Chapter 4 : Molecular Identification

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Molecular Identification, Page 1 of 2

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There are numerous reports on typing or differentiation of strains within the same species of by various genetic fingerprinting methods. These include ribotyping, restriction fragment length polymorphism, amplified fragment length polymorphism, arbitrarily primed polymerase chain reaction PCR (AP-PCR) or randomly amplified polymorphic DNA PCR, enterobacterial intergenic consensus sequence PCR, pulsed-field gel electrophoresis, and multilocus sequence typing. These techniques require more attention than the gene probe and the PCR techniques and are not suitable for routine examination. Essential genes are conserved in bacteria and other organisms but the sequence may vary, and they are used for phylogenetic analysis. Genetic examination of the strains shows rare strains carrying the toxin genes that are reported to be major virulence genes in other bacterial species. Various 16S rRNA gene-targeted oligonucleotide probes to identify have been reported. Some strains of share important virulence genes with and less frequently with . The most frequently used systems are based on the multiplex PCR formats. Practical application of molecular identification techniques is limited mostly to medically important species. Selection of suitable target genes and fine-tuning of their detection conditions are the basis for development of more sophisticated molecular identification systems, including multiplex PCR and real-time PCR.

Citation: Nishibuchi M. 2006. Molecular Identification, p 44-64. In Thompson F, Austin B, Swings J (ed), The Biology of Vibrios. ASM Press, Washington, DC. doi: 10.1128/9781555815714.ch4

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Multiplex Real-Time PCR
Restriction Fragment Length Polymorphism
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1. Abbott, S. L.,, L. S. Seli,, M. Catino, Jr.,, M. A. Hartley, and, J. M. Janda. 1998. Misidentification of unusual Aeromonas species as members of the genus Vibrio: a continuing problem. J. Clin. Microbiol. 36:11031104.
2. Alam, M. J.,, K. Tomochika,, S. Miyoshi, and, S. Shinoda. 2002. Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan. FEMS Microbiol. Lett. 208:8387.
3. Alim, R. A., and, P. A. Manning. 1990. Biotype-specific probe for Vibrio cholerae serogroup O1. J. Clin. Microbiol. 28:823824.
4. Alim, R. A.,, U. H. Stroeher, and, P. A. Manning. 1988. Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the haemolysin of the haemolytic El Tor strain O17 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B. Mol. Microbiol. 2:481488.
5. Aono, E.,, H. Sugita,, J. Kawasaki,, H. Sakakibara,, T. Takahashi,, K. Endo, and, Y. Deguchi. 1997. Evaluation of the polymerase chain reaction method for identification of Vibrio vulnificus isolated from marine environments. J. Food Prot. 60:8183.
6. Arias, C. R.,, E. Garay, and, R. Aznar. 1995. Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water. Appl. Environ. Microbiol. 61:34763478.
7. Aznar, R.,, W. Ludwig,, R. I. Amann, and, K. H. Schleifer. 1994. Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oligonucleotide probes. Int. J. Syst. Bacteriol. 44:330337.
8. Baba, K.,, H. Shirai,, A. Terai,, K. Kumagai,, Y. Takeda, and, M. Nishibuchi. 1991. Similarity of the tdh gene-bearing plasmids of Vibrio cholerae non-01 and Vibrio parahaemolyticus. Microb. Pathog. 10:6170.
9. Banerjee, S. K.,, S. Pandian,, E. C. Todd, and, J. M. Farber. 2002. A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydropho-bic grid membrane filters. J. Food Prot. 65:10491053.
10. Basu, A.,, A. K. Mukhopadhyay,, P. Garg,, S. Chakraborty,, T. Rama-murthy,, S. Yamasaki,, Y. Takeda, and, G. B. Nair. 2000. Diversity in the arrangement of the CTX prophages in classical strains of Vibrio cholerae O1. FEMS. Microbiol. Lett. 182:3540.
11. Baudry, B.,, A. Fasano,, J. Ketley, and, J. B. Kaper. 1992. Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae. Infect. Immun. 60:428434.
12. Bej, A. K.,, D. P. Patterson,, C. W. Brasher,, M. C. Vickery,, D. D. Jones, and, C. A. Kaysner. 1999. Detection of total and he-molysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tlh, tdh, and trh. J. Microbiol. Methods 36:215225.
13. Bi, K.,, S. I. Miyoshi,, K. I. Tomochika, and, S. Shinoda. 2001. Detection of virulence associated genes in clinical strains of Vibrio mimicus. Microbiol. Immunol. 45:613616.
14. Biosca, E. G.,, J. D. Oliver, and, C. Amaro. 1996. Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus. Appl. Environ. Microbiol. 62:918927.
15. Bisharat, N.,, V. Agmon,, R. Finkelstein,, R. Raz,, G. Ben-Dror,, L. Lerner,, S. Soboh,, R. Colodner,, D. N. Cameron,, D. L. Wyk-stra,, D. L. Swerdlow, and, J. J. Farmer III. 1999. Clinical, epi-demiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacter-aemia in Israel. Lancet 354:14211424.
16. Blackstone, G. M.,, J. L. Nordstrom,, M. C. Vickery,, M. D. Bowen,, R. F. Meyer, and, A. DePaola. 2003. Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR. J. Microbiol. Methods 53:149155.
17. Brasher, C. W.,, A. DePaola,, D. D. Jones, and, A. K. Bej. 1998. Detection of microbial pathogens in shellfish with multiplex PCR. Curr. Microbiol. 37:101117.
18. Brauns, L. A.,, M. C. Hudson, and, J. D. Oliver. 1991. Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells. Appl. Environ. Microbiol. 57:26512655.
19. Brown, M. H., and, P. A. Manning. 1985. Haemolysin genes of Vibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains. FEMS Microbiol. Lett. 30:197201.
20. Byun, R.,, B. R. Elbourne,, R. Lan, and, P. R. Reeves. 1999. Evolutionary relationships of pathogenic clones of Vibrio cholerae by sequence analysis of four housekeeping genes. Infect. Immun. 7:111611124.
21. Cabrera-García, M. E.,, C. Vázquez-Salinas, and, E. I. Quiñones-Ramírez. 2004. Serologic and molecular characterization of Vibrio parahaemolyticus strains isolated from seawater and fish products of the Gulf of Mexico. Appl. Environ. Microbiol. 70:64016406.
22. Campbell, M. S., and, A. C. Wright. 2004. Real-time PCR analysis of Vibrio vulnificus from oysters. Appl. Environ. Microbiol. 69:71377144.
23. Cerda-Cuellar, M.,, J. Jofre, and, A. R. Blanch. 2000. A selective medium and a specific probe for detection of Vibrio vulnificus. Appl. Environ. Microbiol. 66:855859.
24. Chen, C.-H.,, T. Shimada,, N. Elhadi,, S. Radu, and, M. Nishibuchi. 2004. Phenotypic and genotypic characteristics of ctx+ strains of Vibrio cholerae isolated from seafood in Malaysia and the significance of the strains in epidemiology. Appl. Environ. Microbiol. 70:19641972.
25. Chow, K. H.,, T. K. Ng,, K. Y. Yuen, and, W. C. Yam. 2001. Detection of RTX toxin gene in Vibrio cholerae by PCR. J. Clin. Microbiol. 39:25942597.
