寡核苷酸芯片检测兽医病原菌耐药性的研究

 利用寡核苷酸芯片（oligonucleotide array）对兽医临床常见病原菌耐药性检测进行了初步研究。设计1对兼并引物扩增3种病原菌（E. coli、S. aureus、M. gallisepticum）的gyrA基因片段，并用cy5-dCTP标记荧光。设计一系列检测病原菌对氟喹诺酮类药物耐药的寡核苷酸探针，将探针固定在APS-PDC法制作的Microarray上，用标记的PCR产物与之杂交，置于Scanarrray4000中扫描，利用ImaGene3.0软件对杂交图像进行分析。研究结果表明，设计的兼并引物能很好地扩增出3种病原菌的gyrA基因片段，与Microarray杂交后在相应的位点出现可检测的杂交信号，样本O78S（禽大肠杆菌）、SS（金黄色葡萄球菌）、S6-10（鸡毒支原体）的ratio比值均≥2，说明这3株是敏感株；样本EDr（犬大肠杆菌）、ECr（猫大肠杆菌）与a1探针（检测GyrA第83位氨基酸发生突变的探针）的ratio比值≤0.5，说明这两株是耐药突变株，均与样本的实际情况相吻合。说明利用Oligonucleotide array能快速准确地检测出GyrA亚基第83、87位发生突变的耐药菌株，是可行、有效、快捷的抗菌药耐药性的监测方法。本研究为进一步研制和开发兽医临床常见病原菌耐药性检测芯片打下初步基础。

Study on Detection of Resistance in Veterinary Pathogen by Using Oligonucleotide Array

Detection of the antimicrobial resistance in common veterinary pathogen by using oligonucleotide array was studied. A pair of primers was designed to amplify fragments of gyrA gene from E. coli, S. aureus and M. gallisepticum, and simultaneously label fluorescence with cy5-dCTP. A series of oligonucleotide probes with which to detect the resistance to fluoroquinolones in the above three bacterial apecies were designed, then dotted and immobilized onto the oligonucleotide array by APS-PDC method. The labelled PCR products were hybridized with the oligonucleotide array, and the signals were scanned and analyzed with a ImaGene 3.0 software. The results showed that the hybridized oligonucleotide array presented detectable signals, and were consistent with the facts. This study demonstrated that the array could distinguish single base mutation between wild types and mutant types of E. coli,S. aureus,M. gallisepticum, and was able to identify differences in the wild strains and mutant strains occurring at positions 83 and 87 of the GyrA subunit. The preliminary results demonstrated that oligonucleotide array provided an accurate, sensitive and feasible tool for the detection of resistance in veterinary bacteria. The present study may provide a basis for further research and development of gene chip in monitoring resistance to antimicrobial agents.