Establishment And Application Of Nested PCR Detection Method For Porcine Circovirus Type 3

Porcine circovirus type 3(PCV3)is a new circovirus that was first discovered in the United States in 2016 and subsequently reported in countries such as South Korea,Brazil,Italy,and Poland.At present,PCV3 has existed in many provinces and cities in China.Studies have shown that PCV3 and PCV2 can cause dermatitis and nephrotic syndrome,reproductive disorders and multi-system inflammation in pigs.Given the severe economic impact of PCV2,further research is needed on this new circovirus to clarify its importance and its role in circovirus-associated diseases.However,at present,the research on the virus is still in its infancy,and it is necessary to carry out relevant detection technology research as soon as possible,and establish a sensitive and rapid diagnosis method for early diagnosis of the disease.This study aims to establish a PCV3 nested PCR detection method to achieve rapid detection of PCV3.It will lay a foundation for understanding the infection status of PCV3 in Anhui Province and the molecular epidemiology of PCV3,and provide a scientific basis for the in-depth study and prevention of PCV3.1.Establishment of PCV3 nested PCR detection methodIn this study,two pairs of nested PCR-specific primers were designed based on the PCV3 gene sequence registered in GenBank(accession number:MF084994),and the PCV3 nested PCR detection method was established by the following experiments:(1)setting 54?,55 Screening of annealing temperatures at different annealing temperatures of?,56 0C,57 0C,58 ?,59 0C,and 60?,and by 100 times,50 times,10 times,3 dilutions of the first round of PCR amplification products Dilution was performed as a second round of PCR template to optimize the PCR reaction conditions.(2)Using this method for PCV3 and porcine circovirus type 2(PCV2),swine fever virus(CSFV),pseudorabies virus(PRV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine epidemic diarrhea virus(PEDV)The detection of five common viruses was tested for specificity.(3)Using the method established in this study,the sensitivity test was carried out on the detection of(10-10?10-1)different dilution standard plasmids simultaneously with the common PCR method.(4)Repeatability tests were carried out by batch-to-batch and batch-to-batch detection of 10-8,10-9,10-10 copies/uL concentration standard plasmids.(5)Using the PCV3 nested PCR established in this study and the commercial kit to detect 78 serum samples simultaneously,Kappa analysis was used to compare the detection results of the two methods to verify the coincidence rate.The results showed that:(1)58? is the optimal annealing temperature of PCV3 nested PCR established in this study.The amplification result obtained by dilating a product with 50-fold dilution for the second expansion is the best.(2)Except for the expected target band amplified by PCV3,none of the five viruses of PCV2,CSFV,PRV,PRRSV and PED appeared.(3)The minimum detection limit of this method is 1.74×10 copies/?L,which is 105 times higher than the sensitivity of ordinary PCR.(4)10-8 and 10-9 copies/uL of the standard template parallel samples have the appearance of the desired band,but when the template concentration is 10-10 copies/uL,3 parallel samples have no target bands;10-8,10-9 copies/uL of the standard template showed the target band in 3 tests,but there was no target band 3 times when the template concentration was 10-10 copies/uL.(5)The positive rates of PCV3 in 78 serum samples detected by nested PCR assay and commercialized real-time PCR were 30.77%and 33.33%,respectively.The Kappa analysis coefficient of the two methods was 0.94(kappa>0.75 is consistent).2.Application of PCV3 nested PCR detection methodThe PCV3 nested PCR detection method established in this study was used to detect PCV3 from 528 different clinical samples from 15 cities in Anhui.The SPSS 22.0 software was used to process the positive rate of PCV3 in different sample sources,different clinical symptoms,different growth stages and different types of samples.The chi-square test was used for data analysis.The results showed that:the total positive rate of PCV3 in 528 clinical samples in 1 5 cities in Anhui was 20.45%,and PCV3 infection occurred in 12 cities.PCV3 was detected in pigs with clinical manifestations of respiratory diseases,reproductive disorders,neurological symptoms,diarrhea,hyperthermia,acute death,and weight loss.The positive detection rate was between 5.00%and 44.44%,with no significant difference;different growth.PCV3 infection was present in the pigs(breastfeeding pigs,nursery pigs,fattening pigs,sows,stillbirths).The positive detection rates were 14.52%,19.35%,25.42%,29.69%,24.24%,respectively.The difference was not significant;PCV3 was The detection rates of diseased tissue samples,serum and feces were 18.72%,33.33%,and 12.50%,respectively.The positive detection rate of serum samples was significantly higher than that of tissue samples;PCV3 was in pig brain,lung,liver,The detection rates in the spleen,kidney and lymph nodes were 21,21%,23.33%,26.67%,20.18%,31.25%,and 47.83%,respectively.The detection rate of PCV3 in lymph nodes was significantly higher than that in the spleen.The results show that:(1)The PCV3 nested PCR detection method established in this study is specific,sensitive and reproducible,and can be used for the detection of clinical samples.(2)PCV3 infection exists in most areas of Anhui;PCV3 is associated with respiratory diseases,reproductive disorders,neurological symptoms,diarrhea,hyperthermia,acute death and other diseases;pigs in all growth stages can be infected with PCV3;serum The positive detection rate of PCV3 in samples was higher than that of tissue samples;PCV3 was distributed in different tissues and organs,and the positive detection rate of lymph nodes was the highest.