结核分枝杆菌耐喹诺酮临床分离株gyrA基因突变的研究

目的　了解结核分枝杆菌喹诺酮 (quinolones)耐药株耐药基因突变情况,评价其应用价值。方法 通过 16SrRNA聚合酶链反应-单链构象多态性 (PCR SSCP)技术分析 77株分枝杆菌临床分离株；通过PCR SSCP和直接测序技术分析结核分枝杆菌的gyrA基因突变的情况。 结果 75株为结核分枝杆菌复合群。 30株喹诺酮敏感株的 gyrA基因的SSCP图谱中,11株与H₃₇R_v相同,19株与卡介苗相同；与卡介苗 gyrA基因的SSCP图谱相同的敏感株 95位密码子为ACC,与H₃₇R_v该图谱相同的敏感株 95位密码子为AGC。 45株喹诺酮耐药株中,34株 (75.6%) gyrA基因SSCP图谱泳动异常,对 25株泳动异常、出现频率较高耐药株测序证实 15株 (44.1%)为 94位密码子GAC→GGC突变；10株 (29.4%)为 90位密码子GCG→GTG的突变。结论 结核分枝杆菌耐喹诺酮与 gyrA基因突变有关,PCR SSCP可能成为测定结核分枝杆菌耐喹诺酮类药基因型的快速、简便的方法。

Study on gyrA mutation in Mycobacterium tuberculosis quinolone-resistant isolates

Objective To observe the mutations of gyrA gene in M.Tuberculosis quinolones-resistant isolates,and to evaluate their clinical value.Methods 77 clinical isolates were identified for their mycobacteria species,and were analyzed M.tuberculosis gyrA genes by PCR-SSCP and PCR-direct sequencing.Results Seventy-five isolates were identified as M.tuberculosis.Of 30 quinolones-sensitive isolates,11 isolates had same gyrA SSCP profiles as H₃₇R_v,19 had same profiles as M.bovis BCG.The drug-sensitive isolates whose gyrA SSCP profiles was identical to H₃₇R_v were AGC at condon 95,the others were ACC at condon 95.Of 45 quinolones-resistant isolates,34 (75.6%) displayed abnormal gyrA SSCP profiles.25 clinicals of 2 kinds of gyrA SSCP profiles which appeared at a high frequency were sequenced.15 isolates had GAC→GGC mutation at condon 94;10 isolates had GCG→GTG mutation at condon 90.Conclusion Quinolones resistances in some M.tuberculosis isolates were due to mutation on gyrA genes.PCR-SSCP method might become a simple and rapid diagnostic test for identification of genotypes of M.tuberculosis quinolones-resistance.