A TaqMan based real-time PCR assay with the internal amplified control for the detection of Enterobacter sakazakii in infant formula

摘要

A TaqMan real-time polymerase chain reaction (PCR) assay for detecting Enterobacter sakazakii (E.sakazakii) in infant formula was evaluated and established with an internal amplification control (recombinant plasmids) to prevent reporting false negative results.Four strains of E.sakazakii and 11 strains of non-E.sakazakii were tested in order to ensure the specificity of the primers and the probes involved in the assay.By using two TaqMan probes, the sensitivity of the TaqMan real-time PCR assay was 8.624 copies/μl (2.7 CFU/ml), and the detection limit was 14.117 copies/μl (3.5 CFU/100 g) for detecting E.sakazakii in infant formula samples which were artificially contaminated with E.sakazakii Among 103 infant formula samples from market, 4 were determined to be positive by real-time PCR assay and 3 by classical method of FDA and these results revealed a high correlation between the two methods (99%).In the mean time, the internal controls were presented in real-time PCR assay to avoid false negative results.