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博碩士論文 etd-0914101-113959 詳細資訊
Title page for etd-0914101-113959
論文名稱
Title
以複合式聚合酵素連鎖反應及限制酵素切割分析鑑定食品媒介之致病性細菌
Rapid Detection and Identification of Foodd-Borne Bacterial Pathogens by Multiplex PCR and Restriction Endonuclease Digestion
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
56
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2001-07-26
繳交日期
Date of Submission
2001-09-14
關鍵字
Keywords
食物中毒、複合式聚合酵素連鎖反應、限制酵素
Restriction Endonuclease Digestion, Food-Borne Baterial Pathogens, Multiplex PCR
統計
Statistics
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The thesis/dissertation has been browsed 5721 times, has been downloaded 11394 times.
中文摘要
中文摘要
本研究選用16S rRNA、virA、tpl、及H1d 等四種目標基因做為鑑定食品媒介之致病性細菌,包括Escherichia coli、Shigella flexneri、Citrobacter freundii、Salmonella typhi、Vibrio cholerae、Vibrio parahaemolyticus;及Staphylococcus aureus等七種試驗細菌。實驗顯示九對本研究所使用的引子均能對目標基因具有特異性,而七種試驗細菌之16S rRNA 基因產物均經定序比對確認,另外,再以示差性PCR分別對E. coli / Shigella及Citrobacter / Salmonella做進一步之鑑別,並以限制酵素AfaI切割分析V. cholerae、V. parahaemolyticus 與 Staphylococcus aureus 16S rRNA之基因產物,確認本研究設計於這三種細菌之引子具備種特異性。實驗顯示本研究複合式聚合酵素連鎖反應之最適引子煉合溫度為59 ℃,因此選用合適之目標基因、特異性高之引子、PCR反應條件及參數使得本研究在32個循環複製之後,複合式聚合酵素連鎖反應之靈敏度可達102 CFU,顯示此方法能在單一次PCR反應中鑑定食品媒介之致病性細菌,且具有靈敏度高、特異性強、及快速偵測之特點。




Abstract
英文摘要
Multiplex PCR amplification of 16S rRNA gene、virA、tpl、and H1d genes was developed enabling simultaneous detection in Escherichia coli,an indicator of fecal contamination and food-borne microbial pathogens,Shigella flexneri、Citrobacter freundii、Salmonella typhi、Vibrio cholerae、Vibrio parahaemolyticus、and Staphylococcus aureus。Each of the nine pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene。The optimized multiplex PCR reaction utilized a primer annealing temperature of 59 ℃and used agarose gel electrophoresis for detection of the PCR-amplified products。Selection of appropriate target genes、oligonucleotide primers 、PCR reaction、and cycling parameters resulted in the amplification of four target genes simultaneously in a single PCR reaction with the sensitivity of detection was 102 CFU after 32 cycles。Multiplex PCR amplification followed by differential PCR for E. coli / Shigella, and Citrobacter / Salmonella,sequenced for the PCR-amplified products of 16S rRNA gene of the seven pathogens in this study,and used restriction endonuclease AfaI to confirm the PCR-amplified products of V. cholerae,V. parahaemolyticus and Staphylococcus aureus,has been shown to be an sensitive,specific,and rapid method to detect food-borne bacterial pathogens。



目次 Table of Contents
目錄

誌謝……………………………………………………………………...I
中文摘要………………………………………………………………..II
英文摘要…………………………………………………………….....III
目錄…………………………………………………………………….IV
一、緒論…………………………………………………………………1
(1)、前言…………………………………………………………...1
  (2)、大腸桿菌(Escherichia coli)………………………………1
  (3)、志賀氏桿菌(Shigella flexneri)……………………………2
  (4)、枸櫞酸桿菌(Citrobacter freundii)…………………………2
(5)、傷寒桿菌(Salmonella typhi)………………………………3
(6)、霍亂弧菌(Vibrio cholerae)………………………………..3
(7)、腸炎弧菌(Vibrio parahaemolyticus)………………………3
(8)、金黃色葡萄球菌(Staphylococcus aureus)………………..4
  (9)、16S核糖體核糖核酸基因(16S ribosomal RNA gene)…..4
(10)、毒力基因(Virulence gene)…………………………………5
(11)、酪氨酸裂解酵素(Tyrosine phenol lyase)…………………5
(12)、鞭毛抗原基因(Flagellin gene)……………………………6
(13)、複合式聚合酵素連鎖反應(Multiplex PCR)……………..6
(14)、差異性聚合酵素連鎖反應(Multiplex PCR)……………..7
(15)、限制片段長度多型性(Restriction fragment length
polymorphism,RFLP)……………………………………8
二、研究目的……………………………………………………………9
三、材料與方法…………………………………………………………10
  (1)、菌株來源………………………………………………………10
  (2)、冷凍乾燥管之開封及菌株之保存與培養…………………..10
  (3)、細菌基因組DNA(genomic DNA)之製備……………….10
(4)、16S核糖體核糖核酸基因序列之比對(alignment)分析..10
(5)、引子(primer)之選擇與特異性(specificity)………….10
(6)、Differential PCR之試驗方法……………………………….10
(7)、Multiplex PCR之特異性試驗………………………………10
(8)、Multiplex PCR之靈敏度(sensitivity)試驗……………..11


