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

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检测产组胺细菌的分子方法。利用纯化蛋白的N端氨基酸序列克隆了光杆菌HDC基因，该基因编码一种可诱导的组氨酸脱羧酶(HisDcase)，该基因参与了大量组胺的细胞外排泄。对克隆的含有HDC基因的染色体片段进行了测序。设计寡核苷酸引物用于组胺假单胞菌和其他几种革兰氏阴性组胺产生菌HDC基因的PCR扩增。对组胺产生菌的最佳聚合酶链式反应条件进行了评价，并以沙门氏菌为例，对TaqMan聚合酶链式反应快速检测细菌进行了评价。该系统利用Taq DNA聚合酶的5‘核酸酶活性来检测目的基因的扩增。这些结果表明，TaqMan是一种可靠、快速的食品行业沙门氏菌检测方法。北海道大学Shinano，Inoue，Yamazaki(北海道大学)利用16S rRNA基因的特定区域，RT-PCR方法能够检测到酸性饮料中一种新型的嗜热嗜酸腐败菌A.acidoterrestris。建立了荧光探针检测产气荚膜梭菌的方法。此外，16S和23S rRNA基因内部间隔区的PCR片段长度分型可以快速、可靠地区分产气荚膜梭菌和梭状芽胞杆菌。本研究开发的从食品中提取DNA/RNA的方法被证明是实用的。

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.