MACRO: A Combined Microchip-PCR and Microarray System for High-Throughput Monitoring of Genetically Modified Organisms

Abstract

The monitoring of genetically modified organisms (GMOs) is a primary step of GMO regulation. However, there is presently a lack of effective and high-throughput methodologies for specifically and sensitively monitoring most of the commercialized GMOs. Herein, we developed a multiplex amplification on a chip with readout on an oligo microarray (MACRO) system specifically for convenient GMO monitoring. This system is composed of a microchip for multiplex amplification and an oligo microarray for the readout of multiple amplicons, containing a total of 91 targets (18 universal elements, 20 exogenous genes, 45 events, and 8 endogenous reference genes) that covers 97.1% of all GM events that have been commercialized up to 2012. We demonstrate that the specificity of MACRO is ~100%, with a limit of detection (LOD) that is suitable for real-world applications. Moreover, the results obtained of simulated complex samples and blind samples with MACRO were 100% consistent with expectations and the results of independently performed real-time PCRs, respectively. Thus, we believe MACRO is the first system that can be applied for effectively monitoring the majority of the commercialized GMOs in a single test.

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

“…the number of dyes that can currently be detected, the occurrence of false positives when the targets to be multiplexed become too numerous and/ or may interact and the loss of sensitivity. Furthermore, high-throughput technologies, as, for instance, NASBA implemented microarray analysis (NAIMA) [23,24], microdroplet PCR implemented capillary gel electrophoresis (MPIC) [25], multiplex ligation detection methods [26] (and references therein), a combined microchip-PCR and microarray system (MACRO) [27], digital PCR [28,29] and next-generation sequencing (NGS) [30][31][32], have been developed and their possible use in GMO detection was demonstrated. These methods are, however, still too costly and/or cumbersome for routine use and often require expensive equipment and/or specialised data analysis tools and staff.…”

Microarray-in-a-tube system in the function of detection human papillomavirus genotypes

“…Taq DNA polymerase, dNTPs and buffer were purchased TaKaRa Taq TM R001B (Dalian, China), Microarray buffer was purchased from Capitalbio Corporation (Beijing, China), Hybridization solution (6× SSC, 50 % Denhardt, and 0.2 % (w/v) SDS [12].…”

GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays

“…The primers were designed based on reported sequences (http:// gmdd.shgmo.org) listed in Table S1 in the Electronic Supplementary Material (ESM). Conventional PCR primers for these 8 targets and 12 GM events (in order to verify the LAMP assay results of practical samples) were described in our previous work [17]. All of the conventional PCR primers were also synthesized by Invitrogen Co., Ltd. (Shanghai, China) and listed in ESM Table S2.…”

“…Since as many as more than 300 GM crop events have been approved and commercialized, development of comprehensive and effective screen-specific methods for fast and cost-effective GMO detection are highly needed. In previous studies, we have utilized CaMV35S promoter, FMV35S promoter, CaMV35S terminator, NOS terminator, cry1Ab/c, pat, bar, NptII, CP4 epsps, and bla as targets for GMO screening using PCR-based methods [16][17][18]. Herein, we report the development of a series of LAMP assays of these eight targets (CaMV35S promoter, FMV35S promoter, NOS terminator, cry1Ac, pat, bar, NptII, and CP4 epsps) for GMO screening.…”