-IV-


(9)、限制酵素之切割(Digestion)分析………………………11
四、結果………………………………………………………………..16
(1)、引子的特異性……………………………………………….16
(2)、Differential PCR之特異性………………………………...16
(3)、限制表現型(restriction phenotype)之特異性…………17
(4)、Multiplex PCR之特異性…………………………………..17
(5)、Multiplex PCR之靈敏度…………………………………..19
五、討論………………………………………………………………..20
六、結論………………………………………………………………..26
七、參考文獻…………………………………………………………..27
八、表…………………………………………………………………..31
九、圖…………………………………………………………………..36













參考文獻 References
七、參考文獻:

1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. Sideman, J. Smith, and K. Struhl. 1987. Current protocols in molecular biology.
p. 2.4.1-2.4.5. New York : John Wiley & Sons, Inc.

2. Bai, Q., and R. L. Somerville. 1998. Integration host factor and cyclic
aMP receptor protein are required for TyrR-mediated activation of tpl in Citrobacter freundii. J. Bacteriol. 180 : 6173-6188.

3. Becker, K., R. Roth, and G. Peters. 1998. Rapid and specific
detection of toxigenic Staphylococcus aureus : use of two muliplex PCR enzyme immunoassays for amplification and hybridization of Staphylococcal enterotoxin genes, exfoliative toxin gene, and toxin shock syndrome toxin I gene. J. Clin. Microbiol. 36 : 2548-2553.

4. Bej, A. K., D. P. Patterson, C. W. Brasher, M. C. L. Vickery, D. D.
Jones, and C. A. Kaysner. 1999. Detection of total and hemolysin-producing Vibrio parahaemolyticus of tl, tdh and trh. 36: 215-225.

5. Brenner, D. J., and S. Falkow. 1971. Molecular relationships among
members of Enterobacteriaceae. Adv. Genet. 16 : 81-118.

6. Chamberlain, J. S., R. A. Gibbs, J. E. Ranier, and C. T. Caskey.
1990. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy, p.272-281. In M. Innis, D. Gelfand, J. Sninski, and T. White (ed.), PCR protocols : a guide to methods and applications. Academic
Press, Orlando, Fla.

7. Chamberlain, J. S., R. A. Gibbs, J. E. Ranier, P. N. Nggyen, and C.
T. Caskey. 1988. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 16 : 11141-11156.

8. Conner, C. P., D. M. Heithoff, S. M. Julio, R. L. Sinshimer, and M. J.Mahan. 1998. Differential patterns of acquired virulence genes
distinguish Salmonella strains. Proc. Natl. Acad, Sci. USA. 95: 4641-
4645.

-27-


9. Edwards, M. C., and R. A. Gibbs. 1994. Multiplex PCR:Advantages,
Development, and Applications. PCR Methods and Application. 4:
565-575

10. Ellwood, M., and M. Nomura. 1982. Chromosomal location of the
genes for rRNA in E. coli K-12. J. Bacteriol. 143 : 1077-1080.

11. Frankel, G., S. M. C. Newton, G. K. Schoolwik, and B. A.
D.Stocker. 1989. Unique sequence in region VI of the flagellin
gene of Salmonella typhi. Mol. Microbiol. 3: 1379-1383.

12. Hala, T. L. 1991. Genetic basis of virulence in Shigella species.
Microbiol. Rev. 55 : 206-224.

13. Hassouna, N., B. Michot, and J. P. Bachellerie. 1984. The complete
nucleotide sequence of mouse 28S rRNA gene. Implications for the
process of size increase of large subunit rRNA in higher eukaryotes.
Nucleic Acids Res. 12 : 3563-3583.

14. Henegariu, O., N. A. Heerema, S. R. Dlouhy, G. H. Vance, and P.
H. Vogt. 1997. Multiplex PCR: Critical parameters and step-by-step
protocol. BioTechniques. 23: 504-511.

15. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and L. A.
Chandler. 1995. Escherichia coli and coliform bacteria. In
Bacteriological Analytical Manual. 8th. ed. pp. 4.01-4.29. Food and
Drug Administration. Washington D. C., U. S. A.

