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研究生:范志民
研究生(外文):Chin-Min Fan
論文名稱:金黃色葡萄球菌新型腸毒素N、O、P基因引子之設計及其應用於食品及臨床菌株腸毒素型之分佈調查
論文名稱(外文):Development of PCR Primers of New Staphylococcal Enterotoxin Genes sen, seo, sep and Its Application of Enterotoxigenic Distribution for Staphylococcus aureus Isolates Obtained from Poisoning Cases and Clinical Samples
指導教授:謝尤敏
指導教授(外文):You-Min Hsieh
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:93
中文關鍵詞:金黃色葡萄球菌腸毒素
外文關鍵詞:enterotoxinStaphylococcus aureus
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金黃色葡萄球菌 ( Staphylococcus aureus )是造成食物中毒的重要病原菌之一,其產生的腸毒素 (enterotoxin ) 依照血清學上的分類可分為SEA、SEB、SEC、SED、SEE五種傳統型腸毒素與中毒性休克症候群毒素 ( TSST-1 ),而近年來有許多的新型腸毒素被發現,如:SEG、SEH、SEI、SEJ、SEK、SEL、SEM、SEN、SEO、SEP、SEQ、SER及SEU等。本研究目的在於設計用於PCR之新型腸毒素基因sen、seo和sep之特異性引子組,並對金黃色葡萄球菌臨床分離株進行新型腸毒素基因的檢測,來探討新型腸毒素基因的分佈情形,且同時使用已發表之TSST-1引子組,對金黃色葡萄球菌臨床菌進行分佈調查,同時了解其分佈與傳統型腸毒素基因的關聯性。
本研究中針對新型腸毒素所設計的引子組可以正確篩選出腸毒素基因sen、seo和sep。利用非金黃色葡萄球菌之葡萄球菌屬菌株及其他的腸內菌進行PCR檢測,結果得知除了金黃色葡萄球菌之實驗室標準菌會有PCR產物( sen, 135 bp; seo, 172 bp; sep, 148 bp )之外,其餘的非金黃色葡萄球菌皆無PCR產物的出現。至於在靈敏度的測試方面,在未進行增殖培養的條件下,引子組的PCR檢測靈敏度可達到N╳103~104 CFU/ml,而另外進行10小時的增殖培養後,引子組的PCR靈敏度更增加到約N╳100 CFU/ml,由以上的結果可得知引子組利用PCR,篩檢金黃色葡萄球菌腸毒素基因sen、seo、sep其靈敏度佳。本研究也另外針對食品樣本來了解引子組檢測的靈敏度,我們選用市售鮮乳及肉品以額外添加的方式加入金黃色葡萄球菌,在添加之前菌株先以TSB預培養10小時,然後以引子組檢測鮮乳及肉品中之金黃色葡萄球菌腸毒素基因sen、seo、sep,而其靈敏度也可達到N╳100 CFU/g,結果表示本研究所設計的特異性引子組,可應用在食品樣品的檢測。
本研究對於帶有金黃色葡萄球菌新型腸毒素基因sen、seo及sep的臨床菌,進行反轉錄聚合鏈鎖反應( RT-PCR ),我們各選11株PCR為正反應的金黃色葡萄球菌,實驗結果顯示,原本帶有腸毒素基因的菌株利用RT-PCR檢測皆會有mRNA的表現。
關於調查金黃色葡萄球菌毒素性休克症候群毒素( TSST-1 )和新型腸毒素( SEN、SEO、SEP )基因的分佈相關性,所採用的臨床菌株的來源,為台中榮民總醫院及疾病管制局第三分局所提供的金黃色葡萄球菌282株,而帶有TSST-1毒素基因的菌株總共佔126株 ( 44.6 % ),其中同時帶有sea或sec的菌株共有48株 (38 %);腸毒素基因sen有40株 ( 14 % ),帶有腸毒素基因seo有44株 ( 15.6 % ),帶有腸毒素基因sep有91株 ( 32 % ),在同時具有sen和seo的29株菌株中,有15株是連同sec一起出現。
我們藉腸毒素基因的分佈情形,可了解腸毒素基因之間的關聯性,例如當檢測出帶有TSST-1毒素基因的菌株,往往也都有腸毒素基因sea或sec的出現,因此TSST-1毒素基因的分佈甚為廣泛,以及本研究所針對的sen,seo和sep腸毒素基因,在臨床及食物中毒分離株的分佈情況中,也發現帶有sep腸毒素基因的菌株數量比帶有sen和seo腸毒素基因的菌株數要多;另外帶有腸毒素基因sen及seo的菌株中,也能發現腸毒素基因seg、sei和sem的存在,這可說明金黃色葡萄球菌通常帶有一種或一種以上的腸毒素基因。
Staphylococcus aureus is one of the important pathogens which cause food poisoning. The staphylococcal enterotoxins (SEs) produced by strains of S. aureus can be divided into SEA, SEB, SEC, SED, SEE, classical enterotoxins, and toxic shock syndrome toxin (TSST-1). Recently, many new type of SEs have been found, for example: SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER and SEU, etc. The purposes of this study is to design specific primers for SEN, SEO and SEP genes, detection and use them for the survey of the distribution of SE genes and TSST of S. aureus isolates in Taiwan.
