臺灣博碩士論文加值系統

仙人掌桿菌 (Bacillus cereus) 是重要的食品病原菌，在台灣地區由1991至2006年之食品中毒案件病因物質中排名第三位，僅次於腸炎弧菌及金黃色葡萄球菌。其致病因子至少包括五種腹瀉型腸毒素以及一種嘔吐型毒素。本研究建立多重組對引子聚合酶鏈反應 (multiplex polymerase chain reaction, multiplex PCR) 方法，同時以12組引子偵測所有的腸毒型腸毒素基因及嘔吐型菌株特異序列，並將方法應用於162株仙人掌桿菌群分離菌株。結果顯示此些菌株可分為10種毒素型別，而且每株菌株至少攜帶一種毒素基因。雖然仙人掌桿菌與蕈狀桿菌之類緣關係相當接近，但超過70%之蕈狀桿菌 (Bacillus mycoides) 菌株沒有攜帶任何已知的毒素基因，且其毒素型別以及毒素基因分布與仙人掌桿菌菌株之間顯著不同，因此推測許多蕈狀桿菌菌株可能較不易導致食品中毒，也顯示檢驗菌株是否攜帶毒素基因與檢驗仙人掌桿菌群菌株同樣重要。傳統之仙人掌桿菌群菌株之分離及鑑定耗時費力，PCR方法雖可快速檢驗但無法精確定量。實務上，有必要進行食品中仙人掌桿菌群之定量檢驗。因此，本研究使用非溶血性腸毒素 (nonhemolytic enterotoxin, Nhe) 之基因nhe作為目標基因，建立即時聚合酶鏈反應 (real-time PCR) 檢驗方法，直接由食品及糞便中定量仙人掌桿菌群菌株，以達快速檢驗之目的。共使用60株仙人掌桿菌群菌株以及28株其他菌株進行即時聚合酶鏈反應方法之評估，並應用於不同之食品基質及糞便檢體。結果顯示此方法具有足夠的線性關係 (r2>0.993)、定量範圍 (米飯及雞肉檢體由102至107 CFU/g、牛奶檢體由103至107 CFU/mL、糞便檢體由104至107 CFU/g) 以及相對定量準確度 (85.5至101.1%)。另為解決定量低污染菌量之問題，結合最確數 (most probable number, MPN) 方法與real-time PCR建立最確數即時聚合酶鏈反應 (MPN real-time PCR) 檢驗方法，將提高靈敏性至100 CFU/mL。Real-time PCR以及MPN real-time PCR方法應用於市售食品檢體，結果顯示皆適合食品中仙人掌桿菌群菌株之檢驗及定量。仙人掌桿菌之Nhe腸毒素表現量在不同菌株之間相差甚多，而Nhe可能是導致食品中毒最主要的致病因子。為探究影響不同仙人掌桿菌菌株表現Nhe之機制，本研究比較Nhe表現量相異菌株組別之生長情形，並以三種軟體評估仙人掌桿菌mRNA表現研究可用之五種候選內部對照基因，再將評估結果用於建立定量nhe mRNA之反轉錄即時聚合酶鏈反應 (real-time reverse transcription PCR) 方法。結果顯示Nhe表現量相異菌株組別之生長曲線並無顯著差異。各候選之內部對照基因在菌株各生長階段皆有穩定的表現，比較Nhe表現量相異菌株組別之nhe mRNA表現量，發現高Nhe表現量之組別顯著高於低Nhe表現量之組別。因此，影響Nhe表現之差異應非導因於菌株生長速率之不同，推測應為nhe基因表現轉錄之層次所調控。

Bacillus cereus foodborne diseases are a major concern worldwide. In Taiwan for the period 1991 to 2006, outbreaks due to B. cereus were exceeded only by Vibrio parahaemolyticus and Staphylococcus aureus. Five different enterotoxins and one emetic toxin of B. cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex polymerase chain reaction (multiplex PCR) with 12 primer pairs was established. The assay was successfully applied to analyze the toxigenic potential of 162 isolated B. cereus group strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of B. mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains although the two species were closely related. The results suggested that many B. mycoides strains might be less prone to cause food poisoning. It also indicated the importance of detecting the toxin genes together with the detection of the species in the B. cereus group. Conventional bacteriological methods for the detection and identification of species of the B. cereus group require individual biochemical confirmation and are laborious and time-consuming. PCR is a choice of rapid detection but can not quantify the contamination level. In practice, it is necessary to quantify the contamination level of B. cereus group cells. We selected nhe coding for Nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated using 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2>0.993), wide dynamic quantification range (102-107 CFU/g for cooked rice and chicken, 103-107 CFU/mL for milk, and 104-107 CFU/g for feces), and adequate relative accuracy (85.5-101.1%). For the low level contaminations, a most probable number (MPN) real-time PCR assay was developed that could detect as low as 100 CFU/mL. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification. The cytotoxicity titers of Nhe components varied considerably and the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates. In order to examine the regulatory mechanism of different Nhe-producing strains, the expression level of their nhe mRNA was determined by real-time reverse transcription PCR. Five candidates of internal controls were evaluated by three softwares. Meanwhile, the growth curves of different Nhe-producing strains were also compared. All studied candidate of internal control genes reached high expression stability. The growth curves showed no significant difference but the nhe mRNA expression level of high Nhe-producing strains was significantly higher than low Nhe-producing strains. The results indicated that the different Nhe expression levels between B. cereus strains may not be due to their growth difference but may be controlled at the transcribed level of nhe gene expression.

目錄
目錄………………………………………………………………… I
圖目錄 …………………………………………………………… V
表目錄 …………………………………………………………… VIII
縮寫表……………………………………………………………… X
中英文對照表…………………………………………………… XI
中文摘要……………………………………………………… XII
英文摘要………………………………………………………… XIV

第一章 文獻回顧……………………………………………………1
1.1 仙人掌桿菌………………………………………………………1
1.2 仙人掌桿菌食品中毒……………………………………………1
1.3 台灣地區仙人掌桿菌食品中毒之發生情形……………………5
1.4 仙人掌桿菌之毒力因子…………………………………………5
1.5 仙人掌桿菌毒力因子之檢驗……………………………………15
1.6 仙人掌桿菌群……………………………………………………17
1.7 即時聚合酶鏈反應………………………………………………20
1.8 基因表現之定量…………………………………………………25

第二章 研究目的及架構……………………………………………30
2.1 研究目的…………………………………………………………30
2.2 研究架構…………………………………………………………31

第三章　以多重組對引子聚合酶鏈反應偵測仙人掌桿菌群………32
3.1 材料………………………………………………………………32
3.1.1 菌株……………………………………………………………32
3.1.2 培養基…………………………………………………………32
3.1.3 藥品、試劑套組及儀器設備…………………………………32
3.2 方法………………………………………………………………36
3.2.1 菌株分離培養…………………………………………………36
3.2.2 引子……………………………………………………………36
3.2.3 DNA抽取及multiplex PCR方法之建立………………………37
3.2.4. Multiplex PCR之靈敏性試驗及特異性試驗………………37
3.2.5Hbl腸毒素之表現型測定………………………………………39
3.2.6PCR產物定序及序列比對………………………………………39
3.2.7 統計分析………………………………………………………39
3.3 結果………………………………………………………………40
3.3.1Multiplex PCR方法之建立……………………………………40
3.3.2 菌株型別………………………………………………………40
3.3.3 腸毒素基因及嘔吐型菌株特異序列之分布…………………45
3.3.4 蕈狀桿菌之腸毒素基因序列…………………………………45
3.3.5 腹瀉型腸毒素表現型之檢驗結果……………………………57
3.3.6 Multiplex PCR之靈敏性及特異性結果……………………57
3.4 討論………………………………………………………………60

第四章　仙人掌桿菌群快速檢驗方法之研究………………………63
4.1 材料………………………………………………………………63
4.1.1 菌株……………………………………………………………63
4.1.2 培養基…………………………………………………………63
4.1.3 藥品、試劑套組及儀器設備…………………………………65
4.2 方法………………………………………………………………67
4.2.1 菌株……………………………………………………………67
4.2.2 引子之設計及評估……………………………………………67
4.2.3 DNA抽取………………………………………………………67
4.2.4 Real-time PCR反應條件……………………………………69
4.2.5 Real-time PCR靈敏性及特異性試驗………………………70
4.2.6 相對定量準確度之評估………………………………………72
4.2.7 MPN real-time PCR…………………………………………72
4.2.8 定序及序列分析………………………………………………75
4.2.9 食品檢體之檢測………………………………………………75
4.2.10 統計分析……………………………………………………75
4.3 結果………………………………………………………………76
4.3.1 引子之評估……………………………………………………76
4.3.2 目標基因序列比對……………………………………………76
4.3.3 Real-time PCR之靈敏性及標準曲線………………………76
4.3.4 Real-time PCR之特異性……………………………………82
4.3.5 Real-time PCR應用於模擬食品檢體及糞便檢體之檢驗…82
4.3.6 相對定量準確度………………………………………………86
4.3.7 MPN real-time PCR…………………………………………86
4.3.8 食品檢體之檢測………………………………………………90
4.4 討論………………………………………………………………93

第五章　以反轉錄即時聚合酶鏈反應進行仙人掌桿菌內部對照基因表現之穩定度評估以及非溶血性腸毒素之表現研究………………97
5.1 材料………………………………………………………………97
5.1.1 菌株……………………………………………………………97
5.1.2 培養基…………………………………………………………97
5.1.3 藥品、試劑套組及儀器設備…………………………………99
5.2 方法………………………………………………………………100
5.2.1 菌株培養、生長曲線與RNA固定……………………………100
5.2.2 引子之設計及評估……………………………………………100
5.2.3 RNA之抽取、純化與定量……………………………………100
5.2.4. cDNA反應條件………………………………………………102
5.2.5 Real-time PCR反應條件……………………………………102
5.2.6 Nhe表現量測定………………………………………………103
5.2.7 統計分析………………………………………………………103
5.3 結果………………………………………………………………105
5.3.1 內部對照基因及目標基因之real-time PCR方法建立……105
5.3.2 內部對照基因表現穩定度之評估結果………………………105
5.3.3 NheA之mRNA的相對表現量……………………………………138
5.3.4 生長曲線………………………………………………………141
5.4 討論………………………………………………………………143
第六章　結論與未來展望……………………………………………149
第七章 參考文獻……………………………………………………151
附錄……………………………………………………………………165

1.行政院衛生署。1992-2006。民國80-95年食品中毒發生狀況 (共15冊)。台北。台灣。中華民國。
2.Agaisse, H., Gominet, M., Okstad, O. A., Kolsto, A. B., and Lereclus, D. 1999. PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol. Microbiol. 32: 1043-1053.
3.Agata, N., Mori, M., Ohta, M., Suwan, S. Ohtani, I., and Isobe, M. 1994. A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole formation in Hep-2 cells. FEMS Microbiol. Lett. 121: 31-34.
4.Agata, N., Ohta, M., Mori, M., and Isobe, M. 1995a. A novel dodecadepsipeptide, cereulide, is an emetic toxic of Bacillus cereus. FEMS Microbiol. Lett. 129: 17-20.
5.Agata, N., Ohta, M., Arakawa, Y., and Mori, M. 1995b. The bceT gene of Bacillus cereus encodes an enterotoxic protein. Microbiology 141: 983-988.
6.Anderson, C. L., Jesen, J. L., Orntoft, T. F. 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64: 5245-5250.
7.Andersson, A., Mikkola, R., Helin, J., Andersson, M. C., and Salkinoja-Salonen, M. 1998. A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores. Appl. Environ. Microbiol. 64: 1338-1343.
8.Aronson, A. I., Beckman, W., and Dunn, P. 1986. Bacillus thuringiensis and related insect pathogens. Microbiol. Rev. 50: 1-24.
9.Asano, S. I., Nukumizu, Y., Bando, H., Hzuka, T., and Yamamoto, T. 1997. Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis. Appl. Environ. Microbiol. 63: 1054-1057.
10.Ash, C. and Collins, M. D. 1992. Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-directed sequencing. FEMS Microbiol. Lett. 94: 75-80.
11.Ash, C., Farrow, J. A. E., Dorsch, M., Stackebrandt, E., and Collins, M. D. 1991. Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rDNA. Int. J. Syst. Bacteriol. 41: 343-346.
12.Baker, J. M. and Griffiths, M. W. 1995. Evidence for increased thermostability of Bacillus cereus enterotoxin in milk. J. Food Prot. 58: 443-445.
13.Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V., and Hood, L. 2002. Coordinate regulation of energy transduction modules in Halobacterium sp. analyzed by a global systems approach. Proc. Natl. Acad. Sci. USA 99: 14913-14918.
14.Bavykin, S. G., Lysov, Y. P., Zakhariev, V., Kelly, J. J., Jackman, J., Stahl, D. A., and Cherni, A. 2004. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. J. Clin. Microbiol. 42: 3711-3730.
15.Beattie, S. H. and Williams, A. G. 1999. Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay. Lett. Appl. Microbiol. 28: 221-225.
16.Beecher, D. J. and MacMillan, J. D. 1990. A novel biocomponent hemolysin from Bacillus cereus. Infect. Immun. 58: 2220-2227.
17.Beecher, D. J. and MacMillan, J. D. 1991. Characterization of the components of hemolysin BL from Bacillus cereus. Infect. Immun. 59: 1778-1784.
18.Beecher, D. J. and Wong, A. C. 1994. Improved purification and characterization of hemolysin BL, a hemolytic dermonecrotic vascular permeability factor from Bacillus cereus. Infect. Immun. 62: 980-986.
19.Beecher, D. J. and Wong, A. C. 1997. Tripartite hemolysin BL from Bacillus cereus. Hemolytic analysis of component interactions and a model for its characteristic paradoxical zone phenomenon. J. Biol. Chem. 272: 233-239.
20.Beecher, D. J., Schoeni, J. L, Wong, A.C. 1995. Enterotoxic activity of hemolysin BL from Bacillus cereus. Infect. Immun. 63: 4423-4428.
21.Bergdoll, M. S. 1988. Ileal loop fluid accumulation test for diarrheal toxins. Methods Enzymol. 165: 306-323.
22.Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A., and Duncan, K. 2002. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 43: 717-731.
23.Brillard, J. and Lereclus, D. 2004. Comparison of cytotoxin cytK promoters from Bacillus cereus strain ATCC 14579 and from a B. cereus food-poisoning strain. Microbiology 150: 2699-2705.
24.Buchanan, R. L., Schultz, F. J. 1992. Evaluation of the Oxoid BCET-RPLA kit for the detection of Bacillus cereus diarrheal enterotoxin as compared to cell culture cytotoxicity. J. Food Prot. 55: 440-443.
25.Buchanan, R. L., Schultz, F. J. 1994. Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin. Lett. Appl. Microbiol. 19: 353-356.
26.Bustin, S. A. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25: 169-193.
27.Bustin, S. A. 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29: 23-39.
28.Carlson, C. R. and Kolsto, A. B. 1993. A complete physical map of a Bacillus thuringiensis chromosome. J. Bacteriol. 175: 1053-1060.
29.Chang, Y. H., Shangkuan, Y. H., Lin, H. C., and Liu, H. W. 2003. PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells. Appl. Environ. Microbiol. 69: 4502-4510.
30.Chen, M. L., and Tsen, H. Y. 2002. Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites. J. Appl. Microbiol. 92: 912-919.
31.Cherif, A., Borin, S., Rizzi, A., Ouzari, H., Boudabous, A., and Daffonchio, D. 2003. Bacillus anthracis diverges from related clades of the Bacillus cereus group in 16S-23S ribosomal DNA intergenic transcribed spacers containing tRNA genes. Appl. Environ. Microbiol. 69: 33-40.
32.Christiansson, A. 1993. Enterotoxin production in milk by Bacillus cereus: a comparison of methods of toxin detection. Neth. Milk Dairy J. 47: 79-87.
33.Claus, D., and Berkeley, R. C. W. 1986. Genus Bacillus Cohn 1872, 174AL. In: Sneath, P. H. A., Mair, N. S., Sharpe, M. E., and Holt, J. G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 2. Williams and Wilkins, Baltimore, MD., pp. 1105-1139.
34.Damgaard, P. H., Larsen, H. D., Hansen, B. M., Bresciani, J., and Jorgensen, K. 1996. Enterotoxin-producing strains of Bacillus thuringiensis isolated from food. Lett. Appl. Microbiol. 23: 146-150.
35.Day, T. L., Tatani, S. R., Notermans, S., and Bennett, R. W. 1994. A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin. J. Appl. Bacteriol. 77: 3-13.
36.Dierick, K., Van Coillie, E., Swiecicka, I., Meyfroidt, G., Devlieger, H., Meulemans, A., Hoedemaekers, G., Fourie, L., Heyndrickx, M., Mahillon, J., 2005. Fatal family outbreak of Bacillus cereus-associated food poisoning. J. Clin. Microbiol. 43: 4277-4279.
37.Ehling-Schulz, M., Fricker, M., and Scherer, S. 2004. Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay. FEMS Microbiol. Lett. 232: 189-195.
38.Ehling-Schulz, M., Fricker, M., Grallert, H., Rieck, P., Wagner, M., Scherer, S. 2006a. Cereulide synthetase gene cluster from emetic Bacillus cereus: structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pX01. BMC Microbiol. 6: 20.
39.Ehling-Schulz, M., Guinebretiere, M., Monthan, A., Berge, O., Fricker, M., Svensson, B., 2006b. Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS Microbiol. Lett. 260: 232-240.
40.Ehling-Schulz, M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Martlbauer, E., and Scherer, S., 2005. Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Appl. Environ. Microbiol. 71: 105-113.
41.Finlay, W. J. J., Logan, N. A. And Sutherland, A. D. 1999. Semiautomated metabolic staining assay for Bacillus cereus emetic toxin. Appl. Environ. Microbiol. 65: 1811-1812.
42.Fletcher, P., and Logan, N. A. 1999. Improved cytotoxicity assay for Bacillus cereus diarrhoeal enterotoxin. Lett. Appl. Microbiol. 28: 394-400.
43.Fortin, N. Y., Mulchandani, A., and Chen, W. 2001. Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7. Anal. Biochem. 289: 281-288.
44.Fukushima, H., Tsunomori, Y., and Seki, R. 2003. Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools. J. Clin. Microbiol. 41: 5134-5146.
45.Gibson, U. E., Heid, C. A., and Williams, P. M., 1996. A novel method for real time quantitative RT-PCR. Gen. Res. 6: 995-1001.
46.Giffel, M. C., Beumer, R. R., Granum, P. E., and Rombouts, F. M. 1997. Isolation and characterization of Bacillus cereus from pasteurized milk in household refrigerators in The Netherlands. Int. J. Food Microbiol. 34: 307-318.
