臺灣博碩士論文加值系統

本研究目的旨在建立專一性探針，可應用於生物晶片的製作，快速篩檢常見的水產養殖病源菌。於NCBI資料庫先搜尋已發表之弧菌、嗜水性產氣單胞菌及愛德華氏菌所有菌株的SSU rRNA序列，應用PHYLIP軟體做養殖池水常見環境微生物類緣遺傳分析種的歧異度，去除序列保守區後，選取變異序列做為探針的雜合目標，結果篩選出探針VA180、VA625、VP450、AH460、AH642、ET996。以CCRC菌種保存中心之常見養殖池水生菌菌株(Aeromonas, Edwardsiella, Achromobacter, Alcaligenes, Flavobacterium, Moraxella, Pasteurella, Pseudomonas, Vibrio等50餘株菌)作為標準菌株與探針雜合，排除雜菌試驗顯示常見的水產養殖環境微生物諸如Achromobacter sp., Alcaligenes sp., Flavobacterium sp., Moraxella sp., Pasterurella haemolytica, Pseudomonas aeruginosa,等皆無法雜合，確立本實驗設計探針具專一性。其中AH460、AH642可以檢測嗜水性產氣單胞菌，而ET996則可單獨偵測到愛德華氏菌。VA180、VA625、VP450可雜合到常見的台灣鹹水養殖池病原菌V. alginolyticus，V. harveyi，V. parahaemolyticus及V. vulnificus。改良後雜合方式不須萃取純DNA、不需要pre-hybridization及blocking reagent、雜合不需要blocking reagent且雜合時間1分鐘即可、後續清洗步驟約三小時亦須要blocking reagent、呈色需要12~16小時。耗時一天半左右。PDMS微流道晶片不需要pre-hybridization及blocking reagent且雜合時間在10分鐘以內即可、後續清洗步驟約10分鐘亦不須blocking reagent、不需要呈色直接由光電檢測即得知結果。耗時約四十分鐘左右。

The purpose of this study is to establish a specific oligonucleotide probe that can be applied for fabrication of a biochip. Sequences of Vibrio, Aeromonas, Edwardsiella 16S rDNA were downloaded from NCBI database. They were analyzed by PHYLIP software to search species diversification. The variation segment other than conservative region of 16S rDNA was picked up as target for probing. Six oligonucleotides VA180, VA625 and VP450、AH460、AH642、ET996 were selected . VA180, VA625 and VP450 could hybridize 16S rDNA of V. alginolyticus, V. harveyi,V. parahaemolyticus and V. vulnificus. But, these probes could not detect 16S rDNA of Achromobacter sp., Alcaligenes sp., Flavobacterium sp., Moraxella sp., Pasterurella haemolytica, and Pseudomonas aeruginosa. But, these probes could not detect 16S rDNA of Achromobacter sp., Alcaligenes sp., Flavobacterium sp., Moraxella sp., Pasterurella haemolytica, and Pseudomonas aeruginosa. Surprisingly, none were detected except Aeromonas and Edwardsiella. In addition, AH460、AH642 was able to detect Aeromonas , while ET996 can only detect Edwardsiella. VA180, VA625 and VP450 can only detect Vibrio sp. Neither needs to extract pure DNA to modify tradition hybridization methodology nor need pre-hybridization and blocking reagent. Hybridization does not need blocking reagent and only cost one minute hybridized. Washing takes 3 hour, and presenting color takes 12~16 hours. All steps take about one and half day. PDMS chip doesn’t need pre-hybridization and blocking reagent. Hybridizing finishes in ten minutes and washing takes about ten minutes and need neither blocking reagent nor pre-hybridization. By photoelectric inspection can receive the result directly. These steps take about forty minutes.

目錄
謝辭 ................................................................................................. i
中文摘要 ............................................................................................... ii
英文摘要 ............................................................................................... iii
目錄 ................................................................................................. iv
表目錄 ................................................................................................. vi
圖目錄 ................................................................................................. vii
壹、前言 ................................................................................................. 1
一、 研究動機與目的..................................................................... 1
二、 文獻探討................................................................................. 2
貳、實驗材料 ...................................................................................... 16
一、 標準菌株................................................................................. 16
二、 購買之標準菌株冷凍乾燥管開管及培養............................. 16
三、 菌種保種................................................................................. 17
四、 探針合成................................................................................. 17
參、實驗方法 ....................................................................................... 18
一、 專一性SSU rRNA寡核苷酸探針建立及演化樹構築....... 18
二、 革蘭氏染色法....................................................................... 19
三、 標準菌株大量DNA萃取..................................................... 20
四、 核酸透析純化....................................................................... 20
五、 標準菌株大量RNA萃取..................................................... 21
六、 DNA & RNA濃度定量........................................................ 22
七、 計數定量............................................................................... 22
八、 點漬(Dotting) ....................................................................... 23
九、 雜合試驗............................................................................... 24
十、 改良傳統雜合試驗............................................................... 26
十一、 螢光探針雜交參數測試....................................................... 27
十二、 PDMS(Polydimethylsiloxane)晶片實做.............................. 30
十三、 統計分析............................................................................... 30
肆、結果 ....................................................................................... 31
一、 建立專一性SSU rRNA寡核苷酸探針................................... 31
二、 SSU rRNA 寡核苷酸探針鑑別水中菌最佳反應條件........... 32
三、 縮短SSU rRNA寡核苷酸探針數檢測A. hydrophila、E. tarda.......................................................................................... 34
四、 養殖池水常見水生菌之排除................................................... 35
五、 快速檢驗細菌的模式............................................................... 36
六、 螢光探針雜交參數測試........................................................... 39
七、 PDMS微流道晶片................................................................... 41
伍、討論 ....................................................................................... 43
一、 自訂SSU rRNA寡核苷酸探針的可行性............................... 43
二、 傳統雜合方式改良................................................................... 44
三、 PDMS流道晶片........................................................................ 46
陸、參考文獻 ....................................................................................... 49
柒、附件
....................................................................................... 101

表目錄
表一 實驗使用之常見養殖微生物16S rRNA序列來源…….…..… 66
表二 細菌最適培養基及培養溫度…………….................................. 67
表三 篩選高度專一性寡核苷酸探針.... ……………......................... 68
表四 實驗菌種來源…………….....…................................................. 69
表五 寡核苷酸探針雜合常見養殖細菌性病原菌.............................. 70
表六 水產環境微生物之型態觀察、氧化酵素及探針雜合................ 71
表七 以E.tarda 估算cfu/mL 可萃取實際DNA量………….…… 72
表八 雜合比較圖……………………….............................................. 73



圖目錄
圖一 convertible filtration manifold system.................. 74
圖二 DIG探針靈敏度測試........................................... 75
圖三 探針雜合菌體之DNA需求量............................. 76
圖四 寡核苷酸探針與標準病源菌株雜合檢測........... 77
圖五 SSU rRNA寡核苷酸探針檢測弧菌專一性測試 78
圖六 以寡核苷酸探針雜合養殖池水檢測比例圓形示意圖................................................................... 79
圖七-1~3 寡核苷酸數目對專一性之影響........................... 80~82
圖八 Nylon membrane 電顯圖..................................... 83
圖九 測試DNA對薄膜飽和度.................................... 84
圖十 測試blocking reagent的必需性........................... 85
圖十一 雜合時間的測試................................................... 86
圖十二-1~2 以細胞粗萃物雜合探針並比較鹼變性的影響力........................................................................... 87~88
圖十三-1~2 Phylip演化分析.................................................... 89~90
圖十四 Alex Fluor 532螢光探針雜合.............................. 91
圖十五 HEX螢光探針雜合.............................................. 92
圖十六-1 UV能量固定......................................................... 93
圖十六-2 熱能量固定........................................................... 94
圖十七 測試螢光探針雜合需要時間............................... 95
圖十八-1 二層型PDMS微流道晶片.................................. 96
圖十九 探針在薄膜上移動現象....................................... 97
圖二十 三層型PDMS微流道晶片.................................. 98
圖二十一-1~2 PDMS chip 操作.................................................. 99~100

尤智立,2003. 嗜高溫纖維分解菌纖維分解酵素的探討。國立中山大學生命科學系碩士論文。
冉繁華, 2003. 益生菌之應用(上)。養殖漁業42: 12-14
李國誥,2003. 養殖水產生物病害防治。行政院農業委員會水產試驗所
李國誥,2004. 水產用藥的應用概要 。養殖漁業經營管理手冊技術篇。
李鍾熙,2005. 台灣科技及資訊委員會生物晶片專案報告。
張正芳,2003. 海水魚常見之疾病與防治。海水魚常見之疾病與防治。行政院農業委員會水產試驗所
張輝盛,2004. 新型高靈敏度之微型懸臂樑生物感測器應用於免疫分析法之研究。國立臺灣海洋大學機械與輪機工程學系碩士論文
陳玉萍, 2003. 嗜水性產氣單胞菌及愛德華氏菌致病基因表現與感染病魚關係之研究。台灣海洋大學水產養殖系碩士論文
陳立民, 2001. 應用16S rRNA寡核苷酸探針偵測淡水養殖池底泥微生物群相的異動。台灣海洋大學水產養殖系碩士論文
陳孝昭, 2002. 利用SSU rRNA及致病基因寡核苷酸探針鑑別淡水養殖池嗜水性產氣單胞菌及愛德華氏菌。台灣海洋大學水產養殖系碩士論文
陳建初, 1994. 水質分析第七版。九大圖書有限公司
曾怡禎，張權英,2001. 利用分子生物方法分析微生物社會的結構。環境保護分子生物科技策略論壇。
鄢靈湘,2003. 以多套式聚合酶連鎖反應快速檢測腸炎弧菌之研究。國立臺灣海洋大學食品科學系。
蔡文城,1996. 微生物學。第三版。藝軒圖書出版社，台北，台灣。
蔡耀欣, 2001. 應用16S rRNA專一性寡核苷酸探針檢定常見水生病原菌－嗜水性產氣單胞菌與愛德華氏菌，及利用聚合酶連鎖反應法檢測致病基因。台灣海洋大學水產養殖系碩士論文
鍾婉瑜,2004. 以聚合酶連鎖反應技術鑑定水產病原弧菌之親源關係 之研究 。國立台灣海洋大學水產養殖系碩士論文。
Abe, A., Ohashi, E., Ren, H., Hayashi, T., Endo, H., 2006. Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus. Microbiol. Res. (in Press)
Achenbach-Richter, L., Gupta, R., Stetter, K.O., Woese, C.R., 1987. Were the original eubacteria thermophiles? Syst. Appl. Microbiol. 9: 34-39.
