Development Of The Aptamer-based Method For The Detection Of Ochratoxin A And Fumonisin B₁

Mycotoxin is a toxic metabolite produced by organisms of fungi. Ochratoxin A(OTA) is a mycotoxin occurred in commodities such as cereals and feeds, while Fumonisin B1(FB1) is a carcinogenic mycotoxin found in corn and corn-originated products. Analytical methods for determination of mycotoxins are essential to ensure the safety of animals and humans health. To this end, methods for the detection of FB1 and OTA were developed using the specific aptamer against FB1 and OTA in this study.Aptamers are single-stranded oligonucleotides that bind to a specific target molecule with high affinity and specificity. PicoGreen is a fluorochrome that selectively binds dsDNA and has characteristics with an excitation maximum at 480 nm. Here, a fluorescent method for detection of OTA and FB1 was developed using the fluorescent dye PicoGreen and specific aptamer towards OTA and FB1 accordingly.We showed that the optimal buffer for mycotoxin detection in the PicoGreen/aptamer system was: 10 mmol/L Tris, 120 mmol/L NaCl, 5 mmol/L KCl, 20 mmol/L CaCl2(pH 8.5) at room temperature. For the detection of OTA, the sequence of the aptamer employed was 5’-GCATCTGATCGGGTG TGGGTGGCGTAAAGG-3’. After OTA binding with its aptamer, a peak fluorescence of PicoGreen was obtained in 3 min in the presence of remaining OTA aptamer, which formed a double-stranded hybridizer DNA with its complementary strand. The excitation and emission wavelengths for PicoGreen detection were 480 nm and 520 nm, respectively. The sensitivity of this aptamer/PicoGreen-based method of OTA was 1 μg/L with a linear range 1 μg/L-200 μg/L. No cross reactions were found against aflatoxin B1, FB1, citrinin and zearalenone. This aptamer and PicoGreen based method showed excellent agreement with the antibody based enzyme-linked immune-absorbent assay(ELISA) with Kappa value 0.885.In the aptamer/fluorescence dye PicoGreen-based method, the sensitivity was 0.1 μg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 μg/L to 1 μg/L. The entire detection procedure for FB1 could be completed within 45 min. No cross reactions were observed with any other mycotoxin against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based ELISA kit and aptamer method was excellent with a kappa value of 0.857.Taken together, the aptamer/PicoGreen-based method developed in this study is more cost-effective, time-saving and useful than ELISA for detection of OTA and FB1, offering advantages over antibodies in detection applications.