Primers with 5' Flaps Improve the Efficiency and Sensitivity of Multiplex PCR Assays for the Detection of Salmonella and Escherichia coli O157:H7

• Published in: Journal of food protection 2013-04, Vol.76 (4), p.668-673
• Main Authors: TIMMONS, Chris, DOBHAL, Shefali, FLETCHER, Jacqueline, MARIA MA, L. I
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Colony Count, Microbial; Deoxyribonucleic acid; DNA; DNA Primers; DNA, Bacterial - analysis; E coli; Efficiency; Epidemics; Escherichia coli; Escherichia coli O157 - isolation & purification; Flora; Food contamination & poisoning; Food Contamination - analysis; Food industries; Food Microbiology; Food quality; Food safety; Foodborne diseases; Fundamental and applied biological sciences. Psychology; General aspects; Humans; Illnesses; Lycopersicon esculentum; Methods of analysis, processing and quality control, regulation, standards; Microbiology; Pathogens; Polymerase chain reaction; Polymerase Chain Reaction - methods; Salmonella; Salmonella - isolation & purification; Salmonella enterica; Sensitivity and Specificity; Time Factors; Tomatoes
• ISSN: 0362-028X; 1944-9097
• DOI: 10.4315/0362-028X.JFP-12-428

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.