47.Gohar, M., Okstad, O. A., Gilois, N., Sanchis, V., Kolsto, A. B., and Lereclus, D. 2002. Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon. Proteomics 2: 784-791.
48.Granum, P. E., Brynestad, S., and Kramer, J. M. 1993. Analysis of food poisoning incidents and non-gastrointestinal infections. Int. J. Food Microbiol. 17: 269-279.
49.Granum, P. E. 1994. Bacillus cereus and its toxins. Soc. Appl. Bacteriol. Symp. Ser. 23: 61S-66S
50.Granum, P. E. 2001. Bacillus cereus. In: Doyle, M. P., Beuchat, L. R., and Montville, T. D. (Eds.), Food Microbiology: Fundamentals and Frontiers. 2nd ed. ASM Press, Washington, DC. pp. 373-381.
51.Granum, P. E., Andersson, A., Gayther, C., Giffel, M. te, Larsen, H., Lund, T., and O’Sullivan, K. 1996. Evidence for a further enterotoxin complex produced by Bacillus cereus. FEMS Microbiol. Lett. 141: 145-149.
52.Granum, P.E., O’Sullivan K., and Lund T., 1999. The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiol. Lett. 177: 225-229.
53.Griffiths, M. W. 1990. Toxin production by psychrotrophic Bacillus spp. present in milk. J. Food Prot. 53: 790-792.
54.Guinebretiere, M. H., Broussolle, V., and Nguyen-The, C. 2002. Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. J. Clin. Microbiol. 40: 3053-3056.
55.Haggblom M. M., Apetroaie, C., Andersson, M. A., Salkinoja-Salonen, M. S. 2002. Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions. Appl. Environ. Microbiol. 68: 2479-2483.
56.Hansen, B. M., and Hendriksen, N. B. 2001. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Appl. Environ. Microbiol. 67: 185-189.
57.Hardy, S. P., Lund T., and Granum, P. E. 2001. CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and its cytotoxic to intestinal epithelia. FEMS Microbiol. Lett. 197: 45-51.
58.Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M., 1996. Real time quantitative PCR. Genome Res. 6: 986-994.
59.Heinrichs, J. H., Beecher, D. J., Macmillan, J. D., and Zilinskas, B. A. 1993. Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus. J. Bacteriol. 175: 6760-6766.
60.Horwood, P. F., Burgess, G. W., and Oakey, H. J. 2004. Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus. FEMS Microbiol. Lett. 236: 319-324.
61.Hoton, F. M., Andrup, L, Swiecicka, I., and Mahillon, J. 2005. The cereulide genetic determinants of emetic Bacillus cereus are plasmid-borne. Microbiol. Comments 151: 2121-2124.
62.Hsieh, Y. M., Sheu, S. J., Chen, Y. L., and Tsen, H. Y. 1999. Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks. J. Appl. Microbiol. 87: 481-490.
63.Hsu, C. F., Tsai, T. Y., Pan, T. M., 2005. Use of the duplex TaqMan PCR system for detection of shiga-like toxin-producing Escherichia coli O157. J. Clin. Microbiol. 43: 2668-2673.
64.Hughes, S., Bartholomew, B., Hardy, J. C., and Kramer, J. M. 1988. Potential application of a HEp-2 cell assay in the investigation of Bacillus cereus emetic syndrome food poisoning. FEMS Microbiol. Lett. 52: 7-12.
65.Isobe, M., Ishikawa, T., Suwan, S., Agata, N., and Ohta, M. 1995. Synthesis and activity of cereulide, a cyclic dodecadepsipeptide ionophore as emetic toxin from Bacillus cereus. Bioorg. Med. Chem. Lett. 5: 2855-2858.
66.Jackson, S. G., Goodbrand, R. B., Ahemd, R., and Kasatiya, S. 1995. Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation. Lett. Appl. Microbiol. 21: 103-105.
67.Jeyaseelan, K., Ma, D., and Armugam, A. 2001. Real-time detection of gene promoter activity: quantitation of toxin gene transcription. Nucleic Acids Res. 29: e58.
68.Johnson, M. R., Wang, K., Smith, J. B., Heslin, M. J., and Diasio, R. B. 2000. Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal. Biochem. 278: 175-184.
69.Kamat, A. S., Nerker, D. P., and Nair, P. M. 1989. Bacillus cereus in some Indian foods, incidence and antibiotic, heat and radiation resistance. J. Food Saf. 10: 31-41.
70.Kaufman, G. E., Blackstone, G. M., Vickery M. C. L., Bej, A. K., Bowers, J., Bowen, M. D., Meyer, R. F., Depaola, A. 2004. Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix. J. Food Prot. 67: 2424-2429.
71.Koo, K., and Jaykus, L. A. 2003. Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set. Appl. Environ. Microbiol. 69: 1082-1088.
72.Kotiranta, A., Lounatmaa, K., and Haapasalo, M. 2000. Epidemiology and pathogenesis of Bacillus cereus infections. Microbes Infect. 2: 189-198.
