Septaplex PCR assay for rapid identification of Vibrio cholerae including detection of virulence and int SXT genes.

In this study, we describe a septaplex PCR assay for rapid identification of Vibrio cholerae including detection of the virulence and intsxt genes. Conditions were optimized to amplify fragments of ISRrRNA (encoding for 16S-23S rRNA gene, Intergenic spacer regions), O1rfb (O1 serogroup specific rfb), O139rfb (O139 serogroup specific rfb), ctxA (cholera toxin subunit A), tcpA (toxin coregulated pilus), and intsxt (sxt integron) simultaneously in a single PCR. The septaplex PCR was evaluated using 211 strains of V. cholerae and six water samples for in situ testing. PCR results were correlated with genotype data obtained by individual PCR and slot-blot assays. The one-step PCR described here can be used to identify V. cholerae accurately and rapidly. Also, the virulence and intsxt genes can be simultaneously detected, providing a useful method for monitoring pathogenic, intsxt-positive and nonpathogenic, intsxt-negative V. cholerae serogroups both in the environment and clinical settings.

[1]  D. Singh,et al.  Molecular Characterization of Vibrio cholerae O139 Bengal Isolated from Water and the Aquatic Plant Eichhornia crassipes in the River Ganga, Varanasi, India , 2003, Applied and Environmental Microbiology.

[2]  R. Colwell,et al.  Development of a Hexaplex PCR Assay for Rapid Detection of Virulence and Regulatory Genes in Vibrio cholerae and Vibrio mimicus , 2002, Journal of Clinical Microbiology.

[3]  A. C. Ghose,et al.  Rapid Method for Species-Specific Identification ofVibrio cholerae Using Primers Targeted to the Gene of Outer Membrane Protein OmpW , 2000, Journal of Clinical Microbiology.

[4]  C. R. Osorio,et al.  A Region of the Transmembrane Regulatory Protein ToxR That Tethers the Transcriptional Activation Domain to the Cytoplasmic Membrane Displays Wide Divergence amongVibrio Species , 2000, Journal of bacteriology.

[5]  J. Chun,et al.  Analysis of 16S-23S rRNA Intergenic Spacer Regions of Vibrio cholerae and Vibrio mimicus , 1999, Applied and Environmental Microbiology.

[6]  M. Nishibuchi,et al.  Identification of Vibrio parahaemolyticus Strains at the Species Level by PCR Targeted to the toxR Gene , 1999, Journal of Clinical Microbiology.

[7]  S K Bhattacharya,et al.  Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. , 1998, FEMS immunology and medical microbiology.

[8]  S. Yamasaki,et al.  Molecular Analysis of Non-O1, Non-O139 Vibrio choleraeAssociated with an Unusual Upsurge in the Incidence of Cholera-Like Disease in Calcutta, India , 1998, Journal of Clinical Microbiology.

[9]  S. Yamasaki,et al.  Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139. , 1998, FEMS immunology and medical microbiology.

[10]  A. Weintraub,et al.  Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR , 1997, Journal of clinical microbiology.

[11]  M. Waldor,et al.  A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139 , 1996, Journal of bacteriology.

[12]  R. Mahajan,et al.  Cluster of cases of clinical cholera due to Vibrio cholerae 010 in east Delhi. , 1996, The Indian journal of medical research.

[13]  M. Albert,et al.  Characterization of Vibrio cgolerae non-O1 serogroups obtained from an outbreak of diarrhea in Lima, Peru , 1995, Journal of clinical microbiology.

[14]  G. Schoolnik,et al.  The light organ symbiont Vibrio fischeri possesses a homolog of the Vibrio cholerae transmembrane transcriptional activator ToxR , 1994, Journal of bacteriology.

[15]  Rita R. Colwell,et al.  Vibrios in the environment: viable but nonculturable Vibrio cholerae , 1994 .

[16]  J. Mekalanos,et al.  Regulation of Cholera Toxin Expression , 1994 .

[17]  R. Hall,et al.  Detecting and biotyping Vibrio cholerae 01 with multiplex polymerase chain reaction , 1993, The Lancet.

[18]  J. Mekalanos,et al.  Vibrio parahaemolyticus has a homolog of the Vibrio cholerae toxRS operon that mediates environmentally induced regulation of the thermostable direct hemolysin gene , 1993, Journal of bacteriology.

[19]  C. Hoge,et al.  Epidemic of diarrhea caused by Vibrio cholerae non-O1 that produced heat-stable toxin among Khmers in a camp in Thailand , 1993, Journal of clinical microbiology.

[20]  R. Sakazaki.  Bacteriology of Vibrio and Related Organisms , 1992 .

[21]  J. Mekalanos,et al.  Safe, live Vibrio cholerae vaccines? , 1988, Vaccine.

[22]  J. Mekalanos,et al.  Cholera toxin transcriptional activator ToxR is a transmembrane DNA binding protein , 1987, Cell.

[23]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[24]  S. van Heyningen.  Cholera toxin. , 1982, Bioscience reports.

[25]  R. Colwell,et al.  Vibrio cholerae, Vibrio parahaemolyticus, and other vibrios: occurrence and distribution in Chesapeake Bay. , 1977, Science.