26. Chowdhury, A.,, M. Ishibashi,, V. D. Thiem,, D. T. Tuyet,, T. V. Tung,, B. T. Chien,, L. von Seidlein,, D. G. Canh,, J. Clemens,, D. D. Trach, and, M. Nishibuchi. 2004a. Emergence and serovar transition of Vibrio parahaemolyticus pandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and 1999. Microbiol. Immunol. 48:319327.
27. Chowdhury, N. R.,, O. C. Stine,, J. G. Morris, and, G. B. Nair. 2004b. Assessment of evolution of pandemic Vibrio parahaemolyticus by multilocus sequence typing. J. Clin. Microbiol. 42:12801282.
28. Chun J.,, A. Huq, and, R. R. Colwell. 1999. Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl. Environ. Microbiol. 65:22022208.
29. Coelho, A.,, H. Momen,, A. C. Vicente, and, C. A. Salles. 1994. An analysis of the V1 and V2 regions of Vibrio cholerae and Vibrio mimicus 16S rRNA. Res. Microbiol. 145:151156.
30. Coleman, S. S.,, D. M. Melanson,, E. G. Biosca, and, J. D. Oliver. 1996. Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification. Appl. Environ. Microbiol. 62:13781382.
31. Coleman, S. S., and, J. D. Oliver. 1996. Optimization of conditions for the polymerase chain reaction amplification of DNA from culturable and nonculturable cells of Vibrio vulnificus. FEMS Microbiol. Ecol. 19:127132.
32. Colodner, R.,, R. Raz,, I. Meir,, T. Lazarovich,, L. Lerner,, J. Kopelowitz,, Y. Keness,, W. Sakran,, S. Ken-Dror, and, N. Bisharat. 2004. Identification of the emerging pathogen Vibrio vulnificus biotype 3 by commercially available phenotypic methods. J. Clin. Microbiol. 42:41374140.
33. Colquhoun, D. J., and, H. Sørum. 2002. Cloning, characterisation and phylogenetic analysis of the fur gene in Vibrio salmonicida and Vibrio logei. Gene 296:213220.
34. Conejero, M. J., and, C. T. Hedreyda. 2003. Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection. J. Appl. Microbiol. 95:602611.
35. Conejero, M. J., and, C. T. Hedreyda. 2004. PCR detection of he-molysin (vhh) gene in Vibrio harveyi. J. Gen. Appl. Microbiol. 50:137142.
36. Crawford, J. A.,, J. B. Kaper, and, V. J. DiRita. 1998. Analysis of ToxR-dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae. Mol. Microbiol. 29:235246.
37. Croci, L.,, E. Suffredini,, L. Cozzi, and, L. Toti. 2002. Effects of depuration of molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae O1, and Vibrio parahaemolyticus. J. Appl. Microbiol. 92:460465.
38. Dalsgaard, A.,, I. Dalsgaard,, L. Hoi, and, J. L. Larsen. 1996. Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus. Lett. Appl. Microbiol. 22:184188.
39. Davis, C. R.,, L. C. Heller,, K. K. Peak,, D. L. Wingfield,, C. L. Goldstein-Hart,, D. W. Bodager,, A. C. Cannons,, P. T. Amuso, and, J. Cattanii. 2004. Real-time PCR detection of the thermostable direct hemolysin and thermolabile hemolysin genes in a Vibrio parahaemolyticus cultured from mussels and mussel homo-genate associated with a foodborne outbreak. J. Food Prot. 67:10051008.
40. De, K.,, T. Ramamurthy,, S. M. Faruque,, S. Yamasaki,, Y. Takeda,, G. B. Nair, and, R. K. Nandy. 2004. Molecular characterisation of rough strains of Vibrio cholerae isolated from diarrhoeal cases in India and their comparison to smooth strains. FEMS Microbiol. Lett. 232:2330.
41. De, K.,, T. Ramamurthy,, A. C. Ghose,, M. S. Islam,, Y. Takeda,, G. B. Nair, and, R. K. Nandy. 2001. Modification of the multiplex PCR for unambiguous differentiation of the El Tor and classical biotypes of Vibrio cholerae O1. Indian J. Med. Res. 114:7782.
42. DePaola, A., and, G. C. Hwang. 1995. Effect of dilution, incubation time, and temperature of enrichment on cultural and PCR detection of Vibrio cholerae obtained from the oyster Cras-sostrea virginica. Mol. Cell. Probes 9:7581.
43. DePaola, A.,, M. L. Motes,, D. W. Cook,, J. Veazey,, W. E. Garthright, and, R. Blodgett. 1997. Evaluation of an alkaline phosphatase-labeled DNA probe for enumeration of Vibrio vulnificus in Gulf Coast oysters. J. Microbiol. Methods 29:115120.
44. Desmarchelier, P. M., and, C. R. Senn. 1989. A molecular epidemi-ological study of Vibrio cholerae in Australia. Med. J. Aust. 150:631634.
45. Di Pinto, A.,, G. Ciccarese,, G. Tantillo,, D. Catalano, and, V. T. Forte. 2005. A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. J. Food Prot. 68:150153.
46. Dorsch, M.,, D. Lane, and, E. Stackebrandt. 1992. Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences. Int. J. Syst. Bacterial. 42:5863.
47. Fields, P. I.,, T. Popovic,, K. Wachsmuth, and, O. Olsik. 1992. Use of the polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic. J. Clin. Microbiol. 30:21182121.
48. Fischer-Le Saux, M.,, D. Hervio-Heath,, S. Loaec,, R. R. Colwell, and, M. Pommepuy. 2002. Detection of cytotoxin-hemolysin mRNA in nonculturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater. Appl. Environ. Microbiol. 68:56415646.
49. Fukushima, H.,, Y. Tsunomori, and, R. Seki. 2003. Duplex realtime SYBR green PCR assays for detection of 17 species of food-or waterborne pathogens in stools. J. Clin. Microbiol. 41:51345146.
50. Gauger, E. J., and, M. Gomez-Chiarri. 2002. 16S ribosomal DNA sequencing confirms the synonymy of Vibrio harveyi and V. carchariae. Dis. Aquat. Organ. 52:3946.
51. Ghosh, C.,, R. K. Nandy,, S. K. Dasgupta,, G. B. Nair,, R. H. Hall, and, A. C. Ghose. 1997. A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-O1/non-O139 Vibrio cholerae. Microb. Pathog. 22:199208.
52. Giglio, S.,, P. T. Monis, and, C. P. Saint. 2003. Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Res. 31:e136.
53. Goldberg, S. L., and, J. R. Murphy. 1985. Cloning and characterization of the hemolysin determinants from Vibrio cholerae RV79 (Hly+), RV79(Hly-), and 569B. J. Bacteriol. 162:3541.
54. Gomez-Gil, B.,, S. Soto-Rodriguez,, A. Garcia-Gasca,, A. Roque,, R. Vazquez-Juarez,, F. L. Thompson, and, J. Swings. 2004. Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms. Microbiology 150:17691777.
55. González, S. F.,, M. J. Krug,, M. E. Nielsen,, Y. Santos, and, D. R. Call. 2004. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray. J. Clin. Microbiol. 42:14141419.
56. González, S. F.,, C. R. Osorio, and, Y. Santos. 2003. Development of a PCR-based method for the detection of Listonella anguillarum in fish tissues and blood samples. Dis. Aquat. Organ. 55:109115.
57. Gooch, J. A.,, A. DePaola,, C. A. Kaysner, and, D. L. Marshall. 2001. Evaluation of two direct plating methods using non-radioactive probes for enumeration of Vibrio parahaemolyticus in oysters. Appl. Environ. Microbiol. 67:721724.
