荧光定量PCR法检测不同状态下铜绿假单胞菌intⅠ1基因的表达  

Detecting intⅠ1 Gene Expression of Pseudomonas Aeruginosa with Fluorescent Quantitative PCR Assay

作　　者：刘蓉[1] 杨莉莉[2] 罗必蓉[1] 唐玲[1] 蒋宏[1] 刘继芬[1] 李明远[3] 

机构地区：[1]成都中医药大学医学技术学院微生物学教研室,成都610041 [2]成都市第三人民医院检验科 [3]四川大学华西基础医学与法医学院微生物学教研室

出　　处：《四川大学学报（医学版）》2009年第1期33-36,共4页Journal of Sichuan University(Medical Sciences)

基　　金：成都中医药大学科研基金(批准号WGY-0601)资助

摘　　要：目的检测铜绿假单胞菌Ⅰ类整合酶基因(intⅠ1基因)在生物膜状态和浮游状态表达水平的差异,探讨整合子的作用机制。方法对3株Ⅰ类整合酶阳性的铜绿假单胞菌分别进行液相培养和生物膜细菌培养,常规方法提取3株细菌两种生长状态的总RNA。采用荧光定量PCR(FQ-PCR)法,以细菌的16srRNA为内参照,分别测定菌株SW07、R07和TH12的浮游细菌和生物膜细菌intⅠ1mRNA的相对表达量,比较3株菌在两种状态下intⅠ1mRNA的表达水平。结果3株铜绿假单菌的生物膜细菌和浮游细菌都检测到intⅠ1基因的表达。3株菌在两种状态下intⅠ1mRNA的表达量不同,生物膜细菌intⅠ1基因的表达水平较高,其中菌株R07、SW07和TH12的生物膜细菌intⅠ1mRNA的表达量分别较浮游细菌高1.4、5.7和128倍。结论在生物膜状态下,铜绿假单胞菌intⅠ1基因的表达上调,本实验结果提示整合子在生物膜细菌中可进行更加活跃的基因盒捕获和积累,整合子和细菌生物膜的形成是导致细菌对抗生素耐药的重要机制。Objective In order to understand the role of integron, fluorescent quantitative polymerase chain reaction （FQ-PCR）was developed to measure the changes in int Ⅰ 1 gene expression of Pseudomonas Aeruginosa in biofilm and planktonic cells. Methods Three clinical strains of P. aeruginosa with int Ⅰ 1 gene （SW07, R07 and TH12） were cultured in planktonic cells and biofilm cells. The total RNA of these cultured bacteria were extracted by the conventional method. The FQ-PCR was developed to measure the changes in int Ⅰ 1 mRNA expression of the P. aeruginosa with bacterial 16s rRNA as an internal control. Results The three clinical strains of P. aeruginosa expressed int Ⅰ 1 mRNA in both biofilm and planktonic cells, but with different levels. The int Ⅰ 1 mRNA expressed by the RO7,SW07 and TH12 strains in the biofilm cells were 1.4, 5.7 and 128 times higher than in the planktonic cells, respectively. Conclusion The int Ⅰ 1 gene expression of P. aeruginosa in the biofilm is up-regulated at mRNA level. The integron may capture and accumulate drug resistance gene cassettes more effectively in the biofilm condition.

关 键 词：铜绿假单胞菌 生物膜 intⅠ 1基因 荧光定量PCR 

分 类 号：R378.99[医药卫生—病原生物学]