title: Rapid detection of Cronobacter sakazakii by real-time PCR based on the cgcA gene and TaqMan probe with internal amplification control.
abstract: Cronobacter sakazakii is a severe virulent strain that is frequently detected in powdered infant formula (PIF). Therefore, it is necessary to develop a fast and specific detection method. The specificity of our newly developed quantitative real-time PCR (qRT-PCR) was validated with DNA from 46 strains. Among them, 12 C. sakazakii strains were correctly amplified, whereas no positive florescent signal was observed from 34 nontarget controls. The detection limit of C. sakazakii was about 110 CFU/mL in broth and 1100 CFU/g in PIF. After enrichment in buffered peptone water for 6 h, our developed qRT-PCR assay could reliably detect C. sakazakii when the inoculation level was as low as 2 CFU/25 g (0.08 CFU/g) in PIF. The growth of C. sakazakii could be inhibited by the presence of Lactobacillus pentosus and Bacillus cereus, which used a longer enrichment period before the isolation was accomplished. However, at 5 and 50 CFU/25 g inoculation levels of C. sakazakii in the presence of 4 × 10(6) CFU L. pentosus/25 g or of 2 × 10(4) CFU B. cereus/25 g, the qRT-PCR assay could detect the presence of Cronobacter even though these artificially spiked samples were negative in culture. Therefore, our results indicated that the qRT-PCR assay could detect samples containing inhibitors and could avoid false negatives by using an internal amplification control.
authors.affiliation: a Research Center of Food Safety and Detection, College of Light Industry and Food Sciences, South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou City, Guangdong Province 510640, People's Republic of China.
authors.forename: Shuangfang
authors.initials: S
authors.lastname: Hu
authors.affiliation: a Research Center of Food Safety and Detection, College of Light Industry and Food Sciences, South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou City, Guangdong Province 510640, People's Republic of China.
authors.forename: Yigang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: b Zhongshan Entry-Exit Inspection and Quarantine Bureau, Room 804, No. 2, Zhongshan 6th Road, Zhongshan, Guangdong Province 528403, People's Republic of China.
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.affiliation: c Department of Microbiology, Guangzhou Center for Disease Control and Prevention, Qide Road No. 2, Guangzhou, Guangdong Province 510440, People's Republic of China.
authors.forename: Xinwei
authors.initials: X
authors.lastname: Wu
authors.affiliation: a Research Center of Food Safety and Detection, College of Light Industry and Food Sciences, South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou City, Guangdong Province 510640, People's Republic of China.
authors.forename: Xinglong
authors.initials: X
authors.lastname: Xiao
authors.affiliation: a Research Center of Food Safety and Detection, College of Light Industry and Food Sciences, South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou City, Guangdong Province 510640, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wu
mesh_terms: D044083:Cronobacter sakazakii / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005516:Food Microbiology; D006801:Humans; D007223:Infant; D041943:Infant Formula / Q000382:microbiology*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase
keywords: Cronobacter sakazakii; LPN; PIF; cgcA; détection rapide; qRT–PCR; rapid detection