针对gyrA基因突变的反向斑点杂交技术快速检测结核分枝杆菌喹诺酮耐药性的研究

Reverse dot blot hybridization of gyrA mutation for rapid detection of Mycobacterium tuberculosis resistance to quinolone

目的建立快速检测结核分枝杆菌喹诺酮耐药性的反向斑点杂交(reverse dot blot hybridization,RDBH)技术,并观察其效果。方法针对结核分枝杆菌gyr A基因序列及常见的突变位点,分别设计1条野生型和7条突变型探针,建立RDBH技术,对临床分离结核分枝杆菌菌株进行喹诺酮耐药性检测,以比例法药敏试验和DNA测序做对照。结果应用比例法药敏试验、DNA测序、RDBH三种方法分别检测59株喹诺酮耐药株和51株喹诺酮敏感株,与比例法相比,RDBH试验灵敏度和特异度分别为69.49%(41/59)、100%(51/51),符合度为83.63%;而RDBH与DNA测序结果比较,敏感度和特异度分别为97.56%(40/41),98.55%(68/69),符合度达98.18%。结论RDBH技术检测结核分枝杆菌喹诺酮耐药具有良好的灵敏度、特异度和符合度。

Objective To establish a reverse dot blot hybridization （RDBH） assay for the rapid detection of Mycobacterium tuberculosis resistance to quinolone. Methods Based on the sequence results of gyrA gene and common mutation sites in M. tuberculosis, one wildtype and seven mutant oligonucleotide probes were designed. Meanwhile, conventional drug susceptibility test （DST） and DNA sequencing were performed to evaluate the effect of RDBH assay. Results A total of 59 quinolone resistant M. tuberculosis isolates and 51 quinolone sensitive M. tuberculosis isolates were detected by using DST, DNA sequencing and RDBH assay. Compared with DST results, the sensitivity and specificity of RDBH assay were 69. 49% （41/59） and 100% （51/51）. Compared with DNA sequencing, the sensitivity, specificity and conformity of RDBH were 97. 56% （40/41）, 98.55% （68/69） and 98.18% ,respectively. Conclusion RDBH is a rapid, simple and efficient assay to detect quinolone resistant M. tuberculosis.