臺灣博碩士論文加值系統

梨形鞭毛蟲(Giardia)及隱孢子蟲(Cryptosporidium)可感染人類及
其他多種生物。此兩類致病性原生動物臨床患者通常會出現發燒、噁
心、腹痛及腹瀉等症狀。免疫力正常的患者病程結束後即可痊癒，免
疫缺損者症狀嚴重可能會導致死亡。含有梨形鞭毛蟲囊孢及隱孢子蟲
卵囊之糞便排於水體後，仍具有相當長的存活時間，若飲用水遭到梨
形鞭毛蟲囊孢及隱孢子蟲卵囊污染時，即有可能造成疾病流行。目前
自來水淨水程序可能無法完全使原蟲達到失活，故可能因淨水程序之
缺失，而造成大規模的感染。
本研究採集五處採樣區共107個糞便檢體，以酵素鍵結免疫呈色
分析法(ELISA)、螢光免疫抗體染色法(IFA)以及聚合酶連鎖反應法
(PCR)檢測牛、猪、羊、雞、鴨與觀賞鳥類之糞便檢體中梨形鞭毛蟲
與隱孢子蟲存在情形，並以DNA定序法得知梨形鞭毛蟲及隱孢子蟲
之種別。實驗結果顯示：梨形鞭毛蟲呈現陽性反應之樣本佔3.7％
(4/107)，並從羊及牛之糞便檢體中檢測出G. lambla；隱孢子蟲呈現陽
性反應之樣本佔5.6％(6/107)，從羊及牛之糞便檢體中檢測出C.
parvum 、C.muris calf 而從猪之糞便檢體檢測出C. andersoni 。
本研究選定次氯酸鈉作為消毒劑，設計六種不同濃度，分別為有
效氯含量6.0％、3.0％、1.5％及自來水消毒常使用之含量2ppm、
1ppm、0.5ppm。利用不同接觸時間10min、30min、60min、120min、
12hr、24hr作為消毒效率分析，求出在不同失活率之條件，並以
DAPI/PI搭配螢光免疫抗體之染色法判別原蟲存活率。研究在不同失
活條件下，梨形鞭毛蟲及隱孢子蟲之失活率及C-T值。實驗結果顯示，
隱孢子蟲在高濃度試驗中，當反應時間2小時，可達到100％失活率；
I
梨形鞭毛蟲則在高濃度試驗中，反應時間4小時，才可達到100％失活
率。在低濃度試驗中。其結果顯示，梨形鞭毛蟲在反應72小時僅可達
到60％之失活率；隱孢子蟲在反應72小時僅可達到65％之失活率。結
果顯示一般自來水之消毒程序無法使原蟲完全失活，雖民眾皆有將水
煮沸後飲用之習慣，因而對人體健康雖無立即性的危害，但仍需訂定
相關議題之規範，防止梨形鞭毛蟲及隱孢子蟲因消毒程序不健全而造
成之感染，使其感染風險降至最低。

Cryptosporidium and Giardia are pathogenic protozoan, which can
infect the gastrointestinal tract of human and animals and cause diarrhea.
The two protozoan parasites cause acute diarrhea in
immunocompromised patients, but the AIDS patients couble be death by
giardiasis and cryptosporidiosis. Their oocysts/cysts show exceptional
resistance to various environment pressures and most disinfectants
commonly used for water. Therefore, they can cause massively
waterborne outbreaks.
In this study, 107 fecal specimens from cattle, pigs, sheep, duck,
chicken and bird were collected from six sampling sites in Taiwan to test
for Cryptosporidium oocysts and Giardia cysts. Enzyme-linked
immunosorbent assay (ELISA) and immunofluorescence assay (IFA)
accompanied by microscopy were defined as presumption test and
confirm test. As for the polymerase chain reaction (PCR) were defined as
complete test in this study. All of the six positive samples coincided with
the Cryptosporidium of PCR and. four positive samples coincided with
the of Giardia PCR Detection results of PCR were analyzed by the
nucleic acid sequencing method and three of Cryptosporidium species
were identified. The most frequently detected was C. calf (n=3) from the
feces of cattle and sheep, followed by C. parvum (n=2) from the feces of
cattle and sheep, and C. andersoni from the feces of pigs. G. lambla was
only one species detected by Giardia from the feces of cattle and sheep.
Cryptosporidium oocysts and Giardia cysts could survive in severe
environments, and is highly resistant to disinfectant. The conventional
water treatment process is unable to reduce the amount of oocysts and
cysts effectively, thus, unable to ensure the safety of the drinking water.
In this study, oocysts and cysts suspended in 6.0, 3.0, 1.5% and 2ppm,
1ppm, 0.5ppm aqueous sodium hypochlorite (Clorox laundry bleach) for
10, 30, 60, 120, 720,or 1440 min at 25℃ were administered infectivity
by DAPI/PI with IFA examination by microscopy. The result showed that
oocysts exposure to high sodium hypochlorite gaining 100% inactivity
while contacting 2hr;cysts gaining 100% inactivity while contacting 4hr.
oocysts exposure to 1ppm sodium hypochlorite gaining 65% inactivity
while contacting 72hr;cysts gaining 60% inactivity while contacting 72hr.
The result indicate that sodium hypochlorite could not gain 100%
inactivity in tap water There is no damage to the human health
immediately, but still need to stipulate the regulation in order to prevent
and lower the risk of infecting protozoan.

目錄
第一章前言......................................................................... .1
1.1 研究動機.....................................................................................1
1.2 研究目的.....................................................................................2
1.3 研究流程圖.................................................................................4
1.4 論文架構.....................................................................................5
第二章文獻回顧...................................................................6
2.1 原生動物之分類.........................................................................6
2.2 隱孢子蟲.....................................................................................7
2.2.1 隱孢子蟲感染史................................................................7
2.2.2 隱孢子蟲生活史................................................................7
2.2.3 隱孢子蟲演化史................................................................8
2.2.3.1 Cryptosporidium parvum.............................................9
2.2.3.2 Cryptosporidium andersoni .......................................10
2.2.3.3 其他型別...................................................................10
V
2.2.3.4 感染人類之型別......................................................10
2.3 梨形鞭毛蟲................................................................................11
2.3.1 梨形鞭毛蟲感染史..........................................................11
2.3.2 梨形鞭毛蟲生活史..........................................................11
2.3.3 梨形鞭毛蟲演化史..........................................................12
2.4 原蟲純化法...............................................................................15
2.4.1 濃聚法..............................................................................15
2.4.2 漂浮法..............................................................................15
2.5 原蟲染色...................................................................................17
2.5.1 免疫螢光抗體..................................................................18
2.5.2 DPAI/PI 染色法................................................................19
2.6 原蟲失活程序之評估試驗......................................................22
2.6.1 消毒劑之作用...................................................................22
2.6.1.1 次氯酸鈉...................................................................22
2.6.2 次氯酸鈉中原蟲失活效率之影響...................................23
2.6.2.1 反應時間與濃度......................................................23
2.7 流式細胞儀...............................................................................24
VI
2.7.1 流式細胞儀工作原理......................................................24
2.7.2 流式細胞儀分析原理......................................................25
2.7.3 電子系統..........................................................................27
2.7.4 分選系統..........................................................................28
2.7.5 流式細胞儀應用於原蟲檢測..........................................29
2.8 MPN PCR ..................................................................................30
第三章研究方法與材料.....................................................33
3.1 研究流程...................................................................................33
3.2 實驗試劑...................................................................................34
3.3 實驗材料...................................................................................34
3.4 實驗儀器...................................................................................35
3.5 原蟲檢測與分析實驗...............................................................36
3.5.1 樣本來源..........................................................................36
3.5.1.1 採樣.........................................................................36
3.5.1.2 正控制組.................................................................37
3.5.1.3 採樣時間.................................................................37
3.5.2 免疫酵素呈色分析法......................................................37
VII
