Automated 5' nuclease PCR assay for identification of Salmonella enterica.
A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [DeltaRn], >0. 6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (DeltaRn, < or =0.5). All 100 rough Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C. Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method for identification of Salmonella, particularly in large reference laboratories.
Hoorfar
J
J
Danish Veterinary Laboratory, Copenhagen, Denmark. jho@svs.dk
Ahrens
P
P
Rådström
P
P
D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012480:Salmonella Infections / Q000175:diagnosis* / Q000382:microbiology; D019779:Salmonella enterica / Q000145:classification* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D005456:Fluorescent Dyes; D019914:Taq Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V