【摘要】 根据已知的耐药基因序列设计出检测四环素耐药基因特异探针7条、β-内酰胺类耐药基因特异探针5条,阳性坐标探针1条,长度为18~21 bp。根据探针序列两侧的保守区域设计了耐药基因扩增引物,引物长度17~20 bp。通过荧光聚合酶链式反应,从耐药菌株中获得四环素耐药基因7个,β-内酰胺类耐药基因5个。经过芯片制作过程优化试验和芯片杂交优化试验,结果表明,将探针以终浓度30μmol/L溶于50%DMSO中,在温度为20℃、湿度为70%的条件下点印于Aldehydeslide表面。扩增出的荧光标记产物经纯化后与2×杂交液混合,再与基因芯片在42℃杂交5h可检测到理想的杂交信号。芯片灵敏度试验结果表明,当模板拷贝数大于1×103个/μL时,可检测到较强的杂交信号。本试验最终研制出灵敏度高、特异性强的耐药基因检测芯片。更多还原

【Abstract】 To design a microarrays for the detection and identification of tetracyclines resistant genes and β-lactam resistant genes,there are 13 oligonucleotide probes and 12 pairs of primers were analyzed,designed and selected.12 samples were amplified and lablled with cy3 fluorescent by touchdown PCR and asymmetric multi-PCR,then the amplicons were hybridized to the microarrays which was spoting probes on it.Through optimizing the preparation of microarrays and optimizing the hybridization of microarrays,the efficient hybridization conditions were as follows:30 μmol/L DNA fragments resolved in 50% DMSO and spoting on the glass slide,then hybridized with purified fluorescent sample in 2×hybridization solution and formamide at 42℃.This genchip was proved to be able to recognize the antibiotic-resistant genes and used for detecting the antibiotic-resistant strains sensitively and specifically.更多还原