Abstract
A multiplex PCR assay was developed for the detection of toxigenic and pathogenicV. cholerae from direct water sources using specific primers targeting diverse genes,viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes,ompW acts as internal control forV. cholerae, thectx gene as a marker for toxigenicity andtcp for pathogenicity. The sensitivity of multiplex PCR was 5 × 104 V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. ToxigenicV. cholerae were artificially spiked in different water samples, filtered through a 0.45 µm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence ofV. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenicpathogenic and nonpathogenicV. cholerae.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- APW:
-
alkaline peptone water
- BHI:
-
brain heart infusion (agar)
- CFU:
-
colony-forming unit
- PBS:
-
phosphate-buffered saline
- PCR:
-
polymerase chain reaction
- TCBS:
-
thiocitrate-bile salt-sucrose (agar)
References
Bej A.K., Mahbubani M.H.: Applications of the polymerase chain reaction in environmental microbiology.PCR Meth.Appl. 1, 151–159 (1992).
Catalan V., Moreno F.G., Vila M.J., Apraiz D.: Detection ofLegionella pneumophila in wastewater by nested polymerase chain reaction.Res.Microbiol. 148, 71–78 (1997).
Čermáková Z., Ryšková O., Plíšková L.: Polymerase chain reaction for detection ofToxoplasma gondii in human biological samples.Folia Microbiol. 50, 341–345 (2005).
Choopun N., Louis V., Huq A., Colwell R.R.: Simple procedure for rapid identification ofVibrio cholerae from the aquatic environment.Appl.Environ.Microbiol. 68, 995–998 (2002).
Faruque S.M., Albert M.J., Mekalanos J.J.: Epidemiology, genetics and ecology of toxigenicVibrio cholerae.Microbiol.Mol.Biol.Rev. 62, 1301–1314 (1998).
Goel A.K., Tamrakar A.K., Kamboj D.V., Singh L.: Direct immunofluorescence assay for rapid environmental detection ofVibrio cholerae O1.Folia Microbiol. 50, 448–452 (2005a).
Goel A.K., Tamrakar A.K., Nema V., Kamboj D.V., Singh L.: Detection of viable toxigenicVibrio cholerae from environmental water sources by direct cell duplex PCR assay.World J.Microbiol.Biotechnol. 21, 973–976 (2005b).
Kaper J.B., Morris J.B. Jr.,Levine M.M.: Cholera.Clin.Microbiol.Rev. 8, 48–86 (1995).
Koenraad P.M.F.J., Giesendorf B.A.J., Henkens M.H.C., Beumer R.R., Quint W.G.V.: Methods for the detection ofCampylobacter in sewage: evaluation of efficacy of enrichment and isolation media, applicability of polymerase chain reaction and latex agglutination assay.J.Microbiol.Meth. 23, 309–320 (1995).
Madico G., Checkley W., Gilman R.H., Bravo N., Cabrera L., Calderon M., Ceballos A.: Active surveillance forVibrio cholerae O1 and vibriophages in sewage water as a potantial tool to predict cholera outbreaks.J.Clin.Microbiol. 34, 2968–2972 (1996).
Morris J.G.: Non-O group IVibrio cholerae: a look at the epidemiology of an occasional pathogen.Epidemiol.Rev. 12, 179–191 (1990).
Nandi B., Nandi R.K., Mukhopadhyay S., Nair G.B., Shimada T., Ghose A.C.: Rapid method for species identification ofVibrio cholerae using primers targeted to the gene of outer membrane protein OmpW.J.Clin.Microbiol. 38, 4145–4151 (2000).
Rao V.K., Sharma M.K., Goel A.K., Singh L., Sekhar K.: Amperometric immunosensor for detection ofVibrio cholerae O1 using disposable screen-printed electrodes.Anal.Science 22, 1207–1211 (2006).
Rudenko N., Golovchenko M., Němec J., Volkaert J., Mallátová N., Grubhoffer L.: Improved method of detection and molecular typing ofBorrelia burgdorferi sensu lato in clinical samples by polymerase chain reaction without DNA purification.Folia Microbiol. 50, 31–39 (2005).
Růžičková V., Voller J., Pantůček R., Petráš P., Doškař J.: Multiplex PCR for detection of three exfoliative toxin serotype genes inStaphylococcus aureus.Folia Microbiol. 50, 499–502 (2005).
Singh D.V., Matte M.H., Matte G.R., Jiang S., Sabeena F., Shukla B.N., Sanyal S.C., Huq A., Colwell R.R.: Molecular analysis ofVibrio cholerae O1, O139, non-O1 and non-O139 strains: clonal relationship between clinical and environmental isolates.Appl.Environ.Microbiol. 67, 910–921 (2001).
Tamrakar A.K., Goel A.K., Kamboj D.V., Singh L.: Surveillance methodology forVibrio cholerae in environmental samples.Internat.J.Environ.Health Res. 16, 305–312 (2006).
Waldor M.K., Mekalanos J.J.: Lysogenic conversion by a filamentous phage encoding cholera toxin.Science 272, 1910–1914 (1996).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Goel, A.K., Ponmariappan, S., Kamboj, D.V. et al. Single multiplex polymerase chain reaction for environmental surveillance of toxigenic—Pathogenic O1 and Non-O1vibrio cholerae . Folia Microbiol 52, 81–85 (2007). https://doi.org/10.1007/BF02932143
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02932143