In this study, PCR primers specific for sen, seo and sep are designed. Non-staphylococcus species and staphylococcus species other than S. aureus would not generate any fales positive results. Moleclar sizes of the PCR products were 135 bp for sen, 172 bp for seo and 148 bp for sep respectively. Detection sensitivity was N╳103~104 CFU/ml, for samples without preincubation, and N╳100 CFU/ml for samples with the preculture step. These primers were also used for the monitoring of sen, seo and sep S.aureus strains in food smples, such as whole milk and pork.
Eleven PCR positive strains of S. aureus were also assayed by RT-PCR. It was found that all these randomly selected S. aureus strains with sen, seo and sep genes were positive by RT-PCR.
To investigate the distribution of TSST-1 and new SEs (SEN , SEO , SEP) genes in S. aureus, 284 isolates obtained from Veternary General Hospital in Taichung and Center for Disease Control, the third branch in Taiwan, were analyzed by PCR. In 126 isolates (44.3 %)found positive for TSST-1 gene, 48 isolates (38%) were found positive for sea and sec. Forty isolates were positive for sen, 44 for seo and 91 for sep. In 29 isolates with sen and seo, 15 strains were with sec.
Results of SE distribution study, indicate that strains with TSST-1 toxin gene are often with sea or sec. The ratio of sep producing S. aureus strains are higher than the sen and seo producing strains. Coexist of seg, sei and sem in strains with sen and seo were found. According to the above facts, S aureus strains are often found with one or more SEs genes.
目 次
頁次
中文摘要………………………………………………………………….…………I
英文摘要……………………………………………………………………………III
Ι、文獻整理………………………………………………………………………1
一、葡萄球菌的特色及所造成的疾病…………………………………….....1
二、金黃色葡萄球菌之檢測……………………………………………….....3
三、金黃色葡萄球菌產生的毒性因子…………………………………….. ...4
四、葡萄球菌的抗生素耐性………………………….………..………………7
五、金黃色葡萄球菌腸毒素分佈情形…………………………………….....7
六、各型腸毒素之介紹…………………………………..................................8
1. A型腸毒素 (SEA)……………………………………………........8
2. B型腸毒素 (SEB)……………………………………………........9
3. C 型腸毒素 (SEC)……………………………………………........9
4. D 型腸毒素 (SED)……………………………………………......10
5. E 型腸毒素 (SEE)……………………………………………….11
6. G 型腸毒素 (SEG)……………………………………………......11
7. H 型腸毒素 (SEH)…………………………………………...........11
8. I 型腸毒素 (SEI)…………………………………..………..........12
9. J型腸毒素 (SEJ)…………………………………………............12
10. K型腸毒素 (SEK)…………………………………………….......12
11. L型腸毒素 (SEL)………………………………………..………..13
12. M型腸毒素 (SEM)………………………………………………..13
13. N,O型腸毒素 (SEN, SEO)……………………….…………………..14
14. P型腸毒素 (SEP)……………………………………….…………….14
15. Q型腸毒素 (SEQ)……………………………………………........15
16. R型腸毒素 (SER)……………………………………………………..15
17. U型腸毒素 (SEU)……………………………………………….........16
七、金黃色葡萄球菌腸毒素之檢測………………………...............................16
Ⅱ、金黃色葡萄球菌新型腸毒素N、O、P基因引子組之設計及其應用於食品及臨床菌株腸毒素型之分佈調查和應用
壹、前言…………………………………………………………………………22
貳、材料與方法……………………………………………….………………..24
一、 實驗材料……………………………………………………………...24
(一) 菌種………………………………………………………………….24
(二) 培養基……………………………………………………………….24
(三) 藥品………………………………………………………………….25
(四) 緩衝液及試劑……………………………………………………….26
(五) 儀器………………………………………………………………….27
