Establishment And Preliminary Application Of PCV2 And PCV3 Dual Fluorescence Quantitative PCR

Porcine circovirus type 2(PCV2)and type 3 have spread worldwide,causing huge losses to the pig industry.PCV2 is capable of causing multiple disease syndromes in infected pigs,while Porcine circovirus type(PCV3)is mainly associated with reproductive disorders and multisystem inflammation in sows.The two are similar in clinical manifestations,often accompanied by mixed infections and co-infections with other viruses and bacteria,which are not easily distinguished.Therefore,there is a need to establish a quick,accurate as well as convenient diagnostic technique for the diagnosis,prevention and control of the disease.In this study,a dual fluorescent quantitative PCR technique for the detection of two types of porcine circoviruses was established by optimizing the primer and probe concentration and PCR reaction conditions,and applied to the diagnosis of clinical cases.At the same time,samples from pigs at different production stages and different health conditions in pig farms of different clinical scale and environmental samples were collected,and virus load analysis and epidemiological investigation were carried out.The results of the study are as follows.1.Establishment of a dual fluorescent quantitative PCR method for PCV2 and PCV3The complete genome sequences of PCV2 and PCV3 were downloaded from the Gen Bank database,and amplify the conserved sequence of the Cap genes of the two viruses by designing the two pairs of PCV2 and PCV3 of atopic primers and probes.The method was established by optimizing the reaction system and procedure of dual fluorescence quantitative PCR.The results showed that the standard curves of the established dual fluorescence quantitative PCR had good linearity and correlation coefficients(R~2)were all greater than 0.990;in order to demonstrate the good specificity of the method,other pathogens with similar clinical symptoms and mixed infections were used as templates,and the results showed that only PCV2 and PCV3 positive templates showed an"S"amplification curve,indicating the good specificity of the method;the sensitivity results showed that PCV2 and PCV3 could still show amplification curves at dilution to 4.32×10~1copies/μL and 1.01×10~1copies/μL,which were more about 100 times more sensitive than the common PCR method;the coefficient of variation of the tests within the same batch group and between different batch groups were less than 1%,which proved that the reproducibility of the results was stable.A total of 146 clinical serum,saliva and tissue samples were selected,and the established dual fluorescence quantitative PCR method was used to compared with the common PCR method,and Cohen’s kappa coefficient was used to assess the concordance between the two.The results showed that the detection results of the two methods for PCV2 infection alone and co-infection were consistent;the detection rate of PCV3 by q PCR was 21.92%(32/146),while the detection rate of PCV3 detected by common PCR was 20.55%(30/146),and the undetected PCV3 positive samples were all samples with Ct values higher than 35;according to Cohen’s kappa coefficient,the concordance between the two methods was high(κ=0.959,95%CI 1.016-0.902,P<0.001),and the above results indicate that q PCR method has a higher positive detection rate compared to the common PCR method.Therefore,the PCV2 and PCV3 dual fluorescence quantitative PCR method established in this study is highly specific,sensitive,reproducible and can be applied to clinical detection.2.Clinical application of the dual fluorescent quantitative PCR method for PCV2 and PCV3A total of 1241 pig source samples and environmental samples from 3 large-scale breeding farms and 16 fattening farms,all of which were immunized with the PCV2 vaccine and tested for PRRSV simultaneously,were tested by an optimized dual fluorescence quantitative PCR method.The results showed that the detection rate of PCV2 was 0.73%(9/1241),the detection rate of PCV3 was 16.84%(209/1241),the co-infection rate of both was 0.24%(3/1241),and the co-infection rate of PRRSV and PCV3 was 1.21%(15/1241).3.Analysis of the prevalence of PCV2,PCV3 and mixed infections in large-scale pig farmsIn this study,the collected samples were statistically analyzed by dividing the group of pigs with the no-clinical sign and the clinical sign,which were based on clinical information and judgment of the health status of the pigs.The results showed that PCV2was only detected in the group of pigs with the no-clinical sign,and the positive rate of PCV3 in this group was significantly higher than that in the group of pigs with clinical sign,and there was also a significant difference in viral load between the two groups,indicating that there was no significant relationship between viral infection and clinical signs.Comparative analysis according to different ages showed that PCV3 was widely present in different age groups,with the highest positivity rate in finisher and replacement breeding in the group of pigs with the no-clinical sign and in nursery and piglets in the group of pigs with the clinical sign.All samples were analyzed by different types,and the results showed that PCV2 was detected only in the sample of oral fluids and swabs and not in the rest of the sample types;PCV3 had the highest positive rate in the sample of oral fluids and swabs,and was detected in the sample of blood,tissues,feces,colostrum and pigsty.Data analysis of pig farms with high testing volume revealed that some pig farms had co-infection of PCV2 and PCV3,and the positive rate of PCV3 in some pig farms were more than 40%,which required timely prevention and control to cut off the transmission route of the virus.In addition,during the testing process,3 samples were found to have mixed infection of PCV2 and PCV3,and 15 samples were detected to have mixed infection of PCV3 and PRRSV.In conclusion,the study established a dual fluorescent quantitative PCR method for PCV2 and PCV3,which can rapidly and accurately differentiate the two viruses and provide technical support for the diagnosis and epidemiological investigation of the disease and its distribution pattern.