Establishment Of Fluorescent Quantitative PCR Method For Detection Of Porcine Circovirus Type 2 And Study On Genomic Genetic Variation Of Tibetan Porcine Circovirus Type 2

Porcine circovirus type 2(PCV-2)is an important pathogen of porcine circovirus-related diseases,which has caused serious harm to pig industry.Tibetan pigs are mainly distribution in The Qinghai-Tibet Plateau and are valuable local breed resources in China.Currently,there are few reports about the PCV-2 infection in Tibetan pigs,and the epidemic situation and molecular characteristics of Tibetan pigs in Ganzi Tibetan Autonomous Prefecture of Sichuan Province(hereinafter referred to as Ganzi Prefecture)are still not clear.The aim of this study was to establish fluorescent quantitative PCR method for detecting PCV-2,and to investigate the prevalence and molecular characteristics of PCV-2 in Tibetan pigs in Ganzi Prefecture.The results are as follows:In our previous study,PCV-2 was identified from diarrhea stool samples of Tibetan pigs by metagenomic technology,but the sample was detected as PCV-2negative by fluorescent quantitative PCR(GB/T 34745-2017).In this study,Based on a 771 bp ORF1 sequence of PCV-2 obtained by metagenomic technology and the PCV-2 ORF1 sequences in Gen Bank,a pair of primers were designed.By optimizing the reaction system and conditions,a fluorescent quantitative PCR assay for detecting PCV-2 was successfully established.The standard curve of the method was Y =-3.336X+35.3,the correlation coefficient R2 was 0.9898,and the amplification efficiency was 99.42%,indicating that the method had good linear relationship and high amplification efficiency.The melting curve showed primer-free dimer and nonspecific amplification peaks.This method only amplified PCV-2 specifically,and did not detect other common unrelated pathogens.The coefficients of variation between and within batches were 0.73%-1.08% and 5.20%-8.16%,respectively.The minimum detection limit was 3.07copies·μL-1.In addition,the detection rate of PCV-2 in diarrhea samples of Tibetan pigs by the proposed method was higher than that by the four quantitative PCR methods,including the national standard detection method.In conclusion,the quantitative PCR method for detecting PCV-2 was successfully established in this study,providing an effective mean for further epidemiological investigation of PCV-2 in Tibetan pigs.From May to June in 2021,85 stool samples of Tibetan pigs with diarrhea and259 stool samples of Tibetan pigs without diarrhea were collected from 13 farms in 7major Tibetan pig breeding counties in Ganzi Prefecture,and PCV-2 was detected by fluorescence quantitative established in this Study PCR.The results showed that the average detection rate of PCV-2 was 77.92% in diarrhea samples and 87.44% in non-diarrhea samples.Field positive rate is up to 100%.The results showed that PCV-2 was widely distributed in Tibetan pigs in Ganzi Prefecture and infection rate was very high.Three genomic sequences of PCV-2 from Tibetan pigs were successfully amplified,including 2 strains of PCV-2D subtype and 1 strain of PCV-2Bsubtype,with the length of 1767 bp.No recombination event was found in the three PCV-2 genomes in this study,and evolutionary analysis showed that the three strains had unique evolutionary trends.This study firstly investigate the infection of PCV-2 in Tibetan pigs in Ganzi Prefecture,and three PCV-2 genomes were obtained,providing a reference for understanding the epidemic and molecular characteristics of PCV-2 in Tibetan pigs.