Anderson,D.P., 1992. Immunostimulant,Adjuvants and Vaccine Carriers in Fish:Application to Aquaculture.
Andrade, H.M., Reis, A.B., Dos Santos, S.L., Volpini, A.C., Marques, M.J., Romanha, A.J., 2006. Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs. Vet. Parasitol. (in Press)
Alm, E.W., Zheng, D.,Stahl, D.A.and Raskin, L.,1996.Characterization of Universal Small-Subunit rRNA Hybridization Probes for Quantitative Molecular Microbial Ecology Studies. Appl. Environ. Microbiol. 62(12): 4504-13.
Austin , B. and Austin , D.A., 1999. Bacterial Fish Pathogens : Disease of Farmed and Wild Fish . Springer, New York .
Barns, S.M., Delwiche, C.F., Palmer, J.D., Dawson, S.C., Hershberger, K.L., Pace, N.R., 1996. Phylogenetic perspective on microbial life in hydrothermal ecosystems, past and present. Ciba found. Symp. 202:24-32.
Bau, M., Castella, G., Bragulat, M.R., Cabanes, F.J., 2006. RFLP characterization of aspergillus niger aggregate species from grapes from europe and israel. Int. J. Food Microbiol. (in Press)
Baumann, P. and Baumann, L., 1977. Biology of the marine enterobacteria: Genera beneckea and photobacterium. Annu. Rev. Microbiol. 31: 39-61.
Borsting, C., Sanchez, J.J., Morling, N., 2004. Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray. Int. J. Legal Med. 118: 75-82.
Brosius,J., Palmer,M.L., Kennedy,P.J., Noller,H.F., 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 75 (10): 4801-4805 .
Bruno, W.J., Socci, N.D., Halpern, A.L., 2000. Weighted neighbor joining: A likelihood-based approach to distance-based phylogeny reconstruction. Mol. Biol. Evol. 17: 189-197.
Busse, H.J., Denner, E.B., Lubitz, W., 1996. Classification and identification of bacteria: Current approaches to an old problem. overview of methods used in bacterial systematics. J. Biotechnol. 47: 3-38.
Campos-Macias, P., Arenas, R., Vega-Memije, M., Kawasaki, M., 2006. Sporothrix schenckii type 3D (mtDNA-RFLP): Report of an osteoarticular case. J. Dermatol. 33: 295-299.
Chang, A.K., Kim, H.Y., Park, J.E., Acharya, P., Park, I.S., Yoon, S.M., You, H.J., Hahm, K.S., Park, J.K., Lee, J.S., 2005. Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis. J. Bacteriol. 187: 6909-6916.
Chatzidaki-Livanis, M., Jones, M.K., Wright, A.C., 2006. Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon. J. Bacteriol. 188: 1987-1998.
Cheng, J., Sheldon, E.L., Wu, L., Heller, M.J., O'Connell, J.P., 1998. Isolation of cultured cervical carcinoma cells mixed with peripheral blood cells on a bioelectronic chip. Anal. Chem. 70: 2321-2326.
Chen, J.D., H. C.,Chen,Y. S.,Tsai,and S.C.,Huang,2006.Use of phylogenetically based hybridization probes for rapid discrimination of Aeromonas hydrophila and Edwardsiella tarda from aquaculture environment. J. Fish. Soc. Taiwan, 33(2) (in Press)
Cohan, F.M., 2002a. Sexual isolation and speciation in bacteria. Genetica 116: 359-370.
Cohan, F.M., 2002b. What are bacterial species? Annu. Rev. Microbiol. 56: 457-487.
Cole, J.R., Chai, B., Farris, R.J., Wang, Q., Kulam, S.A., McGarrell, D.M., Garrity, G.M., Tiedje, J.M., 2005. The ribosomal database project (RDP-II): Sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res. 33: D294-6.
