聚合酶链反应RFLP与SSCP检测伤寒杆菌DNA旋转酶基因变异

Detection of quinolone resistant mutation in gyr A gene of Salmonella typhi by PCR-RFLP, SSCP

DNA旋转酶为喹诺酮类抗菌药物作用靶位 ,其变异为细菌耐药主要原因之一。本文利用 PCR-RFL P、SSCP对伤寒杆菌 2 75 (临床分离敏感株 )及其自发耐药突变株 RG1 DNA旋转酶 A亚单位基因 (gyr A)耐喹诺酮类决定区进行检测 ,并以 DNA序列分析对该变异进行检测。结果表明 ,与伤寒杆菌 2 75相比 ,RG1 gyr A第 2 47位由 T变异为 G,相应于 DNA旋转酶 A亚单位第 83位氨酸由丝氨酸变为丙氨酸。Hinf 对PCR产物酶切分析表明有一 GANTC序列变异 ,SSCP则发现伤寒杆菌 2 75与 RG1 DNA电泳图象有明确差异。结果表明 PCR- RFL P、SSCP检测伤寒杆菌 gyr A变异快速、特异和灵敏。

The mutations of DNA gyrase (the antibacterial target of quinolones) are major reasons of quinolone resistance. The quinolone resistant determining regions (QRDR) of gyr A genes of DNA gyrase subunit A of Salmonella typhi 275 (a clinically isolated quinolone susceptible strain) and its spontaneous mutant RG 1 were examined in this study with PCR RFLP and SSCP. DNA sequence of their gyr A QRDR was also made. Results revealed that the base 274 of gyr A in RG 1 had a change from T to G. PCR RFLP suggested that a hydrolysis point of HinfⅠ disappeared from QRDR of RG 1 and the electrophoretogram of PCR SSCP in RG 1 was totally different from that of 275. These results indicatd that PCR SSCP and RFLP are rapid, sensitive and specific methods in the detection of gyr A mutation in Salmonella typhi .