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

Abstract

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. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for the final evaluation. After the second evaluation, the final amplification curves and melting curves have been achieved.

Development and application of a novel multiplex PCR method for four living modified soybeans

“…According to Shin et al [11], the simultaneous detection method was applied to identify LM corn samples which were collected from the natural environment LMO monitoring to verify its detection efficiency. In this study, the random DNA mixture combinated with four CRM genomic DNAs of LMOs and non-LMOs was used to verify the efficiency of the multiplex PCR analysis.…”

“…[7,8]. Moreover, the detection methods were further elaborated for the multiplex PCR which was applicable to the analysis of LMO monitoring samples [9][10][11].…”

A high-throughput and ultrasensitive identification methodology for unauthorized GMP component based on suspension array and logical calculator

“…As the identification of unauthorized GMP components includes the PCR amplification of different screening elements or event sequences 8–11 , the utilization of high-throughput technologies is essential to save labor and achieve reliable results. Recently, several technologies, including ChIP- 12,13 , sequencing- 8,14 and PCR-based methodologies 15,16 , have been developed to meet various demands, such as gene expression evaluation, SNP identification and multiplex component distinction. Among the different technologies, PCR-based methods, particularly multiplex PCR, are the most easily operated methods with an extremely low threshold for both facilities and operators 15,16 .…”

“…Recently, several technologies, including ChIP- 12,13 , sequencing- 8,14 and PCR-based methodologies 15,16 , have been developed to meet various demands, such as gene expression evaluation, SNP identification and multiplex component distinction. Among the different technologies, PCR-based methods, particularly multiplex PCR, are the most easily operated methods with an extremely low threshold for both facilities and operators 15,16 . However, these methods would be not convenient due to the increase in target amplicons.…”

“…The ME-qPCR is firstly mentioned in 2005 with the definition of MT-PCR. This technology have been optimized and utilized for the area of GMP components by Fu 16 and Wei 17 . Based on the combination of universal primer 18 , the UP-ME-qPCR has been developed to ensure the multiplex amplification more stable and feasible.…”