利用熱休克蛋白基因設計Clostridium tyrobutricum及Listeria monocytogenes之PCR引子組 及其應用
陳志遠、李世傑 ; 曾浩洋
E-mail: [email protected]
摘 要
Listeria monocytogenes 廣泛分布自然界的病原菌，而傳染給人類，其為現代食品衛生上極為重要的一株病原菌，也經常出 現在生的食品，以及許多熟食或加工食品，尤其低溫殺菌的牛乳、乾酪和冰淇淋、煙燻的魚或肉類製品。另外 Clostridium tyrobutyricum 雖然非病原菌，然其常在乾酪及生乳中會分離出來。因該菌會破壞乾酪及牛乳品質，因此快速檢測是很重要 的，因為在傳統檢測方法通常很費力費時。而本研究中建立兩對新穎特異性引子組，分別針對 C. tyrobutyricum 及 L.
monocytogenes 進行檢測。而因為 C. tyrobutyricum 之熱休克蛋白未發表，本研究因此先利用 Clostridium 屬內菌株之已發表 之熱休克蛋白基因比對，分析並選擇基因序列之保守區段設計引子組，嘗試增幅出 C. tyrobutyricum 之熱休克蛋白基因片 段並進行定序確認。之後利用定序結果之熱休克蛋白基因序列，設計引子 CT-1/CT-2，以及 L. monocytogenes 之類熱休克 蛋白基因設計引子 Lm1/Lm2，分別針對 C. tyrobutyricum 及 L. monocytogenes 使用 PCR 增幅進行 PCR 引子組特異性檢測
。使用引子組 CT-1/CT-2 進行檢測，除了 C. tyrobutyricum 會產生 104 bp 之預期產物外，其他菌株，包含其他
Clostridium 屬菌株，皆不會產生偽陽性之干擾。而由 L. monocytogenes 之類休克蛋白基因引子進行 PCR 增幅，可得到 120 bp 之預期產物外，其他菌株，包含其他 Listeria 屬，皆不會產生偽陽性之干擾。當引子組 CT-1/CT-2 應用於檢測鮮乳樣品 中 C. tyrobutyricum 時，在 PCR 增幅前，先進行 24 小時之預培養後，其檢測靈敏度為 N×100 CFU/ml；而應用於檢測乾 酪樣品，其靈敏度為 N×100 CFU/g。而引子組 Lm1/Lm2 應用於食品樣品中 L. monocytogenes 檢測，經 12 小時預培養後
，鮮乳及乾酪樣品的檢測靈敏度均可達到 N×100 CFU/ml 及 N×100 CFU/g。 本研究亦利用 Real-time PCR 方法，以引 子組 CT-1/CT-2 及 Lm1/Lm2 檢測鮮乳及乾酪樣品中之 C. tyrobutyricum 及 L. monocytogenes。當引子組 CT-1/CT-2 應用 於檢測鮮乳樣品中 C. tyrobutyricum 時，在 Real-time PCR 增幅前先進行 24 小時之預培養後，其檢測靈敏度為 N×100 CFU/ml；而應用於檢測乾酪樣品，其靈敏度為 N×100 CFU/g。而引子組 Lm1/Lm2 應用於食品樣品中 L. monocytogenes 檢測，經 12 小時預培養後，鮮乳及乾酪樣品的檢測靈敏度可達到 N×100 CFU/ml 及 N×100 CFU/g。
關鍵詞 : 病原菌 ; 熱休克蛋白基因 ; 特異性 ; 鮮乳 ; 乾酪
目錄
目錄 封面內頁 簽名頁 授權書...iii 中文摘要...iv 英文摘 要...vi 誌謝...vii 目錄...viii 圖目
錄...xi 表目錄...xiii 1.文獻回顧...1 1.1 Clostridium
…...1 1.1.1 Clostridium 分類...1 1.1.2 Clostridium 之特性...1 1.1.3 Clostridium 形 態...2 1.1.4臨床上重要致病性 Clostridium菌株...2 1.1.5 Clostridium tyrobutyricum...3 1.1.6 C.
