Rapid and sensitive detection of aflatoxin in animal feeds and food grains using immunomagnetic bead based recovery and real-time immuno quantitative PCR (Rt-iqPCR) assay

Abstract

Scope and Method of Study:

Aflatoxins are one of the major mycotoxins that are considered as unavoidable natural toxins encountered in food and feedstuffs. Aflatoxins are produced by several strains of Aspergillus fungi and known to be deleterious to human and animal health even at trace levels. Development of highly sensitive analytical methods for detection of aflatoxins could prevent potential health hazards while being in compliance with regulatory limits for the trade of commodities. In this study, we have developed a hitherto unexplored use of real time immuno quantitative PCR (RT iq-PCR) assay for sensitive detection and quantification of aflatoxins in animal feeds, feed grains and foodstuffs. The use of several immuno-PCR approaches to estimate aflatoxin contents in various agricultural commodities is outlined in this work. Our methodology can detect and quantify aflatoxins as low as 0.1 ppb in methanol extractions of complex food and feed matrices.

Findings and Conclusions:

The first part of the immuno-PCR approach developed here involves magnetic bead based recovery of aflatoxin B1 (AFB1) from methanol extractions of food and feed samples and sensitive quantification of AFB1 using real time PCR. The use of different types of magnetic beads as solid supports for the immunoassays was tested to arrive at a bead type showing least nonspecific binding of signal generating marker. Development strategies for reporter DNA labels, conjugation of reporter DNA to secondary detection antibodies and performance of capture antibodies during immunoassays are outlined. We also tested usefulness of different monoclonal and polyclonal antibodies in direct and indirect sandwich assay formats as capture antibodies and verified the preference of detection antibodies tethered with reporter DNA marker. Calibration curves using several dilutions of aflatoxin standards in 60% methanol were developed. Second part of the study involved demonstration of the optimized immuno-PCR approach for detection and quantification of aflatoxins extracted from complex matrices of food and feedstuffs. Methanol extractions of animal feeds, corn feed grains and yellow corn meal were compared and most of the samples tested contained total aflatoxin content of less than 20 ppb. However, an excess antigen hook effect due to high analyte concentrations beyond the detection limit of our immuno-PCR assay (0.1 to 10ppb) was noticed as common occurrence in test samples. To overcome false negative results due to excess aflatoxin contents, we demonstrated the use of dilution protocols enabling the detection of very high levels of aflatoxins in feed extractions. Testing for the reliability of the immuno-PCR assay, samples were spiked with 200 ppb of aflatoxin B1 and about 96% of the spiked aflatoxin was recovered from horse feed extracts which further demonstrated the assay performance and reliability. Considering the significance of estimating trace levels of aflatoxins and their serious implications on animal and human health, we thus developed and tested our quantitative real time immuno-PCR method that could be used as a model system for aflatoxin detection in complex matrices of food or animal feed samples.