Tiisolation And Identification Of PRRSV And Regulation On Expression Of Immunological Factor To PAM

Porcine reproductive and Respiratory syndrome(PRRS)caused by Porcine reproductive and Respiratory syndrome virus(PRRSV)is a highly contagious disease which widely circulated in China's pig population and commonly cause mixed infection with other viruses leading to a serious threat to China's pig industryAlthough there are a variety of detection methods for this pathogen,conventional detection methods of etiology and serology have disadvantages such as time and energy consumption.Therefore,it is of great significance to establish a time-saving,labor-saving and highly automated pathogen detection method.Fluorescence quantitative PCR,developed in the 1990 s,is a new technique for quantitative detection of nucleic acid.SYBR Green-1 that is fluorescent double-stranded DNA(dsDNA)-specific intercalating dyes has been used in real-time PCR extensively.The principle of this method is to add fluorescent dye in PCR;The fluorescence intensity can change during the PCR process,so as to realize real-time monitoring of the PCR process.After the reaction,the nucleic acid molecules in the unknown samples can be precisely quantitatively analyzed by generating standard curves.Because of the advantages of simple operation and low cost of the SYBR green-1 fluorescence quantitative PCR detection method,compared with other quantitative methods,this technology has been successfully applied in many fields of life science research.In this study,specific primers were firstly synthesized according to the PRRSV N protein gene sequence available in GenBank.The length of the amplification fragment was 272 bp,which was cloned into pMD18 T vector and sequenced.The concentration of pMD18T-ORF7 recombinant plasmid was determined and its copy number was calculated.Then,pMD18T-ORF7 was used as the standard substance after gradient dilution,and the real-time fluorescence quantitative PCR detection method SYBR green-1 was established.The correlation coefficient R2 of this PCR detection method was 0.99.The specificity test showed that there is no specific amplification of PRRSV,CSFV,JEV genomic cDNA and PCV2,PRV and PPV genomic DNA of the control group.The detection sensitivity of this method is 3.30 102 copy number/l.The results showed that the coefficient of variation was between 0.76% and 1.50%,and the coefficient of variation was between 0.14% and 0.69% showing good specificity,sensitivity and repeatability of the method.Among the PRRSV positive samples detected by this method,one sample was selected for PRRSV isolation.One strain PRRSV was successfully isolated and named PRRSV-NM12.Gene sequence analysis showed that the virus belongs to American type and JXA1 derived strain.There is the typical 30 aa discontinuous deletion in Nsp2 amino acid sequence.In addition,relative quantitative PCR detection method was used in this study to detect the transcription level changes of SSBP3,ZC3 HAVI and Viperin caused by PRRSV infection,and found that the transcription levels of SSBP3 and ZC3 HAVI mRNA could be slightly reduced after PRRSV after inoculated with PAM.The transcription level of Viperin mRNA was significantly increased.In summary,this study provides a certain theoretical reference for PRRSV detection and prevention and control?...