tyrobutyricum 之快速檢測...3 1.2 Listeria monocytogenes...4 1.2.1李斯特菌之分類...5 1.2.2 L.
monocytogenes 之一般特性...5 1.2.3血清型分類與致病決定位(Virulence determinants)...6 1.2.4 L.
monocytogenes 之快速檢驗方法...6 1.3 熱休克蛋白的介紹...8 1.4 即時 PCR (Real-Time PCR) 介
紹...10 1.5 實驗目的...13 2.材料方法...15 2.1 實驗材料...15 2.1.1 菌株...15 2.1.2 培養基...15 2.1.3 藥品...15 2.1.4 緩衝液及試 劑...16 2.1.5 儀器...17 2.2 實驗方法...18 2.2.1 熱休克蛋白基因分析及 篩選…...18 2.2.2 PCR 引子組設計...19 2.2.3 PCR引子組及寡核?酸引子之合成...20 2.3 聚合?鏈 鎖反應 (PCR)...20 2.3.1 DNA製備...20 2.3.2 C. tyrobutyricum 之 DnaK 基因定序...21 2.3.3 引子組CT-1/CT-2 及 Lm1/Lm2 之 PCR 特異性 試驗...22 2.4 CT-1/CT-2 及 Lm1/Lm2 於食品檢驗之 應用...22 2.4.1 CT-1/CT-2 鮮乳樣品的檢驗應用...22 2.4.1.1直接檢測...22 2.4.1.2 增菌培養
…...23 2.4.2 CT-1/CT-2 乾酪樣品的檢驗應用...24 2.4.3 Lm1/Lm2 鮮乳樣品的檢驗應用...24 2.4.3.1 直接檢驗...24 2.4.3.2 增菌培養...24 2.4.4 Lm1/Lm2 乾酪樣品的檢驗應
用...24 2.5 Real-Time PCR (即時聚合?鏈反應)...25 3.結果與討論...26 3.1 C. tyrobutyriucm 及L.monocytogenes之序列比對26 3.2 聚合?鏈鎖反應 (PCR)...27 3.3 食品檢測之應用…...29 3.3.1 傳統 PCR 檢測靈敏度...29 3.3.2 即時 PCR 檢測靈敏度...30 3.3.3 Real-Time PCR標準曲線之建 立...31 4.結論...33 參考文獻…...71 附錄...79
參考文獻
參考文獻 1. 王怡惠。2000。腸球菌之 groEL 基因定序與菌株鑑定。國立臺 灣大學醫事技術學研究所碩士論文。 2. 石心怡。2002。鼠傷 寒沙門氏菌之致病相關基因、表型特性及 其與李斯特菌之免疫磁珠-PCR檢測。國立中興大學食品科學所 博士論文，台中。 3. 王聖予、
李麗俐、吳秀玲、周啟馥、楊志元、陳建和(2002) 最 新醫用微生物學藝軒圖書出版社。 4. 李世仁。2004。單核細胞增多性李斯特菌之 特性與檢驗。國立 中山大學生物科學系碩士在職專班碩士論文，高雄。 5. 林嘉威。2004。利用類熱休克蛋白基因設計金黃色葡萄球菌 及 利用fliC基因設計鼠傷寒沙門氏菌之PCR引子組及其應用。國 立中興大學食品科學系碩士論文，台中。 6. 蔣育錚。2005。食品病原 菌與金黃色葡萄球菌腸毒素檢測用生 物晶片之發展以及金黃色葡萄球菌之分子分類與即時聚合?鏈 反應。國立中興大學食品暨應用生物 科技學所博士論文，台中。 7. 陳瑋芸。2006。彎曲桿菌及空腸彎曲桿菌之分子鑑定及 PCR 與 生物晶片發展。國文中興大學食品暨應用 生物科技學系碩士論 文，台中。 8. 廖婉雯。2006。金黃色葡萄球菌新型腸毒素 Q、R、U 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。弘 光科技大學生物科技所碩士論文，台中。 9. Alary, R., A. Serin, D. Maury, Ben-Jouira, H., J. P. Sirven, M. F.
