Development Of Digital PCR Assays For Porcine Circovirus

Entry-Exit quarantine animal and plant system need a rapid,sensitive and high productive detection method.Porcine circovirus,as a second type of infectious disease,has three types called porcine circovirus type 1（PCV 1）,PCV 2,PCV 3.It is widely prevalent all over the world.It is necessary to conduct inspection and quarantine on inbound and outbound breeding pigs,pork products,and pork-containing products to prevent the spread of diseases.In this study,two new,sensitive and easy-to-operate digital PCR quantitative detections of porcine circovirus were established.And one of them was applied to the detection of PCV 2 content in feed samples and pig tissues.Both detection methods were compared and verified with the Taq Man fluorescent quantitative PCR method,and the porcine circovirus that meets the requirements of entry-exit inspection and quarantine was highly efficient and sensitive quarantine method.The main content and results of this study are as follows:1.Establishment of PCV 2 dd PCR detection methodIn this study,a more sensitive assay based droplet digital polymerase chain reaction（dd PCR）system for monitoring of porcine circovirus type 2（PCV 2）was developed,targeting its ORF 1 gene.The optimal reaction conditions were confirmed with the final primer and probe concentration of 0.7μmol/L and 0.25μmol/L,respectively,as well as the optimized annealing temperature of 59℃.With only positive signal to PCV 2genome by dd PCR,the system showed high specificity to PCV 1,PCV 3 and other swine pathogens.The limit of detection of the dd PCR assay was confirmed as 2.93copies/μL using serially diluted standard plasmids,which is 10 times more sensitive than the q PCR method.The coefficient of variation of the intra-and inter-assay repeatability test of this method is less than 10%which means this method is reproducible.A panel of field samples including 24 tissue samples and 70 pet feed samples were analyzed by the dd PCR and q PCR,respectively.The results indicated that identical results of 10 positive were found in both assays.However,nineteen pet feed samples were detected as PCV 2 positive by the dd PCR assay,in which 5 samples were negative by q PCR,indicating that the dd PCR assay was a much more sensitive,reliable and powerful method for PCV 2 infection monitoring.2.Establishment of triple Crystal digital PCR method for PCV detectionAccording to the PCV 1 gene sequence（KC447455.1）and PCV 3 gene sequence（NC＿031753.1）as templates,the corresponding specific primers and probes were designed respectively.After the optimized reaction,a triple Crystal digital PCR was established to detect PCV 1,PCV 2,and PCV 3.At the same time,the q PCR method is used for comparison.The results of the established standard curve showed that the correlation coefficient:R²_PCV1=0.9878,R²_PCV2=0.9966,R²_PCV3=0.9833,amplification efficiency:E_PCV1=90%,E_PCV2=98%,E_PCV3=94%.The specificity of this method is good,only specific amplification of related genes,the established triple Crystal digital PCR can detect the minimum copy number of the plasmid standard:PCV 1:4.96 copies/μL,PCV 2:12.29 copies/μL,PCV 3:11.54 copies/μL.The anti-interference test shows that the minimum detection limit of the method is not affected by similar viruses.And the repeatability test showed that the repeatability coefficient of variation within the group was within 10%,and the coefficient of variation between the groups was good when the coefficient of variation was on the order of 10⁶ copies/μL～10¹ copies/μL,and it was relatively at the order of 10⁰ copies/μL,although all tests can be detected.It shows that the established triple Crystal d PCR has good repeatability and has market application potential.