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Development of an automated, DNA amplification technique-based method for detection of food-poisoning bacteria

开发基于 DNA 扩增技术的自动化食物中毒细菌检测方法

基本信息

批准号：
04557024
负责人：
NISHIBUCHI Mitsuaki
金额：
$ 8.26万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

The purpose of this research project was to develop a quick and easy system to confirm food poisoning cases by combining the DNA amplification method or polymerase chain reaction (PCR) method for a particular target gene of the causative bacterium and an automated sample-processing and DNA-detecting equipment. The most important point was to establish an approach to obtain the oligonucleotide primers and amplification conditions which are the key factors determining the specificity and sensitivity of detection. We have adapted an empirical but sure method ; we repeatedly screened many test strains to determine the two parameters for each target gene of the food-poisoning bacterium. We then obtained primer sets and amplification conditions for the following target genes : the thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) genes of Vibrio parahaemolyticus, heat-labile and -stable enterotoxin genes and Vero toxin genes of Escherichia coli, enterotoxin genes and toxic shock syndrome toxin genes of Staphylococcus aureus, and cholera toxin gene of Vibrio cholerae.We also developed a method to detect amplified DNA fragments so that the above PCR can be performed in an automated equipment. The PCR products were labeled with biotin, trapped through affinity with streptoavidin onto the wells of a 96-well microtiter plate. The trapped PCR products were detected by oligonucleotide probes labeled nonisotopically (alkaline phosphatase). The feature of our detection method was that a few copies of the trget gene could be detected in 45 minites which is faster than any other microtiter-based non-isotopic methods reported so far.

该研究项目的目的是通过结合致病菌特定靶基因的DNA扩增方法或聚合酶链反应（PCR）方法以及自动化样本处理和DNA检测设备，开发一种快速简便的系统来确认食物中毒病例。其中最重要的是建立一种方法来获得寡核苷酸引物和扩增条件，这是决定检测特异性和灵敏度的关键因素。我们采用了一种经验性的，但肯定的方法，我们反复筛选了许多测试菌株，以确定两个参数为每个目标基因的食物中毒细菌。然后，我们获得了以下靶基因的引物组和扩增条件：副溶血弧菌的热稳定直接溶血素（tdh）和tdh相关溶血素（trh）基因，大肠杆菌的热不稳定和热稳定肠毒素基因和Vero毒素基因，金黄色葡萄球菌的肠毒素基因和中毒性休克综合征毒素基因，我们还建立了一种检测扩增DNA片段的方法，使上述PCR反应能在自动化设备中进行。用生物素标记PCR产物，通过与链霉亲和素的亲和性将其捕获到96孔微量滴定板的威尔斯孔上。用非同位素标记的寡核苷酸探针（碱性磷酸酶）检测捕获的PCR产物。该检测方法的特点是在45分钟内就可以检测到几个拷贝的trget基因，这比迄今为止报道的任何基于微量滴定的非同位素方法都要快。