58. Guglielmetti, P.,, L. Bravo,, A. Zanchi,, R. Monte,, G. Lombardi, and, G. M. Rossolini. 1994. Detection of the Vibrio cholerae heat-stable enterotoxin gene by polymerase chain reaction. Mol. Cell. Probes 8:3944.
59. Gutacker, M.,, N. Conza,, C. Benagli,, A. Pedroli,, M. V. Bernasconi,, L. Permin,, R. Aznar, and, J. C. Piffaretti. 2003. Population genetics of Vibrio vulnificus: identification of two divisions and a distinct eel-pathogenic clone. Appl. Environ. Microbiol. 69:32033212.
60. Hara-Kudo, Y.,, K. Sugiyama,, M. Nishibuchi,, A. Chowdhury,, J. Yatsuyanagi,, Y. Ohtomo,, A. Saito,, H. Nagano,, T. Nishina,, H. Nakagawa,, H. Konuma,, M. Miyahara, and, S. Kumagai. 2003. Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus O3:K6 in seafood and the coastal environment in Japan. Appl. Environ. Microbiol. 69:38833891.
61. Heidelberg, J. F.,, J. A. Eisen,, W. C. Nelson,, R. A. Clayton,, M. L. Gwinn,, R. J. Dodson,, D. H. Haft,, E. K. Hickey,, J. D. Peterson,, L. Umayam,, S. R. Gill,, K. E. Nelson,, T. D. Read,, H. Tettelin,, D. Richardson,, M. D. Eermolaeva,, J. Vamathevan,, S. Bass,, H. Quin,, I. Dragoi,, P. Sellers,, L. McDonald,, T. Utterback,, R. D. Fleishmann,, W. C. Nierman,, O. White,, S. L. Salzberg,, H. O. Smith,, R. R. Colwell,, J. J. Mekalanos,, J. C. Venter, and, C. M. Fraser. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477484.
62. Hernandez, G., and, J. Olmos. 2004. Molecular identification of pathogenic and nonpathogenic strains of Vibrio harveyi using PCR and RAPD. Appl. Microbiol. Biotechnol. 63:722727.
63. Hill, W. E.,, S. P. Keasler,, M. W. Trucksess,, P. Feng,, C. A. Kaysner, and, K. A. Lampel. 1991. Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters. Appl. Environ. Microbiol. 57:707711.
64. Hiratsu, K.,, K. Yamamoto, and, K. Makino. 1997. Molecular cloning and functional analysis of an rpoS homologue gene from Vibrio cholerae N86. Genes Genet. Syst. 72:115118.
65. Hirono, I.,, T. Masuda, and, T. Aoki. 1996. Cloning and detection of the hemolysin gene of Vibrio anguillarum. Microb. Pathog. 21:173182.
66. Hoge, C. W.,, O. Sethabutr,, L. Bodhidatta,, P. Echeverria,, D. C. Robertson, and, J. G. Morris, Jr. 1990. Use of a synthetic oligo-nucleotide probe to detect strains of non-serovar O1 Vibrio cholerae carrying the gene for heat-stable enterotoxin (NAG-ST). J. Clin. Microbiol. 28:14731476.
67. Holt, J. G.,, N. R. Krieg,, P. H. A. Sneath,, J. T. Staley, and, S. T. Williams (ed.). 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed. Lippincott Williams & Wilkins, Baltimore, Md.
68. Hong, B. X.,, L. F. Jiang,, Y. S. Hu,, D. Y. Fang, and, H. Y. Guo. 2004. Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections. J. Microbiol. Methods 58:403411.
69. Hoshino, K.,, S. Yamasaki,, A. K. Mukhopadhyay,, S. Chakraborty,, A. Basu,, S. K. Bhattacharya,, G. B. Nair,, T. Shi-mada, and, Y. Takeda. 1998. Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. FEMS Immunol. Med. Microbiol. 20:201207.
70. Iida, T.,, A. Hattori,, K. Tagomori,, H. Nasu,, R. Naim, and, T. Honda. 2001. Filamentous phage associated with recent pandemic strains of Vibrio parahaemolyticus. Emerg. Infect. Dis. 7:477478.
71. Iida, T.,, O. Suthienkul,, K. S. Park,, G. Q. Tang,, R. K. Yamamoto,, M. Ishibashi,, K. Yamamoto, and, T. Honda. 1997. Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus. J. Med. Microbiol. 46:639645.
72. Iredell, J. R., and, P. A. Manning. 1994. Biotype-specific tcpA genes in Vibrio cholerae. FEMS Microbiol. Lett. 121:4754.
73. Islam, M. S.,, R. Tasmin,, S. I. Khan,, H. B. Bakht,, Z. H. Mahmood,, M. Z. Rahman,, N. A. Bhuiyan,, M. Nishibuchi,, G. B. Nair,, R. B. Sack,, A. Huq,, R. R. Colwell, and, D. A. Sack. 2004. Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh. Can. J. Microbiol. 50:827834.
74. Ito, H.,, H. Ito,, I. Uchida,, T. Sekizaki, and, N. Terakado. 1995. A specific oligonucleotide probe based on 5S rRNA sequences for identification of Vibrio anguillarum and Vibrio ordalii. Vet. Microbiol. 43:167171.
75. Ji, N.,, B. Peng,, G. Wang,, S. Wang, and, X. Peng. 2004. Universal primer PCR with DGGE for rapid detection of bacterial pathogens. J. Microbiol. Methods 57:409413.
76. Jiang, S.,, W. Chu, and, W. Fu. 2003. Prevalence of cholera toxin genes (ctxA and zot) among non-O1/O139 Vibrio cholerae strains from Newport Bay, California. Appl. Environ. Microbiol. 69:75417544.
77. Jiang, S. C., and, W. Fu. 2001. Seasonal abundance and distribution of Vibrio cholerae in coastal waters quantified by a 16S-23S intergenic spacer probe. Microb. Ecol. 42:540548.
78. Kaper, J. B.,, H. B. Bradford,, N. C. Roberts, and, S. Falkow. 1982. Molecular epidemiology of Vibrio cholerae in the U. S. Gulf Coast. J. Clin. Microbiol. 16:129134.
79. Kaper, J. B.,, J. G. Morris, Jr., and, M. Nishibuchi. 1989. DNA probes for pathogenic Vibrio species, p. 6577 In F. C. Tenover (ed.), DNA Probes for Infectious Diseases. CRC Press, Boca Raton, Fla.
80. Karunasagar, I.,, I. Rivera,, B. Joseph,, B. Kennedy,, V. R. Shetty,, A. Huq,, I. Karunasagar, and, R. R. Colwell. 2003. ompU genes in non-toxigenic Vibrio cholerae associated with aquaculture. J. Appl. Microbiol. 95:338343.
81. Karunasagar, I.,, G. Sugumar, and, I. Karunasagar. 1995. Rapid detection of Vibrio cholerae contamination of seafood by poly-merase chain reaction. Mol. Mar. Biol. Biotechnol. 4:365368.
82. Kaufman, G. E.,, G. M. Blackstone,, M. C. Vickery,, A. K. Bej,, J. Bowers,, M. D. Bowen,, R. F. Meyer, and, A. DePaola. 2004. Realtime PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix. J. Food Prot. 67:24242429.