Cole, J.R., Chai, B., Marsh, T.L., Farris, R.J., Wang, Q., Kulam, S.A., Chandra, S., McGarrell, D.M., Schmidt, T.M., Garrity, G.M., Tiedje, J.M., Ribosomal Database Project., 2003. The ribosomal database project (RDP-II): Previewing a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucleic Acids Res. 31: 442-443.
Cosner, M.E., Jansen, R.K., Moret, B.M., Raubeson, L.A., Wang, L.S., Warnow, T., Wyman, S., 2000. A new fast heuristic for computing the breakpoint phylogeny and experimental phylogenetic analyses of real and synthetic data. Proc. Int. Conf. Intell. Syst. Mol. Biol. 8: 104-115.
Debeljak, N., Horvat, S., Vouk, K., Lee, M., Rozman, D., 2000. Characterization of the mouse lanosterol 14alpha-demethylase (CYP51), a new member of the evolutionarily most conserved cytochrome P450 family. Arch. Biochem. Biophys. 379: 37-45.
DeLong, E.F., 1992. Archaea in coastal marine environments. Proc. Natl. Acad. Sci. U. S. A. 89: 5685-5689.
Di Pinto, A., Ciccarese, G., Tantillo, G., Catalano, D., Forte, V.T., 2005. A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. J. Food Prot. 68: 150-153.
Doyle, L.M., McInerney, J.O., Mooney, J., Powell, R., Haikara, A., Moran, A.P., 1995. Sequence of the gene encoding the 16S rRNA of the beer spoilage organism megasphaera cerevisiae. J. Ind. Microbiol. 15: 67-70.
Dvorak, J., Akhunov, E.D., Akhunov, A.R., Deal, K.R., Luo, M.C., 2006. Molecular characterization of a diagnostic DNA marker for domesticated tetraploid wheat provides evidence for gene flow from wild tetraploid wheat to hexaploid wheat. Mol. Biol. Evol. (in Press)
Euzeby,J.P.,1997. List of Bacterial Names with Standing in Nomenclature: a folder available on the Internet. Int.J.Syst.Bacteriol., 590-592.
Ferrari, M., Cremonesi, L., Bonini, P., Stenirri, S., Foglieni, B., 2005. Molecular diagnostics by microelectronic microchips. Expert Rev. Mol. Diagn. 5: 183-192.
Frankel, G., Riley, L., Giron, J.A., Valmassoi, J., Friedmann, A., Strockbine, N., Falkow, S., Schoolnik, G.K., 1990. Detection of shigella in feces using DNA amplification. J. Infect. Dis. 161: 1252-1256.
Fukunaga, K., Ichitani, K., Kawase, M., 2006. Phylogenetic analysis of the rDNA intergenic spacer subrepeats and its implication for the domestication history of foxtail millet, setaria italica. Theor. Appl. Genet. (in Press)
Gill, P., Jeffreys, A.J., Werrett, D.J., 1985. Forensic application of DNA 'fingerprints'. Nature 318: 577-579.
Gomez-Gil, B., Soto-Rodriguez, S., Garcia-Gasca, A., Roque, A., Vazquez-Juarez, R., Thompson, F.L., Swings, J., 2004. Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms. Microbiology 150: 1769-1777.
Gomez-Leon, J., Villamil, L., Lemos, M.L., Novoa, B., Figueras, A., 2005. Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (ruditapes decussatus) larvae associated with mass mortalities. Appl. Environ. Microbiol. 71: 98-104.
Gong, W., Fu, C.X., Luo, Y.P., Qiu, Y.X., 2006. Molecular identification of sinopodophyllum hexandrum and dysosma species using cpDNA sequences and PCR-RFLP markers. Planta Med. (in Press)
Gundersen, D.E., Lee, I.M., Schaff, D.A., Harrison, N.A., Chang, C.J., Davis, R.E., Kingsbury, D.T., 1996. Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). Int. J. Syst. Bacteriol. 46: 64-75.
Guo, W.L., Gong, L., Ding, Z.F., Li, Y.D., Li, F.X., Zhao, S.P., Liu, B., 2006. Genomic instability in phenotypically normal regenerants of medicinal plant codonopsis lanceolata benth. et hook. f., as revealed by ISSR and RAPD markers. Plant Cell Rep. (in Press)
Gurtler, V. and Stanisich, V., 1996. New Approaches to Typing and Identification of Bacteria using the 16S-23S rDNA Spacer Region. 3 pp.
Gurtner, C., Tu, E., Jamshidi, N., Haigis, R.W., Onofrey, T.J., Edman, C.F., Sosnowski, R., Wallace, B., Heller, M.J., 2002. Microelectronic array devices and techniques for electric field enhanced DNA hybridization in low-conductance buffers. Electrophoresis 23: 1543-1550.
Gutierrez-Cazarez, Z., Qadri, F., Albert, M.J., Giron, J.A., 2000. Identification of enterotoxigenic Escherichia coli harboring longus type IV pilus gene by DNA amplification. J. Clin. Microbiol. 38: 1767-1771.
