Abstract
Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.
Highlights
The amplification and detection of signal from nucleic acid targets to test for the presence of specific genetic markers in sample material are of ever increasing importance in a vast array of application areas
The field of nucleic acid based testing may be termed “molecular diagnostics,” and its central step most often consists of nucleic acid amplification techniques (NAATs)
The assay described here is designed in a duplex format to allow simultaneous amplification and detection of the Roundup Ready 2 Yield (RR2Y) insertion and the endogenous soybean gene, lectin, as an internal control
Summary
The amplification and detection of signal from nucleic acid targets to test for the presence of specific genetic markers in sample material are of ever increasing importance in a vast array of application areas. These include trait detection applications in the agricultural sector, as well as clinical diagnostics, testing for food-borne pathogens, environmental testing, and many others [1,2,3]. PCR-based testing in laboratories is well established and extremely successful, the reliance of PCR on precise thermal control (at relatively high and rapidly changing—or “cycling”— temperatures; Figure 1(a)) has limited its use outside of centralized facilities. The limitations inherent in PCR make this method generally unsuitable for providing cost-effective access of molecular diagnostics to most end-users and have generally limited the adoption of NAAT-based diagnostic devices
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