73.Lechner, S., Mayr, R., Francis, K. P., Prub, B. M., Kaplan, T., Wiebner-Gunkel, E., Stewart, G. S. A. B., and Scherer, S. 1998. Bacillus weihenstephanensis sp. Nov. is a new psychrotolerant speceis of the Bacillus cereus group. Int. J. Syst. Bacteriol. 48: 1373-1382.
74.Lee, P. S., Shaw, L. B., Choe, L. H., Mehra, A., Hatzimanikatis, V., and Lee, K. H. 2003. Insights into the relation between mRNA and protein expression patterns: II. Experimental observations in Escherichia coli. Biotechnol. Bioeng. 84: 834-841.
75.Levin, R.E. 2004. The application of real-time PCR to food and agricultural systems. Food Biotechnol. 18: 97-133.
76.Lindback, T., Fagerlund, A., Rodland, M. S., and Granum, P. E. 2004. Characterization of the Bacillus cereus Nhe enterotoxin. Microbiology 150: 3959-3967.
77.Livak, K. J. and Schmittgen, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods 25: 402-408.
78.Logan, N. A. 2005. International committee on systematics of prokaryotes: Subcommittee on the taxonomy of the genus Bacillus and related organisms. Minutes of the meeting, 30 July 2002, Paris, France. Int. J. Syst. Evol. Microbiol. 55: 977-979.
79.Lund, T., Debuyser, M. L., and Granum, P. E. 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol. Microbiol. 38: 254-261.
80.Lund, T. and Granum, P. E. 1996. Characterization of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiol. Lett. 141: 151-156.
81.Mahler, H., Pasi A, and Kramer J. M. 1997. Fulminant liver failure in association with the emetic toxin of Bacillus cereus. N. Engl. J. Med. 336: 1142-1148.
82.Mantynen, V. and Lindstrom, K. 1998. A rapid PCR-based DNA test for enterotoxic Bacillus cereus. Appl. Environ. Microbiol. 64: 1634-1639.
83.McKillip, J. L. and Drake, M. 2000. Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk. J. Food Prot. 63: 855-859.
84.McKillip, J. L. and Drake, M. 2004. Real-time nucleic acid-based detection methods for pathogenic bacteria in food. J. Food Prot. 67: 823-832.
85.Mikami, T., Horikawa, T., Murakami, T., Matsumoto, T., Yamakawa, A., Murayama, S., Katagiri, S., Shinagawa, K, and Suzuki, M. 1994. An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples. FEMS Microbiol. Lett. 119: 53-57.
86.Mikkola, R., Saris, N. E. L, Grigoriev, P. A., Andersson, M. A., and Salkinoja-Salonen, M. S. 1999. Ionophoretic properties and mitochondrial effects of cereulide-the emetic toxin of B. cereus. Eur. J. Biochem. 263: 112-117.
87.Moravek, M., Dietrich, R., Buerk, C., Broussolle, V., Guinebretiere, M. H., Granum, P. E., Nguyen-the, C., and Martlbauer, E., 2006. Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses. FEMS Microbiol. Lett. 257: 293-298.
88.Nakamura, L. K. 1998. Bacillus pseudomycoides sp. nov. Int. J. Syst. Bacteriol. 48: 1373-1382.
89.Notermans, S., and Tatini, S. 1993. Characterization of Bacillus cereus in relation to toxin production. Neth. Milk Dairy J. 47: 71-77.
90.Okstad, O. A., Gominet, M., Purnelle, B., Rose, M. Lereclus, D., and Kolsto, A. B. 1999. Sequence analysis of three Bacillus cereus loci carrying PlcR-regulated genes encoding degradative enzymes and enterotoxin. Microbiology 145: 3129-3138.
91.O’Mahony, J. and Hill, C. 2004. Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Appl. Environ. Microbiol. 70: 4561-4568.
92.Oslon, S. J., MacKinon, L. C., Goulding, J. S., Bean, N. H., and Slutsker, L. 2000. Surveillance for foodborne-disease outbreaks-United States, 1993-1997. Morb. Mortal. Wkly. Rep. 49/SS-1: 1-51.
93.Pan, T. M., Chiou, C. S., Hsu, S. Y., Huang, H. C., Wang, T. K., Chiu, S. I., Yea, H. L. and Lee, C. L. 1996. Food-borne disease outbreaks in Taiwan, 1994. J. Formosan Med. Assoc. 95: 417-420.
94.Pan, T. M., Wang, T. K., Lee, C. L., Chien, S. W., and Horng, C. B. 1997. Food-borne disease outbreaks due to bacteria in Taiwan, 1986-1995. J. Clin. Microbiol. 35: 1260-1262.
