Establishment And Preliminary Application Of Porcine Circovirus Type 3 Microdrop Digital PCR Method

Porcine circovirus type 3(PCV3)can cause a variety of disorders such as reproductive disorders,dermatitis nephrotic syndrome,cardiac and systemic multiple system failure syndrome,lymphoid tissue lesions,and progressive wasting.Currently,PCV3 is widespread,and it mainly attacks the immune system of pigs and causes co-infection with other important swine diseases.The establishment of a sensitive and accurate molecular diagnostic method can be used for the early diagnosis of the disease,epidemic surveillance and inspection and quarantine of import and export of breeding pigs,which will help the comprehensive prevention and control of the disease.In this paper,a PCV3 droplet digital PCR(ddPCR)detection method is established for the detection of clinical samples of pigs.This method is performed in conjunction with the TaqMan flurogenic quantitative PCR(qPCR)method established for PCV3.The parallel detection and comparison lays the foundation for exploring the transmission characteristics and tissue addiction of PCV3.The main contents of the research in this paper are summarized and expressed as follows:1.Establishment of PCV3 ddPCR detection methodAccording to the nucleotide sequence of the PCV3 ORF2 gene(MF139082.1)published by Gen Bank,design PCR primers to amplify the PCV3 ORF2 gene(about651bp);The recombinant plasmid p MD-PCV3-ORF2 was successfully constructed.According to the nucleotide sequence of PCV3 ORF2 gene in Gen Bank,the accession number is(MF631813.1),design synthetic primers and probes,optimize annealing temperature,primer probe concentration,establish PCV3 droplet digital PCR and TaqMan fluorescence quantitative detection To determine the sensitivity,specificity and repeatability of the two methods.The results showed that the minimum detection limit of ddPCR was 16.35copies/?L,and the minimum detection limit of fluorescent quantitative PCR was 65.4copies /?L.The sensitivity of ddPCR was higher;the two methods did not cross-react with other viral nucleic acids,only The fluorescence signal of PCV3 positive template can be collected.Both methods have strong specificity.The two methods were used to repeat the test.The coefficient of variation(CV%)value of the intra-group and inter-group tests was less than 3%,indicating This method has good repeatability and can be used for the detection of clinical samples.2.Preliminary application of PCV3 ddPCR detection methodThe ddPCR and qPCR detection methods established in this study were used to detect474 clinical samples,including 238 serum samples,47 aborted fetuses,39 semen,62 saliva swabs,53 skin samples,and 35 piglet visceral tissues;The positive rates of ddPCR and qPCR test results in serum samples were 50%(119/238)and 47.05%(112/238)respectively.Kappa analysis was performed on the two methods,and the Kappa coefficient was 0.941(SE = 0.065,95% confidence interval),Indicating that the two detection methods are highly compatible;the positive rates of ddPCR and qPCR in the remaining samples were 61.7%(29/47)and 46.8%(22/47)of aborted fetuses,69.2%(27/39)and58.9% of semen,respectively.(23/39),saliva swabs 69.3%(43/62)and 59.6%(37/62),skin 58.4%(31/53)and 49.1%(26/53),piglet visceral tissue 80%(28/35)and 57.1%(20/35).The total positive detection rate of ddPCR was 7.8% higher than that of qPCR.It shows that ddPCR has high sensitivity accuracy and can be used for detection of latent infection with low virus titer.The results of 61.7%(29/47)fetal abortion,69.2%(27/39)semen,69.3%(43/62)saliva swab,and 58.4%(31/53)skin analysis showed that PCV3 had horizontal transmission and The possibility of vertical transmission.By analyzing the detection results of PCV3 virus in different tissues from 35 suspected tissue samples of 5dead piglets,it was shown that PCV3 had the highest virus content in submandibular lymph nodes.It can be speculated that the main tissue and organ invaded by PCV3 may be lymph nodes,followed by lung,Brain and kidney,the virus exists widely in sick pigs,can invade different tissues and organs,and the virus content in different tissues and organs is different,late infection may cause extensive infection of various organs and eventually cause systemic viremia,PCV3 Provide an important basis for replication and proliferation in sick pigs.