Development of real-time PCR systems based on SYBR Green for the specific detection and quantification of Klebsiella pneumoniae in infant formula

Klebsiella pneumoniae is an emerging opportunistic pathogen that causes severe morbidity and mortality among the newborn and immunocompromised persons. Infant formula has been associated with the K. pneumoniae-related outbreaks in a newborn intensive care unit. In this study, a SYBR Green real-time PCR assay was developed for the rapid identification of K. pneumoniae from infant formulas. 142-bp phoE gene was selected for specificity, and two K. pneumoniae strains and 15 non-K. pneumoniae strains were evaluated. All K. pneumoniae strains tested were phoE-positive to the real-time PCR detection and no cross-reactivity was observed with non-K. pneumoniae strains. Sensitivity of the detections is 1 CFU/25 g infant formulas. And the assays can be accomplished within two business days including pre-enrichment. The results indicate that the method is efficient and can be employed to detect specifically K. pneumoniae from infant formulas with considerable sensitivity. In this study, we also constructed reference standards for quantification of K. pneumoniae by real-time PCR. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the reference standards, indicating that they can be used as quantitative detection of K. pneumoniae. This result is of special significance for applications of predicting the load of K. pneumoniae.