SHARP PCR DNA/RNA sequencing diagnostic technique

Overview

This solution integrates an inhibitor-resistant isothermal amplification method (SHARP PCR) with portable Nanopore sequencing to deliver rapid, in-field molecular diagnostics for plant pathogens. By bypassing traditional DNA purification steps and bulky thermal cycling equipment, the workflow enables genotyping directly from crude plant samples. The approach targets agricultural stakeholders who need fast, cost-effective pathogen detection without relying on centralized laboratory infrastructure.

Technical specifications

Key features:

• Isothermal amplification using the pcrA M6 helicase-based SHARP PCR method, which operates at a constant temperature and eliminates the need for thermal cycling hardware
• Inhibitor-resistant Bst enzyme chemistry that amplifies target genes from crude nucleic acid preparations without prior purification
• 30-second nucleic acid purification as the upstream sample preparation step, dramatically reducing hands-on time
• Portable Nanopore sequencer for amplicon readout, leveraging adaptive sampling to eject already-sequenced reads and reduce per-sample cost
• Barcoded primers enabling multiplexed, temporally separated samples to be pooled and analyzed individually
• End-to-end turnaround of approximately 2.5 hours, with under 15 minutes of operator labor and projected cost below $10 per sample
• Bioinformatics pipeline for downstream genotyping of detected pathogens

Technology readiness level

The underlying SHARP PCR method has been validated at Johns Hopkins University and is being independently confirmed in an academic setting. The 30-second DNA extraction method has been extensively tested in-lab, and the Bst enzyme-based LAMP approach has already been deployed on similarly prepared samples, including successful use by an eight-year-old child with minimal equipment. Nanopore sequencing and adaptive sampling via reverse-polarity filtering are established, commercially proven technologies. The remaining validation steps include reproducing SHARP PCR with the pcrA M6 helicase, designing indexing primers for specific diagnostic targets, confirming compatibility with crude DNA extracts, and developing a user-friendly Nanopore protocol. Estimated timeline is 8–10 months of laboratory research plus 2 months of field trials. The team is seeking licensing access to the pcrA M6 helicase, access to specific plant varieties for primer design, and funding for a student researcher and laboratory supplies.