3.5.3 螢光免疫抗體染色法......................................................38
3.5.4 聚合酶酵素連鎖反應......................................................39
3.5.4.1 DNA 純化萃取......................................................40
3.5.4.2 QIAamp stool Mini Kit(Qiagen)純化DNA ..........40
3.5.4.3 Nuclisens solation Kit 純化DNA..........................41
3.5.4.4 NucleoSpin Tissue 純化DNA ...............................43
3.5.5 引子(Primer)設計.............................................................44
3.5.6 PCR 最佳操作條件...........................................................45
3.5.7 PCR 試劑配置...................................................................46
3.5.8 Nest-PCR 試劑配置..........................................................47
3.5.9 PCR 產物之檢測...............................................................47
3.5.9.1 膠片製作................................................................47
3.5.9.2 電泳的操作及照相................................................48
3.5.10 PCR 產物DNA 純化......................................................48
3.5.11 PCR 產物定序.................................................................50
3.5.12 MPN PCR ........................................................................50
3.5.12.1 MPN-PCR 定量實驗方法.......................................50
VIII
3.5.12.2 MPN-PCR 結果與螢光免疫染色結果對照..........51
3.6 原蟲純化與消毒.......................................................................52
3.6.1 Percoll-Sucrose 梯度澄清法純化糞便檢體....................52
3.6.1.1 配置Percoll Sucrose 標準液...................................52
3.6.1.2 原蟲純化步驟..........................................................52
3.6.1.3 留洗步驟..................................................................52
3.6.1.4 原蟲存活狀態..........................................................53
3.6.2 磁性免疫抗體分離法( IMS) ...........................................53
3.6.3 次氯酸鈉配置..................................................................55
3.6.3.1 低濃度次氯酸鈉之消毒試驗..................................55
3.6.3.2 高濃度次氯酸鈉之消毒試驗..................................55
3.6.3.3 原蟲存活染色判定..................................................55
3.7 流式細胞儀分析........................................................................56
3.7.1 實驗準備藥品..................................................................56
3.7.2 實驗待測樣本前處理......................................................57
3.7.3 實驗方法與分析項目.......................................................58
第四章結果與討論.............................................................63
IX
4.1 豢養動物糞便檢體中梨形鞭毛蟲及隱孢子蟲之檢測..........63
4.1.1 推定試驗－酵素免疫呈色分析(ELISA) ........................64
4.1.2 確定試驗－螢光免疫抗體分析法..................................67
4.1.3 完成試驗－聚合酶連鎖反應及定序結果......................73
4.1.3.1 梨形鞭毛蟲定序結果..............................................73
4.1.3.2 隱孢子蟲定序結果..................................................73
4.1.3.3 核酸定序結果之親緣演化樹狀圖..........................75
4.1.4 聚合酶連鎖反應及螢光免疫抗體分析之結果..............77
4.1.5 MPN 與原蟲數量對照公式建立...................................80
4.2 原蟲純化與染色.......................................................................82
4.2.1 原蟲純化..........................................................................82
4.2.2 原蟲染色..........................................................................85
4.2.2.1 螢光免疫抗體染色..................................................85
4.2.2.2 原蟲存活染色判定..................................................87
4.3 原蟲消毒之失活分析...............................................................89
4.3.1 高濃度次氯酸鈉實驗......................................................89
4.3.1.1 隱孢子蟲反應時間與失活率之關係......................89
X
4.3.1.2 高濃度次氯酸鈉中隱孢子蟲之C-T 值..................91
4.3.1.3 梨形鞭毛蟲反應時間與失活率之關係..................93
4.3.1.4 高濃度次氯酸鈉中梨形鞭毛蟲之C-T 值..............95
4.3.2 低濃度次氯酸鈉實驗......................................................97
4.3.2.1 隱孢子蟲反應時間與失活率之關係......................97
4.3.2.2 低濃度次氯酸鈉中隱孢子蟲之C-T 值..................99
4.3.2.3 梨形鞭毛蟲反應時間與失活率之關係................102
4.3.2.4 低濃度次氯酸鈉中梨形鞭毛蟲之C-T 值............103
4.3.3 流式細胞儀實驗結果....................................................107
4.3.3.1 流式細胞儀分析步驟............................................107
第五章結論....................................................................... 109
第六章建議....................................................................... 111
參考文獻............................................................................... 112
自述....................................................................................... 124
著作....................................................................................... 125
XI
圖目錄
圖1.1 本研究之實驗流程......................................................................4
圖2.1 福馬林-乙醚離心法程序圖.......................................................16
圖2.2 流式細胞儀工作原理................................................................25
圖2.3 樣品的進流與偵測....................................................................26
