应用寡核苷酸芯片技术检测食源性感染常见致病菌  

Application of oligonucleotide array technology in rapid detection of common bacteria of foodborne diseases

作　　者：洪帮兴[1] 江丽芳[1] 胡玉山[1] 方丹云[1] 陶剑平[1] 郭辉玉[1] 

机构地区：[1]中山大学中山医学院微生物学教研室,广州510089

出　　处：《中华检验医学杂志》2005年第2期169-172,共4页Chinese Journal of Laboratory Medicine

基　　金：国家863项目(2002AA215014);广东省自然科学基金资助项目(2002B3100105)

摘　　要：目的建立食源性感染常见致病菌的快速检测方法。方法利用带正电荷尼龙膜为芯片载体,合成寡核苷酸探针,点样于载体制成寡核苷酸芯片,对食源性感染常见致病菌23SrDNA基因片段扩增产物进行杂交检测。结果在同一条件下运用设计的通用引物扩增出16种(属)细菌23SrDNA基因片段。大肠埃希菌、沙门菌、志贺菌、霍乱弧菌、副溶血弧菌、变形杆菌、单核细胞增生李斯特菌、蜡样芽孢杆菌、肉毒梭菌和空肠弯曲菌等10种(属)菌杂交结果显示,高灵敏度和特异性;金黄色葡萄球菌、链球菌、小肠结肠炎耶尔森菌和产气荚膜梭菌等4种(属)菌,未能显示预期的种(属)杂交结果;而应用食源性感染模拟标本,检测肺炎链球菌和绿脓假单胞菌两种与食源性感染无关的致病菌未与芯片上探针出现杂交反应,检出水平可达10CFU/ml。结论建立的寡核苷酸芯片技术在食源性感染常见致病菌的检测上快速准确等优点,为食源性感染的诊治与预防提供了有效的技术手段和方法依据。Objective To construct a rapid and accurate detection method of common pathogenic bacteria in foodborne diseases with the technology of oligonucleotide array.Methods Nylon membrane was used as the array base.Oligonucleotide probes were synthesized and spotted onto the nylon membrane as oligonucleotide array. The 23S rDNA gene amplifiaction products of different pathogenic bacteria were hybridized on the oligonucleotide array.Results With the same amplification and hybridization conditions, the 23S rDNA gene fragments of 16 species of pathogenic bacteria were amplified and hybridized onto the oligonucleotide array. Hybrizdization results manifested that 10 species (E.coli,S.enterica,S.dysenteriae,V.cholerae,V.parahaemolyticus,P.vulgaris,L.monocytogenes,B.cereus,C.botulinum and C.jejune) showed high sensitivity and specificity. Four species (S.aureus,S.pyogenes,Y.enterocolitica and C.perfringens) couldn′t show anticipated results because of improper choice of probes or unsuitable hybridization conditions. The construction of the oligonucleotide array can also be applied in simulated samples. Two species unrelated to foodborne disease showed no hybridization signal on the oligonucleotide array. Sensitivity of this assay mounts to 10 CFU/ml.Conclusion The method of oligonucleotide array has the priority of rapidity and accuracy in the diagnosis of foodborne diseases, and provides an effiencient way in the diagnosis, therapy and control of foodborne diseases.

关 键 词：食源性感染 常见致病菌 寡核苷酸芯片 小肠结肠炎 模拟标本 志贺菌 检出 DNA基因 尼龙膜 片段 

分 类 号：R446.5[医药卫生—诊断学]