Attenuated typhoid vaccine Salmonella typhi Ty21a: fingerprinting and quality control.
Live attenuated vaccines, developed with molecular genetical techniques, require new approaches for their quality control. To develop novel quality control tests that enhanced and extended existing procedures, the attenuated vaccine strain Salmonella typhi Ty21a and its parent strain Ty2 were characterized by pulsed-field gel electrophoresis (PFGE) and direct nucleotide sequence analysis. Mutant and parent strains were distinguished using fingerprints generated by the resolution on PFGE of chromosomal DNA digested with each of the enzymes SfiI, SpeI or XbaI. These fingerprints were stable through multiple in vitro passages of the vaccine strain and were identical from one batch of vaccine to another. It was also possible to distinguish between the mutant and parent strains by direct nucleotide sequence analysis of the galE gene. This analysis identified two base changes in the gene from strain Ty21a: a single base deletion causing a frameshift that would result in a truncated gene product, accounting for the galE phenotype; and a transition that eliminated an AluI restriction site. The consequent change in the AluI fingerprint of the galE gene in strain Ty21a provided a rapid, PCR-based alternative to the use of differential media or biochemical assays for the identification of the vaccine strain.
McKenna
Alison J
AJ
Bygraves
Jane A
JA
Maiden
Martin C J
MCJ
Feavers
Ian M
IM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002238:Carbohydrate Epimerases / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D016521:Electrophoresis, Gel, Pulsed-Field; D004755:Enterobacteriaceae / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011786:Quality Control; D012485:Salmonella typhi / Q000235:genetics / Q000276:immunology*; D012689:Sequence Homology, Nucleic Acid; D014534:UDPglucose 4-Epimerase; D014613:Vaccines, Attenuated / Q000235:genetics*
D004269:DNA, Bacterial; D014613:Vaccines, Attenuated; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase; C020551:galactose epimerase; D005690:Galactose