Abstract
Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ – 53.74 ng) and 78.3 to 94.22% (LOQ – 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.
Highlights
Aflatoxins are highly toxic difuranocoumarin derivatives produced by the genus Aspergillus, mainly by Aspergillus flavus, A. parasiticus, and A. nomius (Yu, 2012; Markov et al, 2013)
SsDNA library, aptamer sequences, primers, and mycotoxins used in this study were obtained from Sigma-Aldrich (India), except mentioned
The single stranded DNA library used in the present study was designed with a randomized region of 40 nucleotides flanked by two known sequences of 20 nucleotides on either side
Summary
Aflatoxins are highly toxic difuranocoumarin derivatives produced by the genus Aspergillus, mainly by Aspergillus flavus, A. parasiticus, and A. nomius (Yu, 2012; Markov et al, 2013). Aflatoxin contamination is a major concern in global food safety, urging many countries to set stringent regulations on their occurrences. The guidelines set by Food Safety and Standards Authority of India limit aflatoxins to 15 and 30 ppb in Immuno Affinity SELEX for Mycotoxin cereals and spices, respectively (FSSAI, 2015). Food and Drug Administration (FDA) has set permissible levels to 20 ppb in food and 300 ppb in livestock feed; whereas, European Union (EU) stipulate an acceptable range upto 2 ppb of AFB1 and 4 ppb of total aflatoxins in cereal products (European Union Commission, 2006; Le et al, 2010)
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