臺灣博碩士論文加值系統

豬霍亂沙門氏菌 ( Salmonella enterica serovar Choleraesuis ) 具有宿主適應性，會造成豬隻副傷寒病症，另外，它會感染人類，並造成敗血症的發生。線毛（fimbriae）是一種位於大多數革蘭氏陰性菌外膜上的一種毛髮狀蛋白質結構物，豬霍亂沙門氏菌具有std線毛基因組，其可轉譯出相似於線毛的結構物。
本實驗室藉由PCR方式，增幅7.4 kb的std線毛基因組，將其選殖至pCR-XL-TOPO質體DNA，委外定序後，分析std線毛基因組之開放性讀框 ( ORF )，進而將此重組DNA送入大腸桿菌HB101，並藉由RT-PCR及西方點墨試驗觀察到S. Choleraesuis在靜置培養液中可以表現stdA線毛基因，進而有StdA蛋白質的產生，而震盪培養液及固態培養基均無法觀察到stdA線毛基因表現或是StdA蛋白質的產生。Std線毛的主要線毛次單位（major fimbrial subunit）為StdA，而其N端的胺基酸序列僅與沙門氏菌屬有同源性，在其他非沙門氏菌屬中則無同源性。因此若能以stdA當作偵測沙門氏菌的目標基因，在S. Choleraesuis之stdA基因上設計一對引子，藉由PCR方式，測試45種沙門氏菌血清型及15種非沙門氏菌菌株，用以做為偵測沙門氏菌的方法。在經PCR反應後，可觀察到這45種沙門氏菌血清型，共268株，均可產生518-bp大小的PCR產物，而15種非沙門氏菌菌株則無任何的PCR產物。而PCR的偵測敏感度為菌體DNA濃度3.4 × 10-1 pg，使用南方點墨試驗再次偵測，其敏感度提高為3.4 × 10-2 pg。並且進行接種試驗，屠體液中非沙門氏菌細菌數量為4 × 107 CFU/ml，當4.4 × 100 CFU/ml的沙門氏菌加上屠體液於培養液中增殖後，可以使用stdA成對引子藉由PCR方式看到stdA的產物，表示stdA成對引子在PCR反應液中存在沙門氏菌及其他菌種時，仍然可以順利進行反應，而不會受到影響。藉由這些實驗，我們了解到針對stdA所設計的成對引子確實具有專一性及高敏感度，能夠確實偵測出沙門氏菌。

Salmonella enterica serovar Choleraesuis is host adapted and causes swine paratyphoid. In addition, S. Choleraesuis is invasive in human and usually causes septicemia without involvement of gastrointestinal tract. Fimbriae are proteinous appendages present on the most of the Gram negative bacteria including Salmonella. The genomic DNA of S. Choleraesuis possesses the std fimbrial gene cluster that has the potential to encode fimbrial appendages. The 7.4 kb DNA fragment containing the S. Choleraesuis std fimbrial gene cluster was PCR amplified and cloned into the pCR-XL-TOPO vector. The open reading frames of the std fimbrial gene cluster were analyzed. The resulting recombinant DNA was transformed into Escherichia coli HB101 strain. RT-PCR and Western blot analysis demonstrated that stdA expression and StdA protein were detected only when S. Choleraesuis strains were grown in static broth, but not on solid agar or shaking broth culture conditions. The amino acid sequence of the N-terminal portion of the StdA, the major fimbrial subunit of Std fimbriae, reveals no homology to that of the non-Salmonella strains and is conserved within Salmonella strains. To test the feasibility if stdA could be used as a target to detect Salmonella, primers internal to stdA in S. Choleraesuis were designed to amplify specific PCR products from the genomic DNA of 45 Salmonella serovars and 15 non-Salmonella strains. The 518-bp stdA derived PCR products were present in 268 strains composing of 45 Salmonella serovars. Non-Salmonella strains yielded no specific DNA products. The limit of detection by PCR was 3.4 × 10-1 pg using the genomic DNA from S. Choleraesuis while Southern hybridization detected to the level of 3.4 × 10-2 pg. The sensitivity of the stdA-specific primer set was demonstrated on a Salmonella-free swab sample from a pork carcass surface, which was then artificially contaminated with different concentrations of S. Typhimurium. Inoculation studies showed that stdA-specific primer set detected Salmonella in samples having initial 4.4 × 100 CFU/ml of Salmonella inoculum prior to the enrichment step in the presence of 4 × 107 CFU/ml of non-Salmonella florae. Results in this study demonstrate that stdA is unique to Salmonella species and is an appropriate PCR target for detecting these microorganisms.