83. Kaufman, G. E.,, M. L. Myers,, C. L. Pass,, A. K. Bej, and, C. A. Kaysner. 2002. Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific north-west outbreaks. Lett. Appl. Microbiol. 34:155161.
84. Keasler, S. P., and, R. H. Hall. 1993. Detection and biotyping Vibrio cholerae O1 with multiplex polymerase chain reaction. Lancet 341:1661.
85. Khan, A. A.,, S. McCarthy,, R. F. Wang, and, C. E. Cerniglia. 2002. Characterization of United States outbreak isolates of Vibrio parahaemolyticus using enterobacterial repetitive intergenic consensus (ERIC) PCR and development of a rapid PCR method for detection of O3:K6 isolates. FEMS Microbiol. Lett. 206:209214.
86. Kim, Y. B.,, J. Okuda,, C. Matsumoto,, N. Takahashi,, S. Hashimoto, and, M. Nishibuchi. 1999. Identification of Vibrio parahaemolyticus at the species level by PCR targeted to the toxR gene. J. Clin. Microbiol. 37:11731177.
87. Kimoto, R.,, T. Funahashi,, N. Yamamoto,, S. Miyoshi,, S. Narimatsu, and, S. Yamamoto. 2001. Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus. Microbiol. Immunol. 45:135142.
88. Kishishita, M.,, N. Matsuoka,, K. Kumagai,, S. Yamasaki,, Y. Takeda, and, M. Nishibuchi. 1992. Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus. Appl. Environ. Microbiol. 58:24492457.
89. Kita-Tsukamoto, K.,, H. Oyaizu,, K. Nanba, and, U. Simidu. 1993. Phylogenetic relationships of marine bacteria, mainly members of the family Vibrionaceae, determined on the basis of 16S rRNA sequences. Int. J. Syst. Bacteriol. 43:819.
90. Kobayashi, K.,, K. Seto,, S. Akasaka, and, M. Makino. 1990. Detection of toxigenic Vibrio cholerae O1 using polymerase chain reaction for amplifying the cholera enterotoxin gene. J. Jpn. Assoc. Infect. Dis. 64:13231329.
91. Koch, W. H.,, W. L. Payne,, B. A. Wentz, and, T. A. Cebula. 1993. Rapid polymerase chain reaction method for detection of Vibrio cholerae in foods. Appl. Environ. Microbiol. 59:556560.
92. Kong, R. Y.,, S. K. Lee,, T. W. Law,, S. H. Law, and, R. S. Wu. 2002. Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR. Water Res. 36:28022812.
93. Kurazono, H.,, A. Pal,, P. K. Bag,, G. B. Nair,, T. Karasawa,, T. Mihara, and, Y. Takeda. 1995. Distribution of genes encoding cholera toxin, zonula occludens toxin, accessory cholera toxin, and El Tor hemolysin in Vibrio cholerae of diverse origins. Microb. Pathog. 18:231235.
94. Kvitt, H.,, M. Ucko,, A. Colorni,, C. Batargias,, A. Zlotkin, and, W. Knibb. 2002. Photobacterium damselae ssp. piscicida: detection by direct amplification of 16S rRNA gene sequences and genotypic variation as determined by amplified fragment length polymorphism (AFLP). Dis. Aquat. Organ. 48:187195.
95. Kwok, A. Y.,, J. T. Wilson,, M. Coulthart,, L. K. Ng,, L. Mutharia, and, A. W. Chow. 2002. Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences. Can. J. Microbiol. 48:903910.
96. Lan, R., and, P. R. Reeves. 1998. Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae. Microbiology 144:12131221.
97. Laohaprertthisan, V.,, A. Chowdhury,, U. Kongmuang,, S. Kalnauwakul,, M. Ishibashi,, C. Matsumoto, and, M. Nishibuchi. 2003. Prevalence and serodiversity of the pandemic clone among the clinical strains of Vibrio parahaemolyticus isolated in southern Thailand. Epidemiol. Infect. 130:112.
98. Lee, C., and, S. F. Pan. 1993. Rapid and specific detection of the thermostable direct haemolysin gene in Vibrio parahaemolyticus by the polymerase chain reaction. J. Gen. Microbiol. 139:32253231.
99. Lee, C. Y.,, C. H. Chen, and, Y. W. Chou. 1995a. Characterization of a cloned pR72H probe for Vibrio parahaemolyticus detection and development of a nonisotopic colony hybridization assay. Microbiol. Immunol. 39:177183.
100. Lee, C. Y.,, S. F. Pan, and, C. H. Chen. 1995b. Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR. Appl. Environ. Microbiol. 61:13111317.
101. Lee, C. Y.,, L. H. Chen,, M. L. Liu, and, Y. C. Su. 1992. Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters. Appl. Environ. Microbiol. 58:34193422.
102. Lee, C. Y.,, G. Panicker, and, A. K. Bej. 2003. Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization. J. Microbiol. Methods 53:199209.
103. Lee, S. E.,, S. H. Shin,, S. Y. Kim,, Y. R. Kim,, D. H. Shin,, S. S. Chung,, Z. H. Lee,, J. Y. Lee,, K. C. Leong,, S. H. Choi, and, J. H. Rhee. 2000. Vibrio vulnificus has the transmembrane transcription activator toxRS stimulating the expression of the hemolysin gene vvhA. J. Bacteriol. 182:34053415.
104. Lee, S. E.,, S. Y. Kim,, S. J. Kim,, H. S. Kim,, J. H. Shin,, S. H. Choi,, S. S. Chung, and, J. H. Rhee. 1998. Direct identification of Vibrio vulnificus in clinical specimens by nested PCR. J. Clin. Microbiol. 36:28872892.
105. Lee, S. K.,, H. Z. Wang,, S. H. Law,, R. S. Wu, and, R. Y. Kong. 2002. Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences. Mar. Pollut. Bull. 44:412420.
106. Le Roux, F.,, M. Gay,, C. Lambert,, J. L. Nicolas,, M. Gouy, and, F. Berthe. 2004a. Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences. Dis. Aquat. Organ. 58:143150.
107. Le Roux, W. J.,, D. Masoabi,, C. M. de Wet, and, S. N. Venter. 2004b. Evaluation of a rapid polymerase chain reaction based identification technique for Vibrio cholerae isolates. Water Sci. Technol. 50:229232.
108. Lin, W.,, K. J. Fullner,, R. Clayton,, J. A. Sexton,, M. B. Rogers,, K. E. Calia,, S. B. Calderwood,, C. Fraser, and, J. J. Mekalanos. 1999. Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc. Natl. Acad. Sci. USA 96:10711076.
109. Lin, Z.,, K. Kumagai,, K. Baba,, J. J. Mekalanos, and, M. Nishibuchi. 1993. Vibrio parahaemolyticus has a homolog of the Vibrio cholerae toxRS operon that mediates environmentally induced regulation of the thermostable direct hemolysin gene. J. Bacteriol. 175:38443855.
110. Linkous, D. A., and, J. D. Oliver. 1999. Pathogenesis of Vibrio vulnificus. FEMS Microbiol. Lett. 174:207214.
111. Lipp, E. K.,, I. N. Rivera,, A. I. Gil,, E. M. Espeland,, N. Choopun,, V. R. Louis,, E. Russek-Cohen,, A. Huq, and, R. R. Colwell. 2003. Direct detection of Vibrio cholerae and ctxA in Peruvian coastal water and plankton by PCR. Appl. Environ. Microbiol. 69:36763680.
