Open AccessDetection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification
Author(s) -
Else Marie Fykse,
Gunnar Skogan,
William A. Davies,
Jaran Strand Olsen,
Janet Martha Blatny
Publication year - 2007
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.01635-06
Subject(s) - vibrio cholerae , biology , nasba , microbiology and biotechnology , escherichia coli , molecular beacon , rna , dna , gene , bacteria , biochemistry , genetics , oligonucleotide
A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection ofVibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA ), the toxin-coregulated pilus (tcpA ; colonization factor), thectxA 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 fromV. 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 ofV. cholerae , otherVibrio species, andBacillus cereus ,Salmonella enterica , andEscherichia coli strains. ThetoxR ,groEL , andhlyA beacons identified allV. cholerae isolates, whereas thectxA andtcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detectedV. cholerae at 50 CFU/ml by using the general markergroEL andtcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for thectxA ,toxR , andhlyA targets. RNA isolated from different environmental water samples spiked withV. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection ofV. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using thetcpA andctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.