縮寫表 Ⅰ
中文摘要 Ⅱ
英文摘要 Ⅳ
第一章 緒論 1
第二章 文獻回顧 3
第一節 沙門氏菌 3
1.1 命名 3
1.2 型態及生化特性 4
第二節 沙門氏菌的致病機轉及沙門氏菌症 5
2.1 致病機轉 5
2.2 沙門氏菌症 6
第三節 沙門氏菌線毛 7
3.1 沙門氏菌線毛分類 8
第四節 std線毛基因 8
第三章 材料與方法 10
第一節 Std線毛分析 10
1.1 std基因組選殖 10
1.2 利用RT-PCR觀察stdA之mRNA表現 13
1.3 利用西方點墨試驗觀察StdA蛋白質表現情形 14
第二節 使用stdA成對引子偵測沙門氏菌 17
2.1 菌株 17
2.2 引子設計 18
2.3 聚合酶鏈鎖反應增幅stdA基因片段 18
2.4 南方點墨試驗 19
2.5 敏感度試驗 21
2.6 使用stdA成對引子偵測豬隻屠體表面之沙門氏菌 22
第四章 結果 24
第一節 Std線毛的分析 24
1.1 std線毛基因組定序 24
1.2 利用RT - PCR觀察stdA之mRNA表現 24
1.3 利用西方點墨試驗觀察StdA蛋白質的表現情形 25
第二節 stdA基因在檢測沙門氏菌的應用 25
2.1 stdA成對引子可用於沙門氏菌的檢測 25
2.2 南方點墨試驗 26
2.3 敏感度試驗 26
2.4 stdA成對引子可用於檢測屠體液中的沙門氏菌 26
第五章 討論 28
第一節 Std線毛表現情形 28
第二節 stdA基因在檢測沙門氏菌的應用 31
參考文獻 35

1.B?黷mler, A. J., F. Heffron, and R. Reissbrodt. 1997. Rapid detection of Salmonella enterica with primers specific for iroB. J. Clin. Microbiol. 35:1224-30.
2.Bahrani-Mougeot, F. K., and M. S. Donnenberg. 2004. Enteropathogenic bacteria, p. 403-414. In M. Schaechter (ed.), The Desk Encyclopedia of Microbiology, USA.
3.Balbont?瀋, R., G. Rowley, M. G. Pucciarelli, J. Lopez-Garrido, Y. Wormstone, S. Lucchini, F. Garcia-Del Portillo, J. C. Hinton, and J. Casadesus. 2006. DNA adenine methylation regulates virulence gene expression in Salmonella enterica serovar Typhimurium. J. Bacteriol. 188:8160-8.
4.Brenner, F. W., R. G. Villar, F. J. Angulo, R. Tauxe, and B. Swaminathan. 2000. Salmonella nomenclature. J. Clin. Microbiol. 38:2465-7.
5.Cano, R. J., S. R. Rasmussen, G. Sanchez Fraga, and J. C. Palomares. 1993. Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods. J. Appl. Bacteriol. 75:247-53.
6.Chan, K., S. Baker, C. C. Kim, C. S. Detweiler, G. Dougan, and S. Falkow. 2003. Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar Typhimurium DNA microarray. J. Bacteriol. 185:553-63.
7.Chang, C. F., L. C. Chang, Y. F. Chang, M. Chen, and T. S. Chiang. 2002. Antimicrobial susceptibility of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis recovered from Taiwanese swine. J. Vet. Diagn. Invest. 14:153-7.
8.Chevrier, D., M. Y. Popoff, M. P. Dion, D. Hermant, and J. L. Guesdon. 1995. Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridisation using covalently immobilised oligonucleotide on a microplate. FEMS Immunol. Med. Microbiol. 10:245-501.
