Modh Harshvardhan, Scheper Thomas, Walter Johanna-Gabriela
Institute of Technical Chemistry Leibniz University of Hannover Hannover Germany.
Eng Life Sci. 2017 Aug 2;17(8):923-930. doi: 10.1002/elsc.201700048. eCollection 2017 Aug.
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) (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.
高灵敏度检测食品毒素非常重要且具有挑战性。赭曲霉毒素A（OTA）经常作为食品污染物存在于受污染的谷物及其衍生物（如面包和啤酒）中。在这项工作中，开发了一种基于靶标诱导解离（TID）的适体辅助实时荧光定量PCR检测方法（适体-qPCR），该方法能够有效检测OTA。适体-qPCR有效地将适体作为核苷酸序列进行扩增的能力与其与相应靶标分子的特异性相互作用结合起来。与常用的基于荧光和比色法相比，qPCR检测核苷酸序列（适体）的灵敏度提高了基于适体检测OTA的灵敏度。在此，将OTA适体固定在涂有d(T)的磁珠（dT磁珠）上。将与适体的OTA结合部分互补的序列用作dT磁珠与适体序列之间的连接子。当加入OTA时，由于TID，适体从dT磁珠上释放出来。所得到的检测方法能够检测低至0.009 ng/mL的OTA，动态范围为0.039-1000 ng/mL。适体-qPCR可以很容易地转移到其他小分子上，使用相应的适体进行高灵敏度检测。