95.Pang, X., Lee, B., Chiu, L., Preiksaitis, J. K., and Monroe, S. S., 2004. Evaluation and validation of real-time reverse transcription-PCR assay using the LightCycler system for detection and quantitation of norovirus. J. Clin. Microbiol. 42: 4679-4685.
96.Panicker, G, Myers, M. L., and Bej, A. K. 2004. Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Appl. Environ. Microbiol. 70: 498-507.
97.Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time PCR. Nucl. Acids Res. 29: 2002-2007.
98.Pfaffl, M. W., Horgan, G. W., and Dempfle, L., 2002. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucl. Acids Res. 30: e36.
99.Pfaffl, M. W., Tichopad, A., Prgomet, C., and Neuvians, T. P. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26: 509-515.
100.Phelps, R. J. and McKillip, J. L. 2002. Enterotoxin production in natural isolates of Bacillaceae outside the Bacillus cereus group. Appl. Environ. Microbiol. 68: 3147-3151.
101.Pichon, J. P., Bonnaud, B., Cleuziat, P., Mallet, F. 2006. Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling. Nucleic Acids Res. 34: e46.
102.Prescott, L. M., Harley, J. P., Klein, D. A. 2002. Genes: structure, replication, and mutation. In: Microbiology. 5th ed. McGraw-Hill, New York. pp. 242-244.
103.Priha, O., Hallamaa, K., Saarela, M., Raaska, L. 2004. Detection of Bacillus cereus group bacteria from cardboard and paper with real-time PCR. J. Ind. Microbiol. Biotechnol. 31: 161-169.
104.Prub, B. M., Dietrich, R., Nibler, B., Martlbauer, E., and Scherer, S. 1999. The hemolytic enterotoxin Hbl is broadly distributed among species of the Bacillus cereus group. Appl. Environ. Microbiol. 65: 5436-5442.
105.Rajkowski, K. T., and Smith, J. L. 2001. Update: food poisoning and other diseases induced by Bacillus cereus. In: Hui, Y. H., Pierson, M. D., and Gorham, J. R. (Eds.), Food-borne Disease Handbook, vol. 1. Marcel Dekker, New York, pp. 61-76.
106.Rhodehamel, E. J., and Harmon, S. M. 1998. Bacillus cereus. In: Bacteriological Analytical Manual, 8th Ed. Revision A. US-FDA.
107.Rodriguez-Lazaro, D., Hernandez, M., Scortti, M., Esteve, T., Vazquez-Boland, J. A., and Pla, M. 2004. Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology. Appl. Environ. Microbiol. 70: 1366-1377.
108.Rodriguez-Lazaro, D., Jofre, A., Aymerich, T., Garriga, M., and Pla, M. 2005. Rapid quantitative detection of Listeria monocytogenes in salmon products: evaluation of pre-real-time PCR strategies. J. Food Prot. 68: 1467-1471.
109.Rowan, N. J., Deans, K., Anderson, J. G., Gemmell, C. G., Hunter, I. S., and Chaithong, T. 2001. Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae. Appl. Environ. Microbiol. 67: 3873-3881.
110.Rudi, K., Nogva, H. K., Moen, B., Nissen, H., Bredholt, S., Moretro, T., Naterstad, K., and Holck, A. 2002. Development and application of new nucleic acid-based technologies for microbial community analyses in foods. Int. J. Food Microbiol. 78: 171-180.
111.Rusul, G. and Yaacob, N. H. 1995. Prevalence of Bacillus cereus in selected foods and detection of enterotoxin using TECRA-VIA and BCET-RPLA. Int. J. Food Microbiol. 25: 131-139.
112.Ryan, P. A., Macmillan, J. D., and Zilliskas, B. A. 1997. Molecular cloning and characterization of the gene encoding the L1 and L2 components of hemolysin BL from Bacillus cereus. J. Bacteriol. 179: 2551-2556.
113.Sails, A. D., Fox, A. J., Bolton, F. J., Wareing, D. R., and Greenway, D. L. 2003. A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture. Appl. Environ. Microbiol. 69: 1383-1390.
114.Sakurai, N., Koike, K. A., Irie, Y., and Hayashi, H. 1994. The rice culture filtrate of Bacillus cereus isolated from emetic-type food poisoning causes mitochondrial sweeling in a HEp-2 cell. Microbiol. Immunol. 38: 337-343.
115.Sarker, M. R., Singh, U., and McClane, B. A. 2000. An update on Clostridum perfringens enterotoxin. J Nat. Toxins 9: 251-266.
116.Schlax, P. J. and Worhunsky, D. J. 2003. Translational repression mechanism in prokaryotes. Mol. Microbiol. 48: 1157-1169.
117.Schoeni J. L., Wong A. C., 2005. Bacillus cereus food poisoning and its toxins. J. Food Prot. 68: 636-648.
118.Seo, K. H., Brackett, R. E. 2005. Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay. J. Food Prot. 68: 59-63.
