Detection of ochratoxin A by aptamer-assisted real-time PCR-based assay (Apta-qPCR)
Detection of ochratoxin A by aptamer-assisted real-time PCR-based assay (Apta-qPCR)
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DOI：
10.1002/elsc.201700048
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发表时间：
2017-08-01
影响因子：
2.7
通讯作者：
Walter, Johanna-Gabriela
中科院分区：
文献类型：
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作者：
Modh, Harshvardhan;Scheper, Thomas;Walter, Johanna-Gabriela
高灵敏度检测食物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和谷物衍生物（例如面包和啤酒）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 PCR 检测 (apta-qPCR)，其特点是有效检测 OTA。 Apta-qPCR 有效地将待扩增的核酸适体（核酸序列）的能力与其与相应靶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的方法相比，qPCR 检测核苷酸序列（适配体）的灵敏度提高了基于适配体的 OTA 检测的灵敏度。此处，OTA 适体被固定在涂有 d(T) 25 的磁珠（dT 珠）上。与适体的 OTA 结合部分互补的序列被用作 dT 珠和适体序列之间的接头。添加 OTA 后，由于 TID，适体从 dT 珠中释放出来。所得测定能够检测 0.009 ng/mL OTA，动态范围为 0.039-1000 ng/mL。 Apta-qPCR 可以轻松转移到其他小分子，使用相应的适体进行高灵敏度检测。
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-timePCR-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 interactionwith 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.