臺灣博碩士論文加值系統

葡萄球菌屬中重要的病原菌，包括Staphylococcus aureus、S. saprophyticus、S. epidermidis、S. haemolyticus及S. xylosus等，葡萄球菌屬的檢測不管在食品中毒或臨床上的檢驗均相當重要。許多研究發現，熱休克蛋白基因hsp60, hsp10 and hsp10-hsp60 (IGR)序列可用來鑑定葡萄球菌屬，可作為Staphylococcus genus鑑定及分類之目標基因。因此，本研究之目的乃為利用熱休克蛋白基因針對S. aureus、S. saprophyticus、S. epidermidis及S. haemolyticus設計特異性之PCR引子組S.aur-F / R，S.sap-F/R，S.epi-F/R，S.hae-F/R，並進行特異性、靈敏度檢測。結果顯示，除了S. aureus、S. saprophyticus、S. epidermidis、S. haemolyticus等目標菌株會產生預期產物外，非葡萄球菌屬與非目標菌株不會受僞陽性之干擾。PCR引子組之靈敏度皆可以達到N×103 cfu/ml 。另外，針對未解序之S. xylosus部份，則加以定序。針對Staphylococcus genus方面，目前針對heat shock protein gene設計一組共通性引子組universal-F/R，對Staphylococcus genus進行genus放大之工作，以利用於後續生物晶片之開發。
本研究開發S. aureus、S. saprophyticus、S. epidermidis、S. haemolyticus 之PCR引子組、DNA探針、生物晶片(Biochip)分子檢測方法，一方面可用於菌株之鑑定，另一方面則可應用於食品樣品中目標菌株之快速檢測，建立短時間內完成葡萄球菌檢測之整合型晶片系統。
另外，乳牛乳房炎一直是乳牛場中感染率最高且影響經濟效
應最大的傳染病，其中包括金黃色葡萄球菌(S. aureus)、無乳鏈球菌(Streptococcus agalactiae)、乳房鏈球菌(Strep. uberis)及Strep. bovis等，本研究亦利用熱休克蛋白基因設計Strep. agalactiae、Strep. uberis、Strep. bovis及S. aureus之特異性引子組，開發多套式PCR(Multiplex PCR）。本研究利用針對目標菌株所設計之SAG3/SAG4、SUB3/SUB4、SBO3/SBO4及SAU3/SAU4引子組進行PCR特異性、靈敏度試驗；在特異性方面，結果顯示，除了S. aureus、Strep. agalactiae、Strep. uberis、Strep. bovis會產生預期產物以外，其他非鏈球菌屬與非目標菌株不會受僞陽性之干擾。在靈敏度試驗反應；針對Strep. agalactiae、Strep. uberis、Strep. bovis及S. aureus，在未增殖培養的條件下，其引子組PCR靈敏度可達N×103 cfu/ml。將引子組利用於檢測牛乳樣品中之靈敏度，結果顯示未增殖培養的條件下，PCR靈敏度可達N×103 cfu/ml；在37℃，10小時增殖條件下， PCR靈敏度可達N×100 cfu/ml。本研究所建立之葡萄球菌屬微生物之檢測方法，將可應用於食品、牛奶等樣品中目標菌株之快速檢測，建立短時間內完成葡萄球菌檢測之整合型晶片系統。另外，以多套式PCR檢測易造成牛乳房炎之病原性微生物，將可提供防疫單位、畜牧業使用並縮短檢測時間。以防治乳房炎為出發點，提升生乳品質，增加酪農業的收益與競爭力。

Abstract
Staphylococcus spp. including S. aureus, S. saprophyticus, S. epidermidis, S. haemolyticus and S. xylosus, etc. are important pathogenic bacteria, Diagnosis of these staphylococcus species isolated from food poisoning cases and clinical samples are important. Recently, studies revealed that heat shock protein gene, hsp60, hsp10 and hsp10-hsp60 (IGR) sequences can be used as a target gene for the identification of Staphylococcus spp. Therefore, the purpose of this study is to design of species specific PCR primers, i.e., S.aurF/R, S.sap-F/R, S.epi-F/R, and S.hae-F/R, respectively, these on the heat shock protein gene (hsp) sequences.
For Staphylococcus genus, universal primers, were also developed to amplify the hsp genes of Staphylococcus genus. Using S.aurF/R, S.sap-F/R, S.epi-F/R, and S.hae-F/R primer sets, bacterial species other than S. aureus, S. saprophyticus, S. epidermidis, S. haemolyticus, including other Staphylococcus. Spp. would not generate any false positive reaction. The detection limit were N × 103 cfu/ml. Such methods can be used for bacteria identification, rapid diagnosis, and these primers may be used on probes into biochip system.
Staphylococcus aureus, Streptococcus agalactiae, Strept. uberis, and Strept. bovis, etc., are common pathogens which may cause Mastitis in dairy herds. Conventional methods for the detection of these bacterial species are time consuming and laborious.
Thus, the development of rapid and reliable methods for the detection of these bacterial species is also important. In this study, we also designed the PCR primer sets, SAU3/SAU4, SAG3/SAG4, SUB3/SUB4 and SBO3/SBO4 from the heat shock protein gene, i.e., hsp70, hsp40, and hsp10, for the specific detection of Strep. agalactiae、Strep. uberis、Strep. bovis and S. aureus, respectively.
Using SAU3/SAU4, SAG3/SAG4, SUB3/SUB4 and SBO3/SBO4 primer sets, bacterial species other than S. aureus、Strep. agalactiae、Strep. uberis、Strep. bovis, including other Strept. spp. would not generate any false positive reaction.
As these PCR primer sets were used for multiplex PCR detection of target cells in pure culture, the detection limit was N × 103 cfu/ml, the detection limit were N × 104 and N × 100 cfu/ml milk depending on whereas the target cells were pre-enriched 10 hr, or not. Thus, the PCR and the multiplex PCR system so established offer a rapid method for detection of bovine mastitis pathogenic bacteria. Such method may be useful for the veterinary inspection agency and the dairy industry.

