Detection Of Ochratoxin A In Grains Based On Aptamer Chemiluminescence System

Ochratoxin A (OTA) is a common source of pollution in cereals, and the range and degree of OTA pollution are relatively wide because it can be produced by many species of fungi. At present, it is very important to establish a new type of fast and efficient detection technology for OTA to overcome some of the shortcomings of existing methods, such as high detection costs, long detection time, demands of expensive instruments and complex operation processes. In this article, silica photonic crystal microspheres (SPCMs) were used as the carrier and the aptamers were used as the basis of the reaction. Two methods for the rapid and efficient detection of OTA in cereals were established using the luminol chemiluminescence method.The uniform size and stable structure of SPCMs were prepared by the laboratory self-assembly platform. Then, four groups were modified on the surface of SPCMs,including: Carboxyl, epoxy, aldehyde and isothiocyanato. Subsequently, the complementary strand of OTA-aptamer modified amino were bound to the carboxylated microspheres. At last, we characterized different microspheres by infrared to confirm the modification of the groups and the complementary strand.We established an aptamer competitive chemiluminescence method to detect OTA, and optimized the key factors in the detection process. The optimal experimental conditions were as follows: OTA-aptamer and its complementary strand were 4000nmol/L; the temperature of competitive reaction was 37?; the time of competitive reaction was 1.5h. According to the optimal conditions, we setup standard curves. The linear range of OTA was 0.001 ?1ng/mL and the detection limit was 0.001ng/mL. The linear equations was y=-4329.22x+3565.50(R2=0.9964). The OTA-aptamer specific test showed that there was no significant cross-reactivity with its structurally similar toxins. The recovery rate test of OTA in rice, wheat and maize was 80.54± 1.45?104.55±5.89, and the results showed that this method was feasible for the detection of OTA compared with ELISA. Finally, this method and ELISA method were used to analyze the content of OTA in 18 different grains. The comparison showed that the approximation of the two is approximative, which is more determine that this method can be used for the detection of OTA content in the actual grains.We also established a DNAzyme-aptamer chemiluminescence method to detect OTA, and optimized the key factors in the detection process. The optimal experimental conditions were as follows: the concentration of Mg2+ was 30nM; the concentration of K+ was 9nM; the reaction time of B2 chain was 30min. According to the optimal conditions, we setup standard curves. The linear range of OTA was 0.1 ?100ng/mL and the detection limit was 0.1ng/mL. The linear equations was y=8161.35x+24303.63(R2=0.9920). The OTA-aptamer specific test showed that there was no significant cross-reactivity with other toxins. The recovery rate test of OTA in rice, wheat and maize were 72.19±4.20-121.58±9.75 and 87.15±0.31?119.45±0.54 in this method and ELISA, respectively, which showed that they were in agreement. The results showed that this method was feasible for the detection of OTA.