112. Liu, C. H.,, W. Cheng,, J. P. Hsu, and, J. C. Chen. 2004. Vibrio al-ginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing. Dis. Aquat. Organ. 61:169174.
113. Lyon, W. J. 2001. TaqMan PCR for detection of Vibrio cholerae O1, O139, non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater. Appl. Environ. Microbiol. 67:46854693.
114. Mallard, K. E., and, P. M. Desmarchelier. 1995. Detection of heat-stable enterotoxin genes among Australian Vibrio cholerae O1 strains. FEMS Microbiol. Lett. 127:111115.
115. Martinez-Picado, J.,, A. R. Blanch, and, J. Jofre. 1994. Rapid detection and identification of Vibrio anguillarum by using a specific oligonucleotide probe complementary to 16S rRNA. Appl. Environ. Microbiol. 60:732737.
116. Masuda, N.,, K. Yasukawa,, Y. Isawa,, R. Horie,, J. Saito,, T. Ishi-guro,, Y. Nakaguchi,, M. Nishibuchi, and, T. Hayashi. 2004. Rapid detection of tdh and trh mRNAs of Vibrio parahaemolyticus by the transcription-reverse transcription concerted (TRC) method. J. Biosci. Bioeng. 98:236243.
117. Matsumoto, C.,, J. Okuda,, M. Ishibashi,, M. Iwanaga,, P. Garg,, T. Rammamurthy,, H.-C. Wong,, A. DePaola,, Y. B. Kim,, M. J. Albert, and, M. Nishibuchi. 2000. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses. J. Clin. Microbiol. 38:578585.
118. McCarthy, S. A.,, A. DePaola,, D. W. Cook,, C. A. Kaysner, and, W. E. Hill. 1999. Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile he-molysin (tlh) gene of Vibrio parahaemolyticus. Lett. Appl. Microbiol. 28:6670.
119. McCarthy, S. A.,, A. DePaola,, C. A. Kaysner,, W. E. Hill, and, D. W. Cook. 2000. Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of Vibrio parahaemolyticus. J. Food. Prot. 63:16601664.
120. Miller, V. L., and, J. J. Mekalanos. 1984. Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc. Natl. Acad. Sci. USA 81:34713475.
121. Miller, V. L., and, J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:25752583.
122. Minami, A.,, S. Hashimoto,, H. Abe,, M. Arita,, T. Taniguchi,, T. Honda,, T. Miwatani, and, M. Nishibuchi. 1991. Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan. Appl. Environ. Microbiol. 57:21522157.
123. Miyagi, K.,, K. Sano,, C. Morita,, S. Imura,, S. Morimatsu,, T. Goto,, Y. Nakano,, K. Omura,, Y. Matsumoto,, K. Maeda,, S. Hashimoto, and, T. Honda. 1999. An improved method for detecting faecal Vibrio cholerae by PCR of the toxin A gene. J. Med. Microbiol. 48:883889.
124. Mizunoe, Y.,, S. N. Wai,, K. Umene,, T. Kokubo,, S. Kawabata, and, S. Yoshida. 1999. Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae. Microbiol. Immunol. 43:513520.
125. Morin, N. J.,, Z. Gong, and, X. F. Li. 2004. Reverse transcription-multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi. Clin. Chem. 50:20372044.
126. Morris, J. G., Jr.,, A. C. Wright,, D. M. Roberts,, P. K. Wood,, L. M. Simpson, and, J. D. Oliver. 1987. Identification of environmental Vibrio vulnificus isolates with a DNA probe for the cytotoxin-hemolysin gene. Appl. Environ. Microbiol. 53:193195.
127. Muniesa-Pere, M.,, J. Jofre, and, A. R. Blanch. 1996. Identification of Vibrio proteolyticus with a differential medium and a specific probe. Appl. Environ. Microbiol. 62:26732675.
128. Nair, G. B.,, P. K. Bag,, T. Shimada,, T. Ramamurthy,, T. Takeda,, S. Yamamoto,, H. Kurazono, and, Y. Takeda. 1995. Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal. J. Clin. Microbiol. 33:21862187.
129. Nair, G. B.,, Y. Oku,, Y. Takeda,, A. Ghosh,, R. K. Ghosh,, S. Chattopadhyyay,, S. C. Pal,, J. B. Kaper, and, T. Takeda. 1988. Toxin profiles of Vibrio cholerae non-O1 from environmental sources in Calcutta, India. Appl. Environ. Microbiol. 54:31803182.
130. Nakaguchi, Y.,, T. Ishizuka,, S. Ohnaka,, T. Hayashi,, K. Yasukawa,, T. Ishiguro, and, M. Nishibuchi. 2004. Rapid and specific detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus using transcription-reverse transcription concerted (TRC) reaction with an automated system. J. Clin. Microbiol. 42:42844292.
131. Nandi, B.,, R. K. Nandy,, S. Mukhopadhyay,, G. B. Nair,, T. Shi-mada, and, A. C. Ghose. 2000. Rapid method for species-specific identification of Vibrio cholerae using primers targeted to the gene of outer membrane protein OmpW. J. Clin. Microbiol. 38:41454151.
132. Nasu, H.,, T. Iida,, T. Sugahara,, Y. Yamaguchi,, K.-S. Park,, K. Yokoyama,, K. Makino,, H. Shinagawa, and, T. Honda. 2000. A filamentous phage associated with recent pandemic Vibrio parahaemolyticus O3:K6 strains. J. Clin. Microbiol. 8:21562161.
133. Nilsson, W. B.,, R. N. Paranjpye,, A. DePaola, and, M. S. Strom. 2003. Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence. J. Clin. Microbiol. 41:442446.
134. Nishibuchi, M.,, S. Doke,, S. Toizumi,, T. Umeda,, M. Yoh, and, T. Miwatani. 1988. Isolation from a coastal fish of Vibrio hollisae capable of producing a hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus. Appl. Environ. Microbiol. 54:21442146.
135. Nishibuchi, M.,, W. E. Hill,, G. Zon,, W. L. Payne, and, J. B. Kaper. 1986. Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus. J. Clin. Microbiol. 23:10911095.
136. Nishibuchi, M.,, M. Ishibashi,, Y. Takeda, and, J. B. Kaper. 1985. Detection of the thermostable direct hemolysin gene and related DNA sequence in Vibrio parahaemolyticus and other Vibrio species by the DNA colony hybridization test. Infect. Immun. 49:481486.
137. Nishibuchi, M.,, J. M. Janda, and, T. Ezaki. 1996. The thermostable direct hemolysin gene (tdh) of Vibrio hollisae is dissimilar in prevalence to and phylogenetically distant from the tdh genes of other vibrios: implications in the horizontal transfer of the tdh gene. Microbiol. Immunol. 40:5965.
138. Nishibuchi, M., and, J. B. Kaper. 1995. Thermostable direct he-molysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium. Infect. Immun. 63:20932099.
139. Nishibuchi, M.,, V. Khaeomanee-iam,, T. Honda,, J. B. Kaper, and, T. Miwatani. 1990. Comparative analysis of the hemolysin genes of Vibrio cholerae non-01, V. mimicus, and V. hollisae that are similar to the tdh gene of V. parahaemolyticus. FEMS Microbiol. Lett. 55:251256.
140. Nordstrom, J. L., and, A. DePaola. 2003. Improved recovery of pathogenic Vibrio parahaemolyticus from oysters using colony hybridization following enrichment. J. Microbiol. Methods 52:273277.