Hara-Kud, Y., Miwa, N., Yamasaki, S., Yatsuyanagi, J., Iwade, Y., Takahash, H., Miyasaka, J., 2005. Quantitative detection of Vibrio vulnificus in seafood. Kansenshogaku Zasshi 79: 931-936.
Hill, A.V. and Jeffreys, A.J., 1985. Use of minisatellite DNA probes for determination of twin zygosity at birth. Lancet 2: 1394-1395.
Hill, W.E., Payne, W.L., Aulisio, C.C., 1983. Detection and enumeration of virulent yersinia enterocolitica in food by DNA colony hybridization. Appl. Environ. Microbiol. 46: 636-641.
H'ng, M.W., Chew, W.Y., Tan, B.K., 2005. Necrotizing fasciitis caused by Vibrio vulnificus: A review of four cases in a singapore tertiary hospital. J. Trauma 59: 482-485.
Ho, S. H., C. Z. Lin, Y. C. Chen and Y. L. Song. 2002. Effects of cecropin peptides on aquatic animal pathogens and shrimp hemocytes. Fish Pathology, 37(1): 7-12
Hurley, C.C., Quirke, A.M., Reen, F.J., Boyd, E.F., 2006. Four genomic islands that mark post-1995 pandemic Vibrio parahaemolyticus isolates. BMC Genomics 7: 104
Jeffreys, A.J., Brookfield, J.F., Semeonoff, R., 1985. Positive identification of an immigration test-case using human DNA fingerprints. Nature 317: 818-819.
Jeffreys, A.J., Wilson, V., Thein, S.L., 1985. Individual-specific 'fingerprints' of human DNA. Nature 316: 76-79.
Jiang, B., Bartlett, M.S., Allen, S.D., Smith, J.W., Wheat, L.J., Connolly, P.A., Lee, C.H., 2000. Typing of histoplasma capsulatum isolates based on nucleotide sequence variation in the internal transcribed spacer regions of rRNA genes. J. Clin. Microbiol. 38: 241-245.
Jung, C.R., Park, M.J., Heo, M.S., 2005. Immunization with major outer membrane protein of Vibrio vulnificus elicits protective antibodies in a murine model. J. Microbiol. 43: 437-442.
Kaine, B.P., Gupta, R., Woese, C.R., 1983. Putative introns in tRNA genes of prokaryotes. Proc. Natl. Acad. Sci. U. S. A. 80: 3309-3312.
Kaneko, T. and Colwell, R.R., 1974. Distribution of Vibrio parahaemolyticus and related organisms in the atlantic ocean off south carolina and georgia. Appl. Microbiol. 28: 1009-1017.
Kapperud, G., Dommarsnes, K., Skurnik, M., Hornes, E., 1990. A synthetic oligonucleotide probe and a cloned polynucleotide probe based on the yopA gene for detection and enumeration of virulent Yersinia enterocolitica. Appl. Environ. Microbiol. 56: 17-23.
Kawatsu, K., Ishibashi, M., Tsukamoto, T., 2006. Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable direct hemolysin produced by Vibrio parahaemolyticus in enrichment cultures of stool specimens. J. Clin. Microbiol. 44: 1821-1827.
Kim, N.J., Sugano, Y., Hirai, M., Shoda, M., 2000. Removal of a high load of ammonia gas by a marine bacterium, Vibrio alginolyticus. J. Biosci. Bioeng. 90: 410-415.
Kita-Tsukamoto, K., Oyaizu, H., Nanba, K., Simidu, U., 1993. Phylogenetic relationships of marine bacteria, mainly members of the family vibrionaceae, determined on the basis of 16S rRNA sequences. Int. J. Syst. Bacteriol. 43: 8-19.
Krengel, S., Kuhnt-Lenz, K., Fetscher, S., Solbach, W., Wolff, H.H., 2003. Vibrio vulnificus sepsis. J. Dtsch. Dermatol. Ges. 1: 959-961.
Kruger, A., Padola, N.L., Parma, A.E., Lucchesi, P.M., 2006. Intraserotype diversity among argentinian verocytotoxigenic escherichia coli detected by random amplified polymorphic DNA analysis. J. Med. Microbiol. 55: 545-549.
Kudo, S., Imai, N., Nishitoba, M., Sugiyama, S., Magariyama, Y., 2005. Asymmetric swimming pattern of Vibrio alginolyticus cells with single polar flagella. FEMS Microbiol. Lett. 242: 221-225.
Kumar, S. and Gadagkar, S.R., 2000. Efficiency of the neighbor-joining method in reconstructing deep and shallow evolutionary relationships in large phylogenies. J. Mol. Evol. 51: 544-553.
Kumar, S., Tamura, K., Nei, M., 1994. MEGA: Molecular evolutionary genetics analysis software for microcomputers. Comput. Appl. Biosci. 10: 189-191.
Lee, C.T., Amaro, C., Sanjuan, E., Hor, L.I., 2005. Identification of DNA sequences specific for Vibrio vulnificus biotype 2 strains by suppression subtractive hybridization. Appl. Environ. Microbiol. 71: 5593-5597.
Lee, K. K., P. C. Liu, W. H. Chuang, 2002, Pathogenesis of Gastroenteritis Caused by Vibrio carchariae in Cultured Marine Fish. Marine Biotechnology, 4: 267-277.
Lee, K.K., Liu, P.C., Huang, C.Y., 2003. Vibrio parahaemolyticus infectious for both humans and edible mollusk abalone. Microbes Infect. 5: 481-485.
Lee, K.-K., P.-C. Liu, C.-Y. Huang, 2003, Vibrio parahaemolyticus infectious for both humans and edible mollusk abalone, Microbes and Infection, 5: 481-485.
Lee, K.K., Yu, S.R., Yang, T.I., Liu, P.C., Chen, F.R., 1996. Isolation and characterization of Vibrio alginolyticus isolated from diseased kuruma prawn, penaeus japonicus. Lett. Appl. Microbiol. 22: 111-114.
Lewis, P.R., Cook, L., Drewitt-Smith, J., McEwen, A.D., Granger, L.V., 2005. Septicaemia secondary to Vibrio vulnificus cellulitis. Commun. Dis. Intell. 29: 305-307.
Liu, C.H., Cheng, W., Hsu, J.P., Chen, J.C., 2004. Vibrio alginolyticus infection in the white shrimp litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing. Dis. Aquat. Organ. 61: 169-174.
Liu, P.C., Lee, K.K., Yii, K.C., Kou, G.H., Chen, S.N., 1996. Isolation of Vibrio harveyi from diseased kuruma prawns penaeus japonicus. Curr. Microbiol. 33: 129-132.
Liu, W.T. and Zhu, L., 2005. Environmental microbiology-on-a-chip and its future impacts. Trends Biotechnol. 23: 174-179.
Lukacs, G.L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., Verkman, A.S., 2000. Size-dependent DNA mobility in cytoplasm and nucleus. J. Biol. Chem. 275: 1625-1629.
Maidak, B.L., Cole, J.R., Lilburn, T.G., Parker, C.T.,Jr, Saxman, P.R., Stredwick, J.M., Garrity, G.M., Li, B., Olsen, G.J., Pramanik, S., Schmidt, T.M., Tiedje, J.M., 2000. The RDP (ribosomal database project) continues. Nucleic Acids Res. 28: 173-174.
Maidak, B.L., Cole, J.R., Parker, C.T.,Jr, Garrity, G.M., Larsen, N., Li, B., Lilburn, T.G., McCaughey, M.J., Olsen, G.J., Overbeek, R., Pramanik, S., Schmidt, T.M., Tiedje, J.M., Woese, C.R., 1999. A new version of the RDP (ribosomal database project). Nucleic Acids Res. 27: 171-173.
Maidak, B.L., Cole, J.R., Parker, C.T.,Jr, Garrity, G.M., Larsen, N., Li, B., Lilburn, T.G., McCaughey, M.J., Olsen, G.J., Overbeek, R., Pramanik, S., Schmidt, T.M., Tiedje, J.M., Woese, C.R., 1999. A new version of the RDP (ribosomal database project). Nucleic Acids Res. 27: 171-173.
Maidak, B.L., Larsen, N., McCaughey, M.J., Overbeek, R., Olsen, G.J., Fogel, K., Blandy, J., Woese, C.R., 1994. The ribosomal database project. Nucleic Acids Res. 22: 3485-3487.
Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R., McCaughey, M.J., Woese, C.R., 1997. The RDP (ribosomal database project). Nucleic Acids Res. 25: 109-111.
Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R., McCaughey, M.J., Woese, C.R., 1996. The ribosomal database project (RDP). Nucleic Acids Res. 24: 82-85.
Martin, C., Roberts, D., van Der Weide, M., Rossau, R., Jannes, G., Smith, T., Maher, M., 2000. Development of a PCR-based line probe assay for identification of fungal pathogens. J. Clin. Microbiol. 38: 3735-3742.
Mayer, W.E., Tichy, H., Klein, J., 1998. Phylogeny of african cichlid fishes as revealed by molecular markers. Heredity 80 ( Pt 6): 702-714.
McGill, T.J., Jurka, J., Sobieski, J.M., Pickett, M.H., Woese, C.R., Fox, G.E., 1986. Characteristic archaebacterial 16S rRNA oligonucleotides. Syst. Appl. Microbiol. 7: 194-197.
Mechichi, T., Stackebrandt, E., Gad'on, N., Fuchs, G., 2002. Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions, and description of Thauera phenylacetica sp. nov.,Thauera aminoaromatica sp. nov., and Azoarcus buckelii sp. nov. Arch. Microbiol. 178: 26-35.
