【摘要】 以氨基糖苷类、四环素类、磺胺类和氯霉素类四大类抗生素的11种耐药基因为靶基因,构建了耐药基因的检测芯片.确定芯片点样温度为25℃,湿度为65%;各样点中心距离为1000μm,样点直径为220μm.芯片点样结束后,室温干燥6 h,60℃水合处理30 s,80℃干燥10 s,254 nm波长紫外交联10min.以532 nm(绿光)波长扫描采集杂交信号,激光扫描分辨率为10μm,激光功能90%,PMTG 85%.选用tetA基因进行杂交动力学研究,确定靶基因的点样浓度为100 ng?μL-1,探针最适浓度为3600 pg?μL-1,系统检测灵敏度为3 pg?μL-1.基因芯片杂交的主要参数为:44℃预杂交2 h,52℃杂交6 h.杂交信号的判读标准为:样点信噪比(S/N)≥1.5及信号强度中位值≥1000.芯片杂交特异性高,重复性好,实现了11种耐药基因的同时检测.更多还原

【Abstract】 A glass-based microarray was developed to detect 11 antimicrobial resistance genes that confer resistance to aminoglycosides,tetracyclines,sulfonamides,and chloramphenicols.DNA samples were arrayed with 220 μm diameter pins at a spacing distance 1000 μm on silane-coated（amine） glass slides under condition of 25℃,65% relative humidity.DNA microarray was dried at room temperature for（6 h,） rehydrated over a 60℃ water bath for 30 s and dried on a heating block at 80℃ for 10 s.Target DNA on the microarray was fixed for 10 min by UV cross-linking at 254 nm.Hybridized microarray was scanned at 10 μm with photomultiplier tube（PMT） at 532 nm（Cy3） with laser power 90% and PMT gain（PMTG） 85%.Hybridization dynamics of the microarray were examined by using tetA gene.As results,target DNA concentration of 100 ng·μL^-1 was used for microarray construction,the optimal concentration of probe was 3600 pg·μL^-1,and the sensitivity of the microarray was 3 pg·μL^-1.Prehybridization of the microarray was carried out at 44℃ for 2 h to block nonspecific binding of（probe（s）,） followed by hybridization at 52℃ for（6 h.） When the signal-to-noise（S/N） was ≥1.5 and median pixel intensity was ≥1000,a spot was scored as positive.The microarray can detect 11 antimicrobial resistance genes at the same time with high specificity and repeatability.更多还原