141. Novais, R. C.,, A. Coelho,, C. A. Salles, and, A. C. P. Vincente. 1999. Toxin co-regulated pilus cluster in non-O1, non-toxigenic Vibrio cholerae: evidence of a third allele of pilin gene. FEMS Microbiol. Lett. 171:4955.
142. Oakey, H. J.,, N. Levy,, D. G. Bourne,, B. Cullen, and, A. Thomas. 2003. The use of PCR to aid in the rapid identification of Vibrio harveyi isolates. J. Appl. Microbiol. 95:12931303.
143. Ogawa, A., and, T. Takeda. 1993. The gene encoding the heat-stable enterotoxin of Vibrio cholerae is flanked by 123-base pair direct repeats. Microbiol. Immunol. 37:607616.
144. O’Hara, C. M.,, E. G. Sowers,, C. A. Bopp,, S. B. Duda, and, N. A. Strockbine. 2003. Accuracy of six commercially available systems for identification of members of the family Vibrionaceae. J. Clin. Microbiol. 41:56545659.
145. Okuda, J.,, E. Hayakawa,, M. Nishibuchi, and, T. Nishino. 1999. Sequence analysis of the gyrA and parC homologues of a wild-type strain of Vibrio parahaemolyticus and its fluoroquinolone-resistant mutants. Antimicrob. Agents Chemother. 43:11561162.
146. Okuda, J.,, M. Ishibashi,, S. Abbott,, J. M. Janda, and, M. Nishibuchi. 1997a. Analysis of the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes in urease-positive strains of Vibrio parahaemolyticus isolated on the west coast of United States. J. Clin. Microbiol. 35:19651971.
147. Okuda, J.,, M. Ishibashi,, E. Hayakawa,, T. Nishino,, Y. Takeda,, A. Mukhopadhyay,, S. Garg,, S. K. Bhattacharya,, G. B. Nair, and, M. Nishibuchi. 1997b. Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan. J. Clin. Microbiol. 35:31503155.
148. Okuda, J.,, T. Nakai,, P. S. Chang,, T. Oh,, T. Nishino,, T. Koitabashi, and, M. Nishibuchi. 2001. The toxR gene of Vibrio (Listonella) anguillarum controls expression of the major outer membrane proteins but not virulence in a natural host model. Infect. Immun. 69:60916101.
149. Okura, M.,, R. Osawa,, A. Iguchi,, E. Arakawa,, J. Terajima, and, H. Watanabe. 2003. Genotypic analyses of Vibrio parahaemolyticus and development of a pandemic group-specific multiplex PCR assay. J. Clin. Microbiol. 41:46764682.
150. Okura, M.,, R. Osawa,, A. Iguchi,, M. Takagi,, E. Arakawa,, J. Terajima, and, H. Watanabe. 2004. PCR-based identification of pandemic group Vibrio parahaemolyticus with a novel group-specific primer pair. Microbiol. Immunol. 48:787790.
151. Osorio, C. R.,, M. D. Collins,, J. L. Romalde, and, A. E. Toranzo. 2004. Characterization of the 23S and 5S rRNA genes and 23S- 5S intergenic spacer region (ITS-2) of Photobacterium damselae. Dis. Aquat. Organ. 61:3339.
152. Osorio, C. R.,, M. D. Collins,, J. L. Romalde, and, A. E. Toranzo. 2005. Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure. Appl. Environ. Microbiol. 71:636645.
153. Osorio, C. R.,, M. D. Collins,, A. E. Toranzo,, J. L. Barja, and, J. L. Romalde. 1999. 16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis. Appl. Environ. Microbiol. 65:29422946.
154. Osorio, C. R., and, K. E. Klose. 2000. A region of the transmembrane regulatory protein ToxR that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among Vibrio species. J. Bacteriol. 182:526528.
155. Osorio, C. R.,, A. E. Toranzo,, J. L. Romalde, and, J. L. Barja. 2000. Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae. Dis. Aquat. Organ. 40:177183.
156. Pal, A.,, T. Ramamurthy,, R. K. Bhadra,, T. Takeda,, T. Shimada,, Y. Takeda,, G. B. Nair,, S. C. Pal, and, S. Chakrabarti. 1992. Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe. Appl. Environ. Microbiol. 58:24852489.
157. Palmer, L. M., and, R. R. Colwell. 1991. Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hybridization and polymerase chain reaction. Appl. Environ. Microbiol. 57:12861293.
158. Panicker, G.,, D. R. Call,, M. J. Krug, and, A. K. Bej. 2004a. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays. Appl. Environ. Microbiol. 70:74367444.
159. Panicker, G.,, M. L. Myers, and, A. K. Bej. 2004b. Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Appl. Environ. Microbiol. 70:498507.
160. Panicker, G.,, M. C. Vickery, and, A. K. Bej. 2004c. Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish. Can. J. Microbiol. 50:911922.
161. Park, S. D.,, H. S. Shon, and, N. J. Joh. 1991. Vibrio vulnificus sep-ticemia in Korea: clinical and epidemiologic findings in 70 patients. J. Am. Acad. Dermatol. 24:397403.
162. Park, T. S.,, S. H. Oh,, E. Y. Lee,, T. K. Lee,, K. H. Park,, M. J. Figueras, and, C. L. Chang. 2003. Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system. Lett. Appl. Microbiol. 37:349353.
163. Rajan, P. R.,, J. H. Lin,, M. S. Ho, and, H. L. Yang. 2003. Simple and rapid detection of Photobacterium damselae ssp. piscicida by a PCR technique and plating method. J. Appl. Microbiol. 95:13751380.
164. Ramamurthy, T.,, M. J. Albert,, A. Huq,, R. R. Colwell,, Y. Takeda,, T. Takeda,, T. Shimada,, B. K. Mandal, and, G. B. Nair. 1994. Vibrio mimicus with multiple toxin types isolated from human and environmental sources. J. Med. Microbiol. 40:194196.
165. Ramamurthy, T.,, A. Pal,, P. K. Bag,, S. K. Bhattacharya,, G. B. Nair,, H. Kurozano,, S. Yamasaki,, H. Shirai,, T. Takeda, and, Y. Uesaka. 1993. Detection of cholera toxin gene in stool specimens by polymerase chain reaction: comparison with bead enzyme-linked immunosorbent assay and culture method for laboratory diagnosis of cholera. J. Clin. Microbiol. 31:30683070.
166. Randa, M. A.,, M. F. Polz, and, E. Lim. 2004. Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR. Appl. Environ. Microbiol. 70:54695476.
167. Rehnstam, A. S.,, A. Norqvist,, H. Wolf-Watz, and, A. Hagström. 1989. Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe. Appl. Environ. Microbiol. 55:19071910.
168. Reich, K. A., and, G. K. Schoolnik. 1994. The light organ symbiont Vibrio fischeri possesses a homolog of the Vibrio cholerae trans-membrane transcriptional activator ToxR. J. Bacteriol. 176:30853088.
169. Rhine, J. A., and, R. K. Taylor. 1994. TcpA pilin sequences and colonization requirements for O1 and O139 Vibrio cholerae. Mol. Microbiol. 13:10131020.
170. Rivera, T. G.,, J. Chun,, A. Huq,, R. B. Sack, and, R. R. Colwell. 2001. Genotypes associated with virulence in environmental isolates of Vibrio cholerae. Appl. Environ. Microbiol. 67:24212429.
