Rapid Extraction of Plant Nucleic Acids by Microneedle Patch for In-Field Detection of Plant Pathogens.

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.

Integrated microneedle-smartphone nucleic acid amplification platform for in-field diagnosis of plant diseases.

We demonstrate an integrated microneedle (MN)-smartphone nucleic acid amplification platform for "sample-to-answer" diagnosis of multiplexed plant pathogens within 30 min. This portable system consists of a polymeric MN patch for rapid nucleic acid extraction within a minute and a 3D-printed smartphone imaging device for loop-mediated isothermal amplification (LAMP) reaction and detection. We expanded the extraction of the MN technology for DNA targets as in the previous study (ACS Nano, 2019, 13, 6540-6549) to more fragile RNA biomarkers, evaluated the storability of the extracted nucleic acid samples on MN surfaces, and developed a smartphone-based LAMP amplification and fluorescent reader device that can quantify four LAMP reactions on the same chip. In addition, we have found that the MN patch containing as few as a single needle tip successfully extracted enough RNA for RT-PCR or RT-LAMP analysis. Moreover, MN-extracted RNA samples remained stable on MN surfaces for up to three days. The MN-smartphone platform has been used to detect both Phytophthora infestans DNA and tomato spotted wilt virus (TSWV) RNA down to 1 pg, comparable to the results from a benchtop thermal cycler. Finally, multiplexed detection of P. infestans and TSWV through a single extraction from infected tomato leaves and amplification on the smartphone without benchtop equipment was demonstrated.

Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of human and plant diseases.

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.

DNA Extraction from Plant Leaves Using a Microneedle Patch.

Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Microneedle patch-based DNA extraction Support Protocol 1: Microneedle patch fabrication Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA.