リアルタイムPCRによる水環境水中の糞便汚染指標細菌の定量

摘要

A rapid and quantitative polymerase chain reaction (PCR) method for determining the numbers of two fecal pollution indicators, E. coli and Enterococcus was developed. The quantitative detection of E. coli and Enterococcus was performed on the basis of the uidA and 23S-rRNA gene sequences, respectively. A linear relationship was observed between the log concentration of each gene and the threshold cycle value obtained from the PCR amplification curve. Using these standard PCR curves, the concentrations of E. coli and Enterococcus were measured in environmental water samples collected from a wastewater treatment plant and four bodies of water located in the northern part of Japan. Measurements by the real-time PCR method were compared with E. coli and Enterococcus counts determined by cultivation methods such as membrane filter and MPN analyses. The PCR method gave quicker results and higher E. coli and Enterococcus counts than the traditional cultivation methods. These higher counts suggested the ability of the PCR method to detect DNA from both culturable and nonculturable or dead bacteria. Regression analysis of these with correlation coefficients of over 0.7.%糞便汚染を厳密に特定するため，大腸菌，翼便性連鎖球菌をターゲットにリアルタイムPCR法を適用して遺伝子レベルでターゲットのみを迅速かつ特異的に検出し定量する方法を検討した。大腸菌のuidA遺伝子と糞便性連鎖球菌の23S-rRNA遺伝子をターゲットにしてリアルタイムPCRの閾値サイクル数と遺伝子コピー数の関係から検量線を作成し，排水処理施設や水域に適用して調査を行つた。PCR法による大腸菌の計数値は，排水処理施設，水域ともほぼ大腸菌群数と糞便性大腸菌群数の中間に位置し，従来の指標との間に密接な関連性が認められた。さらに排水処理施設の処理水では大腸菌，糞便性連鎖球菌ともに他の指標ではほとんど不検出だったのに対し，PCR法では1×10~2～10~4個·100mL~(-1)程度検出され，過大評価につながる可能性が示唆された。リアルタイムPCRによる計数法は，大腸菌，糞便性連鎖球菌共に他の従来指標との間に高い相関性が認められ，新しい迅速な計数法となる可能性が示された。