Establishment And Preliminary Application Of Multiplex TaqMan-MGB Probe Fluorescence Quantitative PCR Detection Method For CSFC,PRV And PPV

Classical swine fever virus(Classical Swine Fever Virus,CSFV),porcine parvovirus(Porcine Parvovirus,PPV),pseudorabies virus(Pseudorabies Virus,PRV)infection can cause reproductive disorders in pigs.The clinical symptoms of reproductive disorders caused by these three viruses are very similar,and it is necessary to confirm the diagnosis through pathogen identification,and then formulate a targeted prevention plan in a timely manner.At present,PCR diagnostic methods for detecting these three pathogens alone have been established,but the methods that can detect these three pathogens at the same time are not perfect.Therefore,it is extremely necessary to establish a detection method with high sensitivity,strong specificity,fast and simple,and can distinguish the three viruses at the same time.In order to establish a multiplex TaqMan-MGB probe fluorescence quantitative PCR detection method for CSFV,PPV and PRV,this study used the software DNAMAN 9 and Primer Premier 5 to analyze the conserved gene sequences of the NCBI accession numbers of the above three viruses,and designed and synthesized 3 specific pairs.The TaqMan-MGB probe fluorescence quantitative PCR detection method was established by optimizing the probe concentration,primer concentration and annealing temperature by using primers and TaqMan-MGB probes.The test results are as follows:(1)A single-plex TaqMan-MGB probe fluorescence quantitative PCR method for CSFV,PPV and PRV was established.Homology analysis was carried out according to the gene sequences of the NCBI accession numbers of the three viruses to determine the relatively conserved genes,and specific primers were designed for the conserved fragments of the E2 gene of swine fever virus,the VP2 gene of porcine parvovirus and the g B gene of pseudorabies virus.The target fragment was amplified by PCR reaction.The lengths of CSFV,PPV and PRV amplified fragments were 323 bp,393 bp and 243 bp,respectively.The ligated product of the target fragment and p UC19 was transformed into competent cell DH5α,and the recombinant plasmid was extracted.The recombinant plasmids were prepared by PCR sequencing,concentration detection and gradient dilution.The standard curves established by using the standard products all had a good linear relationship.The correlation coefficients of the standard curves for CSFV,PPV and PRV were 0.999,0.999 and 0.993,respectively.The results of specificity test and sensitivity test were good.The specificity test showed that only CSFV,PPV and PRV were positive,and other pathogens(JEV,PCV,PRRSV,etc.)did not show specific amplification;the sensitivity test showed that CSFV,PPV and PRV were positive The lowest detection limits were 7.40×10~1 Copies/μL,9.70×10~1 Copies/μL and1.70×10~2 Copies/μL,respectively.The results of the repeatability test showed that the coefficients of variation of the single-plex detection methods for the three viruses were all lower than 3%.(2)Multiplex TaqMan-MGB probe fluorescence quantitative PCR method for CSFV,PPV and PRV was established.The method can simultaneously carry out the differential diagnosis of three viruses,and the method has good specificity and sensitivity.The standard curve has a certain linear relationship:the correlation coefficient of the standard curve of swine fever virus is 0.995,the correlation coefficient of standard curve of pseudorabies virus is 0.998,and the correlation coefficient of standard curve of porcine parvovirus is 0.995.And in the specific detection of various viruses,pathogens other than the three viruses studied in this experiment did not show obvious fluorescent signals,and the minimum detection copy numbers of CSFV,PPV and PRV were3.44×10~1 Copies/μL,1.07×10~1 Copies/μL and 8.59×10~0 Copies/μL.The repeatability of the test was good with a coefficient of variation below 3%.(3)The multiplex TaqMan-MGB probe fluorescence quantitative PCR method established in this study was used to detect the collected 64 clinical samples,and the detection results were compared with the detection results of the national standard PCR method.The results showed that the detection rates of the two methods were identical,indicating that the method established in this study can be applied to production practice.In conclusion,the multiplex TaqMan-MGB probe fluorescence quantitative PCR method established in this study has unique advantages for the rapid diagnosis and differential diagnosis of CSFV,PPV and PRV,and provides technical support for the precise prevention and control of the three infectious diseases.