A rapid and direct real time PCR-based method for identification of Salmonella spp.
The aim of this work was the validation of a rapid, real-time PCR assay based on TaqMan technology for the unequivocal identification of Salmonella spp. to be used directly on an agar-grown colony. A real-time PCR system targeting at the Salmonella spp. invA gene was optimized and validated through a four times repeated blind experiment performed in two different laboratories including 50 Salmonella spp. with representative strains from each of the 5 different Salmonella subgenera and 30 non-Salmonella strains. Both parameters DeltaR(n) (fluorescence intensity of template through a normalized reporter value) and C(T) (cycle at which the fluorescence intensity achieved a pre-established threshold) were analyzed. Overall mean DeltaR(n) and C(T) values for Salmonella strains (2.14+/-0.87 and 15.30+/-0.90, respectively) were statistically different from values for non-Salmonella strains, allowing the establishment of cut-off DeltaR(n) and C(T) values based on 95% confidence intervals that allowed the correct identification of all strains tested in each independent experiment. The accuracy of this assay in terms of inclusivity and exclusivity was 100%. Moreover, the PCR system proved to be especially convenient because the pre-mix containing all PCR reagents except for the bacterial cells could be kept at -20 degrees C for at least 1 month before its use. The optimized TaqMan real-time PCR assay is a useful, simple and rapid method for routine identification of Salmonella spp., irrespective of the particular subgenus.
Rodriguez-Lázaro
David
D
Institute of Food and Agricultural Technology (INTEA), University of Girona, Campus Montilivi s/n, Girona E-17071, Spain.
Hernández
Marta
M
Esteve
Teresa
T
Hoorfar
Jeffrey
J
Pla
Maria
M
D001426:Bacterial Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000145:classification / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; C075518:invA protein, Bacteria