Real Time Quantitative PCR And Droplet Digital PCR For Detection Of Seneca Valley Virus And Preparation Of Reference Material

Seneca Valley virus(SVV)is genetically related to the small RNA virus family and the heart virus genus.Clinically,it can cause blisters-like lesions on the nose and hoof crowns of pigs.Occasionally,diarrhea symptoms are present and the condition is rapid.The mortality rate of newborn piglets is 30%to 70%.There are reports of SVV infection in some pig farms in many provinces in China,although SVV will not cause significant economic losses.However,the clinical symptoms after infection are similar to those after transboundary animal diseases such as foot-and-mouth disease,swine vesicular disease,vesicular stomatitis,and swine vesicular rash,which often cause confusion.In China,SVV was first separated in 2016.At present,corresponding laboratory testing methods have been developed for SVV at home and abroad,such as electron microscope,immunohistochemistry,RT-PCR,etc.dPCR technology is a new technology for absolute quantification of nucleic acids based on qPCR detection methods.Compared with the relative quantitative method of qPCR nucleic acid developed before,the quantitative method is more sensitive and accurate,and the result can be directly judged without the need to establish a standard curve.Testing standard materials is the key to verifying the reliability of testing methods and testing reagents,and is also an important factor in the laboratory quality certification system.Therefore,the use of testing standard substances can provide technical guarantee for precise epidemic prevention and control.The research on animal pathogenic microorganism detection standard materials is still in its infancy in China,there are only a few standard serum,standard antigen and bacterial toxin standard materials for a few pathogens.However,the standard materials for the detection method of swine Seneca virus are very scarce in domestic,and the testing institutions at all levels lack nucleic acid standard materials.In this experiment,specific primers and probes for swine Seneca virus were designed,and a sensitive and specific quantitative PCR method was established.Based on the application of fluorescent quantitative PCR method,a digital PCR instrument was screened,and a digital PCR method of swine Seneca virus was established,and its sensitivity and specificity were tested.The porcine Seneca virus digital PCR method established in this study was used to quantify the isolated and identified porcine Seneca virus,and the homogeneity,stability,and repeatability of the porcine Seneca virus were determined.Then,in conjunction with 8 certified laboratories,the characteristic value of the Seneca virus nucleic acid reference material is determined.The results are as follows:1.Establishment of a double qPCR differential detection method for Seneca virus and foot-and-mouth disease virusThe FMDV and SVV specific primer probes were designed,and the optimal primer probe concentration was optimized for screening and,finally,a qPCR detection method for identifying FMD virus and Seneca virus in the same reaction tube was successfully established.it has good sensitivity and can detect 1.0×10~1 copies/μL at least.The established FMDV and SVV double qPCR detection method is a simple,rapid,specific and sensitive diagnostic method,which can be used for the rapid detection and differential diagnosis of FMDV and SVV.2.Establishment and application of SVV digital PCR methodBased on the SVV quantitative PCR method,And it successfully establishes the SVV ddPCR absolute quantitative detection method.it does not amplify nucleic acids of other pathogens.The sensitivity is good,and a minimum of3 copies/μL can be detected.The coefficient of variation calculated by repeatability is 2.39%.The established digital PCR method has good repeatability and stable and reliable test results.3.Preparation,value and application of porcine Seneca virus nucleic acid standard substanceThrough a series of studies,such as screening and identification of swine Seneca virus candidates,and using the established swine Seneca virus ddPCR absolute quantitative detection method to detect the swine Seneca virus concentration,the predetermined value of the cell virus culture fluid,the initial uniformity test,and so on,the nucleic acid standard substance of swine Seneca virus was prepared and divided into sub-packages.the minimum sample volume when using this standard substance is specified as 200μl.The results showed that the nucleic acid reference material of swine Seneca virus was stable for 9 days at 4℃and1 day at 25℃.Cold chain transportation is required,and the transportation time cannot exceed9d.This standard substance can be stable for six months under-20℃environment,and can be reconstituted 3 times.Eight qualified laboratories were selected to use the ddPCR absolute quantification method for the joint determination of the nucleic acid reference material of swine Seneca virus.From the data of eight laboratory tests,the average value of the nucleic acid standard substance of swine Seneca virus was calculated as 1.6×104 copies/μl,and the standard value was 1.7×104 copies/μl,and the calculated uncertainty was 0.3×10~4 copies/μl.