摘要
目的探讨利用分子生物学方法直接快速检测临床标本中淋球菌gyrA基因突变的可行性。方法设计针对淋球菌gyrA基因的特异扩增引物,运用聚合酶链反应-单链构象多态性技术(PCR-SSCP)对95份泌尿生殖道标本直接进行基因及突变检测,并与测序及药敏试验结果进行对比分析。结果32份分离培养阴性的标本无特异性扩增产物,而63份分离培养阳性标本以及对应的分离菌株均能扩增出168 bp片断。以淋球菌标准株ATCC19424为对照,分离菌株对环丙沙星敏感的5份临床标本与标准株SSCP带型相同;分离菌株对环丙沙星中介或耐药的58份临床标本与标准株SSCP带型均不同,共出现5种突变带型。5份SSCP分析显示,无突变的标本gyrA基因序列未发生改变;16份SSCP分析显示,有突变的标本gyrA基因序列均有改变;具有相同突变的标本SSCP带型完全相同,具有不同突变类型的标本SSCP带型不同。结论PCR-SSCP银染技术可简便、快速、准确地检测临床标本中淋球菌对氟喹诺酮类药物的敏感性。
Objective To investigate the feasibility of directly detecting the gyrA gene mutation of Neisseria gonorrhoea by molecular biotechnology in clinical specimens. Methods The gyrA gene was amplified by PCR in 95 clinical specimens. The PCR products were detected by single strand conformation polymorphism （SSCP） and directly sequenced. The results were compared with those of drug susceptibility test and sequencing analysis. Results The 168 bp fragments could be amplified from 63 positive specimens and corresponding isolated strains. There were no specific amplified products in 32 neagtive specimens. SSCP patterns in 5 specimens whose corresponding isolates were susceptible to ciprofloxacin were the same with wild type strains. SSCP patterns in 58 specimens whose corresponding isolates were intermediate or resistant to ciprofloxacin were different from wild type stains. And there were 5 mutation types all together. Sequencing analysis revealed that PCR products of 5 SSCP negative specimens had no sequence changes, while PCR products of 16 SSCP positive specimens were all found sequence changes. The results of SSCP and sequencing analysis were completely concordant. In addition, specimens with the same mutations had identical SSCP patterns and specimens with different mutations had distinct SSCP patterns. Conclusions PCR-SSCP can rapidly and accurately detect the suscep.tibility of Neisseria gonorrhoea to fluoroquinolone in clinical specimens.
出处
《疾病控制杂志》
2006年第4期343-347,共5页
Chinese Journal of Disease Control and Prevention
基金
湖南省科技厅科研基金资助项目(03JZY3031)
湖南省卫生厅科研基金资助项目(B2003-041)
