Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula

Incidence of Cronobacter sakazakii in Powdered Infant Formula Milk Available in Malaysia

“…5'-TATAGGTTGTCTGCGAAAGCG-3' and Reverse: 5'-GTCTTCGTGCTGCGAGTTTG-3' (Liu et al 2006) was done in a PCR thermal cycler (Gene Amp® PCR Systems 2700, Applied Biosystems, USA) with the following thermal-cycling conditions: Initial incubation at 95°C for 10 min, followed by a 3-step cycling program, performed for 30 cycles: denaturing at 95°C for 30 s, annealing the primers at 60°C for 30 s and 72°C for 45 s for further primer extension. Following amplification, the final extension was performed at 72°C for 10 min and cooled at 4°C.…”

“…The results showed large degree of variation in the specificity of the PCR primers and that not all primers were suitable for accurate detection and identification of Cronobacter (Cawthorn et al 2008). Liu et al (2006) developed a Real-time PCR assay using TaqMan and SYBR Green for detection and enumeration of this pathogen after selective enrichment. The newly developed assays would detect 1.1 cfu/g infant formula and both could be accomplished within 2 days, while standard methods such as conventional culture and biochemical-based assays used to enumerate and identify Cronobacter as 'E.…”

“…The most popular, the 5'-nuclease assay is based on the specific hybridization of a dual-labeled TaqMan probe to the PCR product. SYBR-Green method is based on the binding of the fluorescent dye SYBR-Green into the amplified DNA during the primer annealing and extension steps (Liu et al 2006).…”

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

“…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.…”