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 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 detection of V. cholerae O1 directly from environmental waters in cholera endemic areas and in complementing the identification of toxigenic strains isolated by culture.
Mendes
Carina Lucena
CL
Departamento de Microbiologia, Centro de Pesquisas Aggeu Magalhães, Fiocruz, Avenida Prof. Moraes Rego, s/n, Campus da UFPE, Cidade Universitária, 50670-420 Recife, Pernambuco, Brazil. carina@cpqam.fiocruz.br
Abath
Frederico Guilherme Coutinho
FG
Leal
Nilma Cintra
NC
D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D002772:Cholera Toxin / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D019081:O Antigens / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D041241:Vibrio cholerae O1 / Q000235:genetics / Q000302:isolation & purification*; D014871:Water Microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D019081:O Antigens; C097237:RfbN protein, Vibrio cholerae; D002772:Cholera Toxin