Development of a multiplex single-tube nested PCR (MSTNPCR) assay for Vibrio cholerae O1 detection

A multiplex nested PCR method for detection of Vibrio cholerae O1 using a single tube was developed (MSTNPCR). Firstly, single-tube nested PCR (STNPCR) with primers directed to ctxA gene was standardized, and its detection limit was compared to simple PCR and two-step nested PCR. Secondly, primers d...

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Bibliographic Details
Published in:Journal of microbiological methods 2008-02, Vol.72 (2), p.191-196
Main Authors: Mendes, Carina Lucena, Abath, Frederico Guilherme Coutinho, Leal, Nilma Cintra
Format: Article
Language:English
Subjects:
Bacterial Proteins - genetics
Bacterial Typing Techniques
Bacteriological methods and techniques used in bacteriology
Bacteriology
Biological and medical sciences
Cholera
Cholera Toxin - genetics
Diagnostic
DNA Primers
DNA, Bacterial - genetics
Fundamental and applied biological sciences. Psychology
Microbiology
Multiplex
Nested-PCR
O Antigens - genetics
Polymerase Chain Reaction - methods
Sensitivity and Specificity
Sequence Analysis, DNA
Vibrio cholerae
Vibrio cholerae O1 - genetics
Vibrio cholerae O1 - isolation & purification
Water Microbiology
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Summary:A multiplex nested PCR method for detection of Vibrio cholerae O1 using a single tube was developed (MSTNPCR). Firstly, single-tube nested PCR (STNPCR) with primers directed to ctxA gene was standardized, and its detection limit was compared to simple PCR and two-step nested PCR. Secondly, primers directed to rfbN gene were added to the reaction. The detection limit of the multiplex reaction was determined using V. cholerae O1 DNA and V. cholerae O1 grown in alkaline peptone water (APW). STNPCR was shown to be approximately 100-fold more sensitive than simple PCR and 10 times less sensitive than two-step nested PCR. This drawback is compensated by a lower risk of cross-contamination. The addition of a second target did not impair the detection limit of STNPCR (as little as 1 pg of V. cholerae O1 DNA detected). MSTNPCR could specifically detect up to three V. cholerae O1 cells or colony forming units (cfu) directly from the APW growth. A diagnostic kit consisting of a set of microtubes having the inner primers fixed onto the inside of the tube cap and a set of tubes containing the reaction mixture was evaluated for stability, and it proved to be stable for five months at − 20 °C. Therefore, MSTNPCR would be useful in the detection of V. cholerae O1 directly from environmental waters in cholera endemic areas and in complementing the identification of toxigenic strains isolated by culture.
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2007.11.018