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   全文下載

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

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）以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367 bp，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/hdc-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105與106 CFU/mL，在旗魚肉均質液中之最低檢測靈敏度分別為106與107 CFU/g。另外，以hdc-f/hdc-r引子檢測遭E. aerogenes與R. orithinolytica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；若以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。

It is important to develop rapid methods to detect the presence of histamine producers. In this study, the polymerase chain reac-tion (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 106/107 and hdc-f/hdc-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 detec-tion of Enterobacter aerogenes or Raoultella ornithinolytica after the PCR amplification using hdc-f/hdc-r and 106/107 primers were 105 and 106 CFU/mL in TSB broth, and 106 and 107 CFU/g in marlin homogenates, respectively. The hdc amplification using hdc-f/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. ornithinolytica. 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.