9.Chiu, C. H., L. H. Su, and C. Chu. 2004. Salmonella enterica serotype Choleraesuis: epidemiology, pathogenesis, clinical disease, and treatment. Clin. Microbiol. Rev. 17:311-22.
10.Chiu, C. H., P. Tang, C. Chu, S. Hu, Q. Bao, J. Yu, Y. Y. Chou, H. S. Wang, and Y. S. Lee. 2005. The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen. Nucleic Acids Res. 33:1690-8.
11.Chiu, C. H., T. L. Wu, L. H. Su, C. Chu, J. H. Chia, A. J. Kuo, M. S. Chien, and T. Y. Lin. 2002. The emergence in Taiwan of fluoroquinolone resistance in Salmonella enterica serotype choleraesuis. N. Engl. J. Med. 346:413-9.
12.Chiu, S., C. H. Chiu, and T. Y. Lin. 2004. Salmonella enterica serotype Choleraesuis infection in a medical center in northern Taiwan. J. Microbiol. Immunol. Infect. 37:99-102.
13.Clegg, S., S. Hull, R. Hull, and J. Pruckler. 1985. Construction and comparison of recombinant plasmids encoding type 1 fimbriae of members of the family Enterobacteriaceae. Infect. Immun. 48:275-9.
14.Cohen, H. J., S. M. Mechanda, and W. Lin. 1996. PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp. Appl. Environ. Microbiol. 62:4303-8.
15.Doran, J. L., S. K. Collinson, J. Burian, G. Sarlos, E. C. Todd, C. K. Munro, C. M. Kay, P. A. Banser, P. I. Peterkin, and W. W. Kay. 1993. DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae. J. Clin. Microbiol. 31:2263-73.
16.Fluit, A. C., M. N. Widjojoatmodjo, A. T. Box, R. Torensma, and J. Verhoef. 1993. Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay. Appl. Environ. Microbiol. 59:1342-6.
17.Gray, J. T., P. J. Fedorka-Cray, T. J. Stabel, and T. T. Kramer. 1996. Natural transmission of Salmonella choleraesuis in swine. Appl. Environ. Microbiol. 62:141-6.
18.Hashimoto, Y., Y. Itho, Y. Fujinaga, A. Q. Khan, F. Sultana, M. Miyake, K. Hirose, H. Yamamoto, and T. Ezaki. 1995. Development of nested PCR based on the ViaB sequence to detect Salmonella typhi. J. Clin. Microbiol. 33:3082.
19.Humphries, A., S. Deridder, and A. J. Baumler. 2005. Salmonella enterica serotype Typhimurium fimbrial proteins serve as antigens during infection of mice. Infect. Immun. 73:5329-38.
20.Humphries, A. D., M. Raffatellu, S. Winter, E. H. Weening, R. A. Kingsley, R. Droleskey, S. Zhang, J. Figueiredo, S. Khare, J. Nunes, L. G. Adams, R. M. Tsolis, and A. J. Baumler. 2003. The use of flow cytometry to detect expression of subunits encoded by 11 Salmonella enterica serotype Typhimurium fimbrial operons. Mol. Microbiol. 48:1357-76.
21.Ibanez-Ruiz, M., V. Robbe-Saule, D. Hermant, S. Labrude, and F. Norel. 2000. Identification of RpoS (sigma(S))-regulated genes in Salmonella enterica serovar Typhimurium. J. Bacteriol. 182:5749-56.
22.Jones, B. D., N. Ghori, and S. Falkow. 1994. Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer''s patches. J. Exp. Med. 180:15-23.
23.Khakhria, R., D. Woodward, W. M. Johnson, and C. Poppe. 1997. Salmonella isolated from humans, animals and other sources in Canada, 1983-92. Epidemiol. Infect. 119:15-23.
24.Koneman, E. W., S. D. Allen, W. M. Janda, P. C. Schreckenberger, and J. Washington C. Winn. 1997. color atlas and textbook of diagnostic microbiology, 5 ed, Philadelphia.
25.Kwang, J., E. T. Littledike, and J. E. Keen. 1996. Use of the polymerase chain reaction for Salmonella detection. Lett. Appl. Microbiol. 22:46-51.
26.Lu, L., and W. A. Walker. 2001. Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium. Am. J. Clin. Nutr. 73:1124S-1130S.
