Isolation And Identification Of PRRSV NADC30-Like CJS01 Strain And Establishment Of Its Detection Method

PRRSV NADC30-Like strain is a popular strain that has been started in the past 5 years.Some areas have not yet received attention.Many places in China have caused large economic losses.Therefore,it is important to study the pathogenic characteristics and detection methods of this type of strain for prevention and control of PRRSV.With the internationalization of pig trade,the pig industry is in an unprecedented period of development,which also makes the prevention and control of the disease more difficult.In this study,a strain of PRRSV NADC30-Like was isolated from the samples from Sichuan,and some biological characteristics and bioinformatics analysis were performed on the isolates.At the same time,the NADC30-Like PRRSV SYBR Green I qPCR detection method was established.Two methods for detecting antibodies were established based on the isolate,including IPMA and recombinant N protein indirect ELISA..This study aimed to understand the relationship between recombinant evolution and immunity based on the biological characteristics of the isolate,and to improve the PRRSV detection method.And finally laid the foundation for accurate and reliable diagnosis of PRRSV NADC30-Like strain,and at the same time,better control PRRSV provided a scientific reference.1.NADC30-Like PRRSV SYBR Green I qPCR detection method establishment and applicationIn order to diagnose the NADC30-Like PRRSV pathogen more quickly and accurately,this study established the SYBR Green I qPCR detection method.According to this strain conserved sequence,specific primers was designed,standard products were prepared,and the standard curve equation was established:Ct=-3.143 Lg?copies?+43.032,the copy number had a good linear relationship with the Ct value,R²=0.975,the amplification efficiency was 108.1%,the intra-group coefficient of variation was less than 1.6%,and the sensitivity was 8.85×10² copies/?L.The method had high sensitivity,high specificity,low cost and good application value.2.Isolation and identification of strains and some biological characteristics and bioinformatics analysisIn this study,the above positive samples were screened to ensure that there were no other pathogens,and virus isolation was carried out.The above method was used to identify and identify with IFA,and it was identified as PRRSV.Compared with the common PRRSV strain,the isolate did not produce obvious CPE in the early stage of isolation.CPE was found in the late stage,and the titer was determined to be 10^-4.17 TCID₅₀/0.1 mL.Bioinformatics analysis showed that it was a PRRSV NADC30-Like strain,named CJS01 strain.By measuring the nucleic acid load during the proliferation of the strain on Marc-145 cells,it showed that the virus had good adaptability on the cells,and the virus proliferated to the highest in the DNA for 84 h;the recombinant analysis of the ORF3⁷ gene was carried out.And its antigenic epitope prediction,found that CJS01 strain caused two important nucleotide sites of neutralizing antibody and sub-neutralizing antibody in the carcass in this recombination interval,which may make the immune situation more complicated.3.Establishment of PRRSV NADC30-like strain IPMA methodSince NADC30-Like is a novel PRRSV strain,further research is needed on its antibody production.The IPMA assay has many advantages in terms of time and type of antibody production.In this study,after the initial cell inoculation dose,incubation time were optimized,the concentration of the secondary antibody was 10 times diluted by the inoculum of the virus solution(10^-4.17TCID₅₀/0.1mL),the secondary antibody against HRP-SPA was pressed and a multiple of 1:3000 and serum were diluted at a multiple of 1:30,an IPMA detection method was established based on the isolate CJS01.The results showed that the method had high specificity and low sensitivity,and had potential for exploring research related to antibody production of new strains.4.Establishment and preliminary application of indirect ELISA method based on recombinant N proteinIn order to adapt to detecta large number of clinical samples,the recombinant protein granule pET32a-N of NADC30-Like strain N gene was constructed,and the target protein was identified as soluble protein by SDS-PAGE.Western blotting showed that the protein had good reactogenicity.A series of optimizations were carried out on the expression conditions,indicating that the optimal expression condition was obtained by inducing expression of 2 h at a final concentration of IPTG of 0.2 mmol/L at 37°C.The expressed recombinant protein N was used as an antigen,when the concentration of the antigen was 10?g/mL,the serum to be tested was diluted 40 times and the secondary antibody was diluted3000 times,the effect was ideal and can be performed well.The detection of clinical samples had good specificity and stability.