Miyamoto, T., Miwa, H., Hatano, S., 1990. Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods. Appl. Environ. Microbiol. 56: 1480-1484.
Miyasaka, J., Yahiro, S., Arahira, Y., Tokunaga, H., Katsuki, K., Hara-Kudo, Y., 2005. Isolation of Vibrio parahaemolyticus and Vibrio vulnificus from wild aquatic birds in japan. Epidemiol. Infect. 1-6.
Molyneaux, B.J., Mulcahey, M.K., Stafford, P., Langford, G.M., 2000. Sequence and phylogenetic analysis of squid myosin-V: A vesicle motor in nerve cells. Cell Motil. Cytoskeleton 46: 108-115.
Musselwhite, M.J. and Jeffreys, D.A., 1985. The influence of spatial frequency on the reaction times and evoked potentials recorded to grating pattern stimuli. Vision Res. 25: 1545-1555.
Muyzer, G., Teske, A., Wirsen, C.O., Jannasch, H.W., 1995. Phylogenetic relationships of thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch. Microbiol. 164: 165-172.
O'brien, C.H. and Sizemore, R.K., 1979. Distribution of the luminous bacterium beneckea harveyi in a semitropical estuarine environment. Appl. Environ. Microbiol. 38: 928-933.
Oren, A., 2004. Prokaryote diversity and taxonomy: Current status and future challenges. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 359: 623-638.
Ota, S. and Li, W.H., 2000. NJML: A hybrid algorithm for the neighbor-joining and maximum-likelihood methods. Mol. Biol. Evol. 17: 1401-1409.
Otsuka, J., Terai, G., Nakano, T., 1999. Phylogeny of organisms investigated by the base-pair changes in the stem regions of small and large ribosomal subunit RNAs. J. Mol. Evol. 48: 218-235.
Oyaizu, H., Debrunner-Vossbrinck, B., Mandelco, L., Studier, J.A., Woese, C.R., 1987. The green non-sulfur bacteria: A deep branching in the eubacterial line of descent. Syst. Appl. Microbiol. 9: 47-53.
Pacheco, A.B., Guth, B.E., Soares, K.C., de Almeida, D.F., Ferreira, L.C., 1997. Clonal relationships among Escherichia coli serogroup O6 isolates based on RAPD. FEMS Microbiol. Lett. 148: 255-260.
Painter, J., Crozier, R.H., Crozier, Y.C., Clarke, M.F., 1997. Characterization of microsatellite loci for a co-operatively breeding honeyeater. Mol. Ecol. 6: 1103-1105.
Panicker, G. and Bej, A.K., 2005. Real-time PCR detection of Vibrio vulnificus in oysters: Comparison of oligonucleotide primers and probes targeting vvhA. Appl. Environ. Microbiol. 71: 5702-5709.
Parvathi, A., Kumar, H.S., Bhanumathi, A., Ishibashi, M., Nishibuchi, M., Karunasagar, I., Karunasagar, I., 2006. Molecular characterization of thermostable direct haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters in mangalore, india. Environ. Microbiol. 8: 997-1004.
Perumal, R., Isakeit, T., Menz, M., Katile, S., No, E.G., Magill, C.W., 2006. Characterization and genetic distance analysis of isolates of peronosclerospora sorghi using AFLP fingerprinting. Mycol. Res. 110: 471-478.
Rahman, M., Bhuiyan, N.A., Kuhn, I., Ramamurthy, T., Rahman, M., Mollby, R., Nair, G.B., 2006. Biochemical fingerprinting of Vibrio parahaemolyticus by the PhenePlate system: Comparison between pandemic and non-pandemic serotypes. Epidemiol. Infect. (in Press)
Rhee, J.E., Kim, K.S., Choi, S.H., 2005. CadC activates pH-dependent expression of the Vibrio vulnificus cadBA operon at a distance through direct binding to an upstream region. J. Bacteriol. 187: 7870-7875.
Rosche, T.M., Smith, D.J., Parker, E.E., Oliver, J.D., 2005. RpoS involvement and requirement for exogenous nutrient for osmotically induced cross protection in Vibrio vulnificus. FEMS Microbiol. Ecol. 53: 455-462.
Saitou, N. and Nei, M., 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.
Seo, Y.S., Lee, S.H., Shin, E.K., Kim, S.J., Jung, R., Hahn, T.W., 2006. Pulsed-field gel electrophoresis genotyping of salmonella gallinarum and comparison with random amplified polymorphic DNA. Vet. Microbiol. (in Press)
Shieh, W.Y., Chen, A.L., Chiu, H.H., 2000. Vibrio aerogenes sp. nov., a facultatively anaerobic marine bacterium that ferments glucose with gas production. Int. J. Syst. Evol. Microbiol. 50 Pt 1: 321-329.
