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G03022-Refinement of GMO screening methods by combining existing multiplex PCR approaches with lab-on-a-chip capillary electrophoresis endpoint detection

Project Code: G03022

10/09/2009

The FSA requires high throughput and low cost screening methods to help Public Analysts provide enforcement of the GM Food and Feed Regulation (EC 1829/20030) for GM food and feed ingredients. British Standard qualitative and quantitative methods (BS EN ISO 21569:2005 and BS EN ISO 21570:2005) are available for GMO testing. These use the polymerase chain reaction (PCR) to target DNA sequences within the GM crops but uptake by enforcement laboratories has been limited due to the nature of the equipment or skill base needed to perform the analyses and produce reliable results.

Project G0322 investigated the use of a lab-on-a-chip capillary electrophoresis technology (Agilent 2100 Bioanalyzer) to deliver simple, cost effective methods for routine DNA analyses. It concentrated on the screening for common markers associated with GM crops, the identification of Roundup Ready soya and the detection of several GM maize varieties.

A novel multiplex GMO screening assay was developed to detect small target sequences (<130bp) found in the Nos terminator, lectin (endogenous soya), zein (endogenous maize), cauliflower mosaic virus (CaMV) and the CaMV 35S promoter. The CaMV target was included in the assay to enable differentiation of samples containing GMO ingredients from those samples which contain material derived from cruciferous plants infected with the CaMV. The assay was applied to certified reference materials, processed ingredients and animal feeds. It was further validated in a trial amongst 10 local authority public analysts. They were provided with a detailed SOP and PCR mastermix reagents and asked to analyze a range of materials. Their results indicated that the multiplex assay worked well with little variation in the number of targets detected by the laboratories for each sample. Results also indicated that it could detect down to 0.1% (w/w) levels of GMO.

A second multiplex assay was developed to detect the endogenous soya lectin gene and targets within the Roundup Ready (RR) soya transgene. This assay was used to estimate the levels of RR soya in some soya based ingredients including soya flour, textured vegetable protein (TVP) and animal feed. The levels produced compared well with the levels of Roundup Ready soya measured by Real-time PCR. During this study, an automated DNA extraction instrument, Promega’s Maxwell 16, was also evaluated. Results indicated that the Maxwell 16 was suitable for reliable extraction of DNA from some food and ingredients but the yield was lower than with other methods.

A third multiplex assay was proposed for the detection of 5 different GM maize varieties (Bt176, Bt11, Mon810, GA21, T25) and endogenous maize gene, zein. This used targets and PCR primer sets from existing published assays that were selected for optimal use on the Bioanalyzer. The individual primer sets were obtained and tested. Results indicated that the individual assays detected the intended GM maize varieties with no cross-reactivity, but when the primers were used together in a multiplex assay there was preferential amplification of some GM maize varieties. Further development and optimisation of the assay is required prior to uptake by food enforcement laboratories.

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