新型通用引物多重 PCR 结合焦磷酸测序的多重定性检测。

Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing.

机构信息

Yunnan Institute of Food Safety, Kunming University of Science and Technology, Kunming, Yunnan 650500, China; Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.

Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083, China.

出版信息

Food Chem. 2017 Dec 15;237:773-778. doi: 10.1016/j.foodchem.2017.05.068. Epub 2017 May 15.

DOI:10.1016/j.foodchem.2017.05.068

PMID:28764066

Abstract

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events.

摘要

本研究描述了一种使用焦磷酸测序进行新型多重定性检测的方法。该方法基于通用引物多重 PCR 的原理，仅使用一个测序引物即可实现多个目标的检测。使用不同比例的三种转基因作物的混合样品对该方法进行了验证。根据产物序列设计了 dNTP 分配顺序。在理想条件下，仅需添加 12 轮（ATCTGATCGACT）dNTP，通常只需添加三轮（CAT）即可定性检测 GM 事件，灵敏度低至混合物的 1%。然而，在考虑混合物的情况下，计算信号值可以估计每个 GM 的比例。基于这些结果，我们得出结论，我们的新方法不仅实现了检测，还允许对单个事件进行半定量检测。