Establishment Of Real-time Fluorescence Quantitative PCR Detection Method For PCV2 And PRRSV

PCV2 can cause immune inhibition,multiple pathogens secondary infection and porcine circovirus disease such as postweaning multisystemic wasting syndrome,porcine dermatitis and nephropathy syndrome and so on,which leads to a significant increase in mortality in pig farms and seriously endangers the pig industry.PRRSV mainly attacks pulmonary macrophage and also causes immune inhibition in pigs,resulting in porcine reproductive and respiratory disorders syndrome.In addition,PRRSV is prone to gene mutation which makes disease prevention more difficult and also brings huge economic losses to the pig industry.The results of early monitoring of pig farm antibody showed that the inhibition of PRRSV antibody was correlated with the control of PCV2 and PRRSV and the health level of pig farm.To detect the timing and status of infection of PCV2 and PRRSV in pigs in advance and solve the problem that virus mutation is easy to cause false negative test result,this trial established three SYBR Green Ⅰ fluorescent quantitative PCR methods for detecting PCV2 and PRRSV,respectively,and carried out preliminary application.In this experiment,five pairs(PCV2)and eight pairs(PRRSV)specific primers were designed by comparing the gene sequences of multiple PCV2 and PRRSV in GenBank.The better fragments were selected for PCR amplification for cloning and 5 recombinant plasmids were obtained respectively.Further,three SYBR Green I fluorescent quantitative PCR diagnostic methods of PCV2 and PRRSV were established by optimizing the reaction system and conditions with three of the better primers.The method has good specificity,repeatability and sensitivity of 101 copies/μL.The established method was used to detect 144 samples of serum from pigs and 6 samples of tissues suspected to be infected with PRRSV.Compared with the conventional PCR method,the fluorescence quantitative PCR method established in this experiment has a higher detection rate for PCV2 and PRRSV,and the Ct value of samples is negatively correlated with the brightness of the conventional PCR amplif ied bands.In addition,the results of the six tissue samples showed mixed infection of PCV2 and PRRSV.Compared with the detection results of PCV2,three samples of PRRSV were significantly different in Ct values detected by different detection methods,which presumably dues to the high variability of the PRRSV gene.It can be seen that in order to avoid missed detection and quantitative analysis,it is necessary to use more than two methods for PRRSV detection.The fluorescence quantitative PCR method for the detection of PCV2 and PRRSV has the advantages of rapid,specific,sensitive,stable and reliable which cannot only provide technical means for the monitoring and prevention of PCV2 and PRRSV,but also provide qualitative and quantitative testing technical support for epidemiological investigation,import and export quarantine and other applications.