| Porcine circovirus 2(PCV2)can cause many diseases and syndromes in pigs and seriously damage the immune system,which is one of the important pathogens threatening healthy pig breeding in China.Porcine circovirus 3(PCV3)is similar to PCV2 in clinical symptoms,including reproductive system disorders,systemic inflammation,and diarrhea.There is currently no effective vaccine to control PCV3.Given the high positive rates and the long-term mixed infection of PCV2 and PCV3 in farms all over China,it is very important to develop a rapid and accurate differential diagnosis technology for the prevention and purification of PCV2 and PCV3.Therefore,this study aimed to establish a dual real-time PCR assay for PCV2 and PCV3 for clinical diagnosis.The main research contents and results are as follows:1.Establishment of duplex real-time fluorescence quantitative PCR assay for PCV2 and PCV3In this study,a total of 2764 whole-genome sequences of PCV2 and 453 wholegenome sequences of PCV3 were retrieved from the NCBI Gen Bank database on June 26,2020.Two pairs of primers and probes were designed to amplify the conserved regions of the Rep gene in PCV2 and PCV3,respectively.After the recombinant plasmids,p MD18TPCV2 and PMD18T-PCV3 were constructed as the standard positive plasmids of this method,the reaction system and reaction program were optimized by adjusting the concentration of primers and probes in the reaction system and the annealing time and temperature of the reaction program.Using other infectious diseases with high co-infection rates and similar clinical symptoms as templates,the specificity test of the dual fluorescence quantitative detection method was carried out.The results showed that only PCV2 and PCV3 templates appeared with specific amplification,and no amplification of other pathogenic DNAc Dwhich NA pwhich roves that this assay has good specificity.The positive standard plasmid diluted at 10 times gradient concentration was used as a template for amplification,and the amplification results were drawn into a standard curve.The sensitivity test results showed that the sensitivity of PCV2 and PCV3 could reach a minimum of 10 copies/reaction and 50 copies/reaction,which proved the high sensitivity of the method.Using 5 positive standard plasmids diluted by 10-fold ratio dilution as templates,3 groups of repeated stability tests within the same batch and 5 different batches of repeatability tests were carried out respectively.The results showed that within the batch,the relative standard deviation(RSD)of the Ct values between batches was less than 1%,which proved that the assay had good stability and repeatability.30 PCV2 positive nucleic acids and 30 negative nucleic acids in clinical samples detected by this assay were selected as templates,and the match rate test was carried out with the national standard porcine circovirus type 2 fluorescent PCR detection assay(GB/T35901-2018),and the results showed positive and negative samples was 100%.Using this method,30 PCV3 positive nucleic acids were selected from clinical samples and sent to sequencing companies for genome sequencing.NCBI-BLAST analysis of nucleotide sequence alignment results showed that the homology of PCV3 was over 99.5%,which proved that the positive coincidence rate of PCV3 was 100%.2.Etiological detection of porcine circovirus type 2 and 3 in a certain pig farm in Hubei ProvinceThe prevalence of PCV2 and PCV3 in a local swine farm in Hubei Province was analyzed using the dual real-time fluorescence quantitative detection method of PCV2 and PCV3.In the first half of 2021,106 clinical autopsy materials were collected.The positive rate of PCV2 was 50.0%(53/106),the positive rate of PCV3 was 11.3%(12/106),and the total infection rate was 8.5%(9/106).The positive nucleic acid obtained was confirmed by gene sequencing after amplification.The detection results showed that PCV2 and PCV3 mixed infection did exist in the field,and the infection of PCV2 was persevere it was necessary to take timely control measures.3.Sequencing and genetic evolution analysis of porcine circovirus type 2 and type 3in a pig farm in Hubei ProvinceThe whole genomes of nucleic acids positive for PCV2 and PCV3 were sequenced from a pig farm in Hubei Province.The sequencing results were compared with the nucleotide sequences of different subtypes of PCV2 and PCV3 strains uploaded in NCBI Gen Bank,and the phylogenetic evolutionary tree and homology matrix was constructed based on the sequencing results.On this basis,whole-genome characteristics,genetic evolution analysis,and amino acid sequence alignment analysis of Cap protein were performed.The results showed that the isolates identified in this study belonged to PCV2 b and PCV3 belonged to PCV3a-1.The nucleotide homology of the strains sequenced in this study(PCV2-HB/XN/2021)was 83.5%-99.2% with that of the reference strains from different subgroups inside and outside China from 2001 to 2021.The sequencing strains in this study(PCV3-HB/XN/2021)had a high nucleotide homology with the REFERENCE strains of PCV3 from different sources at home and abroad from 2016 to 2021,ranging from 98.1% to 99.5%.The alignment of amino acid sequences of PCV2 and PCV3 Cap proteins showed that the mutation of amino acid at position 59 of the PCV2 strain sequenced in this study was K,which was the same as that of PCV2 d and PCV2 h,while R/A was found in other PCV2 b.In addition,the different amino acids of Cap proteins of PCV2 strain sequenced in this study were identical to those of Porcine circovirus 2 strain09 GD strain isolated in China in 2009.The PCV3 strain identified in this study had two specific amino acids in addition to the 24 and 27 sites,phenylalanine(F)mutated to tyrosine(Y)at position 104 on the predicted B cell epitope,and threonine(T)at position 156,distinct from serine(S)at position 156 in other subtypes.In conclusion,the dual real-time PCR detection assay of porcine circovirus type 2 and type 3 established in this study can sensitively and specifically detect and differentiate PCV2 and PCV3,which is a useful tool for the diagnosis and epidemiology of the disease in veterinary clinics.The investigation provides technical support for detection. |
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