Rapid detection of Shigella by real-time fluorescent PCR assay using TaqMan-MGB probes

Objective Toestablish an approach for rapid identification of Shigella isolated from patients, food and environment, taking advantage of real-time fluorescent PCR assay featuring TaqMan-MGB probes.MethodsA couple of primers and a TaqMan-MGB probe were specifically designed according to the published sequence of Shigella ipaH gene.PCR amplification system and the reaction condition optimized, a TaqMan-MGB probe-based rapid detection of Shigella using real-time fluorescent PCR was developed.By adding strain samples of known concentrations, the sensibility of assay was evaluated.The specificity was validated using Shigella isolates and standard strains, Escherichia coli, Salmonella，Vibrio parahaemolyticus,and Staphylococcus aureus.Results Good corresponding relationship (Y=-3.93log（X）+37.34，R=0.999）between Ct value and the logarithm of bacterial concentration was present in the TaqMan-MGB probe-based real-time fluorescent PCR assay for Shigella detection.The minimum detectable concentration of Shigella was 30 cfu/ml.All the results of three Shigella standard strains and 30 isolates were positive, while those of 91 other bacterial strains such as Escherichia coli, Salmonella，Vibrio parahaemolyticus, and Staphylococcus aureus, were negative with the Ct value greater than 35 or the amplification curve turning into a smooth line.Compared with conventional identification methods, there was no significant difference（P＞0.05,2=0.27）.However, the entire process of assay cost merely two and ten hours for pure bacteria and food samples.Conclusion The TaqMan-MGB probe-based real time fluorescent PCR assay served as a promising approach for easy, rapid and highly specific and sensitive detection of Shigella.