Rapid multiplex snp-specific nucleic acid detection assay for plant materials

Overview

This solution delivers a rapid, portable, and SNP-specific nucleic acid detection platform designed for plant materials. By integrating cellulose-based nucleic acid extraction and purification with reverse transcriptase loop-mediated amplification (RT-LAMP) coupled to a PfAgo-based detection assay, the platform enables field-deployable testing that can be completed in under 30 minutes. The approach is particularly valuable for agricultural diagnostics, plant pathogen surveillance, crop breeding programs, and phytosanitary screening where speed, portability, and single nucleotide polymorphism (SNP)-level specificity are critical.

The core innovation lies in combining two proven technologies into a streamlined workflow. Cellulose-based extraction eliminates the need for laboratory-grade silica columns and centrifugation, while the PfAgo nuclease — derived from the archaeon Pyrococcus furiosus — enables multiplexable, SNP-specific cleavage without the PAM site constraints that limit CRISPR/Cas systems. This makes the assay well suited for distinguishing closely related pathogen strains or genetic variants in plants.

Technical specifications

Workflow components:

• Nucleic acid extraction and purification using cellulose adsorption combined with simple pestle grinding, completed in approximately 3 minutes
• Amplification via RT-LAMP assay, completed in approximately 20 minutes
• Detection via the PfAgo assay, completed in approximately 5 minutes
• Total time-to-result of under 30 minutes in field conditions with minimal resources

Key technical features:

• Cellulose offers higher nucleic acid adsorption capacity than silica, enabling rapid point-of-care sample preparation
• PfAgo is a DNA-guided nuclease capable of sequence-specific cleavage without PAM site limitations, enabling multiplex detection
• RT-LAMP provides isothermal amplification at a single temperature, removing the need for thermal cycling equipment
• The assay has been validated for SNP differentiation, demonstrated by distinguishing the SARS-CoV-2 Delta variant from wild type
• Compatible with RNA and DNA targets through the RT-LAMP step

Technology readiness level

The individual components are well established. The RT-LAMP-PfAgo assay has been validated for simultaneous detection of SARS-CoV-2 genes with limits of detection at sub-genome-copy concentrations, for SNP-level variant differentiation, and for compatibility with wastewater-derived nucleic acid samples. Cellulose-based nucleic acid extraction and purification is a proven technique for plant samples and is commercially available in dipstick-based kits.

The current project focuses on integrating and optimizing these two established technologies specifically for plant material workflows. Remaining work includes designing RT-LAMP primers, guide DNAs, and reporters for plant pathogen targets; optimizing extraction and washing buffer formulations to eliminate RT-LAMP-PfAgo inhibitors; and validating sensitivity and specificity using plant samples with and without target nucleic acids. The technology is positioned at an early-to-mid stage of development for the plant diagnostics application, with a clear pathway toward field validation and eventual deployment.