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研究生:林孟瑄
研究生(外文):Meng-Hsuan Lin
論文名稱:以PMA-qPCR定量金黃色葡萄球菌之分析方法評估
論文名稱(外文):Quantification of Staphylococcus aureus by propidium monoazide in combination with real-time quantitative PCR
指導教授:張靜文張靜文引用關係
口試委員:吳佩芝李書安
口試日期:2015-07-30
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:環境衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:115
中文關鍵詞:金黃色葡萄球菌即時定量聚合酶連鎖反應propidium monoazide
外文關鍵詞:Staphylococcus aureusreal-time quantitative PCRpropidium monoazide
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Staphylococcus aureus (金黃色葡萄球菌)是一種普遍存在於環境中之菌種。在人體免疫力低下時可能藉由空氣傳播以及接觸到受污染之物體而導致感染,其致病特性已被證實,加以抗藥特性導致治療困難,使得此菌已然成為職業場所與醫院院內感染重要病原菌之一。然而傳統以培養法進行細菌之定性與定量過程繁複且所需耗費時間較長;除此之外,此分析方法無法定量具有活性但不可培養(viable but non-culturable (VBNC))之金黃色葡萄球菌,可能低估實場中具有感染風險之實際活菌濃度。有鑑於此,本研究欲利用核酸染劑 (propidium monoazide,PMA)可準確定量活菌之特性,結合即時定量聚合酶連鎖反應偵測快速、敏感度高之優點,進行定量空氣中活性金黃色葡萄球菌方法建立,評估定量活菌之qPCR條件與最佳PMA濃度,觀察其線性關係範圍,期望能找出PMA-qPCR之最佳條件並將其應用於實場環境S. aureus濃度偵測。
首先以培養法與BacLight染劑搭配螢光顯微鏡,觀察金黃色葡萄球菌經由加熱處置(90℃、20分鐘)是否能製備出後續實驗所需死菌,結果顯示,加熱法(90℃、20分鐘)處置後已無可培養之金黃色葡萄球菌,且顯微鏡之觀察也發現此加熱處置可使絕大部分活菌轉變成細胞膜破損之死菌狀態。接著評估不同回溶液體之PMA與不同光照來源對於定量活菌S. aureus效果,不同回溶液體之PMA選擇了對細胞較無傷害性之以滅菌去離子水回溶PMA,其定量活菌與抑制死菌效果均優於以20% DMSO回溶之PMA (P<0.05)。本研究亦評估了500W鹵素燈與60W PhAST Blue系統兩種光照來源對於PMA效能之影響,由qPCR結果發現,添加PMA後分別以此兩種燈源進行光照並不會顯著影響PMA定量活菌效能,兩者光源表現無統計顯著差異 (P>0.05),因此後續選擇較多研究使用且一次可分析較多樣本數之500W鹵素燈進行PMA光照處置。
選定PMA與燈源材料後,再分別以相位差/螢光顯微鏡與qPCR搭配選定之PMA,檢視PMA染劑對於活菌與死菌之定量影響,其結果顯示,PMA濃度1.5 μg/mL即可有效侵入破損細胞內與DNA形成共價鍵結,抑制死菌被qPCR定量,使添加PMA之死菌組細菌濃度下降並與對照組(PMA=0 μg/mL)達統計顯著差異 (P<0.05);相對之下,PMA較不會侵入金黃色葡萄球菌之活菌細胞內,且1.5與2.3 μg/mL之PMA未顯著降低活性S. aureus濃度 (P>0.05),均可穩定應用於菌液濃度3–8 log cells/mL之範圍,以X軸為對照組之S. aureus濃度(log cells/mL)、Y軸為添加PMA後qPCR偵測結果之S. aureus濃度(log cells/mL)進行作圖並觀察兩者對應線性關係,其線性分別為0.97與0.96。綜合上述結果,本研究選擇抑菌效果佳,於單純活菌組與活/死菌混合菌液組ND樣本數較少且可穩定定量活性金黃色葡萄球菌之1.5 μg/mL PMA為最佳PMA濃度。
本研究與過往研究不同之處為不再只是探討單一S. aureus濃度下PMA最佳適用條件,除了重新將歷年定量S. aureus之qPCR條件進行調整與測試外,亦評估出較適用於定量活菌之以水回溶PMA與不同光源之影響,研究成果較適合應用於細菌濃度變化範圍廣泛之實際環境中。


Staphylococcus aureus, considered as one of the infectious bioaerosols, may be transmitted to the humans by airborne route. S. aureus plays an important role in nosocomial infections and has been detected in the residence, community and poultry feeding industry. Long-term uses of the antibiotics have caused the emergence of methicillin-resistant Staphylococcus aureus (MRSA) that was resulted from the drug resistance of S. aureus. Detecting and quantifying this pathogen via appropriate analytical method is necessary to identify the exposure risks of people, adopt effective intervention strategies and evaluate the performances of control measures.
Monitoring a specified pathogen in environment may be accomplished via the analyses of samples with culture assay and a series of isolation/identification processes. However, such analytical methods are complex, time-consuming, limited by the number of workable colonies and can only detect culturable cells but not the cells with viable but not culturable (VBNC) state; therefore, the microbial quantification tends to be falsely determined. The real-time quantitative polymerase chain reaction (qPCR) detects the DNA of microorganism of a specific target and is not affected by the nonculturability of target cells. Thus, this qPCR method overcomes the drawback of the culture assay. However, the qPCR may overestimate the number of viable cells because it cannot differentiate the dead cells from viable ones.
To address the issue that both culture assay and qPCR may not accurately quantify the exposure level of S. aureus, this study is initiated to develop a qPCR-based method coupled with nucleic acid dye that can accurately quantify viable S. aureus. Different types and concentrations of nucleic acid dye (propidium monoazide) are evaluated and the detection limit of this assay are determined. Furthermore, this analytical method will be validated in environments. It is expected that this study will help to detect and quantify the exposure of S. aureus in environments precisely.
According to the experiments by culture assays and BacLight/epifluorescence microscopy, our data show heating cells at 90℃ for 20 min resulted in a complete loss of cell culturability and significant damages on the integrity of cell membrane. Further examination of PMA-treated cells under light and epifluorescence microscopes indicate that PMA penetrated cells pre-heated at 90℃ for 20 min (represented as dead S. aureus).

However, it is noted that PMA (23 μg/mL) did not penetrate the membrane of some alive cells.
The performance of five different concentrations of PMA (1.5, 2.3,10, 23, 46 μg/mL) coupled with the qPCR was further assessed. For heat-inactivated S. aureus, PMA significantly inhibited the DNA amplification in qPCR. As for live S. aureus, only the PMA of 1.5 and 2.3 μg/mL showed comparable DNA concentrations between PMA-treated and –untreated cells (P>0.05). The PMA of 1.5 and 2.3 μg/mL were tested for the linearity, and obtained the respective R2 values of 0.97 and 0.96 for live cells in the range of 103-108 cells/mL.


