Establishment Of A Molecular Biology Identification And Fluorescence Quantitative Detection Method For Porcine Circovirus Type 3 In The Aksu Region Of Xinjiang

Porcine circovirus type 3(PCV3),a newly discovered pathogen,has been widely prevalent within pig herds farmed in many countries around the world since it was first reported in the USA in 2016,seriously jeopardising the development of the pig industry.In response to this situation,rapid and accurate detection of this virus is of great importance to prevent and control the spread of this disease in pig herds.To understand the infection status of PCV3 in pigs in large-scale pig farms in the Aksu region of Xinjiang,a total of 584 whole blood samples were collected from four large-scale pig farms(including one breeding farm and three fattening farms)in the Aksu region between 2020 and 2021,and the pathogen of porcine circovirus type 3 was detected by PCR using specific primers for the Cap gene of PCV3.The test results showed that a total of 44 PCV3 positive samples were detected in four large-scale pig farms in the Aksu area of Xinjiang,with an overall positive rate of 7.53%(44/584),of which 3.25%(19/584)were positive for PCV3 infection in breeding farms and 4.28%(25/584)were positive for infection in fattening farms;by testing different farm types and different growth stages of pigs The data analysis showed that breeding sows and piglets were susceptible to PCV3,and there was a correlation between PCV3 infection and growth stage;by sequencing the PCR products of PCV3 positive samples,two PCV3 Cap gene sequences,AKesucap-1(fattening farms)and AKesucap-2(breeding farms),were obtained,which were compared with domestic and foreign PCV3 Cap gene sequences.The homology analysis showed that AKesucap-1 was 100% homologous to CN/Xinjiang-AK16/2018 strain and AKesucap-2 was 99.6%homologous to NWHEB21 and PCV3-CN-ZJ-QZ-2-2017.PCR amplification was performed using specific primers designed for PCV3 whole genome segmentation,using positive sample DNA from breeding farms susceptible to PCV3 as a template.After cloning,identification and sequencing,the whole genome sequence of PCV3 was spliced to obtain the full length of 2000 bp,which was named PCV3-XJAKesu.The sequence was homologous to eight foreign PCV3 reference strains and six domestic PCV3-XJAKesu had the highest homology of 99.4% with the Chinese strain NWHEB21 and both belonged to the PCV3 3c subgroup.A pair of PCV3-specific primers and fluorescent probes were designed for the conserved region of PCV3 Cap gene,a PCV3 Cap gene positive plasmid standard was constructed,and a Taq Man fluorescent quantitative PCR assay for PCV3 was established using the standard as a template.The standard curve of the assay showed good linearity between 3.72×1010-3.72×103 copies/?L,where the correlation coefficient was 0.998 and the sensitivity of the assay was 3.72×103 copies/?L.The coefficient of variation was less than 1% by intra-group and inter-group reproducibility tests,indicating that the method has good reproducibility.Also,the method is free of crossover amplification reactions and is 10 times more sensitive than conventional PCR methods.The q PCR method established in this experiment was used to test 53 porcine whole blood samples.The positive detection rate of this q PCR method was 3.7% higher than that of the traditional PCR technique.