| PRRSV has a wide range of epidemics and strong variability.The main domestic strains are divided into two categories.One is CH-1a and VR-2332,and the other is JXA1 and CHSX1401.To understand the PRRSV variation,the researchers established a NSP2 or ORF5 single-plex PCR method based on the ORF5 or NSP2 gene sequences.Single-gene PCR is highly sensitive.Single-plex PCR analyzes the sequence variation of one gene at a time.When researchers conduct multi-gene analysis,single-plex PCR is more cumbersome than multiplex PCR,and multiplex PCR can amplify multiple genes at a time.Perform analysis to simplify the steps of the multi-gene analysis method.This experiment established the NSP2-ORF5 dual PCR method of PRRSV,and the amplification product can analyze the variation of two gene sequences simultaneously.A set of NSP2-ORF5 double PCR method was established and the clinical application was carried out with reference to the blue ear pathogenic mutant strain and the classical strain gene sequence and related primer sequences.The specificity test by CH-1a,JXA1-R,JEV,PEDV,TGEV,PRV,CSFV NSP2-ORF5 double PCR method,the specificity of this method is good,and the lowest concentration of PRRSV cDNA detection is 5.95×10-3?g/?L.This method was used to detect blue-positive samples of clinical samples(identification of detection primers).A total of 8 ORF5 sequences of PRRSV and 7 NSP2 sequences of PRRSV were identified.The gene sequence of the strain and the highly pathogenic blue ear virus subgroup(subcluster ?)The genetic distance is the closest and the gene homology is the highest.The NSP2-ORF5 double PCR method established in this experiment has good specificity and high sensitivity.This method can be used to analyze the variation of NSP2 and ORF5 gene sequences in clinical samples,and can effectively analyze the variation of PRRSV strains.At present,prevention and control of PRRSV infection are mainly through comprehensive prevention and control through measures such as vaccine immunization,drug care,and biosafety.The PRRSV vaccine mainly includes VR-2332,CH-1a classical strain and JXA1,TJM-F92 and other mutant strain vaccines.In the prevention and control of PRRSV infection,the safety of PRRSV live vaccines of different strains(immunosuppression,genetic recombination,detoxification There is a big difference in infection,etc.).In this experiment,PRRSV vaccine(classical strain and mutant strain)was used to infect PAM in vitro,qRT-PCR was used to detect the expression of PAM immune-related factor mRNA,and the expression of PAM immune function-related factor mRNA in classical strain PRRSV vaccine and mutant strain PRRSV vaccine was obtained.The difference.Purchase healthy pig lungs(PCR detection of PRRSV,PCV2,Mhp negative),and separate and purify PAM by lavage.PAM was divided into three groups: blank group control group,JXA1R-PRRSV vaccine infection group,and VR2332-PRRSV vaccine infection group,with three replicates in each group.PAM was obtained at 37 °C 5% CO2 for 0 h,12 h,and 24 h,respectively.The expression of immune-related molecules and cytokine mRNA was detected by qRT-PCR.The results showed that PRRSV was infected with PAM for 12 h.In the VR-2332 group and JXA1-R group,IL-1?,IL-1?,IL-6,TNF?,and MHC II mRNA expression levels were up-regulated compared with the blank group,of which VR The expression of TNF?,IL-6,IL-1?,IL-1?,and MHC II mRNA in-2332 group was higher than that in JXA1-R group,but the mRNA expression of IL-10 in VR-2332 group was lower than that in JXA1-R group.The expression of IL-1? and IL-1? mRNA in VR-2332 group and JXA1-R group was still higher than that in PRRSV group.The expression of TNF?,IL-10 and TLR2 mRNA in VR-2332 group was higher than that in the VR-2332 group.In the JXA1-R group,the mRNA expression levels of IL-1? and IL-1? in the VR-2332 group were lower than those in the JXA1-R group.PAM in vitro infection of PRRSV vaccine strain test,classic strain VR2332 vaccine and highly pathogenic blue ear JXA-1 strain vaccine have different effects on PAM proinflammatory cytokines and immunosuppressive cytokines mRNA transcription,high pathogenic blue ear The JXA-1 strain was infected with PAM,and the expression of immune-related cytokine mRNA was lower than that of the classic VR-2332 strain.It is suggested that the classical strain PRRSV has higher immunosuppression of PAMs than JXA1-R.The NSP2-ORF5 double PCR method was established to analyze the variation of PRRSV strain and provide a reference for PRRSV prevention and control.The PRRSV vaccine(classic strain and mutant strain)infects PAM in vitro,detects the expression level of related factors,and understands the difference in expression of PAM-related immune work factors between the classical strain PRRSV vaccine and the mutant PRRSV vaccine,and provides a basis for evaluating vaccine safety... |
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