title: Mining for sensitive and reliable species-specific primers for PCR for detection of Cronobacter sakazakii by a bioinformatics approach.
abstract: Although several studies have reported PCR assays for distinguishing Cronobacter sakazakii from other species in the genus, reports regarding assay sensitivity and specificity, as well as applications for food testing, are lacking. Hence, the objective of this study was to develop a sensitive and reliable PCR-based method for detection of C. sakazakii by screening for specific target genes. The genome sequence of C. sakazakii in the GenBank database was compared with that of other organisms using BLAST. Thirty-eight DNA fragments unique to C. sakazakii were identified, and primers targeting these sequences were designed. Finally, 3 primer sets (CS14, CS21, and CS38) were found to be specific for C. sakazakii by PCR verification. The detection limit of PCR assays using the 3 pairs of primers was 1.35 pg/μL, 135 fg/μL, and 135 fg/μL, respectively, for genomic DNA, and 5.5×10(5), 5.5×10(3), 5.5×10(3) cfu/mL, respectively, using pure cultures of the bacteria, compared with 13.5 pg/μLand 5.5×10(5) cfu/mLfor primer set SpeCronsaka, which has been previously described. Cronobacter sakazakii were detected in artificially contaminated powdered infant formula (PIF) by PCR using primer sets CS21 and CS38 after 8h of enrichment. The detection limit was 5.5×10(-1) cfu/10g of PIF. Thus, the PCR assay can be used for rapid and sensitive detection of C. sakazakii in PIF.
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Chen
authors.initials: C
authors.lastname: Qiming
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Tingting
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Bie
authors.initials: B
authors.lastname: Xiaomei
authors.affiliation: Department of Nutrition and Food Sciences, University of Maryland, College Park 20742.
authors.forename: Lu
authors.initials: L
authors.lastname: Yingjian
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Lu
authors.initials: L
authors.lastname: Fengxia
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China.
authors.forename: Zhai
authors.initials: Z
authors.lastname: Ligong
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, People's Republic of China. Electronic address: fmb@njau.edu.cn.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhaoxin
mesh_terms: D019295:Computational Biology; D044083:Cronobacter sakazakii / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D057225:Data Mining; D005516:Food Microbiology; D016680:Genome, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D017931:DNA Primers
keywords: Cronobacter sakazakii; PCR detection; powdered infant formula; species-specific primers