以宿主專一性聚合酶連鎖反應及指標生物法應用於河川糞便污染評估

Abstract

目前我國判斷水體是否有受到糞便污染乃以培養計數法測得之指標微生物量值做為判斷基準，但是單由指標微生物之數值無法判斷糞便污染之來源。本研究欲建立新的微生物追蹤法 (microbial source tracking, MST) -宿主專一性聚合酶連鎖反應 (宿主專一性PCR)，檢測水體是否受到糞便污染，並找出糞便污染之來源。本研究採用16對已發展成熟之宿主專一性引子，並與57個糞便樣品進行交叉試驗，篩選出11對適用於台灣環境之宿主專一性引子 (目標物種分別為人、豬、牛、羊及雞)。最後將宿主專一性PCR應用於實際河川監測，檢測台北水源集水區 (北勢溪、南勢溪及新店溪) 之21個測站的水樣是否有受到糞便污染，確認其污染來源。另外同時分析傳統指標微生物及河川水質參數，並進一步比較參數間之關係。由傳統指標微生物分析結果顯示，在北勢溪的測站坪林堰、大林橋及水源橋，南勢溪的烏來溫泉區附近之測點及整條新店溪測點其指標微生物皆有明顯升高之趨勢，可知上述之河域可能有受到糞便污染。而由宿主專一性PCR的分析結果則可進一步得知在北勢溪流域所受到的糞便污染物種種類較多，有人、反芻動物及雞之糞便污染訊號，且在北勢溪8個測站皆有測到雞之糞便污染，可見雞之糞便污染在此北勢溪流域有一定的影響。而在南勢溪及新店溪流域則是以人之糞便污染較為主，除了測站-福山其他測站皆有人之糞便污染。水質分析結果顯示SS、氨氮及總磷與水中的指標微生物有正相關性 (P < 0.05)，pH則與指標微生物呈負相關性 (P < 0.05)。在北勢溪流域土地利用多為茶園、營地及幾家養鹿場，另外，上游農家也有養殖家禽，因此在北勢溪有人、反芻動物及雞之糞便污染，而在南勢溪及新店溪由於為觀光勝地加上居住人口密集，因此在此區人之糞便污染較為嚴重。而氮、磷為微生物生長主要營養鹽之一，因此與指標微生物之數量會有正相關性。經由土地利用調查之結果與宿主專一性PCR檢測結果相比較，其結果皆相符合，證實本實驗挑選出之11對宿主專一性引子 (HF183、Hum163、Hum181、PF163、Pig-2-Bac、RumD1、Bac3、Cow-Bac2、CP2-9、CB-R2-42及CP3-49) 確實有潛力做為應用於台灣河川污染之監測，並可以有效指出糞便污染之來源。

Fecal pollution is frequently detected in the Taipei drinking water drainage basin by traditional indicator microbial cultures. However, it is difficult to defind the origin of fecal pollution by culturing methods. To identify the possible fecal contamination sources in the watershed, this study used microbial source tracking (MST) method identify fecal source by host-specific polymerase chain reaction (host-specific PCR). Sixteen pairs of host-specific primers from varies studies were tested for the suitability in detecting fecal pollution in Taipei water source catchment (including Pei-Shih River, Nan-Shih river, and Sin-Dian River). After cross examination of 57 feces from 8 different species collected in Taiwan, 11 pairs of host-specific primers were selected for the following 21 water samples analysis from the Taipei water source catchment. Water quality parameters (including pH, dissolved oxygen, conductivity, temperature, ammonia, nitrate, nitrite, total phosphorus, suspended solid, turbidity, and total organic carbon) and indicator microorganism (including total coliforms, fecal coliforms, Escherichia coli, and Enterococcus) were also determined. The traditional cultivation results shown that fecal pollution were dectected in several sample locations. From the MST experiments, human was the major fecal source in Nan-Shih River and Sin-Dian River. In Pei-Shih River, human, chicken, and ruminate were the major sources of fecal pollution. The microbial indicator concentration positively correlated to suspended solid, ammonia, and total phosphorus (p < 0.05) and negatively correlated to pH level (p < 0.05). Tea plantations and camp grounds are the major human activities performed in Bei-Shih River; and deer farm and chicken farm are also found in this area. Therefore, human, chicken, and ruminate may be the major source of fecal pollutions. The results of host-specific PCR were well corresponded with land usage. The results from this study may provide the knowledge in tracking fecal pollution for watershed management.