171. Robert-Pillot, A.,, A. Guenole, and, J. M. Fournier. 2002. Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA-DNA hybridization. FEMS Microbiol. Lett. 215:16.
172. Rosche, T. M.,, Y. Yano, and, J. D. Oliver. 2005. A rapid and simple PCR analysis indicates there are two subgroups of Vibrio vulnificus which correlate with clinical or environmental isolation. Microbiol. Immunol.49:381389.
173. Rubin, E. J.,, W. Lin,, J. J. Mekalanos, and, M. K. Waldor. 1998. Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage. Mol. Microbiol. 28:12471254.
174. Ruimy, R.,, V. Breittmayer,, P. Elbaze,, B. Lafay,, O. Boussemart,, M. Gauthier, and, R. Christine. 1994. Phylogenetic analysis and assessment of the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas deduced from small-subunit rRNA sequences. Int. J. Syst. Bacterial. 44:416426.
175. Sahu, G. K.,, R. Chowdhury, and, J. Das. 1997. The rpoH gene encoding CT homolog of Vibrio cholerae. Gene 189:203207.
176. Salles, C. A.,, H. Momen,, A. C. P. Vicente, and, A. Coelho. 1993. Vibrio cholerae in South America: polymerase chain reaction and zymovar analysis. Trans. R. Soc. Trop. Med. Hyg. 87:272.
177. Sarkar, B.,, T. Bhattacharya,, T. Ramamurthy,, T. Shimada,, Y. Takeda, and, G. B. Nair. 2002. Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup. Epidemiol. Infect. 129:245251.
178. Saulnier, D.,, J. C. Avarre,, G. Le Moullac,, D. Ansquer,, P. Levy, and, V. Vonau. 2000. Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of syndrome 93 in New Caledonia. Dis. Aquat. Organ. 40:109115.
179. Shangkuan, Y. H.,, Y. S. Show, and, T. M. Wang. 1995. Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1.J. Appl. Bacteriol. 79-264273.
180. Shi, L.,, S. Miyoshi,, M. Hiura,, K. Tomochika,, T. Shimada, and, S. Shinoda. 1998. Detection of genes encoding cholera toxin (CT), zonula occludens toxin (ZOT), accessory cholera enterotoxin (ACE) and heat-stable enterotoxin (ST) in Vibrio mimicus clinical strains. Microbiol. Immunol. 42:823828.
181. Shinoda, S.,, T. Nakagawa,, L. Shi,, K. Bi,, Y. Kanoh,, K. Tomochika,, S. Miyoshi, and, T. Shimada. 2004. Distribution of virulence-associated genes in Vibrio mimicus isolates from clinical and environmental origins. Microbiol. Immunol. 48:547551.
182. Shirai, H.,, H. Ito,, T. Hirayama,, Y. Nakamoto,, N. Nakabayashi,, K. Kumagai,, Y. Takeda, and, M. Nishibuchi. 1990. Molecular epidemiologic evidence for association of thermostable direct he-molysin (TDH) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis. Infect. Immun. 58:35683573.
183. Shirai, H.,, M. Nishibuchi,, T. Ramamurthy,, S. K. Bhattacharya,, S. C. Pal, and, Y. Takeda. 1991. Polymerase chain reaction for detection of the cholera enterotoxin operon of Vibrio cholerae. J. Clin. Microbiol. 29:25172521.
184. Shyu, Y. C., and, F. P. Lin. 1999. Cloning and characterization of manganese superoxide dismutase gene from Vibrio parahaemolyticus and application to preliminary identification of Vibrio strains. IUBMB Life 48:345352.
185. Singh, D. V.,, S. R. Isac, and, R. R. Colwell. 2002. Development of a hexaplex PCR assay for rapid detection of virulence and regulatory genes in Vibrio cholerae and Vibrio mimicus. J. Clin. Microbiol. 40:43214324.
186. Somerville, C. C., and, R. R. Colwell. 1993. Sequence analysis of the β-N-acetylhexosaminidase gene of Vibrio vulnificus: evidence for a common evolutionary origin of hexosaminidases. Proc. Natl. Acad. Sci, USA 90:67516755.
187. Sparagano, O. A.,, P. A. Robertson,, I. Purdom,, J. Mclnnes,, Y. Li,, D. H. Yu,, Z. J. Du,, H. S. Xu, and, B. Austin. 2002. PCR and molecular detection for differentiating Vibrio species. Ann. N. Y. Acad. Sci. 969:6065.
188. Stine, O. C.,, S. Sozhamannan,, Q. Gou,, S. Zheng,, J. G. Morris, Jr., and, J. A. Johnson. 2000. Phylogeny of Vibrio cholerae based on recA sequence. Infect. Immun. 68:71807185.
189. Sugimura, I. I.,, T. Sawabe, and, Y. Ezura. 2000. In situ polymerase chain reaction visualization of Vibrio halioticoli using alginate lyase gene alyVG2. Mar. Biotechnol. (New York) 2:7479.
190. Tada, J.,, T. Ohashi,, N. Nishimura,, H. Ozaki,, S. Fukushima,, J. Takano,, M. Nishibuchi, and, Y. Takeda. 1992a. Non-isotopic microtitre plate-based assay for detecting products of poly-merase chain reaction amplification: application to detection of the tdh gene of Vibrio parahaemolyticus. Mol. Cell. Probes 6:489494.
191. Tada, J.,, T. Ohashi,, N. Nishimura,, Y. Shirasaki,, H. Ozaki,, S. Fukushima,, J. Takano,, M. Nishibuchi, and, Y. Takeda. 1992b. Detection of thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Mol. Cell. Probes 6:477487.
192. Takahashi, H.,, Y. Hara-Kudo,, J. Miyasaka,, S. Kumagai, and, H. Konuma. 2005. Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus. J. Microbiol. Methods 61:7785.
193. Takeda, T.,, Y. Peina,, A. Ogawa,, S. Dohi,, H. Abe,, G. B. Nair, and, S. C. Pal. 1991. Detection of heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio cholerae O1. FEMS Microbiol. Lett. 64:2327.
194. Taniguchi, H.,, H. Hirano,, S. Kubomura,, K. Higashi, and, Y. Mizuguchi. 1986. Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin from Vibrio parahaemolyticus. Microb. Pathog. 1:425432.
195. Terai, A.,, K. Baba,, H. Shirai,, O. Yoshida,, Y. Takeda, and, M. Nishibuchi. 1991. Evidence for insertion-sequence-mediated spread of the thermostable direct hemolysin gene among Vibrio species. J. Bacteriol. 173:50365046.
196. Theron, J.,, J. Cilliers,, M. Du Preez,, V. S. Brozel, and, S. N. Venter. 2000. Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure. J. Appl. Microbiol. 89:539546.
197. Thompson, C. C.,, F. L. Thompson,, K. Vandemeulebroecke,, B. Hoste,, P. Dawyndt, and, J. Swings. 2004. Use of recA as an alternative phylogenetic marker in the family Vibrionaceae. Int. J. Syst. Evol. Microbiol. 54:919924.
198. Thompson, F. L.,, B. Hoste,, K. Vandemeulebroecke, and, J. Swings. 2003. Reclassification of Vibrio hollisae as Grimontia hollisae gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 53:16151617.
