Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of...

Aflatoxin B(1) is an unavoidable natural mycotoxin that enters the food chain by contamination of food grains and feedstuffs, potentially posing carcinogenic risks to animal and human health. Immuno-PCR methods have the potential to address the need of meeting the regulatory limits by detecting trace levels of toxins present in food and animal feeds. This paper describes a real-time immuno-quantitative PCR (RT-iqPCR) assay for quantification of aflatoxin B(1) suspended in methanol:water solution that can also serve as an extraction solvent. Immuno-PCR approaches were examined including direct vs. indirect sandwich assays using monoclonal vs. polyclonal antibodies. Our best approach was obtained using monoclonal antibodies to capture aflatoxin in solution prior to immobilizing the F(c) portion of the capture antibodies onto to protein G magnetic beads. This was followed by the addition of a polyclonal 'signal antibody' tethered with an oligonucleotide template for a subsequent PCR assay. The RT-iqPCR assay described herein leads to the sensitive detection and quantification of aflatoxin B(1) from 10ppb down to 0.1ppb with high correlation (r(2)=0.97) and efficiency (99.5%). The approach also detected the high-dose 'hook effect' phenomenon (excess antigen) which was overcome by the use of dilution protocols to eliminate false negatives that may occur at levels above quantification limits of the assay. The RT-iqPCR approach discussed here is presented as a model system that could easily be adapted for aflatoxin detection in a variety of food or animal feed samples using a simple methanol:water solution as an extraction solvent.