27.Mahon, J., and A. J. Lax. 1993. A quantitative polymerase chain reaction method for the detection in avian faeces of salmonellas carrying the spvR gene. Epidemiol. Infect. 111:455-64.
28.McClelland, M., K. E. Sanderson, J. Spieth, S. W. Clifton, P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, F. Du, S. Hou, D. Layman, S. Leonard, C. Nguyen, K. Scott, A. Holmes, N. Grewal, E. Mulvaney, E. Ryan, H. Sun, L. Florea, W. Miller, T. Stoneking, M. Nhan, R. Waterston, and R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852-6.
29.Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607-25.
30.Nicholson, B., and D. Low. 2000. DNA methylation-dependent regulation of pef expression in Salmonella typhimurium. Mol. Microbiol. 35:728-42.
31.Norris, T. L., R. A. Kingsley, and A. J. Bumler. 1998. Expression and transcriptional control of the Salmonella typhimurium Ipf fimbrial operon by phase variation. Mol. Microbiol. 29:311-20.
32.Popoff, M. Y., J. Bockemuhl, and L. L. Gheesling. 2004. Supplement 2002 (no. 46) to the Kauffmann-White scheme. Res. Microbiol. 155:568-70.
33.Rahn, K., S. A. De Grandis, R. C. Clarke, S. A. McEwen, J. E. Galan, C. Ginocchio, R. Curtiss, 3rd, and C. L. Gyles. 1992. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol. Cell. Probes. 6:271-9.
34.Reeves, M. W., G. M. Evins, A. A. Heiba, B. D. Plikaytis, and J. J. Farmer, 3rd. 1989. Clonal nature of Salmonella typhi and its genetic relatedness to other salmonellae as shown by multilocus enzyme electrophoresis, and proposal of Salmonella bongori comb. nov. J. Clin. Microbiol. 27:313-20.
35.Rexach, L., F. Dilasser, and P. Fach. 1994. Polymerase chain reaction for Salmonella virulence-associated plasmid genes detection: a new tool in Salmonella epidemiology. Epidemiol. Infect. 112:33-43.
36.Sansonetti, P. J., and A. Phalipon. 1999. M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process. Semin. Immunol. 11:193-203.
37.Svensson, M., B. Stockinger, and M. J. Wick. 1997. Bone marrow-derived dendritic cells can process bacteria for MHC-I and MHC-II presentation to T cells. J. Immunol. 158:4229-36.
38.Tauxe, R. V. 1997. Emerging foodborne diseases: an evolving public health challenge. Emerg. Infect. Dis. 3:425-34.
39.Thorns, C. J., and M. J. Woodward. 2000. Fimbriae of Salmonella, p. 35-55, Salmonella in Domestic Animals.
40.Tinker, J. K., and S. Clegg. 2001. Control of FimY translation and type 1 fimbrial production by the arginine tRNA encoded by fimU in Salmonella enterica serovar Typhimurium. Mol. Microbiol. 40:757-68.
41.Townsend, S. M., N. E. Kramer, R. Edwards, S. Baker, N. Hamlin, M. Simmonds, K. Stevens, S. Maloy, J. Parkhill, G. Dougan, and A. J. Baumler. 2001. Salmonella enterica serovar Typhi possesses a unique repertoire of fimbrial gene sequences. Infect. Immun. 69:2894-901.
42.Vassiliadis, P., V. Kalapothaki, D. Trichopoulos, C. Mavrommatti, and C. Serie. 1981. Improved isolation of Salmonellae from naturally contaminated meat products by using Rappaport-Vassiliadis enrichment broth. Appl. Environ. Microbiol. 42:615-8.
43.Vugia, D. J., M. Samuel, M. M. Farley, R. Marcus, B. Shiferaw, S. Shallow, K. Smith, and F. J. Angulo. 2004. Invasive Salmonella infections in the United States, FoodNet, 1996-1999: incidence, serotype distribution, and outcome. Clin. Infect. Dis. 38 Suppl 3:S149-56.
44.Way, J. S., K. L. Josephson, S. D. Pillai, M. Abbaszadegan, C. P. Gerba, and I. L. Pepper. 1993. Specific detection of Salmonella spp. by multiplex polymerase chain reaction. Appl. Environ. Microbiol. 59:1473-9.