199. Thompson, F. L.,, T. Iida, and, J. Swings. 2004. Biodiversity of vibrios. Microbiol. Mol. Biol. Rev. 68:403431.
200. Trucksis, M.,, J. E. Galen,, J. Michalski,, A. Fasano, and, J. B. Kaper. 1993. Accessory cholera enterotoxin (Ace), the third toxin of a Vibrio cholerae virulence cassette. Proc. Natl. Acad. Sci. USA 90:52675271.
201. Uchimura, M.,, K. Koiwai,, Y. Tsuruoka, and, H. Tanaka. 1993. High prevalence of thermostable direct hemolysin (TDH)-like toxin in Vibrio mimicus strains isolated from diarrhoeal patients. Epidemiol. Infect. 111:4953.
202. Varela, P.,, G. D. Pollevick,, M. Rivas,, I. Chinen,, N. Binsztein,, A. C. Frasch, and, R. A. Ugalde. 1994. Direct detection of Vibrio cholerae in stool samples. J. Clin. Microbiol. 32:12461248.
203. Varela, P.,, M. Rivas,, N. Binsztein,, M. L. Cremona,, P. Herrmann,, O. Burrone,, R. A. Ugalde, and, A. C. Frasch. 1993. Identification of toxigenic Vibrio cholerae from the Argentine outbreak by PCR for ctx A1 and ctx A2-B. FEBS Lett. 315:7476.
204. Venkateswaran, K.,, N. Dohmoto, and, S. Harayama. 1998. Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp. Appl. Environ. Microbiol. 64:681687.
205. Vicente, A. C. P.,, A. Coelho, and, C. A. Salles. 1997. Detection of Vibrio cholerae and V. mimicus heat-stable toxin gene sequence by PCR. J. Med. Microbiol. 46:15.
206. Vital Brazil, J. M.,, R. M. Alves,, I. N. Rivera,, D. P. Rodrigues,, D. K. Karaolis, and, L. C. Campos. 2002. Prevalence of virulence-associated genes in clinical and environmental Vibrio cholerae strains isolated in Brazil between 1991 and 1999. FEMS Microbiol. Lett. 215:1521.
207. Vuddhakul, V.,, A. Chowdhury,, V. Laohaprertthisan,, P. Pun-grasamee,, N. Patararungrong,, P. Thianmontri,, M. Ishibashi,, C. Matsumoto, and, M. Nishibuchi. 2000a. Isolation of Vibrio parahaemolyticus strains belonging to a pandemic O3:K6 clone from environmental and clinical sources in Thailand. Appl. Environ. Microbiol. 66:26852689.
208. Vuddhakul, V.,, T. Nakai,, C. Matsumoto,, T. Oh,, T. Nishino,, C.-H. Chen,, M. Nishibuchi, and, J. Okuda. 2000b. Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of Vibrio hollisae from the environment and its identification. Appl. Environ. Microbiol. 66:35063514.
209. Waldor, M. K., and, J. J. Mekalanos. 1994. Vibrio cholerae O139 specific gene sequences. Lancet 343:1366.
210. Waldor, M. K., and, J. J. Mekalanos. 1996. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272:19101914.
211. Wang, R. F.,, W. W. Cao, and, C. E. Cerniglia. 1997. A universal protocol for PCR detection of 13 species of foodborne pathogens in foods. J. Appl. Microbiol. 83:727736.
212. Welch, T. J., and, D. H. Bartlett. 1998. Identification of a regulatory protein required for pressure-responsive gene expression in the deep-sea bacterium Photobacterium species strain SS9. Mol. Microbiol. 27:977985.
213. Wiik, R.,, E. Stackebrandt,, O. Valle,, F. L. Daae,, O. M. Rodseth, and, K. Andersen. 1995. Classification of fish-pathogenic vibrios based on comparative 16S rRNA analysis. Int. J. Syst. Bacterial. 45:421428.
214. Wimpee, C. F.,, T. L. Nadeau, and, K. H. Nealson. 1991. Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification. Appl. Environ. Microbiol. 57:13191324.
215. Wright A.,, J. G. Morris, Jr.,, D. R. Maneval, Jr.,, K. Richardson, and, J. B. Kaper. 1985. Cloning of the cytotoxin-hemolysin gene of Vibrio vulnificus. Infect. Immun. 50:922924.
216. Wright, A. C.,, Y. Guo,, J. A. Johnson,, J. P. Nataro, and, J. G. Morris, Jr. 1992. Development and testing of a nonradioactive DNA oligonucleotide probe that is specific for Vibrio cholerae cholera toxin. J. Clin. Microbiol. 30:23022306.
217. Wright, A. C.,, R. T. Hill,, J. A. Johnson,, M.-C. Roghman,, R. R. Colwell, and, J. G. Morris, Jr. 1996. Distribution of Vibrio vulnificus in the Chesapeake Bay. Appl. Environ. Microbiol. 62:717724.
218. Wright, A. C.,, G. A. Miceli,, W. L. Landry,, J. B. Christy,, W. D. Watkins, and, J. G. Morris, Jr. 1993. Rapid identification of Vibrio vulnificus on nonselective media with an alkaline phos-phatase-labeled oligonucleotide probe. Appl. Environ. Microbiol. 50:541546.
219. Wu, M. H., and, R. A. Laine. 1999. Sequence of the V. parahaemolyticus gene for cytoplasmic N, N’-diacetylchitobiase and homology with related enzymes. J. Biochem. (Tokyo) 125:10861093.
220. Yam, W. C.,, M. L. Lung,, K. Y. Ng, and, M. H. Ng. 1989. Molecular epidemiology of Vibrio cholerae in Hong Kong. J. Clin. Microbiol. 27:19001902.
221. Yamamoto, K.,, T. Honda,, T. Miwatani,, S. Tamatsukuri, and, S. Shibata. 1992. Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Can. J. Microbiol. 38:410416.
222. Yamasaki, S.,, S. Garg,, G. B. Nair, and, Y. Takeda. 1999a. Distribution of Vibrio cholerae O1 antigen biosynthesis genes among O139 and other non-O1 serogroups of Vibrio cholerae. FEMS Microbiol. Lett. 179:115121.
223. Yamasaki, S.,, T. Shimizu,, K. Hoshino,, S. T. Ho,, T. Shimada,, G. B. Nair, and, Y. Takeda. 1999b. The genes responsible for O-anti-gen synthesis of Vibrio cholerae O139 are closely related to those of Vibrio cholerae O22. Gene 237:321332
224. Yamasaki, S.,, H. Shirai,, Y. Takeda, and, M. Nishibuchi. 1991. Analysis of the gene of Vibrio hollisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus. FEMS Microbiol. Lett. 80:259264.
225. Yoh, M.,, K. Miyagi,, Y. Matsumoto,, K. Hayashi,, Y. Takarada,, K. Yamamoto, and, T. Honda. 1993. Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene. J. Clin. Microbiol. 31:13121314.


Generic image for table

Target genes for molecular identification of

Citation: Nishibuchi M. 2006. Molecular Identification, p 44-64. In Thompson F, Austin B, Swings J (ed), The Biology of Vibrios. ASM Press, Washington, DC. doi: 10.1128/9781555815714.ch4
Generic image for table

PCR methods for simultaneous detection of pathogenic species, including the genus

Citation: Nishibuchi M. 2006. Molecular Identification, p 44-64. In Thompson F, Austin B, Swings J (ed), The Biology of Vibrios. ASM Press, Washington, DC. doi: 10.1128/9781555815714.ch4

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