NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

Abstract

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

Four new SYBR®Green qPCR screening methods for the detection of Roundup Ready®, LibertyLink®, and CryIAb traits in genetically modified products

“…Several multiplex qPCR formats are available reducing the number of analyses and facilitating high throughput but requiring multi-channel detection devices and often including costly detection probes for at least some of the targets [8][9][10]. In other cases, the applied chemistries are quite complex or are to be combined with other technologies that are less appropriate for routine applications [11][12][13][14][15][16]. Several of these approaches involve the use of PCR with multiple targets and consecutive detection and identification of the amplification products using microarrays [11][12][13].…”

“…In other cases, the applied chemistries are quite complex or are to be combined with other technologies that are less appropriate for routine applications [11][12][13][14][15][16]. Several of these approaches involve the use of PCR with multiple targets and consecutive detection and identification of the amplification products using microarrays [11][12][13]. Apart from requiring additional costly array analysis equipment, these approaches are often prone to variable quality of the array chips making them less suitable for routine or enforcement purposes [17].…”

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.…”

Event-specific Real-time RPA Detection of Transgenic Rice Kefeng 6

“…Currently some nucleic acid isothermal amplification technologies have been used for nucleic acid testing, such as loop-mediated isothermal amplification (LAMP) (Guan et al, 2010;Chenet al, 2011;, nucleic acid sequence-based amplification (NASBA) (Morisset et al, 2008) and more. Compared with those isothermal amplification technologies, the most significant characteristic of RPA is the greatly shortened detection time and the reduced reaction temperature.…”