Identification of Salmonella by real-time PCR based on TaqMan-MGB probe

Objective:To develop a TaqMan-MGB probe-based real-time PCR assay for Salmonella detection.Methods:Based on invH gene sequence of Salmonella,the specific Taqman-MGB probes were designed.TaqMan-MGB probe-based real-time PCR for Salmonella detection was established and optimized.Specificity and sensitivity analysis tests were performed with kinds of bacteria strains,food samples and invH gene that was constructed to pMD18-T vector.Results:There was significant linear relationship(r=0.997) between Ct value and logarithm of template concentration in the standard curve of positive referent.The sensitivity of this assay was 7.1×10-2 copies/μl.By this assay,73 Salmonella strains were identified as positive while 86 non-Salmonella strains such as Shigella,Staphylococcus aureus,Vibro parahaemolyticus were identified as negative.6 assays to 2 Salmonella strains,1 Escherichia coli strains were performed each other day successively.Variation coefficient of its Ct value is lower than 4%.There is no significant difference between results of conventional method and this real-time PCR assay to 65 raw meat products and 30 bulk cooked meat products.To complete the whole assay,it only took 1.5 hours to pure bacteria culture while 9.5 hours to food samples.Conclusion:The TaqMan-MGB probe-based real-time PCR assay for Salmonella detection is excellent in specificity and sensitivity.It is a useful,rapid,simple method and may have a significant application prospect.