Detection of Salmonella typhimurium from rectal swabs of experimentally infected beagles by short cultivation and PCR-hybridization.
A rapid and sensitive cultivation and PCR-hybridization procedure for the detection and identification of Salmonella typhimurium was evaluated over a 42-day period with eight experimentally infected beagles. Rectal swabs were taken at several times postinfection, inoculated into selenite-cystine broth, and plated onto Hektoen-Enteric Enteric agar immediately after incubation for 4 and 24 h. PCRs and hybridizations were also conducted with each sample, and the results were compared with those of standard culture techniques to evaluate the efficiency of the PCR-hybridization procedure. The PCR-hybridization procedure was more sensitive than standard culture techniques at each enrichment incubation (P < 0.05). In addition, the PCR-hybridization procedure was significantly better than culture up through 3 days postinfection (P < 0.05). A nonspecific amplified product, relatively close in size to the 457-bp specifically amplified product, did not hybridize to an internal oligonucleotide probe or to a random-primed labeled probe. Subsequent sequence information revealed that the product had very little similarity to the 457-bp product but had significant similarity to an Escherichia coli aldehyde dehydrogenase gene. This study indicated that a cultivation and PCR-hybridization procedure is significantly better than culture for the identification of S. typhimurium. Additionally, the results confirm the importance of determining specificities of PCR products beyond the gel electrophoresis level by hybridization with a specific probe.
Stone
G G
GG
Department of Veterinary Diagnostic Investigation, Kansas State University, Manhattan 66506, USA.
Oberst
R D
RD
Hays
M P
MP
McVey
S
S
Galland
J C
JC
Curtiss
R
R
Kelly
S M
SM
Chengappa
M M
MM
D000818:Animals; D001431:Bacteriological Techniques / Q000706:statistics & numerical data; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D003951:Diagnostic Errors; D004195:Disease Models, Animal; D004285:Dogs; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012007:Rectum / Q000382:microbiology; D012480:Salmonella Infections / Q000175:diagnosis* / Q000382:microbiology; D012486:Salmonella typhimurium / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial