【摘要】 目的研究携带类整合子的铜绿假单胞菌在浮游状态和生物被膜状态类整合酶基因(intI1)mRNA的表达水平,探讨生物被膜中整合子相关的基因转移的分子机制。方法采用竞争RT-PCR方法定量检测intI1在两株临床分离的铜绿假单胞菌(PA10和PA39)生物被膜菌和浮游菌中mRNA的表达水平。首先用PCR方法从类整合子阳性铜绿假单胞菌全基因组DNA中扩增竞争体DNA(cDNA),再以cDNA为模板体外转录合成竞争体RNA(cRNA);再将不同稀释度的cRNA分别与铜绿假单胞菌的浮游菌和生物被膜菌的总RNA提取物进行竞争RT-PCR,产物经电泳分析,以已知cRNA的量确定待测样本中intI1mRNA的表达量。结果两株铜绿假单胞菌在其浮游状态和生物被膜状态都有intI1mRNA表达;两菌的浮游菌和生物被膜菌intI1mRNA表达量分别近似相等;但两菌生物被膜菌intI1mRNA的表达量都较其浮游菌高(约15倍)。结论铜绿假单胞菌在生物被膜状态下类整合子intI1mRNA的表达上调是耐药性广泛传递和多重耐药性形成的可能原因之一。更多还原

【Abstract】 Objective To study the molecular mechanism of integron related gene transfer in biofilm and aqueous culture of P.aeruginosa by investigating the expression level of intI1 mRNA in class 1 integron positive strains.Methods A competitive reverse transcription-PCR(cRT-PCR)method was designed to quantify class 1 integrase mRNA production in two clinical P.aeruginosa strains called PA10 and PA39 when they were grown in biofilms and planktonic culture.In brief,competitive DNA(cDNA)was obtained via PCR from the genomic DNA of class 1 integron positive strains.Then the competitive RNA(cRNA)was amplified from the cDNA.Finally the serials diluted cRNA were mixed separately with the total RNA extracted from the biofilm and planktonic culture of P.aeruginosa and the competitive RT-PCR were conducted.After electrophoresis,the expression level of intI1 mRNA was quantified by comparing with the amount of the cDNA.Results The PA10 and PA39 strains produced intI1 mRNA both in their biofilms and planktonic cells.Furthermore the expression levels of intI1 mRNA from the two strains were of approximation in their biofilm and plankton stage respectitively,while the quantities of intI1 mRNA expression in their biofilm stage were about 15 times higher than those in their planktonic stage.Conclusion The integrase gene is up-regulated at mRNA level in P.aeruginosa biofilm,which may be one of the reseans for the spread of antibiotic resistance and the formation of multidrug resistance.更多还原