Development Of A Double Droplet Digital Polymerase Chain Reaction For Porcine Circovirus Type 2 And Type 3

Porcine circovirus type 2 is one of the recognized pathogenic bacteria that cause serious harm to the pig industry in the world.It can be mixed with a variety of other pathogens to cause Post-weaning multi-systemic wasting syndrome,Porcine Dermatitis and Nephropathy Syndrome,Proliferative and necrotizing Pneumonia and other diseases,It has a great impact on the pig industry all world contries.Porcine circovirus type 3,a new virus discovered in the United States in 2016,is rapidly spreading in pig countries around the world,with significant implications for the pig industry in the future.PCV2 and PCV3 both belong to porcine circovirus,which cause similar clinical manifestations and often appear mixed infection.In 7 provinces of China,the mixed infection rate of Hunan,Hubei and Jiangsu is 42.9%.At present,immunological diagnostic methods and molecular biological diagnostic methods have been established,including common detection methods such as Enzyme-linked immunosorbent assay,PCR and Real-time fluorescence quantitative PCR(RT q PCR).Most of these methods include the disadvantages of time-consuming,low sensitivity,easily contaminated and so on.The purpose of this experiment is to establish a fast and efficient method for quantitative detection of PCV2 and PCV3 simultaneously,it can be used for specific quantitative detection of low concentration samples such as diseased tissue or serum.The specific research contents are as follows.1.Establishment of detection method of PCV2 and PCV3 by droplet digital PCR(dd PCR).We selected the conserved sequences of PCV2 and PCV3(Gen Bank NO.KC823059.1 and KY778776)and vector p UC59 to synthesize plasmids,and designed primers with primer premier 5 software.By optimizing the ratio of annealing temperature and primer probe concentration,the detection methods of PCV2 and PCV3 by dd PCR were established.The specificity of the reaction system was explored through the specificity experiment.The sensitivity and repeatability of the detection methods of PCV2 and PCV3 by dd PCR were compared with that of PCV2 and PCV3 by RT-q PCR.The results showed that when the concentration ratio of the primer probe was 400 nm:200 nm and the annealing temperature was 56?,the effect was the best.It had no specific amplification for other nucleic acid samples,and the sensitivity could reach 0.1 fg/?L.Dd PCR can detect as low as 1.1 copies/?L of p UC59-PCV2 and 1.03 copies/?L of p UC59-PCV3.The correlation coefficients(R~2)of q PCR and dd PCR standard curves of PCV2 were 0.9941 and 0.9762,respectively,and the amplification efficiency of dd PCR was 83.41%.The correlation coefficients(R2)of q PCR and dd PCR standard curves of PCV3 were 0.965 and 0.9575,respectively,and the amplification efficiency of dd PCR was 80.75%.The Intre-assay variation CV(%)of PCV2 of dd PCR and Real-time fluorescent quantitative PCR were less than 5%,and the Intra-assay variation CV(%)of PCV3 of dd PCR and RT-q PCR were less than 6.06%,indicating that the four detection methods established in this experiment have good repeatability and stability.2.Establishment of detection method of PCV2 and PCV3 by double dd PCR.Each micro-reaction system contains two sets of primer probes,which are used to detect with two different reporting group probes,FAM and VIC respectively.PCV2and PCV3 can simultaneously and efficiently quantify,improve detection efficiency and reduce detection cost.The sensitivity of detection method of PCV2 and PCV3 by double dd PCR and double RT q PCR in this experiment can reach 0.1 fg/?L,in which the copy number of p UC59-PCV2 and p UC59-PCV3 detected by double dd PCR is1.1 copies/?L and 1.32 copies/?L respectively.The correlation coefficient(R2)of PCV2 and PCV3 standard curve detected by double q PCR is 0.9907 and 0.9682,respectively.The correlation coefficients(R2)of PCV2 and PCV3 were 0.9651 and0.9638,respectively,and amplification efficiency was 83.9%and 78.8%,respectively.The results of variance analysis show that the four F values are less than their f crit values,then the F values are not significantly different at the level of a=0.05.The results showed that there was no significant difference between the double dd PCR and the double RT q PCR and the single detection.Furthermore,they all show a good linear relationship.3.Actual sample testing and kit development.95 pigs suspected to be infected with PCV2 were detected by q PCR and dd PCR.The positive rate of q PCR was 60%(57/95),the positive rate of dd PCR was 62.10%(59/95).The kappa coefficient of the two methods is 0.9766,which shows that the two methods have good consistency.Among them,dd PCR can make positive or negative judgment through the specific copy number for the suspected results detected by q PCR,and play a unique advantage in low concentration detection samples.In this chapter,we also developed two kinds of double detection kits,which have good specificity and sensitivity,can meet the detection requirements of various environments and conditions,and save costs,shorten the detection time.In one reaction process,we can detect PCV2 and pcv3simultaneously,and will have a broad market prospect.