Abstract
Plant diseases pose a significant threat to global food security. Molecular diagnosis currently plays a crucial role in mitigating the negative impacts of plant diseases by accurately identifying the disease-causing pathogens and revealing their genotypes. However, current molecular assays are constrained to the laboratory because of the cumbersome protocols involved in plant nucleic acid extraction. To streamline this, we have developed a polymeric microneedle (MN) patch-based nucleic acid extraction method, which can be applied to various plant tissues and easily performed in field settings without using bulky laboratory equipment. The MN patch instantly isolates both host and pathogen’s DNA and RNA from plant leaves by two simple steps: press and rinse with a buffer solution or nuclease-free water. The MN-extracted DNA and RNA are purification-free and directly applicable to downstream molecular assays such as polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Here, we describe the fabrication procedures of the MN patch and demonstrate the application of the MN method by extracting Phytophthora infestans DNA and tomato spotted wilt virus (TSWV) RNA from infected tomato leaves. After MN extraction, we directly utilize the MN-extracted nucleic acid samples to run PCR, RT-PCR, LAMP, or RT-LAMP reactions to amplify various biomarker genes, such as the ribulose-bisphosphate carboxylase (rbcL) gene of host tomato DNA, internal transcribed spacer (ITS) region of P. infestans DNA, and nucleocapsid (N) gene of TSWV RNA. Furthermore, this simple and rapid nucleic acid method can be integrated with portable nucleic acid amplification platforms such as smartphone-based microscopy devices to achieve “sample-to-answer” detection of plant pathogens directly in the field.
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Acknowledgments
The authors thank Dr. Jean Ristaino and Dr. Anna Whitfield (Department of Entomology and Plant Pathology, NC State University) for providing P. infestans- and TSWV-infected tomato leaves. The authors also gratefully acknowledge the funding support from the NCSU Chancellor’s Faculty Excellence Program, the Kenan Institute for Engineering, Technology & Science (KIETS), NCSU Game-Changing Research Incentive Program for the Plant Science Initiative (GRIP4PSI), USDA (No. 2019-67030-29311), USDA APHIS Farm Bill grant (No. 3.0096), and BASF.
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Paul, R., Ostermann, E., Wei, Q. (2022). Rapid Extraction of Plant Nucleic Acids by Microneedle Patch for In-Field Detection of Plant Pathogens. In: Luchi, N. (eds) Plant Pathology. Methods in Molecular Biology, vol 2536. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2517-0_4
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DOI: https://doi.org/10.1007/978-1-0716-2517-0_4
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