目 次
頁次
中文摘要………………………………………………………………1
英文摘要………………………………………………………………3
目次…………………………………………………………..………..I
表次……………………………………………………….………….VI
圖次……………………………………………………….……….. VII
第一章、文獻整理
第一部分、葡萄球菌屬
一、葡萄球菌屬之特色及其分類…………………………………….5
二、葡萄球菌屬病原菌之簡介…………………………………….…5
第二部份、鏈球菌屬
一、鏈球菌屬之特性及其分類…………………………………….…8
二、鏈球菌屬病原菌之簡介……………………………………….…9
三、葡萄球菌屬與動物疾病之關係………………………………....11
四、分子檢測技術在葡萄球菌屬之應用............................................11
五、熱休克蛋白的介紹………………………………………………12
六、熱休克蛋白的分類………………………………………………13

第二章、利用熱休克蛋白基因設計Staphylococcus aureus、Staphylococcus saprophyticus、Staphylococcus haemolyticus及Staphylococcus epidermidis之PCR引子組及其應用
壹、前言………………………………………………………………20
貳、材料與方法
一、實驗材料……………………………………………………….…22
(一) 菌株…………………………………………………………….....22
(二) 培養基………………………………………………………….....22
(三) 藥品…………………………………………………………….....22
(四) 緩衝液及試劑………………………………………………….....23
(五) 儀器………………………………………………….....................23
二、實驗方法…………………………………………………..............23
(一) PCR引子組設計…………………………………………………..23
(二) 聚合酶鏈鎖反應(PCR)……………………………………….…..24
1. 葡萄球菌的保存與鑑定………………………………….................24
2. 葡萄球菌DNA之製備…………………………………………....24
3. S.aur-F / S.aur-R、S.sap-F/S.sap-R、S.epi-F/S.epi-R、 S.hae-F/S.hae-R之PCR特異性引子組之應用…………………………………………..25
(1)直接檢測………………………………………………….….…..25
參、結果與討論
S. aureus、S. saprophyticus、S. epidermidis、S. haemolyticus特異性引子組之設計與應用
一、菌株的選定與PCR引子組之設計………………………………26
二、聚合酶鏈鎖反應 (PCR)…………………………………………..27
(1)葡萄球菌PCR引子組特異性試驗……………….…………....27
(2) PCR引子組靈敏度試驗……………………………………….28
肆、結論……………………………………………………………...…29

第三章、利用熱休克蛋白基因設計常見Staphylococcus spp.檢測用生物晶片系統之發展
壹、前言………………………………………………………...……….49
貳、材料與方法
一、實驗材料…………………………………………………………...50
(一) 菌株…………………………………………………………..…...50
(二) 藥品…………………………………………………………..…...51
(三) 培養基……………………………………………………..……...52
(四) Probe…………………………………………………………..…...52
(五) 儀器…………………………………………………………….....52
二、實驗方法
(一) PCR引子組及生物晶片探針之製作與探針之佈放……….……52
(二) DNA 製備（DNA extraction）……………………….…………53
（三）PCR 聚合酶連鎖反應（DNA amplification）……………………53
（四）雜合試驗……………………………………………………..53
（五）呈色試驗……………………………………………………..54
（六）結果判讀………………………………………………..……54
參、結果與討論…………………………………………………....55
第四章、利用熱休克蛋白基因設計Streptococcus agalactiae, Streptococcus uberis , Streptococcus bovis及Staphylococcus aureus檢測用Multiplex PCR引子組及其應用
壹、前言.................................................................................................64
貳、材料與方法………………………………………………………..65
一、實驗材料…………………………………………………………..65
(一) 菌株…………………………………………………………….....65
(二) 培養基………………………………………………………….....66
(三) 藥品…………………………………………………………….....67
(四) 緩衝液及試劑………………………………………………….....67
(五) 儀器…………………………………………………………….....68
二、實驗方法
(一) PCR引子組之設計 ………………………………………………69
(二) 多套式聚合酶鏈鎖反應 (Multiplex PCR ) ……………….……69
SAG3/SAG4、SUB3/SUB4、SBO3/SBO4及SAU3/SAU4四對引子組之多套式PCR特異性試驗及靈敏度試驗
(1)特異性…………………………………………………………….70
(2)靈敏度………………………………………………………….…70
(3)多套式PCR應用於食品樣品中Streptococcus agalactiae、 Streptococcus uberis、Streptococcus bovis 及Staphylococcus aureus檢測之應用………………………………………………………………….70
1.鮮乳樣品直接檢測………………………………………………......71
2.増菌培養……………………………………………………………...71
參、結果與討論…………………………………………….………….71
一、多套式PCR (Multiplex PCR) 特異性之探討…………………...71
二、多套式PCR引子組之檢測靈敏度……………………...….……71
三、多套式PCR應用於食品檢測之應用…………………………….67
1. PCR引子組靈敏度試驗…………………………………………..67
肆、結論……………………………………………………….………72
第五章、參考文獻……………………………………………….……..86

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