Development of a portable DNA extraction and cross-priming amplification (CPA) tool for rapid in-situ visual diagnosis of plant diseases
文献类型： 外文期刊
作者： Li, Jie 1 ; Du, Juan 1 ; Li, Shengzhican 1 ; Dong, Jiali 1 ; Ying, Jiahan 1 ; Gu, Yuehao 1 ; Lu, Jie 2 ; Zeng, Xinyu 3 ; Kear, Philip 4 ; Dou, Daolong 1 ; Wang, Xiaodan 1 ;
作者机构： 1.China Agr Univ, Coll Plant Protect, Dept Plant Pathol, MOA Key Lab Pest Monitoring & Green Management, Beijing 100193, Peoples R China
2.Heilongjiang Acad Agr Sci, Biotechnol Res Inst, Harbin 150028, Peoples R China
3.Chongqing Three Gorges Acad Agr Sci, Chongqing 404100, Peoples R China
4.CIP China Ctr Asia Pacific CCCAP, Beijing 100081, Peoples R China
5.Nanjing Agr Univ, Acad Adv Interdisciplinary Studies, Coll Plant Protect, Nanjing 210095, Peoples R China
关键词： DNA extraction device; Steel microneedle array; Cross-priming amplification; Phytophthora infestans; Pathogen detection; Sample-to-answer
期刊名称：PHYTOPATHOLOGY RESEARCH （ 影响因子：3.4； 五年影响因子：4.2 ）
ISSN： 2096-5362
年卷期： 2023 年 5 卷 1 期
页码：
收录情况： SCI
摘要： Plant pathogens cause severe losses to crop yields and economic returns in agriculture. Despite plant tissue DNA extraction of typically constituting a preliminary step in nucleic acid-based molecular diagnostics, such lab-based methods can be time-consuming and arduous to complete many samples. To mitigate these challenges, we developed an inexpensive portable DNA extraction technique that is lightweight and suitable for deployment in sampling locations, such as fields. It includes a DNA extraction device fabricated with a Steel Microneedle Array (SMA) and a simple high-efficiency DNA extraction buffer. As a result, DNA extraction times can be reduced to within similar to 1 min, and the eluted DNA is demonstrated to be suitable for subsequent molecular biological analyses without requiring additional purification. Cross-priming amplification (CPA) technology was first established to detect Phytophthora infestans, which achieves sensitivity attainment of 10(-7) ng/mu L. The detection result can be conveniently estimated with naked-eye visual inspection using fluorescent dsDNA binding dye. CPA was demonstrated to be more feasible than PCR-based approaches and performed well in species-specific and practicability tests. This study elucidates a novel integrated pathogen detection technique coupled with SMA-Device extraction and a modified visual CPA assay to establish and verify various field-based samples infected with multiple pathogens. Altogether, the total sample-to-answer time for pathogen detection was reduced to similar to 1.5 h, making field-based analysis affordable and achievable for farmers or extension workers inside and outside the laboratory.
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