Gautier, and P. Joudrier. 2002. Comparison of simplex and duplex real-time PCR for the quantification of GMO in maize and soybean. Food Control. 13(4/5): 235-244. 10. Ahmed, F. E., 2002. Detection of genetically modified organisms in foods. Trends in Biotechnol. 20(5): 215-223. 11.
Bohnert, M., Dilasser, F., Dalet, C., Mengaud, J. and Cossart, P., 1992. Use of specific oligonucleotides for direct enumeration of Listeria
monocytogenes in food samples by colony hybridization and rapid detection by PCR. Res Microbiol. 143:271-280. 12. Borucki MK, Kim SH, Call DR, Smole SC, Pagotto F., 2004 Selective discrimination of Listeria monocytogenes epidemic strains by a mixed-genome DNA microarray compared to discrimination by pulsed-field gel electrophoresis, ribotyping, and multilocus sequence typing. J Clin Microbiol. 42(11):5270-6. 13.
Borucki MK, Call DR., 2003. Listeria monocytogenes serotype identification by PCR. J Clin Microbiol. 41(12):5537-40. 14. Brousseau R, Hill JE, Prefontaine G, Goh SH, Harel J, Hemmingsen SM., 2001. Streptococcus suis serotypes characterized by analysis of chaperonin 60 gene sequences.
Appl Environ Microbiol. 67(10):4828-33. 15. Borucki MK, Krug MJ, Muraoka WT, Call DR., 2003. Discrimination among Listeria
monocytogenes isolates using a mixed genome DNA microarray. 16. Borucki MK, Reynolds J, Call DR, Ward TJ, Page B, Kadushin J., 2005.
Suspension microarray with dendrimer signal amplification allows direct and high-throughput subtyping of Listeria monocytogenes from genomic DNA. J Chin Microbiol.43(7):3255-9. 17. Brehm-Stecher BF, Hyldig-Nielsen JJ, Johnson EA., 2005. Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria. Appl Environ Microbiol. 71(9):5451-7. 18. Borucki MK, Kim SH, Call DR, Smole SC, Pagotto F., 2004. Selective discrimination of Listeria monocytogenes epidemic strains by a mixed-genome DNA
microarray compared to discrimination by pulsed-field gel electrophoresis, ribotyping, and multilocus sequence typing. J Clin Microbiol.
42(11):5270-6. 19. Call DR, Borucki MK, Besser TE., 2003. Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes. J Clin Microbiol. 41(2):632-9. 20. Chang YH, Shangkuan YH, Lin HC, Liu HW., 2003. PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells. Appl Environ Microbiol. 69(8):4502-10. 21. Dasgupta, A. P., Hull, R. R., 1989. Late blowing of Swiss cheese: incidence of Clostridium tyrobutyricum in manufacturing milk. Hull Australian Journal of Dairy Technology. 44:22, 82-87. 22. Datta, A. R., Wentz, B. A. and Russell, J. 1990. Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes. Appl. Environ. Microbiol. 56:3874-3877. 23. Deneer, H. G., and Boychuk, I., 1991. Species-specific detection of Listeria monocytogenes by DNA amplification. Appl. Environ. Microbiol. 57:606-609. 24. De Valk, Vaillant, H. V. and Goulet, V., 2000.
Epidemiology of human Listeria infections in France. Bull. Acad. Natl. 184:267-274. 25. Doumith M, Buchrieser C, Glaser P, Jacquet C, Martin P., 2004. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J Clin Microbiol. 42(8):3819-22. 26. Ellingson, J.L., Anderson, J.L., Carlson, S.A., and Sharma, V.K. 2004. Twelve hour real-time PCR technique for the sensitive and specific detection of Salmonella in raw and ready-to-eat meat products. Mol. Cell Probes. 18(1):51-7. 27. Flekna G, Stefanic P, Wagner M, Smulders FJ, Mozina SS, Hein I., 2007.
Insufficient differentiation of live and dead Campylobacter jejuni andListeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR. Res Microbiol. 158(5):405-12. 28. Goh SH, Potter S, Wood JO, Hemmingsen SM, Reynolds RP, Chow AW., 1996. HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. J Clin Microbiol.
