运用PCR-RFLP技术研究耐喹诺酮类铜绿假单胞菌gyrA基因的突变

A study on gyrA gene mutation of quinolone-resistant Pseudomonas aeruginosa by PCR-RLFP

目的研究临床分离的耐喹诺酮类铜绿假单胞菌（Pseudomonas aeruginosa,PA）gyrA基因突变情况。方法以铜绿假单胞菌gyrA基因序列为靶序列,用PCR-RFLP、DNA测序等方法,对铜绿假单胞菌gyrA基因突变进行研究。结果在57株耐喹诺酮类铜绿假单胞菌中,有35株（61.4%）菌gyrA基因的83位出现突变,其突变方式为Thr83（ACC）→Ile（ATC）,其余22株（38.6%）耐喹诺酮类铜绿假单胞菌未发现基因位点突变。33株喹诺酮类敏感临床分离铜绿假单胞菌菌株未发现上述突变。gyrA基因的PCR扩增产物SacⅡ酶切片段与它们测序结果一致。结论 gyrA基因83位氨基酸密码子突变（Thr-83→Ile）是临床分离铜绿假单胞菌耐喹诺酮类药物的主要机制之一。

Objective To explore the transgenation information of gyrA gene in quinolone-resistant Pseudomonas aeruginosa.Methods The gyrA gene sequence and parC gene sequence in Pseudomonas aeruginosa were analyzed by polymerase chain reaction-restriction fragment length polymorphisms（PCR-RFLP） and DNA sequencing.Results Among 57 strains of quinolones-resistant Pseudomonas aeruginosa,35 strains（61.4%） showed a mutation in gyrA codon with way to Thr83（ACC）→Ile（ATC）,22 strains（38.6%） had no gyrA gene mutation;33 quinolones-susceptible strains had no gyrA gene mutation,but directive sequencings were the same results by PCR-RFLP.Conclusions GyrA mutation with Thr83（ACC）→Ile（ATC） may be one of the quinolones resistance mechanism of Pseudomonas aeruginosa isolated from clinical samples.