Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs—standard single, multiplex and construct-specific PCR assays

Abstract

Vegetative insecticidal protein (Vip), a unique class of insecticidal protein, is now part of transgenic plants for conferring resistance against lepidopteron pests. In order to address the imminent regulatory need for detection and labeling of vip3A carrying genetically modified (GM) products, we have developed a standard single PCR and a multiplex PCR assay. As far as we are aware, this is the first report on PCR-based detection of a vip3A-type gene (vip-s) in transgenic cotton and tobacco. Our assay involves amplification of a 284-bp region of the vip-s gene. This assay can possibly detect as many as 20 natural wild-type isolates bearing a vip3A-like gene and two synthetic genes of vip3A in transgenic plants. The limit of detection as established by our assay for GM trait (vip-s) is 0.1%. Spiking with nontarget DNA originating from diverse plant sources had no inhibitory effect on vip-s detection. Since autoclaving of vip-s bearing GM leaf samples showed no deterioration/interference in detection efficacy, the assay seems to be suitable for processed food products as well. The vip-s amplicon identity was reconfirmed by restriction endonuclease assay. The primer set for vip-s was equally effective in a multiplex PCR assay format (duplex, triplex and quadruplex), used in conjunction with the primer sets for the npt-II selectable marker gene, Cauliflower mosaic virus 35S promoter and nopaline synthetase terminator, enabling concurrent detection of the transgene, regulatory sequences and marker gene. Further, the entire transgene construct was amplified using the forward primer of the promoter and the reverse primer of the terminator. The resultant amplicon served as a template for nested PCR to confirm the construct integrity. The method is suitable for screening any vip3A-carrying GM plant and food. The availability of a reliable PCR assay method prior to commercial release of vip3A-based transgenic crops and food would facilitate rapid and efficient regulatory compliance.

Development of insect-resistant transgenic cotton with chimeric TVip3A* accumulating in chloroplasts

“…There is a growing concern about the potential threat of insects developing resistance to Bt endotoxins (Cry1Ab, Cry1Ac, Cry1F, and their combinations), especially with the widespread adoption of Bt crops over recent years (Kurtz et al 2007;Sena et al 2009;Singh et al 2008). To date, many resistant insect strains have emerged in the laboratory, greenhouse, and/or field conditions (Cao et al 2002;Ferre and Van Rie 2002;Shelton et al 2002;Tabashnik et al 2003;Zhao et al 2002).…”

“…To date, many resistant insect strains have emerged in the laboratory, greenhouse, and/or field conditions (Cao et al 2002;Ferre and Van Rie 2002;Shelton et al 2002;Tabashnik et al 2003;Zhao et al 2002). To meet the insectdamage challenge in plants, various management techniques have been implemented, such as high dose/refuge strategy, gene stacking, and special expression methodologies (Jackson et al 2007;Kurtz et al 2007;Sena et al 2009;Singh et al 2008). However, the most important strategy should be to search for novel insecticidal genes, especially those encoding proteins with different insecticidal mechanisms compared with Bt endotoxin proteins.…”

Visual and Rapid Detection of Two Genetically Modified Soybean Events Using Loop-mediated Isothermal Amplification Method

“…In particular, conventional and real-time quantitative polymerase chain reaction (PCR) methods have been widely accepted as standards for identifying and determining the GM contents in food and feeds because of their high efficiency, sensitivity, and stability (Bellocchi et al 2008;Holst-Jensen et al 2003;Singh et al 2008;Marmiroli et al 2008;Zel et al 2008). Several conventional and quantitative real-time PCR methods for GM soybean GTS 40-3-2 have been developed and validated (Yang et al 2008;Taverniers et al 2005;Berdal and Holst-Jensen 2001;Huang and Pan 2005).…”

Qualitative and Quantitative Molecular Testing Methodologies and Traceability Systems for Commercialised Bt Cotton Events and Other Bt Crops Under Field Trials in India

“…A hexaplex PCR method simultaneously targeting the commonly used marker genes, viz., nptII, aadA, hpt, bar, pat and uidA has been developed as an efficient tool for screening of GM crops (Randhawa et al 2009a). Multiplex PCR assays have also been successfully employed for detection of various GM crops that are under different stages of testing in containment or field trials in India such as insect resistant cotton with (vip) 3A-type gene (Singh et al 2008), GM potato expressing AmA1 gene for better protein quality (Randhawa et al 2009b, c), GM tomato with osmotin gene for salinity and drought tolerance (Randhawa et al 2009d). Real-time PCR is a precise, robust and accurate quantification method (Bonfini et al 2002;Zhang et al 2003).…”