Smartphone-integrated RPA-CRISPR/Cas12a detection system with microneedle sampling for early point-of-care diagnosis of potato late blight.

چکیده اصلی

Potato late blight, caused by the oomycete pathogen Phytophthora infestans (P. infestans), is one of the most devastating diseases threatening global potato production. Conventional plant disease detection methods rely on a labor-intensive and time-consuming workflow and require bulky and expensive benchtop equipment, limiting their in-field applications. Here, we report a portable RPA-CRISPR/Cas12a-based diagnostic platform integrated with a polyvinyl alcohol (PVA) microneedle (MN) patch, which allows rapid in-field sampling, and smartphone-based fluorescence acquisition and analysis to detect P. infestans in potato at the early stage. The PVA MN enables leaf sampling rapidly within 1 min, and yields efficient DNA extraction of 56.3 ± 4.2 ng/mg, which is ∼3-fold higher than the traditional CTAB method (18.1 ± 2.1 ng/mg). The RPA-CRISPR/Cas12a isothermal assay achieved specific detection of P. infestans with no cross-reactivity against closely-related species Phytophthora sojae or Phytophthora capsici. The smartphone-based point-of-care test (POCT) system demonstrates a detection limit of 4 pg/μL for P. infestans genomic DNA, which is comparable to that acquired with commercial laboratory equipment. The method enables early-stage diagnosis of potato late blight as early as Day 2 post-inoculation, with detection rates of 37.5% on Day 2 and 75% on Day 3, prior to the development of visible symptoms on leaves. This portable "sample-to-result" platform provides a promising strategy for rapid, field-deployable early diagnosis and surveillance of plant disease.

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