大肠杆菌四环素类抗生素主要耐药基因PCR检测方法的建立

Establishment on PCR method for the main drug resistant genes of tetracyclines antibiotics of E.coli

本研究通过对tet(A)、tet(B)、tet(C)和tet(M)基因的序列分析,利用Primer Premier 5.0软件设计并合成了4对特异性引物,建立了四环素类抗生素主要耐药基因的PCR检测方法。通过反复试验确定这4对特异性引物的最佳退火温度均为55℃。灵敏性试验和特异性试验表明这4对特异性引物能够在核酸浓度分别为1.36、10.8、0.206和136 pg/μL时扩增出清晰地目的基因,并具有较高的特异性。稳定性与重复性试验表明本方法具有良好的稳定性。应用所建立的方法对25株辽宁地区猪源大肠杆菌中四环素类耐药菌株主要耐药基因进行检测分析,辽宁省猪源大肠杆菌中对四环素类的耐药性可能主要与tet(A)、tet(B)介导的主动外排机制有关,其次与tet(M)介导的核糖体保护机制有关。

Based on the analysis of tet(A),tet(B),tet(C)and tet(M)gene sequences of E.coli,four pairs of specific primers were designed by software premier5.0and and synthesized.We established a series of PCR methods for the main drug resistance genes of tetracyclines antibiotics of E.coli.By repeated experiments we determined the optimum annealing temperature of the method was55℃,sensitivity and specificity tests showed that the method can detect the clear target genes of nucleic acids concentration were1.36,10.8,0.206and136pg/μL respectively,and has a high specificity.Stability and repeatability tests showed that the method had good stability.Meanwhile,through detection and analysis of the sequences of the main drug resistance genes of tetracyclines antibiotics of E.coli which isolated from25clinical positive samples,we concluded that the drug resistance of E.coli in Liaoning Province may be mainly related to the proactive external mechanism of tet(A)and tet(B),followed by the nuclear glycogen protection mechanism mediated by tet(M).