以巢式PCR及變性梯度凝膠電泳鑑別技術應用於環境水體中棘阿米巴原蟲與退伍軍人桿菌調查

Identification of Acanthamoeba and Legionella by Nested PCR with DGGE analysis in aquatic environment

董景嘉(Jing-Jia Tung)

指導教授 ： 許昺慕

國立中正大學/理學院/地球與環境科學系應用地球物理與環境科學研究所/碩士(2015年)

摘要

棘阿米巴原蟲與奈氏阿米巴原蟲是自由型阿米巴原蟲，其廣泛生存於各種環境水體之中，甚至是在經過毒處理過後的水體中，也能發現其存在。有些阿米巴原蟲種類，會對動物或人類帶有致病性，並且作為退伍軍人桿菌的宿主，進而提高感染退伍軍人症的風險。本研究目的為探討台灣環境水體中自由型阿米巴原蟲與退伍軍人桿菌分佈情形，其次為利用多步驟放大Nested PCR，將對環境樣本使用直接濃縮法偵測不到棘阿米巴原蟲。但是在培養法上能分離出的樣本作多步驟放大及在環境樣本偵測不到嗜肺性退伍軍人桿菌而在培養法上有分離出嗜肺性退伍軍人桿菌的樣本，去評估何種引子及放大步驟，最適合放大其PCR訊號。最後利用變性梯度凝膠電泳(DGGE)去探討其環境樣本經過多步驟放大Nested PCR後的圖譜狀況。
本研究於全台灣主要溪流、水庫、溫泉區及朴子溪流域共採集475個樣本並利用直接濃縮法與培養法來檢測環境水樣中棘阿米阿原蟲、奈式阿米巴原蟲及退伍軍人桿菌。定序結果顯示，環境水體中主要檢出種別為A.polyphaga; 奈式阿米巴原蟲主要檢出型別為N.clarki及N.australiensis; 退伍軍人桿菌以Legionella spp.為主而在朴子溪水體中有分離出嗜肺性退伍軍人桿菌。多步驟放大Nested PCR方法評估中2~3次擴增放大其PCR訊號，最後利用變性梯度凝膠電泳(DGGE)搭配，棘阿米巴原蟲方面以擴增3次其解析度最佳; 退伍軍人桿菌方面經過擴增2~3次Nested PCR其圖譜顯示以2次擴增方法的歧異度最高，DGGE圖譜中也能區分退伍軍人桿菌及嗜肺性退伍軍人桿菌，在不同型別的嗜肺性退伍軍人桿菌的圖譜上也能分出其型別。
本研究本研究環境水體除溫泉水樣沒有偵測到致病性蟲種外，其他水體
檢測出的棘阿米巴主要以A. polyphaga為主，是帶有感染風險的蟲種之一;
奈氏阿米巴並無偵測到N. fowleri的存在; 退伍軍人桿菌則大都為
Uncultured Legionella spp.而水樣中嗜肺性退伍軍人桿菌檢出雖不高但
還是有一定比率的感染風險。本研究對環境水體做2至3步驟的擴增放大PCR能有效放大其棘阿米巴原蟲及嗜肺性退伍軍人桿菌的微弱訊號，而在DGGE圖譜上能區別出嗜肺性退伍軍人桿菌與非嗜肺性退伍軍人桿菌。本研究的多步PCR-DGGE較直接PCR-DGGE增強>109倍(約109~1018倍)訊號，更可以讓在環境中存量極低的物種被偵測出。

關鍵字

嗜肺性退伍軍人桿菌 ； 奈氏阿米巴原蟲 ； 棘阿米巴原蟲 ； Nested PCR ； 變性梯度凝膠電泳

並列摘要

Acanthamoeba and Naegleria are free living-amoeba. They are widely distributed in a variety of aquatic environment even after disinfect. Some amoeba species of animals and human will cause disease, and as Legionella host to increase the risk of infection Legionella’s disease. The first purpose of this study is to investigate the distribution of free-living amoeba and Legionella in the environment al water in Taiwan. Second, using Nested PCR to detect the environmental Legionella pneumophila of direct concentration method in negative and cultivation method in positive and evaluating which primer is the best to use. Finally, using DGGE to observe the samples pattern.
In this study, a total of 475 samples were collected from streams, reservoirs, hot spring area and Putsu River in Taiwan. All the samples were investigated by direct concentration method and cultivation method to analyze the existence of Acanthamoeba, Naegleria and Legionella sp.. The results show that the most frequently species of free-living amoeba in environmental water is A. polyphaga,N.clarki and N. australiensis. The positive samples of Legionella species are mainly Legionella spp. and Legionella pneumophila.This study observed the Legionella pneumophila in environmental sample and discriminate type by Nested PCR with DGGE, and increasing about 109~1018 PCR detection signal.

並列關鍵字

Legionella pneumophila ； Naegleria ； Acanthamoeba ； Nested PCR ； DGGE

參考文獻

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