PCV2 Isolation And Identification And Establishment Of LAMP-CRISPR/Cas12a Nucleic Acid Detection Method

Porcine circovirus 2(PCV2)causes multisystem failure syndrome and dermatitis nephrotic syndrome in pigs after weaning,a virus that can cause potential harm during pig growth and cause serious economic losses to the global swine industry.Therefore,rapid detection of PCV2 plays a key role in the prevention of postweaning multisystemic wasting syndrome(PMWS)in weaned pigs.PCV2 can currently be divided into 8 different genotypes,namely PCV2a to PCV2h,and PCV2 continues to evolve from the change in the prevalence of PCV2 genotype.Firstly,ORF2 gene sequence amplification,sequencing and analysis were carried out on PCV2-positive disease materials in some provinces in China in 2022-2023 to verify the genetic variation of PCV2.Based on this,the PCV2 strain of Shandong was successfully isolated from suspected PCV2 disease material.Finally,a detection method combining ring-mediated isothermal amplification method with the regularly spaced short palindromic repeat Cas12a system of aggregation was established for the detection of porcine ring ORF2 gene.1.The epidemiological investigation of PCV2 in some provinces in China in 2022-2023 was carried out,the ORF2 sequence amplification and sequencing of PCV2-positive disease materials were carried out by the PCR method,and the PCV2 ORF2 gene sequence and the amino acid sequence encoded by DNA Star and MEGA-X bioinformatics software were used to perform genetic evolutionary analysis.The results showed that the nucleotide homology between the eight PCV2 ORF2 genes was 92.2%～99.6%,compared to 83.1%-99.6%with the domestic and foreign reference strains announced on GenBank,respectively.There were obvious variations in the ORF2 gene of the eight PCV2 sequences and the amino acid sequences encoded by them.Eight PCV2 genotypes belonged to PCV2b and PCV2d,with 1 and 7 strains,respectively.2.The treated tissue disease material was isolated and cultured by PK15 cells for PCV2,and it was identified by indirect immunofluorescence and PCR,and the identification results showed that the isolated and cultured strain was PCV2,and the TCID50 of the strain was 10-3.64/0.1mL.3.Using pMD 18T to construct a positive standard plasmid,LAMP-specific primers for PCV2 and crRNA for Cas12a were designed,and the results showed that the plasmid was successfully constructed,the LAMP primers could be used to amplify the target sequence and be used for detection,and the relevant tests verified the feasibility of the designed crRNA.4.The PCV2 detection method based on LAMP-CRISPR/Cas12a was established and optimized,and the specificity,sensitivity,repeatability and compliance of the newly established detection method were evaluated.The results showed that crRNA3 was relatively the best in the five crRNAs designed,and the optimal reaction concentrations of Casl2a protein and crRNA in the detection system were 150nM and 100nM,respectively,and there was no cross-reactivity between the method and the positive samples of ASFV,PCV3,PRV and CSFV,and the lower limit of detection was as low as 1 00copies/μL,and the method had good reproducibility,the intra-batch coefficient of variation was less than 5%,and the inter-batch coefficient of variation was less than 10%.The compliance rate between this method and qPCR is high.In summary,this study established a method for detecting PCV2 based on LAMP-CRISPR/Cas12a nucleic acid that detects PCV2 under constant temperature conditions without professional instruments,has good specificity,high sensitivity,a single operation,low equipment requirements and a fast detection speed,and develops a new technical method for the clinical detection of PCV2.