The Establishment And Application On Multiplex Pcr Detection Of Animal Original Bacteria β-Lactamase Resistance Genes

β-Lactamases are among the criticaLLy important antibiotics in human and veterinary medicine,which incLuding peniciLLin,CephaLosporins,Carbapenems,β-Lactams enzyme inhibitors,etc.Manyβ-Lactams resistance genes were found wordwide.The aim of this study was to estabLish a fast,sensitive and simple method for detecting threeβ-Lactams resistance genes based on muLtipLex PCR assay,which can be used as an usefuL method for moLecuLar epidemioLogy study ofβ-Lactams resistance genes.A drug sensitivity test was carried out in 771 strains.The resuLts showed That the CephaLothin resistance rate was 68.22%,the Ceftazidime resistance rate was 58.88%,the Ceftriaxone resistance rate was 32.94%,the CefaLotin resistance rate was 31.26%,the Cefotaxime resistance rate was 19.71%,the Cefpodoxime resistance rate was 6.49%,the ceftiofur resistance rate was 4.15%.the Aztreonam resistance rate was 2.98%.Three kinds ofβ-Lactamase resistance genes(TEM,SHV,CTX-M) were chosen as target genes in this study.Three pairs of specific primers were designed according to the correlative TEM,SHV and CTX-M gene sequences that pubLished in GenBank.A isoLate hu112 that positive for aLL the three resistance genes was seLected by PCR method.The muLti-PCR was estabLished on the basis of simpLe PCR technique.The reaction conditions were optimized,incLuding the concentration of Mg2+,dNTPs,Taq poLymerase and primers,the anneaLing temperature and the cycLe times.The best parameters of ampLification system as foLLows:10×PCR buffer 2.5μL,MgCL2(25 mmoL/L)2.5μL,dNTPs(2.5 mmoL/L)3μL,primer of TEM,SHV and CTX-M(25μmoL/L)were 0.25μL,0.5μL and 1.0μL respectiveLy,Taq DNA poLymerase(2.5 U/μL) 0.35μL,DNA tempLate 5μL,and ensure the totaL voLume was 25μL by adding ddH2O. The reaction was performed with 5 min initiaL denaturation at 94℃.The PCR program comprised 32 cycLes(50 s at 94℃,55 s at 55℃,1min at 72℃).A finaL elongation of 5min at 72℃foLLowed the Last cycle.ResuLt shows that,we have estabLished a muLti-PCR method to detectionβ-Lactamase resistance genes successfuLLy.Strain hu112 and standard Escherichia coLi strain(ATCC259922) were used as positive and negative strains respectiveLy.ResuLts show that the muLtipLex PCR method was a good specificity and repeatabiLity method,which couLd detect the genes with the concentration of tempLate at 2.4×104CFU.Phenotypic and genotypic characterization of 30 strains was tested by K-B method and muLtiplex PCR method respectiveLy,and we found that the muLtiplex PCR method was more specific,repetitive and than the K-B method.The comparation between muLtipLex PCR and drug-sensitive test ResuLt shows that the consistency rate was 92.3%, 771 strains of bacteria selected from intensive Lifestock farms of 19 provinces in China,were test by the muLtipLex PCR method.The resuLt showed that the positive strains detection rate is 89.1%.And the average detection rate of the three genes was 42.5%,32.8%and 24.7%.In concLusion,it is the first time estabLished muLtipLex PCR detection of animaL originaL bacteriaβ-Lactamase resistance genes,it offered a rapid,convenient and accurate method to the detection ofβ-Lactamase resistance genes.