1998 Fiscal Year Final Research Report Summary
Studies on rapid molecular method for detection of new histamine-producing bacteria and new acidophilic spoilage bacteria.
| Project/Area Number |
08556034
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Fisheries chemistry
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| Research Institution | Tokyo University of Fisheries |
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Principal Investigator |
FUJII Tateo Tokyo University of Fisheries ; Department of Food Science and Technology, Professor, 水産学部, 教授 (30093305)
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| Co-Investigator(Kenkyū-buntansha) |
SHINANO H. Hokkaido University ; Professor, 水産学部, 教授(元) (10001603)
YAMAZAKI K Hokkaido University ; Associate Professor, 水産学部, 助教授 (40250500)
INOUE T. Hokkaido University ; Professor, 水産学部, 教授 (60002086)
YOKOMOTO Y. Perkin Elmer, Japan ; Reseacher, 研究員
KIMURA B. Tokyo University of Fisheries ; Associate Professor, 水産学部, 助教授 (50262340)
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| Project Period (FY) |
1996 – 1998
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| Keywords | histamine / histamine-producing bacteria / rapid detection / molecular method / TaqMan method / PCR / Alicyclobacillus acidoterrestris / Clostridium perfringens |
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| Research Abstract |
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.
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