Objective To establish a method using TaqMan-MGB probe and fluorescence real-time polymerase chain reaction (RT-PCR) for rapid detection of Enterobacter sakazakii.
Methods A pair of specific primers and one TaqMan-MGB probe were designed based on the nucleic acid sequence of rpsU gene. After optimization of PCR reaction conditions and amplification system, a TaqMan-MGB probe real-time fluorescence PCR method for detection of Enterobacter sakazakii was developed. The sensibility of this method was evaluated by using solutions containing Enterobacter sakazakii strains with known concentration, and the specificity was evaluated using strains such as Enterobacter sakazakii, Escherichia coli, and Salmonella enterica. Meanwhile, comparison between this protocol and the conventional detection method was performed by using infant formula powder.
Results High corresponding relationship (R2=0.999) between the CT values and the logarithm of bacterial concentrations was presented in this proposed detection method. The minimum detectable concentration of Enterobacter sakazakii attained 1000CFU/mL. After enrichment for 8 h at 36℃, the lowest detection limit was 1 CFU/mL. The CT values of all 11 strains of Enterobacter sakazakii were less than 30, whereas the CT values of 30 other bacteria strains including Escherichia coli, Acinetobacter baumannii, and Citrobacter were all higher than 30, and the related amplification curves were even smooth straight lines. The CT values remain stable and the coefficient of variation was lower than 5% after five other-day back-to-back tests. There were no significant differences between the results detected by this proposed assay and by conventional isolation and culture in the test results of 140 different batches of infant formula powder and 192 items of infant cereal food supplement (χ2=0.624, P>0.05).
Conclusion The strengths of the TaqMan-MGB probe realtime fluorescence PCR method for detection of Enterobacter sakazakii include high specificity, high sensitivity, easy to operate, and therefore it is suitable for the rapid detection of Enterobacter sakazakii in infant formula milk powder and other food.
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