食源性致病菌96微孔板DNA诊断芯片的研制

Development of a 96-microwell plate DNA chip for detection of foodborne bacteria

目的:建立一种能同时检测12种常见食源性致病菌的基于微孔板阵列技术的检测平台,以满足食物中毒处置中能快速、准确提供实验室结果的要求。方法:针对12种最常见的食源性致病菌,设计种特异性的PCR引物和寡核苷酸探针,按照5×5的阵列格式将探针点制到96微孔板的微孔内,建立稳定的微孔杂交体系,采用链霉亲和素碱性磷酸酶和化学显色底物NBT/BCIP来检测特异性的PCR杂交产物。结果:经标准菌株验证已建立的食源性致病菌微孔板DNA诊断芯片,获得比较敏感、特异和稳定的实验结果;同时经食物中毒样品检测应用,与传统的细菌分离培养和生化鉴定的结果相符。结论:本研究建立的能同时检测12种常见食源性致病菌的基于微孔板阵列技术的检测平台具有快速、准确、自动化和高通量等特点,作为应对食物中毒等突发公共卫生事件的实用检测技术具有重要意义。

Objective：To develop a 96-microwell plate DNA chip for simultaneous detection of 12 major foodborne bacteria.Methods： Specific PCR primers and oligonucleotide probes were designed according to the conservative genes of 12 major foodborne bacteria,the DNA probes were spotted and immobilized in the wells of the plate in 5×5 array format.Stable hybridization system between PCR products and oligonucleotide probes in the microwell was established.Alkaline phosphatase-conjugated streptavidin and NBT/BCIP were used to detect the hybridized PCR products.Results： Standard bacteria strains were used to validate the novel DNA chip and highly sensitive,specific and stable experiment results were obtained.Using this diagnostic system,the causal pathogen that identified from an incident of food poisoning was consistent with that obtained using conventional bacterial culture and biochemical identification.Conclusion： The novel diagnostic system allows rapid,accurate,automated and high-throughput bacterial detection and is especially valuable for quick response to such public health emergencies like food poisoning.