34(4):818-23. 29. Gilot, P., Hertmans, C., Yde, M., Gigi, J., Janssens, M., Genicot, A., Andre, P. and Wauters, G. 1997. Sporadic case of listeriosis associated with the consumption of a Listeria monocytogenes-contaminated 'Camembert' cheese. J. Infect. 35:195-197. 30. Herman LM, De Block JH, Waes GM., 1995. A direct PCR detection method for Clostridium tyrobutyricum spores in up to 100 milliliters of raw milk. Appl Environ Microbiol. 61(12):4141-6. 31. Ingham SC, Hassler JR, Tsai YW, Ingham BH., 1998. Differentiation of lactate-fermenting, gas-producing Clostridium spp. isolated from milk. Int J Food Microbiol.43(3):173-83. 32. J. Mainil, C. Duchesnes, S. Pelkonen, L. Dubreuil, M.G. Menozzi., 2004. Identification, typing and antibiotic resistance of the genus Clostridium. 33. Jolly C, Morimoto RI. 2000. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst. 92(19): 1564-1572. 34. Klijn N, Nieuwenhof FF, Hoolwerf JD, van der Waals CB, Weerkamp AH., 1995. Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification. Appl Environ Microbiol. 61(8):2919-24. 35. Kozak, J., Balmer, T., Byrne, R. and Fisher, K.,1996. Prevalence of Listeria monocytogenes in foods: incidence in dairy products. Food control 7:215-221. 36. Klein, P. G. and Juneja, V. K., 1997. Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR. Appl. Environ. Microbiol. 63:4441-4448. 37. Kim J, Demeke T, Clear RM, Patrick SK., 2006. Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella Typhimurium in artificially inoculated wheat grain. Int J Food Microbiol.15;111(1):21-5. 38. Kupfahl C, Geginat G, Hof H., 2006. Lentinan has a stimulatory effect on innate and
adaptive immunity against murine Listeria monocytogenes infection. Int Immunopharmacol. 6(4):686-96. 39. Liming, S.H., and Bhagwat, A.A.
2004. Application of a molecular beacon-real-time PCR technology to detect Salmonella species contaminating fruits and vegetables. Int. J. Food Microbiol. 95(2):177-87. 40. Le Bourhis AG, Saunier K, Dore J, Carlier JP, Chamba JF, Popoff MR, Tholozan JL., 2005. Development and validation of PCR primers to assess the diversity of Clostridium spp. in cheese by temporal temperature gradient gel electrophoresis. Appl Environ Microbiol. 71(1):29-38. 41. Le Bourhis AG, Dore J, Carlier JP, Chamba JF, Popoff MR, Tholozan JL., 2007. Contribution of C. beijerinckii and C.
sporogenes in association with C. tyrobutyricum to the butyric fermentation in Emmental type cheese. Int J Food Microbiol.113(2):154-63. 42.
Lopez-Enriquez L, Rodriguez-Lazaro D, Hernandez M., 2007. Quantitative Detection of Clostridium tyrobutyricum in Milk by Real-Time PCR.
Appl Environ Microbiol. 73(11):3747-51. 43. McLauchlin, J. 1996. The relationship between Listeria and listeriosis. Food Control 7:187-193. 44.
Meng, J. and Doyle, M. P. 1998. Emerging and evolving microbial foodborne pathogens. Bull. Inst. Pasteur 96:151-164. 45. Marston, E. L., J. W.
Sumner, and R. L. Regnery., 1999. Evaluation of intraspecies genetic variation within the 60 KDa heat-shock protein gene (groEL) of Bartonella species. Int. J. Syst. Bacteriol. 49: 1015-1023. 46. Mukhopadhyay A, Mukhopadhyay UK., 2007. Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes. J Microbiol Methods. 68 (1):193-200. 47.
Magliulo M, Simoni P, Guardigli M, Michelini E, Luciani M, Lelli R, Roda A., 2007. A Rapid Multiplexed Chemiluminescent Immunoassay for the Detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes Pathogen Bacteria. J Agric Food Chem. 55 (13):4933-4939. 48. Peterkin, P. I., Idziak, E. S. and Sharpe, A. N.1991. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe. Appl. Environ. Microbiol. 57:586-591. 49.
Pinner, R. W., Schuchat, A., Swaminathan, B., Hayes, P, S., Deaver, K. A., 38、Weaver, R. E., Plikaytis, B. D., Reeves, M., Broom, C. V., Wenger, J. D., and the Listeria Study Group. 1992. Role of foods in sporadic listeriosis. II. Microbiologic and epidemiologic investigation. J. Am.
Med. Assoc. 267:2046-2050. 50. Pirkkala, L., P. Nykanen, and L. Sistonen., 2001. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J. 15:1118-1131. 51. Paillard D, Dubois V, Duran R, Nathier F, Guittet C, Caumette P, Quentin C., 2003. Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments. Appl Environ Microbiol. 2003.69 (11):6386-92. 52. Rijpens, N. P., Jannes, G., Van Asbroeck, M., Herman, L. M., Rossau, R.1995. Simultaneous detection of Listeria spp. and Listeria monocytogenes by reverse hybridization with 16S-23S rRNA spacer probes. Mol Cell Probes.9:423-32. 53.
Rodriguez-Lazaro D, Hernandez M, Pla M., 2004. Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay. FEMS Microbiol Lett. 233 (2):257-67. 54. Rodriguez-Lazaro D, Pla M, Scortti M, Monzo HJ, Vazquez-Boland JA., 2005. A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control. Appl Environ Microbiol. 71 (12): 9008-12. 55. Saywell, B. A., McGee, M. A., Veitch, C. M., 1977. The incidence of Clostridium tyrobutyricum in a factory milk supply. NZ J. Dairy Sci. Technol. 12, 274-275. 56. Somer, L. and Kashi, Y., 2003. A PCR method based on 16S rRNA Sequence for simultaneous detection of the genus Listeria and the species Listeria monocytognes in food products. J. food Protection. 66 :1658-1665. 57. Schmid M, Walcher M, Bubert A, Wagner M, Wagner M, Schleifer KH., 2003. Nucleic acid-based, cultivation-independent detection of Listeria spp and genotypes of L. monocytogenes. FEMS Immunol Med Microbiol. 35(3):215-25. 58. Taege, A. J. 1999. Listeriosis: recognizing it, treating it, preventing it. Cleve.
Clin. J. Med. 66:375-380. 59. Tominaga T., 2006. Rapid discrimination of Listeria monocytogenes strains by microtemperature gradient gel electrophoresis. J Clin Microbiol. 44 (6):2199-206. 60. Van Netten, P., Perales, I., van de Moosdijk, A., Curtis, G. D. and Mossel, D. A. 1989.
Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Int. J. Food Microbiol.
8:299-316. 61. Verheij, M., R. Bose, X. H. Lin, B. Yao, W. D. Jarvis, S. Grant, M. J. Birrer, E.Szabo, L. I. Zon, J. M. Kyriakis, A.
Haimovitz-Friedman, Z. Fuks, and R. N. Kolesnick., 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis.
Nature. 380:75-79. 62. Vaitilingomm, M., Pijnenburg, H., Gendre, F., and Brignon, P. 1999. Real-time quantitive PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods. J. Agric. Food Chem. 47: 5261-5266. 63. Volokhov D, Rasooly A., Chumakov K., and Chizhikov V., 2002. Identification of Listeria Species by Microarray-Based Assay. J. of Cinical Microbiology. 4720 –4728. 64. Wang, R. F., Cao, W. W. and Johnson, M. G. 1992. 16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods. Appl. Environ. Microbiol. 58:2827-2831. 65. Wang RS and Chow Aw., 2002. Identification of enteric pathogens by heat shock protein 60 kDa (HSP60) gene sequences. FEMS Microbiol Lett. 206(1):107-13. 66. Wang, X., Jothikumar, N., and Griffiths, M.W. 2004. Enrichment and DNA extraction protocols for the simultaneous detection of Salmonella and Listeria monocytogenes in raw sausage meat with multiplex real-time PCR. J. Food Prot. 67 (1):189-92.
