MPIC: A High-Throughput Analytical Method for Multiple DNA Targets

Abstract

We describe the development of a novel combined approach for high-throughput analysis of multiple DNA targets based on multiplex Microdroplet PCR Implemented Capillary gel electrophoresis (MPIC), a two-step PCR amplification strategy. In the first step, the multiple target DNAs are preamplified using bipartite primers attached with universal tail sequences on their 5'-ends. Then, the preamplified templates are compartmentalized individually in the microdroplet of the PCR system, and multiple targets can be amplified in parallel, employing primers targeting their universal sequences. Subsequently, the resulting multiple products are analyzed by capillary gel electrophoresis (CGE). Using genetically modified organism (GMO) analysis as a model, 24 DNA targets can be simultaneously detected with a relative limit of detection of 0.1% (w/w) and absolute limit of detection of 39 target DNA copies. The described system provides a promising alternative for high-throughput analysis of multiple DNA targets.

A novel quadruplex real-time PCR method for simultaneous detection of Cry2Aeand two genetically modified cotton events (GHB119 and T304-40)

“…Due to their high specificity, sensitivity, operability and repeatability, nucleic acid-based methods (such as PCR, real-time PCR, isothermal amplification techniques and others) are widely employed for the detection of GMO events [2,3]. As the quantity of exogenous genes and GM crop events increase, several multiplex methods for simultaneous detection of more than one genetic element have been developed, such as multiplex PCR, microarray analysis, suspension array analysis, microdroplet-based PCR and others [4-6]. These techniques are especially useful for detecting GM events in complex or mixed samples that contain multiple exogenous insertions.…”

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.…”

How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed

“…Apart from real-time PCR, new alternative and advanced technologies have been proposed including the use of high-throughput systems or platforms for the detection of multiple targets, for example, microarrays, MIPC, PCR combined with capillary gel electrophoresis (fingerprinting), and next generation sequencing ([12, 18, 35–38] and references therein). However, at the present stage they are often more expensive, difficult to standardise and validate, and require extensive, specialised work and equipment.…”