Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension).
Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 μL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods.
Department of Biomedical Engineering, University of Wisconsin-Madison, 1111 Highland Ave., Madison, WI 53705, USA.
Lindsay N
LN
Strotman
Guangyun
G
Lin
Scott M
SM
Berry
Eric A
EA
Johnson
David J
DJ
Beebe
D000818:Animals; D001628:Beverages / Q000382:microbiology; D002417:Cattle; D047013:Clostridium botulinum type A / Q000235:genetics / Q000302:isolation & purification; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D005374:Filtration / Q000379:methods*; D005516:Food Microbiology; D008280:Magnetics; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction; D013500:Surface Tension
D017931:DNA Primers; D004247:DNA