Multiplexed field detection of GMO and snps from plants using RPA or qpcr

Overview

This solution addresses the need for rapid, on-site genetic testing of plant material to detect genetically modified organisms (GMOs) and single nucleotide polymorphisms (SNPs). Two complementary detection methods are developed in parallel: an isothermal recombinase polymerase amplification (RPA) assay and a direct qPCR assay. Both are designed for minimal or no sample preparation, with prefilled reaction tubes that reduce liquid handling in the field. The approach targets industry requirements for fast, portable, and reliable molecular diagnostics that can be performed during sample transit or directly at the collection site.

Technical specifications

RPA-based detection:

• Isothermal amplification using three core enzymes (recombinase, single-stranded DNA-binding protein, and strand-displacing polymerase)
• Single-tube reaction format compatible with fluorescent TwistAmp exo probes for multiplex detection
• Capable of detecting both DNA and RNA targets when reverse transcriptase is added
• Designed for use with T8 (2-plex) or T16 (3-plex) isothermal instruments and validated on a Mic4 qPCR machine
• Short reaction time with tolerance to inhibitors and sequence mismatches

qPCR-based detection:

• Closed-tube multiplex qPCR using commercial direct PCR plant kits such as Direct qPCR ProbesMaster
• Compatible with hydrolysis probes or molecular beacons for 4-plex detection
• Mature, quantitative technology with high sensitivity and specificity
• Designed for the Mic4 qPCR machine (weight 2 kg, dimensions 130 x 150 x 150 mm, 220 W average power, 35–99 °C range)
• Total run time of 60 to 90 minutes, suitable for in-transit testing

Shared advantages:

• Minimal or no sample preparation required
• Prefilled reaction tubes reduce field handling steps
• Portable, lightweight instrumentation suitable for field deployment

Known limitations:

• RPA reagents are relatively expensive and available from limited commercial sources
• RPA is prone to non-specific amplification and offers poor quantitative separation
• Primer specificity challenges may arise in multiplex RPA reactions

Technology readiness level

The project is at an early-to-mid development stage. Both the RPA-based and qPCR-based multiplex detection methods are being designed and tested at Natural Resources Institute Finland (Luke). Sample material and target genetic information are provided by the industry partner. The study is planned to run for approximately one year, covering design, laboratory validation, and comparative assessment of both methods. Field-ready instrumentation has been identified, and the workflow is being optimized for minimal-handling, closed-tube operation. Commercial deployment will require further validation against the full panel of target sequences and confirmation of performance under real field conditions.