Rapid, Specific Detection of Enterobacter sakazakii in Infant Formula Using a Real-Time PCR Assay

Abstract

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5' nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3' end and the primase (dnaG) gene 5' end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.

Real-time PCR targeting OmpA gene for detection of Cronobacter spp. in powdered infant formula

“…using real-time PCR assay The traditional bacterial culture method for the identification and enumeration of Cronobacter spp. is laborious and time-consuming; it generally requires steps of enrichment and biological tests that take 6-7 days to complete (8). To minimize the risk caused by this pathogen, the development of a rapid, sensitive, and specific method for early detection is of the utmost importance.…”

“…Recently, a number of molecular-based techniques for the rapid detection of Cronobacter spp. have been reported, including PCR assays (12)(13)(14), the DuPont Qualicon BAX system (15), real-time PCR (5,8,11), and oligonucleotide arrays (14). Real-time PCR assays using the TaqMan probe have been increasingly exploited, and these assays have replaced ordinary PCR methods in many cases.…”

“…Real-time PCR assays using the TaqMan probe have been increasingly exploited, and these assays have replaced ordinary PCR methods in many cases. During the last few years, 3 real-time PCR methods based on the16S rRNA gene (11), the 16S-23S rDNA internal transcribed spacer region (5), and the rpsU-dnaG intergenic sequence (8) were used to detect Cronobacter spp.…”

“…These procedures take 6-7 days to confirm a definite result. The ordinary PCR method tends to be more rapid than biochemical methods and has been used for the detection of these pathogens (6,7), but its practical application has been limited by several factors, including low sensitivity, low specificity, and ease of contamination during gel electrophoresis (8).…”

In-House Validation of Rapid Detection PCRs for Bacterial Pathogens Causing Infant Diarrhea

“…(ttrRSBCA locus) [6], Shigella spp. and enteroinvasive E. coli (EIEC) (ipaH) [7], enteropathogenic E. coli (EPEC) (eae) [8], enterotoxigenic E. coli (ETEC) (eltA) [4], Shiga toxin-producing E. coli (STEC) (stx 1 and stx 2 ) [9], E. coli O157 (rfbE O157 ) [9], Cronobacter sakazakii (MMS operon) [10], Campylobacter jejuni/C. coli (cadF) [3], Vibrio cholerae (toxR) [4] and Clostridium difficile (tcdB) [4] by real-time PCR.…”