实时荧光定量PCR检测多重耐药大肠杆菌AcrA基因mRNA表达水平

Real time quantitative PCR for detecting mRNA expression of AcrA gene of E.coli isolates and induced by single drug

体外单一药物诱导标准大肠杆菌ATCC25922,获得了耐氯霉素(CHL,MIC≥256 mg/L)、耐环丙沙星(CIP,MIC≥256 mg/L)、耐水杨酸钠(SAL,MIC≥275 mg/L)、耐四环素(T,MIC≥512 mg/L)的菌株。选取ATCC25922株,4株中度耐药菌SAL(175)、CHL(64)、T(64)、CIP(128),4株高度耐药菌SAL(275)、CHL(256)、T(512)、CIP(256),和临床分离的3株耐药大肠杆菌H1、H9、H10,通过实时荧光定量RT-PCR方法检测外输泵AcrA基因的mRNA表达水平。系统自动分析软件显示,Ct值与标准质粒浓度的对数之间存在良好的线性关系,回归系数为0.992。检测结果表明:H10、CHL(256)、CIP(256)拷贝数最高,达1015拷贝/μL;T(512)、H9、T(128)、SAL(275)、H1为1014拷贝/μL;CHL(128)、T(64)、CHL(64)为1013拷贝/μL;ATCC25922为1012拷贝/μL。不同程度耐药株AcrA基因的表达量不同,临床分离株和诱导的各高耐药菌株均比ATCC25922株表达量高,且差异极显著(P≤0.01)。这说明不同耐药程度的11株菌cDNA的量存在差异,AcrA基因转录水平与耐药水平成正相关。

E.coli ATCC25922 strains were induced by in vitro sole medicine to obtain chloramphenicol-resistant strain （CHL, MIC≥ 256 mg/L）, ciprofloxacin-resistant strain （ CIP, MIC ≥256 mg/L）, natrium salicylicum-resistant strain （SAL, MIC≥ 275 mg/L） and tetracycline-resistant strain （T, MIC≥512 mg/L）. ATCC25922, four higher drug-resistant strains including SAL （275）, CHL（256） ,T（512）, CIP（256）, four midrange drug-resistant strains including SAL（175）, CHL（64） ,T（64）, CIP（128） and three strains including H1, H9, H10 from ten clinical strains, the mRNA expression of AcrA gene of which were detected by realtime fluorescent quantitative PCR. The restdts showed there was a good linear ftmction in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen with the regression value of 0.992. The highest copies/μL of H10, CHL （256） and CIP（256） were 10^15;the copies/μL of T（512） ,H9,T（128） ,SAL （275） and H1 strains reached 10^14; the copies/μL of CHL（128） .T（64）. CHL（64） reached 1013, ATCC25922 reached 10^12. The copies of different strains mRNA expression quantity were distinct. Each higher drug-resistant strain from the clinical separation and induction must be higher than the ATCC2592 in expression quantity. It demonstrated that the expression quantity for 11 different levels of drug-resistant strains existed difference significantly（P 〈 0.01） and that the gene transcription level revealed a positive correlation with the drug-resistant level.