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Studies on rapid molecular method for detection of new histamine-producing bacteria and new acidophilic spoilage bacteria.

新型组胺产生菌和新型嗜酸腐败菌的快速分子检测方法研究

基本信息

批准号：
08556034
负责人：
FUJII Tateo
金额：
$ 12.61万
依托单位：
Tokyo University of Fisheries
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

1. Fujii and Kimura(Tokyo University of Fisheries), Yokomoto(Perkin Elmer, Japan)The purpose of this study was to establish a rapid molecular method for detecting histamine-producing bacteria, the causative agent of scombroid poisoning. The Photobacterium histaminum hdc gene encoding an inducible histidine decarboxylase(HisDcase), that is involved in a large scale excretion of histamine extracellularly, has been cloned by using the N-terminal amino acid sequence of the purified protein. The cloned chromosomal fragment containing the hdc gene was sequenced. Oligonucleotide primers were designed for the PCR amplification of hdc genes of P.histaminum and several other gram-negative histamine-producing bacteria. The optimal PCR conditions for detecting histamine-producing bacteria were evaluated.Also, the TaqMan PCR assay for rapid detection of bacteria was evaluated, using Salmonella. This system uses the 5' nuclease activity of Taq DNA polymerase which digests an internal fluorogenic probe to monitor the amplification of the target gene. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry.2. Shinano, Inoue, Yamazaki (Hokkaido University)Using specific region of 16S rRNA gene, RT-PCR method enabled the detection of A.acidoterrestris, a new type of thermoacidophilic spoilage bacterium in acidic beverages. A PCR method for Clostridium perfringens with fluorogenic probes was also developed. Additionally, PCR fragment length typing of the internal spacer region between the 16S and 23S rRNA genes was found to be rapid and reliable to distinguish C.perfringens among clostridia. DNA/RNA extracting methods from food, developed in this study, were proved to be practical.

1. Fujii和Kimura（东京水产大学），Yokomoto（Perkin Elmer，日本）本研究的目的是建立一种快速的分子检测方法，用于检测鲭鱼中毒的病原体--组胺产生菌。组胺发光杆菌hdc基因编码的诱导型组氨酸脱羧酶（HisDcase），这是参与了一个大规模的组胺胞外排泄，已被克隆通过使用纯化的蛋白质的N-末端氨基酸序列。对克隆的含有hdc基因的染色体片段进行测序。设计寡核苷酸引物，PCR扩增组胺假单胞菌和其他几种革兰氏阴性组胺产生菌的hdc基因。对组胺产生菌的PCR检测条件进行了优化，并以沙门氏菌为例，对TaqMan PCR快速检测方法进行了评价。该系统利用Taq DNA聚合酶的5'核酸酶活性，其通过插入内部荧光探针来监测靶基因的扩增。这些结果表明TaqMan是一种可靠、快速的食品工业沙门氏菌检测方法. Shinano，Inoue，Yamazaki（北海道大学）RT-PCR方法利用16 S rRNA基因的特定区域，能够检测酸性饮料中新型嗜热嗜酸腐败菌A.acidoterrestris。建立了一种以荧光探针为探针的产气荚膜梭菌PCR检测方法。此外，发现16 S和23 S rRNA基因之间的内部间隔区的PCR片段长度分型可快速可靠地区分梭菌中的产气荚膜梭菌。本研究建立的食品中DNA/RNA提取方法具有实用性。