目錄
中文摘要 i
Abstract iii
目錄 v
表目錄 ix
圖目錄 xi
第一章 前言 1
第二章 文獻回顧 2
2.1 環境中金黃色葡萄球菌 2
2.2 環境中活性金黃色葡萄球菌之分析方法 3
2.3 核酸染劑結合即時定量聚合酶連鎖反應法 6
2.3.1 S. aureus 之核酸染劑應用 9
2.3.2 非S. aureus 之核酸染劑應用 11
2.3.3 PMA定量活性細菌之影響因子 14
2.3.3.1 PMA不同回溶液體與濃度 14
2.3.3.2 暗反應時間 15
2.3.3.3 光照來源、時間 15
2.3.3.4 環境因子 15
第三章 研究目的 17
第四章 研究架構 18
第五章 材料與方法 20
5.1 實驗菌株 20
5.2培養基與緩衝溶液 20
5.2.1 Luria-Bertani (LB) broth 20
5.2.2 Tryptic Soy Agar (TSA) 20
5.2.3 TE 緩衝溶液 20
5.2.4 Phosphate buffer saline (PBS) 磷酸緩衝液 21
5.3金黃色葡萄球菌引子與qPCR條件測試 21
5.3.1即時定量聚合酶連鎖反應之S. aureus引子對 22
5.3.1.1 S. aureus引子對搜尋 22
5.3.1.2 S. aureus引子對專一性測試 23
5.3.2 S. aureus qPCR 條件測試 23
5.3.2.1引子合成 24
5.3.2.2 DNA標準品合成與檢量線製作 24
5.3.2.3不同升溫條件測試 25
5.3.2.4 不同qPCR試劑濃度測試 27
5.3.2.5 不同template DNA量之測試 28
5.4 製備與建立金黃色葡萄球菌檢量線 29
5.4.1 S. aureus之培養與處置 29
5.4.2 建立S. aureus之檢量線 29
5.5 加熱處理 31
5.5.1 加熱處置S. aureus對可培養性之影響 31
5.5.2 加熱處置S. aureus對細胞膜之影響 32
5.6 以PMA處理活性及非活性金黃色葡萄球菌 34
5.6.1 不同回溶液體之PMA對於金黃色葡萄球菌之影響 34
5.6.2不同光源對於PMA定量金黃色葡萄球菌之影響 37
5.6.3以顯微鏡搭配PMA觀察活性及非活性金黃色葡萄球菌 39
5.6.4 PMA搭配qPCR於定量活性S. aureus之適用性與條件 40
5.6.5 PMA搭配qPCR於定量S. aureus活/死菌混合菌液之適用性 42
5.6.6 DNA萃取、qPCR分析與定量 44
5.7 資料分析及統計 44
第六章 結果 45
6.1選定最佳定量金黃色葡萄球菌之引子對 45
6.2 S. aureus qPCR 條件測試 57
6.2.1 最佳升溫條件 57
6.2.2 最佳試劑條件 58
6.2.3 最佳template DNA量 59
6.2.4 總結論 60
6.2.5 qPCR QA/QC檢量線及偵測下限 61
6.3 S. aureus檢量線建立 62
6.4 加熱處置之成效 63
6.4.1 以培養法檢視加熱對S. aureus可培養性之影響 63
6.4.2 以螢光顯微鏡結合BacLight染劑檢視加熱對S. aureus細胞膜之影響 64
6.5 以PMA-qPCR處理金黃色葡萄球菌之活菌與死菌 65
6.5.1 不同回溶液體之PMA對於金黃色葡萄球菌之影響 65
6.5.2 不同光源對於PMA定量金黃色葡萄球菌之影響 67
6.5.3 以顯微鏡搭配PMA觀察金黃色葡萄球菌之活菌與死菌 68
6.5.4 PMA-qPCR於定量S. aureus之適用性與條件 69
6.5.4.1 PMA-qPCR於定量死菌S. aureus 71
6.5.4.2 PMA-qPCR於定量活菌S. aureus 75
6.5.4.3 PMA搭配qPCR於定量S. aureus活/死菌混合菌液之適用性 80
第七章 討論 81
7.1 最佳核酸染劑條件選擇 81
7.1.1 核酸染劑種類 81
7.1.2 暗反應時間 81
7.1.3 光照來源與光照強度 82
7.1.4 光照時間 83
7.1.5 最佳核酸染劑濃度 85
7.1.5.1 以qPCR搭配PMA定量非活性S. aureus: 85
7.1.5.2 以qPCR搭配PMA定量活性S. aureus 87
7.2 以顯微鏡搭配PMA觀察活性及非活性S. aureus 90
7.3 以qPCR搭配PMA定量活性S. aureus組線性表現 91
7.4 PMA-qPCR無法完全抑制死菌之探討 94
第八章 結論及建議 96
8.1 結論 96
8.2 研究限制 97
8.3 未來研究之建議 97
附錄 99
參考文獻 103


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