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Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification

文献类型： 外文期刊

作者： Fu, Wei 1 ; Zhu, Pengyu 1 ; Wei, Shuang 2 ; Du Zhixin 3 ; Wang, Chenguang 1 ; Wu, Xiyang 4 ; Li, Feiwu 5 ; Zhu, Shuifang 1 ;

作者机构： 1.Chinese Acad Inspect & Quarantine, Inst Plant Quarantine, Beijing Econ Technol Dev Area, Ronghuananlu 11, Beijing 100176, Peoples R China

2.Shantou Entry Exit Inspect & Quarantine Bur, 126 Jinsha Rd, Shantou 515041, Guangdong, Peoples R China

3.Guangxi Entry Exit Inspect & Quarantine Bur, 38 Binhu Rd, Nanning 530028, Guangxi, Peoples R China

4.Jinan Univ, Dept Food Sci & Engn, Guangzhou 510632, Guangdong, Peoples R China

5.Jilin Acad Agr Sci, Inst Agr Stand & Testing Technol, 1363 Shengtai St, Changchun 130033, Jilin, Peoples R China

关键词： ME-qPCR;GMO;Multiplex;High-throughput detection;Highly sensitive

期刊名称：ANALYTICAL AND BIOANALYTICAL CHEMISTRY （ 影响因子：4.142； 五年影响因子：3.863 ）

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年卷期：

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收录情况： SCI

摘要： Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples.

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