二、 實驗方法……………………………………………………………...27
(一) PCR引子組之設計與合成…………………………………………..27
(二) 聚合鏈鎖反應 ( PCR )……………………………………………27
1、 葡萄球菌的保存與鑑定……………………………………....28
2、 葡萄球菌DNA之製備………………………………………..28
(1) Phenol/ chloroform 法…………………………………………28
(2) 商業套組法 ( Viogene, Blood & Tissue Genomic Mini )……..28
3、 非葡萄球菌DNA之製備 ( Viogene, Blood & Tissue Genomic Mini )………………………………………………………….29
4、 引子組SEN1/2、SEO1/2、SEP1/2之PCR特異性試驗……..29
5、 引子組SEN1/2、SEO1/2、SEP1/2之PCR靈敏度試驗……..30
(1) 直接檢測……………………………………………………….30
(2) 二次活化……………………………………………………….30
(三) SEN、SEO、SEP特異性引子組於食品檢測之應用………………31
1、 豬肉樣品之檢測應用…………………………………………31
(1) 豬肉中生菌數之計算………………………………………….31
(2) 肉品中凝固正反應之葡萄球菌總菌數……………………32
(3) 檢測之靈敏度………………………………………………….32
2、 鮮乳樣品的檢測應用………………………………………..32
(1) 鮮乳樣品中生菌數與S. aureus之計算………………………..32
(2) 菌培養……………………………………………………….32
(四) 反轉錄聚合鏈鎖反應 ( Reverse transcription-Polymerase Chain Reaction; RT-PCR )…………………………………………………33
(五) 金黃色葡萄球菌之腸毒素之分布情形…………………………….33
參、結果與討論……………………………………………………………….34
金黃色葡萄球菌新型腸毒素N、O、P特異性引子組之設計與應用
一、標準菌的選定與PCR引子組之設計…………………………………34
二、聚合鏈鎖反應 ( PCR )……………………………………………..35
(一) PCR引子組特異性試驗…………………………………………..35
(二) PCR引子組靈敏度試驗………………………………………......35
三、反轉錄聚合鏈鎖反應 ( Reverse transcription PCR )……………...36
金黃色葡萄球菌腸毒素之分佈
一、 中毒型休克症候群毒素(TSST-1)基因之分佈情形…………………38
二、 新型腸毒素基因之分佈……………………………………………...38
三、 中毒性休克症候群毒素及新型腸毒素基因之相關性與分布關係...38
肆、結論……………………………………………………………………….42
Ⅲ、參考文獻……………………………………………………………….......77








表 次
頁次
表1-1、民國70年至93年台灣地區食品中毒案件病因物質分類表................18
表1-2、金黃色葡萄球菌相關的超級抗原位置…………………………………19
表1-3、金黃色葡萄球菌各腸毒素胺基酸序列相似性之比較………………….20
表1-4、金黃色葡萄球菌腸毒素之主要特性…………………………………….21
表2-1、本實驗用於篩選實驗室標準菌株之腸毒素基因之PCR引子組……….44
表2-2、本實驗所使用之PCR引子組…………………………………………….45
表2-3、本研究所使用之PCR反應條件.................................................................46
表2-4、本研究所使用之非金黃色葡萄球菌葡萄球菌屬標準菌株………………47
表2-5、金黃色葡萄球菌腸毒素標準菌…………………………………………….48
表2-6、金黃色葡萄球菌腸毒素標準菌偵測sen,seo和sep腸毒素基因…………49
表2-7、以PCR檢測腸毒素型S. aureus之靈敏度……………………………….50
表2-8、以PCR技術檢測1992-1993年台中榮總臨床檢體分離之金黃色葡萄球菌腸毒素型基因布………………………………………………………………….51
表2-9、以PCR技術檢測2003年台中榮總臨床檢體分離之金黃色葡萄球菌腸毒素型基因分布……………………………………………………………………….53
表2-10、以PCR技術檢測2003年疾病管制局食品中毒案件臨床檢體球菌分離之金黃色葡萄腸毒素型基因布……………………………………………………55
表2-11、PCR引子組檢測金黃色葡萄球菌臨床分離株腸毒素基因之分布……61表2-12、腸毒素基因sen, seo and sep與傳統型毒素基因(sea~see)之分佈…….62
圖 示
頁次
圖2-1、利用 (a)SEN1/SEN2,(b)SEO1/SEO2及(c)SEP1/SEP2引子組對實驗室標準菌及非金黃色葡萄球菌之PCR增幅作用…………………………………….63
圖2-2、SEN1/SEN2引子組對實驗室標準菌株STA67之靈敏度……………..64
圖2-3、SEO1/SEO2引子組對實驗室標準菌株STA34之靈敏度……………..65
圖2-4、SEP1/SEP2引子組對實驗室標準菌株HYT59之靈敏度……………..66
圖2-5、SEN1/SEN2引子組檢測牛乳及豬肉中腸毒素sen基因之靈敏度……67
圖2-6、SEO1/SEO2引子組檢測牛乳及豬肉中腸毒素seo基因之靈敏度……68
圖2-7、SEP1/SEP2引子組檢測牛乳及豬肉中腸毒素sep基因之靈敏度……..69
圖2-8、利用(a)SEN1/SEN2, (b)SEO1/SEO2及(c)SEP1/SEP2引子組對金黃色葡萄球菌之RT-PCR增幅作用...................................................................................70
圖 2-9、定序結果 (STA67)與N型腸毒素基因序列之比對...............................71
圖 2-10、定序結果 (STA34)與O型腸毒素基因序列之比對………………….72
圖 2-11、定序結果 (HYT59)與P型腸毒素基因序列之比對………………….73
圖 2-12、PCR結果與N型腸毒素基因序列之比對……………………………..74
圖 2-13、PCR結果與O型腸毒素基因序列之比對……………………………..75
圖 2-14、PCR結果與P型腸毒素基因序列之比對……………………………..76
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