Polymerase Chain Reaction for the Detection of Histamine-Producing Bacteria Isolated from Taiwanese Foods

以聚合酶連鎖反應法檢測台灣食品中分離之組織胺生產菌

龔賢鳳(Hsien-Feng Kung) ； 李聰明(Tsong-Ming Lee) ； 林仲聖(Chung-Saint Lin) ； 洪堂耀(Tang-Yao Hong) ； 蔡永祥(Yung-Hsiang Tsai)

《藥物食品分析》 20卷1期 (2012/03) Pp. 76-84+170

摘要

魚肉中組織胺的產生主要係來自細菌分泌組胺酸脫羧（histidine decarboxylase）將組胺酸行脫羧作用所致，將組織胺生產菌快速並正確的檢測出來是重要的研發方向。本研究參考國外文獻之引子（primer）針對組胺酸脫羧基因（hdc）此PCR增幅法分析，具組織胺生產性之革蘭氏陽性菌以JV16HC/JV17HC引子增幅片段為367bp，具組織胺生產性之革蘭氏陰性菌以106/107以及hdc-f/hdc-r兩組primer放大之片段分別為534 bp與709 bp。從台灣食品中分離出之7株革蘭氏陽性組織胺生產菌與15株革蘭氏陰性組織胺生產菌，皆可以此PCR法檢測出hdc增幅片段。其次，針對革蘭氏陰性菌Enterobacter aerogenes與Raoultella orithinolytica為代表，進行兩種引子hdc－f/hde-r與106/107之最低檢測靈敏度分析，得知在TSB培養液中兩引子之最低檢測靈敏度分別為10^5與10^6 CFU/mL，在旗魚肉均實液中之最低檢測靈敏度分別為10^6與10^7 CFU/mL。另外，以hdc－f/hdc－r引子檢測遭E. aerogenes與R. orithinolytic汙染之TSBH培養液與旗魚肉均質液，得知可較HPLC檢出組織胺早2小時增幅出菌的hdc放大片段；若此106/107引子檢測則與HPLC檢出組織胺相同時間增幅出菌的hdc放大片段。因此，以此PCR方法可以容易偵測出食品中之組織胺生產菌。

關鍵字

組織胺 ； 聚合酶連銷反應 ； 組織胺生產菌 ； 組胺酸脫羧酶基因

並列摘要

It is important to develop rapid methods to detect the presence of histamine producers. In this study, the polymerase chain reaction (PCR) assay yielded a 367-bp DNA amplification fragment from histidine decarboxylase (hdc) of gram-positive bacteria using JV16HC/JV17HC primer, and 534-bp and 709-bp DNA amplification fragments from histidine decarboxylase of gram-negative bacteria using 106/107and hde-f/hde-r primers, respectively. Seven gram-positive histamine-producing strains and 15 gram-negative histamine-producing strains isolated from Taiwanese foods successfully produced the PCR amplification products. The minimum levels of detection of Enterobacter aerogenes or Raoultella ornithinolytica after the PCR amplification using hde-f/hde-r and 106/107 primerswere 10^5 and 10^6 CFU/mL in TSB broth, and 10^6 and 10^7 CFU/g in marlin homogenares, respectively. The hdc amplification using hdc-I/hdc-r primer was detected 2 h earlier than the HPLC detection of histamine in TSBH broth or marlin homogenates inoculated E. aerogenes or R. ornithinolyt icu. However, the detection of hdc amplification using the 106/107 primer and that of histamine by HPLC occurred at the same sampling time. Therefore, the PCR method could be easily used to detect potential histamine-producing bacteria in foods.

並列關鍵字

Histamine ； PCR ； histamine-forming bacteria ； histidine decarboxylase gene (hdc)