Detection of ochratoxin A by aptamer-assisted real-time PCR-based assay (Apta-qPCR)

Abstract

Detection of food toxins with high sensitivity is very important and challenging. Ochratoxin A (OTA) is frequently present as food contaminant in contaminated grains and grain derivatives such as bread and beer. In this work, a target-induced dissociation (TID) based aptamer-assisted real-time PCR-based assay (apta-qPCR) is developed that features effective detection of OTA. Apta-qPCR effectively combines the capabilities of aptamer to be amplified, being a nucleotide sequence, with its specific interaction with the corresponding target molecule. Compared to commonly used fluorescence-based and colorimetric methods, the sensitivity of qPCR to detect a nucleotide sequence (aptamer) has ameliorated the sensitivity of the aptamer-based detection of OTA. Here, the OTA aptamer was immobilized on the magnetic beads coated with d(T)25 (dT beads). A sequence complementary to the OTA-binding portion of the aptamer was used as a linker between dT beads and the aptamer sequence. When OTA was added, the aptamer was released from the dT beads due to TID. The resulting assay was able to detect 0.009 ng/mL OTA with a wide dynamic range of 0.039–1000 ng/mL. Apta-qPCR can be easily transferred to other small molecules for highly sensitive detection using corresponding aptamers.