臺灣博碩士論文加值系統

乳牛乳房炎是乳牛養殖過程中一種常見的多發性疾病，每年因乳房炎而造成生乳體細胞數過高、乳質異常、異常乳之廢氣及牛因病淘汰等重大的經濟損失，估計年損失十億元左右，所以為世界各地酪農業所必須克服之重要課題。乳房炎主要病因為病原微生物侵入乳腺內感染而引起。病原菌主要有鏈球菌(Streptococcus spp.)、金黃色葡萄球菌(Staphylococcus aureus)、大腸桿菌(E. coli) 等。其中，以鏈球菌最常見，是引起乳房炎最普遍的病源之一，佔引起乳房炎的絕大多數。而鏈球菌中又以無乳鏈球菌（streptococcus agalactiae）感染最多，其次則為Streptococcus dysgalactiae，Streptococcus uberis 及 Streptococcus bovis。
目前已有許多分子分類方法用於微生物之快速檢測，其中以聚合酶鏈鎖反應法（polymerase chain reaction, PCR）最為常見。而近年來heat shock protein 被廣泛應用在微生物快速檢測，如：Staphylococcus aureus等，因此本研究之目的乃利用熱休克蛋白基因Hsp70、Hsp40、Hsp10設計Strep. agalactiae、Strep. uberis、Strep. bovis之特異性PCR引子組SAG1/SAG2、SUB1/SUB2、SBO1/SBO2，並進行特異性檢測。結果顯示，除了鏈球菌Strep. agalactiae、Strep. uberis、Strep. bovis會產生預期目標產物外，其他非鏈球菌屬菌株皆不會產生僞陽性之干擾。當PCR引子組SAG1/SAG2、SUB1/SUB2、SBO1/SBO2 應用於檢測牛奶樣品中鏈球菌時，傳統PCR靈敏度可以達到N×103 CFU/ ml，而在增值10小時後，其傳統PCR靈敏度可以達到N×100 CFU/ ml。real-time PCR 方法上，其靈敏度也可達N×103 CFU/ ml但較傳統PCR更容易在檢測上辨識。將自行設計之特異性PCR引子組所增幅出來之PCR產物進行序列比對，結果顯示整段序列均具有特異性。
本研究所建立之微生物檢測PCR方法，可快速、定量的檢測易造成牛乳房炎之病原性微生物，以提供防疫單位、畜牧業使用。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。

Staphylococcus aureus, pathogenic E. coli, Streptococcus agalactiae, Strepto. uberis, and Strepto. bovis, etc., are common pathogens which may cause Mastitis in dairy herds. Conventional methods for the detection of these bacterial species need the use of selection and differentiation medium followed by biochemical and serological identification steps. Such process is time consuming and laborious.
Thus, the development of rapid and reliable methods for the detection of these bacterial species is important. In this study, we designed the PCR primer sets, SAG1/SAG2, SUB1/SUB2, and SBO1/SBO2 from the heat shock protein gene, i.e., HSP70, HSP40, and HSP10, for the specific detection of Strep. agalactiae、Strep. uberis、Strep. bovis, respectively. Using SAG1/SAG2, SUB1/SUB2, and SBO1/SBO2 primer sets, bacterial species other than Strep. agalactiae、Strep. uberis、Strep. bovis, including other Strept. spp. would not generate any false positive reaction.
As those PCR primer sets were used for Conventional and Real-time PCR detection of target cells in milk, the detection limit were N × 103, and N × 100 CFU/ per ml milk if the target cells were without/with the pre-enrichment step for 10 hr, respectively. In this study, these PCR primer sets for the conventional and real-time PCR methods so established will offer a rapid and quantitative method for detection of bovine mastitis pathogenic bacteria. Such method can be used by government agency for animals disease monitoring and bovine farming industry.

目 次
頁次
中文摘要…………………………………………………………………………I
英文摘要…………………………………………………………………………III
第一章、 文獻整理........................................................................................1
一、乳房炎之定義及分類......................................................................1
二、乳房炎所造成的原因與發病機制..................................................2
三、鏈球菌引起乳房炎的種類及特性...................................................3
四、本省中部地區潛在性乳房炎病原菌的分析調查...........................4
五、檢測乳房炎常用之方法...................................................................5
（1）物理檢查..................................................................................5
（2）前乳檢查（Strip Test）...........................................................5
（3）加州乳房炎試驗（California Mastitis Test；CMT）............5
（4）導電度測定 (Electrical Conductivity)………………………6
六、熱休克蛋白（heat shock proteins）................................................7
（1）前言..........................................................................................7
（2）熱休克蛋白的分類..................................................................8
七、分子生物學技術在鏈球菌之應用..................................................10
八、聚合酶鏈鎖反應（polymerase chain reaction；PCR）................10
九、即時聚合酶鏈鎖反應（Real-time PCR）.....................................11
十、研究目的.........................................................................................14
目 次
頁數
第二章、利用熱休克蛋白基因設計牛乳房炎感染檢測用PCR引子組及其應用
壹、前言..............................................................................................23
貳、材料與方法..................................................................................25
一、實驗材料...............................................................................25
(一) 菌種................................................................................25
(二) 培養基............................................................................25
(三) 藥品................................................................................26
(四) 緩衝液及試劑................................................................27
(五)儀器 ……………………………………………………28
二、實驗方法 ………………………………………………….29
(一) PCR引子組之設計........................................................29
(二) 聚合酶鏈鎖反應 ( PCR ) ……………………………29
1. 鏈球菌及其他非鏈球菌DNA之製備 ……………..29
2. 引子組SAG1/2、SUB1/2、SBO1/2之PCR特異性試驗
………………………………………………………….30



目 次
頁數
(三) Strep. agalactiae、Strep. uberis、Strep. bovis
特異性引子組於食品檢測之應用………………………….30
1. 鮮乳樣品的檢測應用 ……………………………...30
（1）鮮乳樣品中生菌數與Streptococcus spp.之計算..30
（2）直接檢測…………………………………………30
（3）増菌培養 ………………………………………...31
(四) Real-Time PCR （即時聚合酶鏈反應）……………..31
叁、結果與討論...................................................................................32
一、標準菌的選定與PCR引子組之設計..................................32
二、聚合酶鏈鎖反應 ( PCR ) ………………………………....33
(1)鏈球菌PCR引子組特異性試驗......................................33
三、食品檢測之應用...................................................................33
(1) PCR引子組靈敏度試驗 ……………………………….33
(2)即時PCR（Real-Time PCR）靈敏度試驗 ....................34
(3)real-time PCR標準曲線之建立........................................35
肆、結論.............................................................................................36
第三章、參考文獻....................................................................................64

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