Ensayo anidado de la reacción en cadena de la polimerasa específico para los genes de la toxina del cólera.

The active ingredient of the live vaccine candidate CV638 is the genetically attenuated strain Vibrio cholerae 638, which was obtained from the wild type toxigenic V. cholerae strain C7258, by deleting the cholera toxin genes (ctxAB). The use of live attenuated vaccines requires assays that ensure the stability of the attenuating mutation; as well as the lack of wild type toxigenic infectious cells. The present study was aimed at designing a nested PCR assay specific for ctxAB genes and evaluating its detection limit in lots of CV638 contaminated with known amounts of the toxigenic strain C7258Kn. One primer pairs specific for ctxAB genes were designed and PCR conditions respect to annealing temperature and MgCl2 concentration was established. The detection limit of the nested PCR assay of V. cholerae toxigenic bacteria was determined in CV638 preparations contaminated with increasing amounts of toxigenic vibrions. The detection limit was evaluated statistically and it was found to be ~ 50 colony forming units (CFU) of the toxigenic strain per CV638 dose, value lower than the smaller infective dose informed for V. cholerae (10³ CFU). Once validated, the described nested PCR assay could be used as a purity control assay during the quality control testing of CV638. [ABSTRACT FROM AUTHOR]