Silva, A.E., Villanueva, W.J., Knidel, H., Bonato, V.C., Reis, S.F., Von Zuben, F.J., 2005. A multi-neighbor-joining approach for phylogenetic tree reconstruction and visualization. Genet. Mol. Res. 4: 525-534.
Southern, E.M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.
Stackebrandt, E., Ludwig, W., Weizenegger, M., Dorn, S., McGill, T.J., Fox, G.E., Woese, C.R., Schubert, W., Schleifer, K.H., 1987. Comparative 16S rRNA oligonucleotide analyses and murein types of round-spore-forming bacilli and non-spore-forming relatives. J. Gen. Microbiol. 133: 2523-2529.
Sugauchi, F., Mizokami, M., Orito, E., Ohno, T., Hayashi, K., Kato, T., Tanaka, Y., Kato, H., Ueda, R., 2000. Molecular evolutionary analysis of the complete nucleotide sequence of hepatitis B virus (HBV) in a case of HBV infection acquired through a needlestick accident. Clin. Infect. Dis. 31: 1195-1201.
Tada, J., Ohashi, T., Nishimura, N., Shirasaki, Y., Ozaki, H., Fukushima, S., Takano, J., Nishibuchi, M., Takeda, Y., 1992. Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol. Cell. Probes 6: 477-487.
Takahashi, K. and Nei, M., 2000. Efficiencies of fast algorithms of phylogenetic inference under the criteria of maximum parsimony, minimum evolution, and maximum likelihood when a large number of sequences are used. Mol. Biol. Evol. 17: 1251-1258.
Tanabe, T., Naka, A., Aso, H., Nakao, H., Narimatsu, S., Inoue, Y., Ono, T., Yamamoto, S., 2005. A novel aerobactin utilization cluster in Vibrio vulnificus with a gene involved in the transcription regulation of the iutA homologue. Microbiol. Immunol. 49: 823-834.
Tao, S.C., Gao, H.F., Cao, F., Ma, X.M., Cheng, J., 2003. Blocking oligo--a novel approach for improving chip-based DNA hybridization efficiency. Mol. Cell. Probes 17: 197-202.
Thompson, F.L., Gevers, D., Thompson, C.C., Dawyndt, P., Naser, S., Hoste, B., Munn, C.B., Swings, J., 2005. Phylogeny and molecular identification of Vibrios on the basis of multilocus sequence analysis. Appl. Environ. Microbiol. 71: 5107-5115.
Thompson, F.L., Iida, T., Swings, J., 2004. Biodiversity of Vbrios. Microbiol. Mol. Biol. Rev. 68: 403-31, table of contents.
Valladares, S., Sanchez, C., Martinez, M.T., Ballester, A., Vieitez, A.M., 2006. Plant regeneration through somatic embryogenesis from tissues of mature oak trees: True-to-type conformity of plantlets by RAPD analysis. Plant Cell Rep. (in Press)
van Belkum, A., 2006. Bergey's manual of systematic bacteriology (volume 2, parts A-C, 2nd edition). FEMS Immunol. Med. Microbiol. 46: 476.
Vossbrinck, C.R., Maddox, J.V., Friedman, S., Debrunner-Vossbrinck, B.A., Woese, C.R., 1987. Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes. Nature 326: 411-414.
Ward, L.N. and Bej, A.K., 2006. Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes. Appl. Environ. Microbiol. 72: 2031-2042.
Weagant, S.D., Jagow, J.A., Jinneman, K.C., Omiecinski, C.J., Kaysner, C.A., Hill, W.E., 1999. Development of digoxigenin-labeled PCR amplicon probes for use in the detection and identification of enteropathogenic yersinia and shiga toxin-producing Escherichia coli from foods. J. Food Prot. 62: 438-443.
Weisburg, W.G., Giovannoni, S.J., Woese, C.R., 1989. The deinococcus-thermus phylum and the effect of rRNA composition on phylogenetic tree construction. Syst. Appl. Microbiol. 11: 128-134.
Welsh, J. and McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18: 7213-7218.
Woese, C.R., 1987. Bacterial evolution. Microbiol. Rev. 51: 221-271.
Woese, C.R., Gutell, R., Gupta, R., Noller, H.F., 1983. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbiol. Rev. 47: 621-669.
Wong, H.C., Liu, S.H., Chen, M.Y., 2005. Virulence and stress susceptibility of clinical and environmental strains of Vibrio vulnificus isolated from samples from taiwan and the united states. J. Food Prot. 68: 2533-2540.
Wong, P.N., Mak, S.K., Lo, M.W., Lo, K.Y., Tong, G.M., Wong, Y., Wong, A.K., 2005. Vibrio vulnificus peritonitis after handling of seafood in a patient receiving CAPD. Am. J. Kidney Dis. 46: 87-90.
Xu, W.B., Guo, Q.S., Wang, C.L., 2006. RAPD analysis for genetic diversity of chrysanthemum morifolium. Zhongguo Zhong Yao Za Zhi 31, 18-21.