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Applied and Environmental Microbiology, March 2007, p. 1457-1466, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.01635-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification{triangledown}

Else M. Fykse,1* Gunnar Skogan,1 William Davies,2 Jaran Strand Olsen,1 and Janet M. Blatny1

Norwegian Defence Research Establishment, P.O. Box 25, N-2027 Kjeller, Norway,1 Department of Molecular Biosciences, Oslo University, P.O. Box 1050, N-0316 Oslo, Norway2

Received 14 July 2006/ Accepted 20 December 2006

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.

* Corresponding author. Mailing address: Norwegian Defence Research Establishment, P.O. Box 25, N-2027 Kjeller, Norway. Phone: 47 63 80 78 45. Fax: 47 63 80 75 09. E-mail: else-marie.fykse{at}ffi.no.

{triangledown} Published ahead of print on 12 January 2007.

Applied and Environmental Microbiology, March 2007, p. 1457-1466, Vol. 73, No. 5
0099-2240/07/$08.00+0     doi:10.1128/AEM.01635-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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