Rapid Method for Species-Specific Identification of Vibrio cholerae Using Primers Targeted to the Gene of Outer Membrane Protein OmpW

Journal of Clinical Microbiology - Tập 38 Số 11 - Trang 4145-4151 - 2000
Bisweswar Nandi1, Ranjan K. Nandy1,2, Sibabrata Mukhopadhyay1, G. Balakrish Nair2, Toshio Shimada3, A.M. Ghose1
1Department of Microbiology, Bose Institute, Calcutta 700 054,1 and
2National Institute of Cholera and Enteric Diseases, Calcutta 700 010,2 India, and
3National Institute of Infectious Diseases, Tokyo 162, Japan3

Tóm tắt

ABSTRACT The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in Vibrio cholerae and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254 V. cholerae strains tested were positive for ompW and 229 (∼98%) of 233 were positive for toxR . None of the 40 strains belonging to other Vibrio species produced amplicons with either ompW - or toxR -specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using ompW - and toxR -specific probes in DNA dot blot assay. While the V. cholerae strains reacted with ompW probe, only one ( V. mimicus ) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other Vibrio species (e.g., V. mimicus , V. splendidus , V. alginolyticus , V. fluvialis , V. proteolyticus , V. aestuarianus , V. salmonicida , V. furnissii , and V. parahaemolyticus ) showed weak to strong reactivity to the toxR probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the ompW sequence is highly conserved among V. cholerae strains belonging to different biotypes and/or serogroups. All of these results suggest that the ompW gene can be targeted for the species-specific identification of V. cholerae strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of ompW and ctxA genes which should be of considerable value in the screening of both toxigenic and nontoxigenic V. cholerae strains of clinical as well as environmental origin.

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Tài liệu tham khảo

10.1128/jcm.35.6.1633-1635.1997

10.1128/jcm.31.5.1315-1317.1993

10.1128/AEM.65.5.2202-2208.1999

10.1128/jcm.33.10.2715-2722.1995

10.1128/jcm.14.6.631-639.1981

10.1006/mpat.1996.0105

10.1111/j.1574-695X.1998.tb01128.x

10.1093/nar/18.8.2180

10.1111/j.1365-2958.1992.tb02203.x

10.1128/CMR.8.1.48

10.1016/0140-6736(93)90792-F

10.1128/JCM.37.4.1173-1177.1999

10.1099/00207713-43-1-8

10.1128/jb.175.12.3844-3855.1993

10.1016/0092-8674(87)90430-2

10.1002/elps.1150190539

10.1093/oxfordjournals.epirev.a036052

10.1128/JB.182.2.526-528.2000

Ottemann K. M. Mekalanos J. J. Regulation of cholera toxin expression Vibrio cholerae and cholera: molecular to global perspectives. Wachsmuth I. K. Blake P. A. Olsvik O. 1994 177 185 ASM Press Washington D.C.

10.1128/JB.181.11.3578-3581.1999

Popovic T. Fields P. I. Olsvik O. Detection of cholera toxin genes Vibrio cholerae and cholera: molecular to global perspectives. Wachsmuth I. K. Blake P. A. Olsvik O. 1994 41 51 ASM Press Washington D.C.

10.1128/jb.176.10.3085-3088.1994

Rudra S. Mahajan R. Mathur M. Kathuria K. Talwar V. Cluster of cases of clinical cholera due to Vibrio cholerae O10 in east Delhi.Indian. J. Med. Res.10319967173

10.1099/00207713-44-3-416

Sakazaki R. Bacteriology of Vibrio and related organisms Cholera. Barua D. Greenough W. B. III 1992 37 55 Plenum Medical Book Company New York N.Y

Sambrook J. Fritsch E. F. Maniatis T. Appendix E: commonly used techniques in molecular cloning Molecular cloning: a laboratory manual. Nolan C. 1989 E1 E39 CSH Laboratory Press New York N.Y

Sengupta T. K. Identification and immunological characterisation of cell surface proteins responsible for intestinal adhesion and colonisation of diarrhoeagenic non-O1 Vibrio cholerae. Ph.D. thesis. 1995 University of Calcutta Calcutta India

10.1128/JCM.36.3.756-763.1998

10.1007/BF01571061

10.1073/pnas.95.24.14464

Wilson K. Preparation and analysis of DNA Current protocols in molecular biology Ausubel M. Brent R. Kingston R. E. Moore D. D. Seidman J. G. Smith J. A. Struhl K. 1 1994 2.4.1 2.4.2 John Willey & Sons, Inc. New York, N.Y

Yamai S. Okitsu T. Shimada T. Kaatsube Y. Serogroup of Vibrio cholerae non-O1/non-O139 with specific reference to their ability to produce cholera toxin and addition of novel serogroups.J. Jpn. Infect. Dis.71199710371045

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Journal of Clinical Microbiology

Tập 38 Số 11

4145-4151

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