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

本课题旨在将病原菌特定靶基因的DNA扩增法或聚合酶链反应（PCR）法与自动化样品处理和DNA检测设备相结合，开发一种快速简便的食物中毒病例确诊系统。最重要的是建立寡核苷酸引物的获取方法和扩增条件，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但可靠的方法；我们反复筛选了许多测试菌株，以确定每个食物中毒细菌靶基因的两个参数。我们获得了以下目标基因的引物组和扩增条件：副溶血性弧菌热稳定型直接溶血素（tdh）及其相关溶血素（trh）基因、大肠杆菌热不稳定型肠毒素基因和Vero毒素基因、金黄色葡萄球菌肠毒素基因和中毒性休克综合征毒素基因、霍乱弧菌霍乱毒素基因。我们还开发了一种检测扩增DNA片段的方法，使上述PCR可以在自动化设备中进行。PCR产物用生物素标记，通过与链亲和素的亲和力被捕获到96孔微滴板的孔中。捕获的PCR产物用非同位素标记的寡核苷酸探针（碱性磷酸酶）检测。我们的检测方法的特点是在45分钟内可以检测到目标基因的几个拷贝，这比目前报道的任何其他基于微滴度的非同位素方法都要快。

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.