Abstract 2405 Reconstructed SHARP-PCR system for point of care diagnosis (POC) for plant disease

Plant pathogens incur substantial yield and economic losses in crops. One such pathogen is the Phytophthora infestans, commonly known as potato late blight, which causes economic losses upwards of $210 million, with an additional $77 million for chemical treatment of infected crops. The early detection of plant pathogens is imperative to ensure crop viability and mitigate significant economic loss. Polymerase Chain Reaction (PCR) remains the most common method of DNA amplification to detect plant pathogens. However, conventional PCR is time-consuming and relies on thermocycling instruments, which is not ideal for agricultural field testing. Isothermal DNA amplification methods maintain a constant temperature to amplify DNA, creating a streamlined approach for pathogen detection. However, existing techniques of isothermal PCR - such as Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), and Helicase-Dependent Amplification (HDA) - are highly complex and generate inconsistent products – sometimes yielding shorter products or requiring additional sequencing. A distinct isothermal technique, SHARP, utilizes an engineered DNA helicase called PcrA M6 and single-stranded DNA binding protein (SSB), which replaces thermocyclers for traditional PCR while preserving the versatility of products. In this study, we introduce an innovative reconfigured SHARP system suitable for field applications, enabling Point-of-Care (POC) diagnosis of plant pathogens. To reconstitute the SHARP reaction, we expressed and purified the PcrA M6 helicase and optimized the primer sequences. Using an ethidium bromide gel electrophoresis system, we successfully identified a detectable concentration as low as 10 fg/μL, suggesting that our re-engineered system is capable of detecting minute quantities of plant pathogens. These findings underscore the potential of our re-engineered SHARP-PCR system for early detection of plant pathogens. Future investigations will involve optimizing the parameters of the SHARP reaction, improving the DNA extraction method, and applying the reconstructed system for field applications.