119.Shinagawa, K., Konuma, H., Sekita, H., and Sugii, S. 1995. Emesis of rhesus monkeys induced by intragastric administration with the HEp-2 vacuolation factor (cereulide) produced by Bacillus cereus. FEMS Microbiol. Lett. 130: 87-90.
120.Simone, E., Goosen, M., Notermans, S. H. W., and Borgdorff, M. W. 1997. Investigations of foodborne diseases by food inspection services in The Netherlands, 1991-1994. J. Food Prot. 60: 442-446.
121.Simonen, M., and Palva, I. 1993. Protein secretion in Bacillus species. Microbiol. Rev. 57: 109-137.
122.Slamti, L., Perchat, S., Gominet, M., Vilas-Boas, G., Fouet, A., Mock, M., Sanchis, V., Chaufaux, J., Goohar, M., and Lereclus, D. 2004. Distinct mutantions in PlcR explain why some strains of the Bacillus cereus group are nonhemolytic. J. Bacteriol. 186: 3531-3538.
123.Sutherland, A. D. 1993. Toxin production by Bacillus cereus in dairy products. J. Dairy Res. 60: 569-574.
124.Sutherland, A. D. and Limond, A. M. 1993. Influence of pH and sugars on the growth and production of diarrhoeagenic toxin by Bacillus cereus. J. Dairy Res. 60: 575-580.
125.Suzuki, T., Higgins, P. J., and Crawford, D. R. 2000. Control selection for RNA quantitation. Biotechniques. 29: 332-337.
126.Szabo, R. A., Speirs, J. L. and Akhtar, M. 1991. Cell culture detection and conditions for production of a Bacillus cereus heating: a provisional serotyping scheme. J. Med. Microbiol. 8: 543-550.
127.Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T., Igout, A., and Heinen, E. 1999. Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75: 291-295.
128.Tsai, T.Y., Lee, W.J., Huang, Y.J., Chen, K.L., and Pan, T.M., 2006. Detection of viable enterohemorrhagic Escherichia coli O157 using the combination of immunomagnetic separation with the reverse transcription multiplex TaqMan PCR system in food and stool samples. J. Food Prot. 69: 2320-2328.
129.Tsen, H. Y., Chen, M. L., Hsieh, Y. M., Sheu, S. J., and Chen, Y. L. 2000. Bacillus cereus group strains, their hemolysin BL activity, and their detection in foods using a 16S RNA and hemolysin BL gene-targeted multiplex polymerase chain reaction system. J. Food Port. 63: 1496-1502.
130.Turnbull, P. C. B., Kramer, J. M., Jorgensen, K., Gilbert, R. J., and Melling, J. 1979. Properties and production characteristics of vomiting, diarrheal, and necrotizing toxins of Bacillus cereus. Am. J. Clin. Nutr. 32: 219-228.
131.Vaisanen, O. M., Mwaisumo, N. J., and Salkinoja-Salonen, M. S. 1991. Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature and fatty acid analysis. J. Appl. Bacteriol. 70: 315-324.
132.Vandecasteele, S. J., Peetermans, W. E., Merckx, R., and Van Eldere, J. 2001. Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J. Bacteriol. 183: 7094-7101.
133.Vandesompele, J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., and Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3: Research 0034.
134.Vidal, R., Vidal, M., Lagos, R., Levin, M., and Prado, V. 2004. Multiplex PCR for diagnosis of enteric infections associated with diarrheagenic Escherichia coli. J. Clin. Microbiol. 42: 1787-1789.
135.Vilas-Boas, G., Sanchis, V., Lereclus, D., Lemos, M. V. F., and Bourguet, D. 2002. Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis. Appl. Environ. Microbiol. 68: 1414-1424.
136.Weber, T., Marahiel, M.A., 2001. Exploring the domain structure of modular nonribosomal peptide synthetases. Structure 9: R3-R9.
137.Wong, H., Chang, M., and Fan. J. 1988. Incidence and characterization of Bacillus cereus isolates containing dairy products. Appl. Environ. Microbiol. 54: 699-702.
138.Wong, M. L. and Medrano J. F. 2005. Real-time PCR for mRNA quantitation. Biotechniques. 39: 75-85.
139.Yamane, K., Bunai, K., and Kakeshita, H. 2004. Protein traffic for secretion and related machinery of Bacillus subtilis. Biosci. Biotechnol. Biochem. 68: 2007-2023.
140.Yuan, J. S., Reed, A., Chen, F., and Stewart, C. N. 2006. Statistical analysis of real-time PCR data. BMC Bioinformatics. 7: 85.
141.Zangenberg, G., Saiki, R., and Reynolds, R. 1999. Multiplex PCR: Optimization guidelines, In: Innis, M. A., Gelfand, D. H., and Sninsky, J. J. (Eds.), PCR Applications. Academic Press, San Diego, CA, USA. pp. 73-94.