title: Development and validation of a detection system for wild-type Vibrio cholerae in genetically modified cholera vaccine.
abstract: Orochol, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intact ctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol.
authors.affiliation: Official Medicines Control Laboratory Biologika and R&D Unit, Division of Biologicals, Swiss Federal Office of Public Health, Bern, Switzerland.
authors.forename: E
authors.initials: E
authors.lastname: Studer
authors.forename: U
authors.initials: U
authors.lastname: Candrian
mesh_terms: D002772:Cholera Toxin / Q000235:genetics / Q000276:immunology; D022121:Cholera Vaccines / Q000592:standards*; D016001:Confidence Intervals; D017931:DNA Primers; D004340:Drug Contamination; D020319:Oligodeoxyribonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D019444:Vaccines, DNA / Q000592:standards*; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D022121:Cholera Vaccines; D017931:DNA Primers; D020319:Oligodeoxyribonucleotides, Antisense; D019444:Vaccines, DNA; D002772:Cholera Toxin