基于随机聚合酶链反应的病原菌基因芯片检测技术

Detection of pathogenic bacteria using DNA microarray based on random PCR

目的建立一种基于随机聚合酶链反应的病原细菌基因芯片筛查检测技术。方法用生物学软件分析7种病原菌特异性基因序列的保守性区域,利用Oligo 6.0软件设计针对靶细菌的一系列探针,制备检测用基因芯片。细菌基因组DNA在随机引物扩增中掺入氨基丙烯-dUTP,产物偶联荧光染料后与芯片上探针杂交,通过芯片扫描仪和图像分析软件对结果进行判断。选取19种病原菌基因组DNA进行芯片特异性验证和灵敏度评价,使用问号钩端螺旋体对应靶探针进行基因芯片检测方法重复性验证,并制备问号钩端螺旋体模拟水污染样本进行芯片检测。结果在均一的杂交条件下4种靶细菌均能得到相应特异性杂交图谱,其他非目的细菌均为阴性结果,3种靶细菌基因组DNA最低检测浓度为14.43～363.4 pg/μl,芯片重复性变异系数CV值<15%,最低可检测含问号钩端螺旋体7×105条/ml模拟水污染样本。结论初步建立的随机聚合酶链反应结合芯片技术的检测方法可用于多种病原菌筛查检测,为细菌高通量筛查与鉴定技术提供了新的思路和实验依据。

Objective To establish a DNA microarray technology for screening and identifying pathogenic bacteria based on random PCR.Methods The specific genes of 7 different bacteria were selected and the relative gene sequences were analyzed by the Clustal X program.A series of probes were designed at the conserved regions of specific genes using the Oligo 6.0 software.The olignucleotide probes were synthesized and spotted on DNA microarray.The random PCR was used to amplify bacterial genomic DNA,and aminoallyl-dUTP was incorporated into the products during PCR.The products labeled with fluorescent dye were hybridized with DNA microarray spotted with the specific probes.Hybridized slides were scanned with the scanner and the images were analyzed with software program GenePix Pro 5.1.The specificity and sensitivity of DNA microarray platform were assessed with 19 pathogenic bacteria DNA,and the reproducibility was investigated by the DNA of Leptospira interrogans.Furthermore,the simulated water sample of L.interrogans was deteted.Results The corresponding specific hybridization maps of 4 targeted bacteria were obtained under the same hybridization conditions,the positive signals of the other non-targeted bacteria were not observed,the detection limit of the 3 targeted bacteria DNA was 14.43-363.4 pg/μl.The CV value of the reproducibility of assay was less than 15%,and the detection limit of the simulated water sample of L.interrogans was 7×105/ml.Conclusion DNA microarray based on random PCR technology can be used for the detection of multiple pathogenic bacteria,and the DNA microarray offers new ideas and reference for high throughput detection and identification of bacteria.