圖2.4 信號的收集與濾片的配置........................................................27
圖2.5 分選系統....................................................................................29
圖2.6 Fredslund 之MPN-PCR 方法示意圖.........................................32
圖3.1 原蟲檢測之實驗流程................................................................33
圖3.2 綠色螢光與紅色螢光強度關係圖............................................59
圖3.3 細胞顆粒大小與細胞顆粒複雜之關係圖................................60
圖3.4 紅色螢光強度圖........................................................................60
圖3.5 綠色螢光強度圖........................................................................61
圖4.1 梨形鞭毛蟲及隱孢子蟲檢測流程圖........................................63
圖4.2 糞便檢體樣本中不同隱孢子蟲型別之親緣演化樹狀圖........76
XII
圖4.3 MPN-PCR 與隱孢子蟲卵囊數之對照圖...................................81
圖4.4 MPN-PCR 與梨形鞭毛蟲孢囊數之對照圖...............................81
圖4.5 原蟲純化回收率評估實驗........................................................83
圖4.6 糞便檢體中檢測出梨形鞭毛蟲(放大倍率：100 倍) ..............85
圖4.7 糞便檢體中檢測出梨形鞭毛蟲(放大倍率：400 倍) ..............86
圖4.8 糞便檢體中檢測出隱孢子毛蟲(放大倍率：100 倍) ..............86
圖4.9 失活之梨形鞭毛蟲DAPI/PI 染色之結果(放大倍率：400 倍).........88
圖4.10 仍保有活性之梨形鞭毛蟲DAPI/PI 染色之結果(放大倍
率：1000 倍) ............................................................................88
圖4.11 以6.0％有效氯含量之消毒劑進行隱孢子蟲消毒實驗之
結果...........................................................................................90
圖4.12 以3.0％有效氯含量之消毒劑進行隱孢子蟲消毒實驗之
結果...........................................................................................90
圖4.13 以1.5％ 有效氯含量之消毒劑進行隱孢子蟲消毒實驗
之結果......................................................................................91
圖4.14 高濃度次氯酸鈉中隱孢子蟲之C-T 值..................................92
圖4.15 以6.0％ 有效氯含量之消毒劑進行梨形鞭毛蟲消毒實
XIII
驗之結果..................................................................................94
圖4.16 以3.0％ 有效氯含量之消毒劑進行梨形鞭毛蟲消毒實
驗之結果..................................................................................94
圖4.17 以1.5％ 有效氯含量之消毒劑進行梨形鞭毛蟲消毒實
驗之結果..................................................................................95
圖4.18 高濃度次氯酸鈉中梨形鞭毛蟲之C-T 值..............................96
圖4.19 在濃度2ppm 次氯酸鈉下隱孢子蟲消毒結果.......................98
圖4.20 在濃度1ppm 次氯酸鈉下隱孢子蟲消毒結果.......................98
圖4.21 在濃度0.5ppm 次氯酸鈉下隱孢子蟲消毒結果....................99
圖4.22 低濃度次氯酸鈉中隱孢子蟲之C-T 值................................100
圖4.23 在濃度2ppm 次氯酸鈉下梨形鞭毛蟲消毒結果.................102
圖4.24 在濃度1ppm 次氯酸鈉下梨形鞭毛蟲消毒結果.................103
圖4.25 在濃度0.5ppm 次氯酸鈉下梨形鞭毛蟲消毒結果..............103
圖4.26 低濃度次氯酸鈉中梨形鞭毛蟲之C-T 值............................104
XIV
表目錄
表2.1 隱孢子蟲感染宿主之關係................................................. 9
表2.2 梨形鞭毛蟲種別與其主要感染宿主........................................14
表2.3 梨形鞭毛蟲感染宿主之分類....................................................14
表2.4 以DAPI/PI 染劑進行原蟲染色之染色特徵............................21
表2.5 DAPI/PI 染色結果與原蟲卵囊存活狀態之相關性..................21
表3.1 隱孢子蟲聚合酵素連鎖之引子................................................45
表3.2 梨形鞭毛蟲聚合酵素連鎖之引子............................................45
表3.3 隱孢子蟲之引子操作條件........................................................46
表3.4 梨形鞭毛蟲之引子操作條件....................................................46
表3.5 PCR 反應用量.............................................................................47
表4.1 豢養動物梨形鞭毛蟲之檢出率................................................66
表4.2 豢養動物隱孢子蟲之檢出率....................................................66
表4.3 不同區域梨形鞭毛蟲之檢出率................................................66
表4.4 不同區域隱孢子蟲之檢出率....................................................66
XV
表4.5 豢養動物梨形鞭毛蟲IFA 之檢出率........................................69
表4.6 豢養動物隱孢子蟲IFA 之檢出率............................................69
表4.7 根據區域梨形鞭毛蟲IFA 之檢出率........................................69
表4.8 根據區域隱孢子蟲IFA 之檢出率............................................69
表4.9 酵素免疫呈色法檢測結果呈陽性反應之幼獸及成獸糞便
檢體其梨形鞭毛蟲之檢出率....................................................70
表4.10 酵素免疫呈色法檢測結果呈陽性反應之幼獸及成獸糞便
檢體其隱孢子蟲之檢出率........................................................71
表4.11 梨形鞭毛蟲核酸定序之結果..................................................75
表4.12 隱孢子蟲核酸定序之結果......................................................75
表4.13 豢養動物糞便檢體中之梨形鞭毛蟲以螢光免疫抗體及
聚合酶連鎖反應進行檢測之結果..........................................79
表4.14 豢養動物糞便檢體中之隱孢子蟲以螢光免疫抗體及聚
合酶連鎖反應進行檢測之結果..............................................79
表4.15 不同採樣地區的糞便檢體中梨形鞭毛蟲以螢光免疫抗
體及聚合酶連鎖反應進行檢測之結果..................................79
表4.16 不同採樣地區的糞便檢體中隱孢子蟲以螢光免疫抗體
XVI
及聚合酶連鎖反應進行檢測之結果......................................80
表4.17 原蟲純化回收率......................................................................84
表4.18 隱孢子蟲在低濃度次氯酸鈉中相同C-T 值比較表............101
表4.19 梨形鞭毛蟲在低濃度次氯酸鈉中相同C-T 值比較............105
表4.20 經流式細胞儀分流後之原蟲回收率及失活率....................108

112
參考文獻
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