Study On Detection Of Antibiotic-Resistant Genes Of Pathogenic Bacteria By Microarrays

We developed DNA microarrays which could identify and diagnosistetracyclines antibiotic-resistant genes and β-lactam antibiotic-resistant genes .There are three parts included in this experiment, such as constructing thestandard samples ,optimizing the preparation of microarrays and optimizing thehybridization of microarrays.First of this study,on the basis of bioinformatics of tetracyclinesantibiotic-resistant genes and β-lactam antibiotic-resistant genes,PCR was appliedfor rapid screening of resistant genes.36 primer pairs targeting these genes weredesigned.And by whole gene sequence from Genebank, we applied software ofDNAman , DNAstar and Blastn in NCBI to analysis, design, verify and selectoligo probes about 20bp, which could detect tetracyclines resistance genes andβ-lactam antibiotic-resistant genes.Tetracyclines antibiotic-resistant genes andβ-lactam antibiotic-resistant genes were amlified by PCR, and then cloned intoPGEM-T and tranformed into the competent cell of E.Coli. By selecting thepositive clones, we obtained 19 plasmids which contained the determinantsequence of tetracyclines antibiotic-resistant genes and β-lactamantibiotic-resistant genes. As standard sample, the plasmids were used inexperiment of optimization of microarrays.There were so many factors that could affect immobilization andhybridization, such as slide, spotting solution dissolving DNA, DNAconcentration immobilized on glass surface, spotting temperature and humidity,method of DNA immobilized on glass surface, method of labeling sample,hybridization solution, hybridization temperature, hybridization time, etc. Factorswere changed respectively, and optimal values were selected by comparing thoseof different groups. Different standard samples were used to test the sensitivityand specificity of microarrays.On the basis of results, the efficient hybridizationcondition is as follows:30pmol/μl probes resolved in 50%DMSO and spotting onglass slide ,then hybridize with purified fluorescent sample in 2×hybridizationsolution and formamide at 42℃ in five hours.Standard sample was diluted to different template copys, which were used totest sensitivity of microarrays. We applied standard samples with different numberof mutative bases to test speciality .The results showed that the oligo chipdeveloped in the experiment could detect the samples that contained more than1000 template copys.