基于荧光染料PicoGreen和核酸适配体的伏马毒素B1检测方法

Development of an aptamer/fluorescence dye PicoGreen-based method for detection of fumonisin B1

摘要:

伏马毒素B1是主要存在于玉米及玉米制品中的一种可以引起癌症的霉菌毒素。针对霉菌毒素精准检测技术的开发对于保障食品安全至关重要。本研究利用核酸适配体与伏马毒素B1结合后不再结合其互补核酸序列的选择性以及PicoGreen与双链DNA结合的特异性，开发了一种快速检测伏马毒素B1的适配体方法。PicoGreen与双链DNA反应15 min后激发产生的荧光达到峰值 (激发波长为480 nm，发射波长为520 nm)。该方法的最低检测限为0.1 μg/L (0.1 ppb)，线性范围为0.1?1 μg/L (0.1?1 ppb)，整个检测流程可在40 min内完成。特异性试验显示伏马毒素B1适配体与黄曲霉毒素B1、赭曲霉毒素、桔毒素和玉米赤霉烯酮等常见霉菌毒素无交叉反应。结果表明适配体方法与基于抗体的检测伏马毒素B1商品化ELISA试剂盒相当，Kappa值为0.857。由于核酸适配体比抗体成本低，检测时间短，因此基于核酸适配体的方法比基于抗体的ELISA方法更具有推广应用价值。

Abstract:

Fumonisin B1 (FB1) is a carcinogenic mycotoxin found in commodities such as corn and corn-originated products. An aptamer-based method for detection of FB1 was developed using the fluorescent dye PicoGreen, which can recognize and bind double-stranded DNA. A peak fluorescence of PicoGreen was obtained in 15 min in the presence of FB1 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 was 0.1 μg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 to 1 μg/L. The entire detection procedure for FB1 could be completed within 40 min. No cross reactions were observed with any other mycotoxins against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based enzyme-linked immunosorbent assay (ELISA) kit and aptamer method was excellent with a kappa value of 0.857. Taken together, this aptamer/PicoGreen-based method is more cost-effective, time-saving and useful than ELISA for detection of FB1.