基因芯片技术检测3种食源性致病微生物方法的建立

Detection of Three Foodborne Pathogenic Microorganisms by DNA Microarray

摘要:

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。

Abstract:

A rapid, sensitive and specific method of DNA microarray combined with multiplex PCR that allow the simultaneous detection and identification of Shigella dysenteriae, Salmonella spp. and E. coli O157. The invasion-associated plasmid antigen H of S. dysenteriae(ipaH), Salmonella spp. enterotoxin gene(stn) and Escherichia coli O157 Shiga-Toxin gene(slt) were used as target genes, and then three pairs of primers and captured oligonucleotide probes were designed and synthesized. The multiplex PCR products were hybridized with DNA microarray, which contained specific probes of three foodborne pathogenic microorganisms. Genomic DNAs from 26 bacterial strains were detected, and only 3 index bacteria were positive. The detection limit of this assay was around 8 pg genomic DNA. In addition, this method was applied to detect artificially contaminated food samples, and the detection limit was 50 CFU/mL for non-cultured samples. These results suggested that detection of pathogenic microorganisms by DNA microarray is an effective procedure with high specificity and sensitivity. The DNA microarray assay developed in this study could provide an informative supplement to conventional microbiological methods for routine monitoring of food.