The Selection Of Anti-PRRSV Gene Markers And Study Of Assay Kit For The Related Molecule Mark

PRRS is one of the serious infectious disease puzzles pig-breeding industry at present. Because PRRSV have very high genetic variability and can escape the host immune system. The traditional means of prevention, such as vaccination, drug therapy and nutritional measures control the spread of the disease in some degree, but failed to eliminate it fundamentally. So developing disease-resistant breeding research and enhancing the pig’s own resistance to PRRS is a new direction.In this study, we did the following works,(1)Analyse the frequencies of genotypes in five genes(MBL2,DUSP1,GBP2,Mx1,NID2) and their association with relative risk after pigs infected PRRSV in two populations pigs(Yorkshire, Landrace X Yorkshire)respectively, and did the ANOVA analysis for Death-born Percentage(DP,include stillbirth and mummy) between different genotypes in five genes in two populations pigs respectively. (2)We developed a kit which can check the C1654A SNP in CD 169 gene. This SNP was associated with the resistance of PRRS that we had evaluated. The aim of this study were to (1)Selecting PRRS-resistant molecular markers,(2)Providing the appropriated Kit for Molecular marker-assisted selection(MAS).The main results were descried as following:1. AS-PCR was developed to detected 295 A/G substitution at extron 6 of MBL2 gene.2. The PRRS relative risk associated with this gene polymorphism indicated that the relative risk of AG genotype may be 0.37 times compared with GG genotype (OR=0.37,P=0.039) in Yorkshire population and the relative risk of AA genotype may be 0.11 times compared with GG genotype(OR=0.11,P=0.04) in Landrace X Yorkshire population. The result of ANOVA analysis for DP indicated that the DP of AA genotype (0.14) was lower than GG genotype (0.44). So we hypothesis the A allele is a PRRS resistant gene. 3. AS-PCR was developed to detected 308 C/T substitution at extron 4 of DUSP1 gene.PCR-HpyCH4Ⅲ-RFLP was developed to detect 161 A/G substitution at extron3 of GBP2 gene.PCR-Pvu Ⅱ-RFLP was developed to detect 41 A/G substitution at extronl of Mxl gene. PCR-Taq Ⅰ-RFLP was developed to detect 25 T/C substitution at extron24 of NID2 gene.4. We analysed PRRS resistance of such SNPs(DUSP1-extron 4-C208T, GBP2-extron3-1161G, Mx1-extronl-A41G, NID2-extron24-T25C).The results indicated that these SNPs have not effection on PRRS resistance.5. Developed a kit for detecting C1654A SNP of CD 169 gene, and searched the component of reagent, specificity, sensitivity, and shelf life of the kit. The results indicated that the specificity and repeatability of the kit were very good, and it can amplified 0.5ng/uL DNA at least. The kit was still work 4 months later which store -20℃ condition, and the result was better if the Taq DNA Polymerase was separated from other reagents.