16. Jones, M. E., M. B. Avison, E. Damdinsuren, A. P. Macgowan,
and P. M. Bennett. 1994. Heterogenecity at the β-lactamase
structural gene ampC amongst Citrobacter spp. Assessed by PCR
analysis: potential for typing at a molecular level. J. Med. Microbiol.
41: 209-214.

17. Kearn, A. M., P. R. Seiders, J. Wheeler, R. Ereewan, and M.
Steward. 1999. Rapid detection of methicillin-resistant Staphylococci by multiplex PCR. J. Hospital Infection. 43: 33-37.



-28-


18. Kring, N. R., and J. G. Holt. 1994. Bergey’s manual ofsystematic
bacteriology, vol. 1. Williams & Wilkins, Baltimore, London.

19. Lederberg, J., and T. Iino. 1956. Genetics. 41 : 744-757.

20. Metherell, L. A., C. Hurst, and I. J. Bruce. 1997. Rapid, sensitive,
microbial detection by gene amplification using restriction
endonuclease target sequences. Molecular and Cellular Probes.
11: 297-308.

21. Popoff, M. Y., and L. Minor. 1997. Antigenic formulas of the
Salmonella serovars, 7th revision. WHO Collaborating Centre for
Reference and Research on Salmonella. Institute Pasteur, Paris,
France.

22. Sansonetti, P. J., D. J. Kopecko, and S. B. Formal. 1982.
Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect. Immun. 35: 852-860.

23. Sabat, G., P. Rose, W. J. Hickey, and J. M. Harkin. 2000. Slective
and sensitive method for PCR amplification of Escherichia coli 16S
rRNA genes in soil. Appl. Environ. Microbiol. 66 : 844-849.

24. Schmidt, H., M. Montag, J. Bockemuhl, J. Heesemann, and H.
Karch. 1993. Shiga-like toxin II-related cytotoxins in Citrobacter
freundii strains from humans and beef samples. Infect. Immun.
61 :534-543.

25. Schmitz, F. J., M. Steiert, B. Hofmann, A. C. Fluit, J. Verhoef, H.
P. Heinz, M. E. Jones. 1998. Detection of Staphylococcal genes
directly from cerebrospinal and peritoneal fluid samples using a
multiplex polymerase chain reaction. Eur. J. Clin. Microbiol. Infect.
Dis. 17: 272-274.

26. Shangkuan, Y. H., Y. S. Show, and T. M. Wang. 1995. Multiplex
PCR to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1. J. Appl. Bacteriol. 79 : 264-273.



-29-


27 Sharma, N. K., C. E. Rees, and C. E. Dodd. 2000. Development of a
single-reaction multiplex PCR toxin typing assay for Staphylococcus
aureus strains. Appl. Environ. Microbiol. 66 : 1347-1353.

28. Singh, D. V., M. M. Matte, G. R. Matte, S. Jiang, F. Sabeena, B.
N. Shukla, S. C. Sanyal, A. Hug, and R. R. Colwell. 2001.
Molecular analysis of Vibrio cholerae O1, O139, non-O1, and
non-O139 strains : clonal relationships between clinical and
environmental isolates. Appl. Environ. Microbiol. 67 : 910-921.

29. Song, J. H., H. Cho, M. Y. Park, D. S. Na, H. B. Moon, and C. H.
Pai.1993. Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction. J. Clin. Microbiol. 31 : 1439-1443.

30. Suggs, S. B., T. Hirose, T. Miyake, E. H. Kawashima, M. J.
Johnson, K. Itakara, R. B. Wallace. 1981. Use of synthetic oligodeoxy-ribonucleotides for the isolation of specific clone DNA sequences. ICN-UCLA Symp. Mol. Cell. Biol. 23 : 683-693.

31. Villalobo, E., and A. Torres. 1998. PCR for dectection of Shigella
spp. in mayonnaise. Appl. Environ. Microbiol. 64 : 1242-1245.

32. Way, J. S, K. L. Josephson, S. D. Pillai, M. Abhaszadegan, C. P.
Gerba, and I. L. Pepper. 1993. Specific detection of Salmonella
spp. by multiplex polymerase chain reaction. Appl. Environ.
Microbiol. 59 : 1473-1479.

33. Wei, L. and T. M. Joys. 1985. Covalent structure of three phase-1
filament proteins of Salmonella. J. Mol. Biol. 186: 791-803.

34. Woese, C. R., R. Gutell, R. Gupta, and H. Noller. 1983. Detailed
analysis of the higher order structure of 16S like ribosomal
ribonucleic acids. Microbiological Rev. 47: 621-669.

35. Zhu, Q., C. K. Lim, and Y. N. Chan. 1996. Detection of Salmonella
typhi by polymerase chain reaction. J. Appl. Bacteriol. 80 : 244-251.



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