耐氟喹诺酮类病原菌gyrA基因突变的寡核苷酸芯片检测

根据大肠杆菌、沙门菌、无乳链球菌和鸡毒霉形体的gyrA基因序列,设计了通用引物和11条寡核苷酸探针;利用点样仪将探针点在基片上,制成寡核苷酸芯片;采用PCR荧光标记靶基因,与芯片杂交,用荧光扫描仪检测信号;同时以PCR-测序法进行gyrA基因突变的检测。结果,PCR反应体系能特异性地扩增出靶基因;寡核苷酸芯片能同时检测不同病原菌GyrA第83、87位发生的突变,芯片检测结果与测序结果较为一致。结果表明,使用寡核苷酸芯片技术检测病原菌耐氟喹诺酮类基因突变是可行的;研究结果为基因芯片技术应用于兽医临床耐药性检测提供了基础。

Detection of gyrA mutations in fluoroquinolone-resistant pathogens by the oligonucleotide microarray

The universal primers and 11 oligonucleotide probes were designed according to gyrA gene's sequences of Escherichia coli,Salmonella,Streptococcus agalactiae and Mycoplasma gallisepti-cum.The oligonucleotide probes were spotted onto the slide to make oligonucleotide microarray by the spotter.The DNA fragments of gyrA gene were labeled with fluorescence by PCR,then the PCR product was applied to the microarray for hybridization,and a fluorescent scanner was used to record hybridization signals.DNA sequencing analysis was simultaneously used to detect the gyrA gene mutations.Results showed that the target genes were specifically amplified by PCR.Mutations in gyrA at amino acid position 83 and 87 cused by fluoroquinolone resistance were detected by the developed oligonucleotide microarray.The microarray results were in agreement with the outcomes of DNA sequencing.The result showed that oligonucleotide microarray analysis was able to detect rapidly mutations in fluoroquinolone-resistant pathogens,and the study provided a basis for detection of antimicrobial resistance by microarray in clinic samples.