Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica

doi:10.1016/S2095-3119(20)63459-1

Journal of Integrative Agriculture ›› 2021, Vol. 20 ›› Issue (10): 2696-2705.DOI: 10.1016/S2095-3119(20)63459-1

所属专题：植物病理合辑Plant Protection—Plant Pathology；植物病毒合辑Plant Virus

收稿日期:2020-06-09 出版日期:2021-10-01 发布日期:2021-08-09

Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica

CHEN Wei-yu^1*, YU Jia^{2, 3*}, XU Heng², LU Xin-yu², DAI Ting-ting⁴, TIAN Yue-e⁵, SHEN Dan-yu², DOU Dao-long²

¹Nanjing Plant Protection and Quarantine Station, Nanjing 210036, P.R.China
² Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R.China
³ College of Chemical and Biological Engineering, Taiyuan University of Science and Technology, Taiyuan 030024, P.R.China
⁴ College of Forestry, Nanjing Forestry University, Nanjing 210037, P.R.China
⁵ Department of Plant Protection, Henan University of Science and Technology, Luoyang 471000, P.R.China

Received:2020-06-09 Online:2021-10-01 Published:2021-08-09
Contact: Correspondence SHEN Dan-yu, Tel: +86-25-84396355, E-mail: shendanyu@njau.edu.cn; Dou Dao-long, Tel: +86-25-84396973, E-mail: ddou@njau.edu.cn
About author:CHEN Wei-yu, E-mail: chinesecheng@163.com; YU Jia, E-mail: yujiauwm@163.com; * These authors contributed equally to this study.
Supported by:
This work was funded by grants from the Fundamental Research Funds for the Central Universities, China (KYT202001 and JCQY201901) and the Special Fund for Agro-scientific Research in the Public Interest, China (201503112).

摘要/Abstract

摘要：

由寄生疫霉（Phytophthora parasitica）引起的柑橘根腐病是柑橘生产上普遍发生、对柑橘危害较为严重的一种病害。本研究的目的是建立一种简易快速的结合侧流层析技术判定结果的重组酶聚合酶扩增（lateral flow strip-based recombinase polymerase amplification, LF-RPA）方法检测寄生疫霉，为诊断和防治寄生疫霉引起的柑橘根腐病提供技术支撑。以Ypt1基因为检测的靶标序列，通过多序列比对分析，设计寄生疫霉的特异性引物和探针，建立并优化LF-RPA检测方法，并与简易核酸提取技术相结合，对该检测方法的特异性、灵敏度和实际应用效果进行评估。对LF-RPA体系中的温度、引物和探针比例、时间进行了优化，使得LF-RPA在40°C孵育温度下反应20分钟后即可肉眼判断检测结果。特异性试验中，LF-RPA能特异性地检测出不同来源的寄生疫霉，而其它亲缘关系相近的卵菌病原菌均未检测出。灵敏度试验结果显示，LF-RPA的最低检测灵敏度为1 pg。为了使LF-RPA检测方法更适宜基层使用，对四种不同的简易核酸提取技术进行了比较评估，发现基于PEG-NaOH的简易核酸提取技术更适合本研究。将LF-RPA与PEG-NaOH核酸提取技术相结合，不需要特定的仪器设备，只需要能维持40°C的保温杯，即可在30分钟内完成从发病植株的核酸提取到结果判定的整个检测过程。利用该方法，成功地从接种寄生疫霉的柑橘叶片、枝条和果实上检测出寄生疫霉；此外也从田间采集的10份柑橘发病样品中检测出其中3份样品含有寄生疫霉本研究成功建立了一种针对寄生疫霉的LF-RPA快速检测方法，该方法与简易核酸提取技术相结合，非常适宜于基层使用，为柑橘根腐病的快速诊断奠定了技术基础。

Abstract:

Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation, affecting both yield and quality. Thus, rapid and accurate detection of P. parasitica plays an important role in disease management. The aim of this study was to develop a simple diagnostic method to detect P. parasitica infection by combining recombinase polymerase amplification and lateral flow strips (LF-RPA). To establish the LF-RPA assay of P. parasitica, the primers and probe designed based on the Ypt1 gene were tested for specificity to P. parasitica, which showed no cross-reactivity with DNAs of other related oomycete species. The LF-RPA assay detected the amount of genomic DNA of P. parasitica which was as low as 1 pg. To make the LF-RPA assay useful in low-resource settings, four simplified DNA extraction methods were compared, after which the LF-RPA assay was applied, with no specialized equipment, to analyze a diverse range of citrus tissues by using a simplified PEG-NaOH method for DNA extraction. This method was successful in detecting P. parasitica in infected plant samples within 30 min. Combining the LF-RPA assay and a simplified DNA extraction method could be a potential detection test for P. parasitica, especially in areas with limited resources.

Key words: citrus disease , Phytophthora parasitica , recombined polymerase amplification , equipment-free detection

. [J]. Journal of Integrative Agriculture, 2021, 20(10): 2696-2705.

CHEN Wei-yu, YU Jia, XU Heng, LU Xin-yu, DAI Ting-ting, TIAN Yue-e, SHEN Dan-yu, DOU Dao-long. Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica[J]. Journal of Integrative Agriculture, 2021, 20(10): 2696-2705.

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Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica

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