The Research And Application On Multiplex PCR Detection Kit Of Animal Original Bacteria Choramphenicols Resistance Genes

Three kinds of Choramphenicols resistance genes(cat1,cmlA,flor) were chosen as target genes in this study. According to the principle of multiplex PCR primer design, three pairs of specific primers were designed . A strain (SLPE3-1) carried the three kinds of genes was screened by PCR method and the PCR amplified fragments were sequenced. Therefore, the stain was adopted to be positive control in this study.The multiplex PCR reaction system and parameters ,including concentration of Mg²⁺,dNTPs, Taq polymerase and primers, as well as cycle parameters of annealing temperature, cycle times, were optimized. The results show that the optimal reaction system and parameters were as follows: 25μl system consisted of 2.5μ110×PCR Buffer,MgC12(25mmol/L)3μl,dNTP(2.5mmol/L) 4μl,Taq DNA polymerase 0.2μl(2.5U), primers catl 0.2μl,cmlA 0.9μl, flor 0.4μl, template 2.5μl, deionized water 9.8μl; Multiplex PCR amplification consisted of a 1st cycle of 5min of denaturation at 94℃,followed by 32 cycles at 94℃for lmin,54℃for 1min and 72℃for 1min, and a final extension step of 5min at 72℃.Then stored at 4℃.The kit of specificity, sensibility, repeatability and shelf life were tested. The result show that the specificity and repeatability of the kit were 100%. The kit could detect the genes with the concentration of template at 2.4×10⁴CFU/ml and the shelf life of the kit was after 6 month when stored at -20℃. Drug-resistance genes of 33 strains were test by the kit, the result of which was compared with the result of drug-resistance test, and the coincidence rate of the two methods was 91%.417 strains of bacteria, which isolated from intensive lifestock farms of 21 provinces in China, were test by the kit. The results showed that the highest detection rate of catl, cmlA and flor gene was in Gansu (60.7%),Guangdong (63.2%) and Shanxi (65.0%)province, respectively, and that average detection rate of the three genes was 41.596,35.7% and 37.2%, respectively.In conclusion, it is the first time established multiplex PCR detection kit of animal original bacteria Choramphenicols resistance genes at home and broad. Consequently, it offered a rapid, convenient and accurate method to the detection of Choramphenicols resistance genes.