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Vaccimonitor
versão On-line ISSN 1025-0298
Resumo
MARRERO-DOMINGUEZ, Karen; LEDON-PEREZ, Talena e FANDO-CALZADA, Rafael. Development of a PCR assay to detect cholera toxin genes (ctxAB) in preparations of attenuated live vaccine candidate against cholera CV638. Vaccimonitor [online]. 2016, vol.25, n.2, pp. 30-36. ISSN 1025-0298.
The live oral vaccine candidate against cholera CV638 is produced under Good Manufacturing Practices. The active ingredient of this vaccine candidate is the genetically modified strain Vibrio cholerae 638; developed by researchers at the National Center of Scientific Research, from the strain V. cholerae serogroup O1 biotype El Tor C7258 (Peru, 1991), by removing cholera toxin genes (ctxAB). Since the strain 638 lacks these genes in its genome, the presence of ctxAB in vaccine preparations would be given by a contamination with a toxigenic V. cholerae strain. The present study aimed at designing a specific PCR to detect ctxAB genes in DNA isolated from preparations of the vaccine candidate CV638, artificially contaminated with toxigenic V. cholerae strain. Sensitivity of the optimized PCR assay was 1 pg of genomic DNA from toxigenic strain of V. cholerae C7258, corresponding to ~200 genomic copies. The sensitivity of the PCR method for detecting toxigenic strains in CV638 vaccine preparations contaminated with a toxigenic strain was ~7 x 103 colony forming units of the toxigenic strain per dose of CV638. Once validated, this method could be used in the quality control of the production of the live vaccine candidate CV638.
Palavras-chave : V. cholerae 638; vaccines; cholera toxin; PCR.