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

Journal of Microbiological Methods
Carina Lucena MendesNilma Cintra Leal

Abstract

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 degrees C. Therefore, MSTNPCR would be useful in the d...Continue Reading

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Citations

Jun 30, 2009·Applied and Environmental Microbiology·Christopher J GrimRita R Colwell
Jan 25, 2011·Journal of Microbiological Methods·Nicola PuglieseAntonio Camarda
Jun 7, 2008·Current Opinion in Biotechnology·Ingrid Brettar, Manfred G Höfle
Mar 28, 2013·TheScientificWorldJournal·Carina Lucena Mendes-MarquesNilma Cintra Leal
May 22, 2009·Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases·F A M O CaririN C Leal
Jul 17, 2019·BMC Infectious Diseases·Bhagya DeepachandiYamuna Siriwardana
Nov 5, 2020·Critical Reviews in Food Science and Nutrition·Sharmin Quazi BonnyMohd Rafie Johan
Feb 12, 2020·Heliyon·Diala Shatleh-RantisiYaqoub Ashhab

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