Study On Detection Of Chloramphenicols-Resistance Genes And Sulfonamides-Resistance Of Pathogenic Salmonella And E.coli By Multiplex PCR

According to NCCLS guidelines in America in 1999, the MIC values of Sulfonamide (SD), Sulfamethoxzole (SMZ)and Chloramphenicol(CAM), Thiphenicol(TPC) , Florfenicol(FFC) of the 33 strains of pathogenic Salmonella and 103 strains of pathogenic E.col isolated from swine and poultry were detected by disc dilution method. The results showed that the resistance rates of Salmonellas to SD, SMZ, CAM, TPC and FFC were 57.6%, 48.5%, 66.7%, 63.6% and 33.3% respectively, the resistance rates of E.cols to SD, SMZ, CAM, TPC and FFC were 57.6%, 48.5%, 66.7%, 63.6% and 33.3% respectively.According to the resistance genes sequences published in Genebank, two sets of multiplex PCR primers were designed to detect for Sulfonamides-resistant genes and Chloramphenicols-resistant genes. The detection for 33 strains of Salmonella and 103 strains of E.coli show that the rate of coincidence of the tow multiplex PCR assays result with common PCR assays is 98.5% at least, the rate of coincidence of tow multiplex PCR assays result with MIC assays is 73.3% ~ 100%. The sensitive experiment show the multiplex PCR assays can detect the bacilus at 106CFU/ml. It only takes 7 hours to the complete the whole test.The PCR products of Sull gene from CQCS0304-2 strain, Sul3 gene from SLPE 1-1 strain, Cat1 gene from CQCS0304-2 strain, CmlA gene from PJPS 2-2 strain, Flor gene from CQCS0304-4 strain were determined, and compared with the correspond genes in Genebank. The Homologic rates were 98.4% at lesst. These sequences determined were deposited in Genebank as Accessions of AY617069, AY617070, AY617066, AY617067 and AY617068 resperatively.