DEVELOPMENT OF RAPID IDENTIFICATION METHOD OF PATHOGENIC VIBRIOS

弧菌属细菌是自然水环境中常见的栖息地，包括霍乱弧菌、副溶血性弧菌等致病菌种。由于存在许多与致病菌种相似的弧菌，从自然环境中分离到的致病菌株的鉴定需要很多天的时间。此外，对于败血症患者的标本，简单快速的方法是绝对必要的，因为只有立即使用抗生素治疗才能挽救患者的生命。免疫学或遗传学方法在特异性方面表现出色，能够使快速鉴定成为可能，而无需耗时的生化测试。例如，聚合酶链式反应(PCR)不需要培养细菌。创伤弧菌是一种引起胎儿败血症的病原性弧菌，可引起肝脏功能障碍等基础疾病，本研究旨在建立一种简便、快速的创伤弧菌鉴定方法。为了获得足够数量的免疫原，对溶血素的纯化方法进行了改进和建立。用纯化的溶血素制备的抗血清证实了该种溶血素的特异性。合成了聚合酶链式反应的引物，并将其应用于与酶联免疫吸附试验相结合的聚合酶链式反应。尽管有一些血液成分对聚合酶链式反应有干扰，但预处理法(红细胞裂解和洗涤)排除了干扰。证实了该方法对创伤弧菌的特异性。本文还对创伤弧菌和副溶血弧菌的溶血素作用机理及铁载体、铁捕获剂、副溶血弧菌的免疫学检测方法进行了研究。

Bacteria of genus Vibrio are normal habitant in natural aquatic environments, and include some pathogenic species such as Vibrio cholerae, V.parahaemolyticus. Because of existence of many vibrios species similar to the pathogenic species, many days are necessary for identification of the pathogenic strain isolated from natural environment. Furthermore, simple and rapid method is absolutely necessary for specimens from septicemia patients, because only immediate antibiotics treatment can save life of the patient. Immunological or genetical methods are excel in the specificity and able to make possible rapid identification without time-consuming biochemical tests. For example, polymerase chain reaction (PCR) does not need cultivation of the bacterium. The present study is performed to establish simple and rapid identification methods of V.vulnificus, a pathogenic vibrio causing fetal septicemia to patients having underlying disease such as hepatic dysfunction.Hemolysin of V.vulnificus was chosen as a tool of immunological and genetical methods. To obtain enough amount of immunogen, purification method of the hemolysin was modified and established. Specificity of the hemolysin in the species was confirmed with antiserum prepared with the purified hemolysin. Primers for PCR are synthesized and applied for ED-PCR which is a PCR combined with a enzyme-linked immunosorbent assay. Although some blood components interfered the PCR,a pretreatment (lysis of erythrocytes and washing) eliminated the interfere. Specificity of the PCR for V.vulnificus was demonstrated. Mechanism of hemolysin action and immunologocal detection method of siderophores, chelator for acquisition of iron, of V.vulnificus and V.parahaemolyticus were also studied.