Establishment And Application Of Multiplex Quantitative PCR And Droplet Digital PCR For Porcine Atypical Pestivirus

Atypical porcine pestivirus can cause whole-body muscle tremor,splayed legs,dyskinesia,and obstruction of suckling in affected piglets,which can lead to severe death.Related studies have shown that the virus is the main cause of congenital tremor A-II in piglets.APPV is currently reported in 14 countries on four continents,causing huge economic losses to global pig farming.Among them,the piglet infection rate is high,most of the adult pigs are invisible infection,and there are co-infection of many viruses related to pigs.Therefore,the establishment of an efficient and sensitive molecular detection method is helpful for early detection and diagnosis of the virus and effectively controls the spread of APPV.This study relies on the TaqMan fluorescent quantitative PCR platform for molecular detection and the third-generation new droplet digital PCR detection platform with absolute quantification and ultra-high sensitivity.It has established a variety of APPV molecular detection methods and applied them to clinical samples.Detection,virus co-infection research and virus tissue tropism research,and comparison and verification of multiple molecular detection methods established in this study,in order to obtain efficient and sensitive detection methods that meet clinical needs,this study achieved the following results:1.Establishment and Application of Multiple TaqMan Quantitative PCR Method for Piglet Congenital Tremor-associated VirusBased on the analysis and comparison of APPV NS3 gene,CSFV E2 gene,PCV-3ORF2 gene and PTV VP1 gene,corresponding specific primers and probes were designed.The specificity,sensitivity,and reproducibility of the established multiplex TaqMan fluorescent quantitative PCR detection method were evaluated.The results show that the established detection method has a good linear relationship;the coefficient of variation of the repeatability test is less than 2.1%,which has obvious advantages in terms of repeatability and stability;specific detection of multiple viruses,only the positive template has a fluorescent signal,and the specificity is strong.The minimum number of copies detected by the multiplex TaqMan fluorescent quantitative PCR method is CSFV 48 copies/?L,APPV 9.2x10~2copies/?L,PTV-1 17 copies/?L,and PCV-3 56 copies/?L,with the lowest sensitivity 100 times higher than ordinary PCR.Using this method to test 273 clinical suspected samples of large-scale pig farms in some areas of Sichuan,the results showed that APPV,CSFV,PTV-1 and PCV-3 positive rates were 20.5%,2.9%,1.5%and 10.6%,respectively.Co-infection of the above four viruses exists in large-scale pig farms in various regions of Sichuan.Statistics on APPV infection in different swine herds revealed that the highest infection rate in piglets was 24.3%,and there were many recessive infections in adult pigs and detoxification through various channels.In summary,the multiplex TaqMan fluorescent quantitative PCR detection method established in this study has the characteristics of strong specificity,high sensitivity,and good reproducibility,which can be used for clinical detection and epidemiological investigation of related viruses.2.The establishment and application of porcine atypical plague virus droplet digital PCR methodIn order to establish a more sensitive and efficient APPV detection method,a pair of specific primers and probes were designed and synthesized based on the APPV gene sequence uploaded in Gen Bank,and the amount of primer probes and the reaction conditions were optimized.The results showed that the correlation coefficient R~2=1 of TaqMan fluorescence quantitative RT-PCR detection method,the amplification efficiency E=96.6%,and the minimum detection limit was 92 copies/?L.The correlation coefficient of dd PCR method R~2=0.999,and the minimum detection limit was 0.15 copies/?L.The detection of 135 clinical samples found that the detection sensitivity was dd PCR>RT-q PCR>RT-PCR in order.Compared with the other two methods,dd PCR has obvious advantages in sensitivity and specificity.After piglets were naturally infected with APPV,the established dd PCR method was used to detect the distribution of APPV in different tissues and organs.It was found that the virus was mainly distributed in the central nervous system tissues and lymph nodes.In combination with histopathological sections,congestive enlargement occurred in the central nervous system tissues and lymph nodes.Tissue sections showed obvious neuronal cell necrosis and formation of cavities in damaged tissues and organs.Pathological damage was related to the distribution of the virus.