Direct Real-Time PCR with Ethidium Monoazide: A Method for the Rapid Detection of Viable Cronobacter sakazakii in Powdered Infant Formula

Abstract

The goal of this study was to establish a rapid assay for the specific detection of viable Cronobacter sakazakii in powdered infant formula (PIF). Samples were subjected to treatment multiple times with ethidium monoazide with a concentration gradient (gEMA) prior to PCR to discriminate viable from dead C. sakazakii cells. To improve the current detection limits, we developed a new buffer for direct quantitative real-time PCR (DqPCR) without DNA isolation. Using 17 PIF samples, our rapid assay was compared with the new U.S. Food and Drug Administration (FDA) method published in the Bacteriological Analytical Manual in 2012. Although both the new FDA method and our rapid assay, which consists of DqPCR combined with gEMA (gEMA-DqPCR), produced negative results for all 17 PIF samples, 5 of the 17 PIFs were positive by DqPCR when they were not treated with EMA. Furthermore, for PIF samples artificially contaminated with viable C. sakazakii, gEMA-DqPCR successfully detected between 1 and 9 CFU of viable C. sakazakii in 300 g of PIF within 9 h, including a 6-h preincubation. Our results indicate that multiple EMA treatments are required to avoid false-positive results due to the contamination of commercial PIF with dead or injured C. sakazakii cells. Our rapid assay may also improve the sensitivity of the screening portion required by the new FDA method published in the Bacteriological Analytical Manual in 2012.