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<p>The quantity and variety of GM crops in markets continues to expand; thus, creation of simple, rapid and effective GMO detection tests is crucial for its monitoring to prevent unauthorised entry, and compliance with safety standards and regulatory obligations. GM crops can be identified employing DNA testing like PCR and qPCR, protein based tests like ELISA, in-field sampling strategies in instances where regulatory frameworks demand transparency. Real-time PCR assays stand out as the most efficient methods routinely deployed for the identification of genetically modified plant material. In this context, we outline the development and validation of three new screening assays targeting the frequently used GM elements: <i>Cauliflower mosaic virus</i> (CaMV) 35&#xa0;S promoter (P35S), <i>Figwort mosaic virus</i> (FMV) 34&#xa0;S promoter (pFMV), and nopaline synthase terminator (T-<i>Nos</i>) from <i>Agrobacterium tumefaciens</i>. The optimised real-time PCR assays demonstrated a high specificity for the targeted elements and were sensitive enough to detect as little as ten copies/µl of synthetic template in background of plant nucleic acid. Additionally, these assays were successfully duplexed with plant internal control without compromising sensitive detection of the target element. The findings from this study indicate that the three duplexed assays together will serve as a robust and sensitive preliminary screening test for GMOs. Our tests will be particularly valuable for the preliminary screening for GMO incursions anywhere in the world, with a special focus on soy and maize plants. All three assays developed from the current study were developed to meet international accreditation standards and approved to use through International Accreditation New Zealand (IANZ - ISO 17025).</p>

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Development and validation of robust real-time PCR assays for genetically modified plants using CaMV 35 S, FMV 34 S, and T-Nos markers

  • 期刊论文

  • 发表日期：

    2025年7月23日

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  • Esha Arshad,
  • Rebijith K. Balan,
  • Michael Gemmel,
  • Sherly George,
  • Chandan Pal,
  • Sathish Puthigae

摘要

The quantity and variety of GM crops in markets continues to expand; thus, creation of simple, rapid and effective GMO detection tests is crucial for its monitoring to prevent unauthorised entry, and compliance with safety standards and regulatory obligations. GM crops can be identified employing DNA testing like PCR and qPCR, protein based tests like ELISA, in-field sampling strategies in instances where regulatory frameworks demand transparency. Real-time PCR assays stand out as the most efficient methods routinely deployed for the identification of genetically modified plant material. In this context, we outline the development and validation of three new screening assays targeting the frequently used GM elements: Cauliflower mosaic virus (CaMV) 35 S promoter (P35S), Figwort mosaic virus (FMV) 34 S promoter (pFMV), and nopaline synthase terminator (T-Nos) from Agrobacterium tumefaciens. The optimised real-time PCR assays demonstrated a high specificity for the targeted elements and were sensitive enough to detect as little as ten copies/µl of synthetic template in background of plant nucleic acid. Additionally, these assays were successfully duplexed with plant internal control without compromising sensitive detection of the target element. The findings from this study indicate that the three duplexed assays together will serve as a robust and sensitive preliminary screening test for GMOs. Our tests will be particularly valuable for the preliminary screening for GMO incursions anywhere in the world, with a special focus on soy and maize plants. All three assays developed from the current study were developed to meet international accreditation standards and approved to use through International Accreditation New Zealand (IANZ - ISO 17025).