Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella.
Amplification of nucleotide sequences within the invA gene of Salmonella typhimurium was evaluated as a means of detecting Salmonella. A collection of 630 strains of Salmonella comprising over 100 serovars, including the 20 most prevalent serovars isolated from animals and humans in Canada, was examined. Controls consisted of 142 non-Salmonella strains comprising 21 genera of bacteria. Cultures were screened by inoculating a single colony of bacteria directly into a polymerase chain reaction (PCR) mixture which contained a pair of primers specific for the invA gene. The specific PCR product was a 284 bp DNA fragment which was visualized in 2% agarose gels. With the exception of two S. litchfield and two S. senftenberg strains, all Salmonella strains were detected. In contrast, none of the non-Salmonella strains yielded the specific amplification product. Non-specific amplification of a few non-Salmonella strains resulted in a product that was distinctly different in size from the specific 284 bp product. Specificity of amplification was further confirmed by demonstration of hybridization of a 32P-labelled invA gene fragment only to the specific 284 bp product. The detection of 99.4% of Salmonella strains tested and the failure to specifically amplify DNA from non-Salmonella strains confirm that the invA gene contains sequences unique to Salmonella and demonstrate that this gene is a suitable PCR target, with potential diagnostic applications.
Rahn
K
K
Agriculture Canada, Health of Animals Laboratory, Guelph, Ontario.
De Grandis
S A
SA
Clarke
R C
RC
McEwen
S A
SA
Galán
J E
JE
Ginocchio
C
C
Curtiss
R
R
Gyles
C L
CL
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D012486:Salmonella typhimurium / Q000235:genetics*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial