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目的建立实时荧光定量PCR法检测铜绿假单胞菌MexAB-OprM mRNA水平的表达.探讨中草药穿心莲内酯对铜绿假单胞菌外排泵MexAB-OprM表达情况的影响。方法克隆铜绿假单胞菌mexAB-oprM操纵元中mexB基因和30S rRNA基因rpsL,分别构建相应质粒作为实时荧光定量PCR DNA标准品。以不同浓度穿心莲内酯作用铜绿假单胞菌PAO1株,采用实时荧光定量PCR法检测PAO1菌株中mexB和rpsL基因mRNA的表达水平。结果成功构建标准曲线质粒,50、100、150和200μg/mL穿心莲内酯作用后PAOI mexB基因mRNA表达量分别为0.04±0.03、0.06±0.07、0.09±0.03和0.04±0.03,对照组为0.24±0.04,差异有统计学意义(P<0.05),穿心莲内酯组间差异无统计学意义(P>0.05)。结论穿心莲内酯可在mRNA水平抑制外排泵Mex- AB-OprM的表达,这可能是其抗感染机制之一。
Objective To establish a real-time fluorescent quantitative PCR assay for the expression of MexAB-OprM mRNA in Pseudomonas aeruginosa. To investigate the effect of andrographolide on the expression of MexAB-OprM in Pseudomonas aeruginosa efflux pumps. Methods The mexB gene and 30S rRNA gene rpsL were cloned from the mexAB-oprM operator of Pseudomonas aeruginosa. The corresponding plasmids were constructed as real-time fluorescent quantitative PCR DNA standards. Different concentrations of andrographolide acted on Pseudomonas aeruginosa strain PAO1. Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of mexB and rpsL genes in PAO1 strain. Results The standard curve plasmids were successfully constructed. The mRNA expression levels of PAOI mexB gene in the 50, 100, 150 and 200 μg/mL andrographolide groups were 0.04±0.03, 0.06±0.07, 0.09±0.03 and 0.04±0.03, respectively, and the control group was 0.24±. 0.04, the difference was statistically significant (P<0.05), and the difference between the andrographolide group was not statistically significant (P>0.05). Conclusion Andrographolide can inhibit the expression of efflux pump Mex-AB-OprM at the mRNA level, which may be one of its anti-infection mechanisms.