title: Primers with 5' flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7.
abstract: Foodborne illnesses caused by Salmonella enterica and Escherichia coli O157:H7 are worldwide health concerns. Rapid, sensitive, and robust detection of these pathogens in foods and in clinical and environmental samples is essential for routine food quality testing, effective surveillance, and outbreak investigations. The aim of this study was to evaluate the effect on PCR sensitivity of adding a short, AT-rich overhanging nucleotide sequence (flap) to the 5' end of PCR primers specific for the detection of Salmonella and E. coli O157:H7. Primers targeting the invA gene of Salmonella and the rfbE gene of E. coli O157:H7 were synthesized with or without a 12-bp, AT-rich 5' flap (5'-AATAAATCATAA-3'). Singleplex PCR, multiplex PCR, and real-time PCR sensitivity assays were conducted using purified bacterial genomic DNA and crude cell lysates of bacterial cells. The effect of background flora on detection was evaluated by spiking tomato and jalapeno pepper surface washes with E. coli O157:H7 and Salmonella Saintpaul. When targeting individual pathogens, end-point PCR assays using flap-amended primers were more efficient than nonamended primers, with 20.4 and 23.5% increases in amplicon yield for Salmonella and E. coli O157:H7, respectively. In multiplex PCR assays, a 10- to 100-fold increase in detection sensitivity was observed when the primer flap sequence was incorporated. This improvement in both singleplex and multiplex PCR efficiency and sensitivity can lead to improved Salmonella and E. coli O157:H7 detection.
authors.affiliation: National Institute for Microbial Forensics and Food and Agricultural Biosecurity, Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, Oklahoma 74078, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Timmons
authors.forename: Shefali
authors.initials: S
authors.lastname: Dobhal
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Fletcher
authors.forename: Li Maria
authors.initials: LM
authors.lastname: Ma
mesh_terms: D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial