A rapid and direct real time PCR-based method for identification of Salmonella spp

• Published in: Journal of microbiological methods Vol. 54; no. 3; pp. 381 - 390
• Main Authors: Rodrı́guez-Lázaro, David, Hernández, Marta, Esteve, Teresa, Hoorfar, Jeffrey, Pla, Maria
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.09.2003; Elsevier Science
• Subjects: Bacterial Proteins - genetics; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Microbiology; Pathogen identification; Polymerase Chain Reaction - methods; Real-time PCR; Reproducibility of Results; Salmonella; Salmonella enterica - classification; Salmonella enterica - genetics; Sensitivity and Specificity; Sequence Analysis, DNA; Pathogen identification; Real-time PCR; Salmonella; Polymerase chain reaction; Bacteria; Identification; Real time; Enterobacteriaceae
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/S0167-7012(03)00071-X

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 Δ R 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 Δ R 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 Δ R 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 °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.