplification efficiency is100%,40cycles of real-timefluorescent quantitative PCR minimum detectable103copies/μ l samples. The positive plasmidstandard in standard curve data and curve fitting is good.With the recombinant plasmid standard10~5,10~4,10~3copies/μ l as template, repetitive experiments were performed to detect system, results show that the coefficient of variation of thegroup repeated the experiment in0.490-1.374%between, between group repeatability coefficient ofvariation between0.120-0.826%, the results show no significant difference in Ct values, very smalldiscrete degree, the scope in the statistical confidence interval, show that the detection system isstable, with good repeatability.Was detected by real-time fluorescence quantitative PCR detection method JSRV the sheeplung samples of11local sheep. According to the Ct value in the range of20-30confidence interval,defined as positive. Test results show that, samples from Australia and Luobei, Jidong, Jia Musi,test results were negative; while the positive rate of Inner Mongolia is45%, the positive rate ofQigihar is37%; the positive rate of Zhaodong samples is80%; the positive rate of Lanxi samples is20%; the positive rate of Mudanjiang samples is40%; the positive rate of Shangzhi samples is20%;the positive rate of Nenjiang is100%. From the test results can be seen preliminary, Australia,Luobei, Jidong and Jia Musi no JSRV infection at.In this experiment, the sample number and the competitive ELISA uniform number, resultsshowed that, RT-PCR was detected in Inner Mongolia sample positive ratio ELISA results in morethan3, respectively8,41and57samples, the positive rate was50%; the positive ratio of Qigiharsample ELISA test results of2, were numbered33and46samples, the positive rate was46%; thepositive ratio of Zhaodong sample ELISA test results of1, No.4samples, the positive rate was80%. And, these differences in the sample of RT-PCR amplification curve,"S" type curve issmooth, on peak position earlier, so we can exclude the false positive may, therefore, many of thesesamples were detected by RT-PCR test can determine the detection rate is slightly higher than thecompetitive ELISA method, sensitivity also slightly higher, accuracy better.In this study, using real-time fluorescence quantitative PCR technology, established thedetection method of OPA, for the detection of the disease and after making kit provides a goodpractical technology and research base.