甘薯卷叶病毒的RPA检测方法的建立

许泳清; 李华伟; 张鸿; 李国良; 林赵淼; 邱永祥; 邱思鑫

doi:10.19303/j.issn.1008-0384.2021.08.008

甘薯卷叶病毒的RPA检测方法的建立

doi: 10.19303/j.issn.1008-0384.2021.08.008

福建省农业科学院作物研究所/农业农村部南方薯类科学观测实验站/福建省特色旱作物品种选育工程技术研究中心，福建　福州　350013

基金项目: 福建省科技计划公益类专项（2020R1031008）；国家现代农业产业技术体系项目（CARS-10-B14）；福建省种业创新与产业化工程项目（zycxny2021005）；农业高质量发展超越“5511”协调创新工程项目（KXXYJBG0039）；福建省农业科学院农业科技创新联盟专项（CXLM2021001）

详细信息

作者简介:
许泳清（1980−），女，副研究员，研究方向：薯类作物脱毒培养与病毒检测（E-mail：123071729@qq.com）

通讯作者:
邱思鑫（1974−），男，博士，研究员，研究方向：植物病理学（E-mail: qiusixin@faas.cn）

中图分类号: S 435
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出版历程
- 收稿日期: 2021-05-24
- 修回日期: 2021-06-30
- 网络出版日期: 2021-08-10
- 刊出日期: 2021-08-28

A Rapid Detection Method on RPA of Sweet Potato Leaf Curl Virus

Crop Research Institute , Fujian Academy of Agricultural Sciences/Scientific Observation and Experimentation Station on Tuber and Root Crops in South China, Ministry of Agriculture/Dry Crop Engineering Research Center of Fujian Province, Fuzhou, Fujian　350013, China

摘要

摘要: 目的建立快速检测甘薯卷叶病毒重组酶聚合酶（RPA）等温扩增方法，为甘薯卷叶病毒（sweet potato leaf curl virus，SPLCV）提供快速、简便的检测方法。方法根据甘薯卷叶病毒AV1基因的保守区段设计RPA 检测用引物，并对引物进行筛选，对反应条件和反应体系进行优化，建立SPLCV的RPA检测方法。结果建立的甘薯卷叶病毒RPA方法快速简便，39 ℃反应20 min完成检测；灵敏度高，最低检测限为10 pg·μL⁻¹；特异性强，与感染番茄黄化卷叶病毒（TYLCD），甘薯羽状斑驳病毒（SPFMV）、甘薯G病毒（SPVG）、甘薯退绿矮化病毒（SPCSV）等4种 DNA 病毒均无交叉反应。结论建立的RPA 检测方法具有快速、灵敏、特异性强、不需要特殊仪器等优点，适合田间甘薯卷叶病毒样品的快速检测。
- 甘薯 /
- 卷叶病毒（SPLCV） /
- 重组酶聚合酶等温扩增方法 /
- 检测
Abstract: Objective A rapid isothermal amplification method for detecting the recombinant enzyme polymerase (RPA) in sweet potato leaf curl virus (SPLCV) was established. Method A set of primers for RPA detection was designed based on the AV1 gene selected from SPLCV sequence. Reaction conditions for the detection was optimized and verified for applicability. Result The newly established methodology took merely 20m to complete the nucleic acid amplification at 39℃, which greatly improved the detection efficiency．More importantly, the method was specific toward SPLCV and free of any cross reaction with DNAs of other pathogens, such as TYLCD, SPFMV, SPVG, or SPCSV. It also showed a high repeatability and a minimum detection limit on RPA of 10 pg·μL⁻¹. Conclusion The newly developed method for SPLCV identification based on the detection of RPA in sweet potatoes was rapid, accurate, specific, repeatable, and easy to operate in the field.
- Sweet potato /
- sweet potato leaf curl virus（SPLCV） /
- RPA /
- detection methodology

HTML全文

图 1 甘薯卷叶病毒RPA引物筛选

注：M：DL2 000 DNA marker；1～9：9对甘薯卷叶病毒RPA引物扩增结果。

Figure 1. Primers screening on RPA of SPLCV

Note: M: DL2 000 DNA marker; 1-9: detection results on 9 amplified RPA primers of SPLCV.

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图 2 甘薯卷叶病毒RPA特异性检测

注：M：DL2 000 DNA marker；1：甘薯卷叶病毒；2：番茄黄化卷叶病毒；3：甘薯羽状斑驳病毒；4：甘薯G病毒；5：甘薯退绿矮化病毒。

Figure 2. Specificity on detecting RPA of SPLCV

Note: M: DL2 000DNA marker; 1: SPLCV; 2: Tomato yellow leaf curl virus; 3: Sweet potato feathery mottle virus; 4: Sweet potato virus G; 5: Sweet potato chlorotic stunt virus.

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图 3 甘薯卷叶病毒RPA和PCR灵敏度比较

注：M：DL2 000 DNA marker；1～8：甘薯卷叶病毒DNA质量浓度分别为10 ng·μL⁻¹、1 ng·μL⁻¹、100 pg·μL⁻¹、10 pg·μL⁻¹、1 pg·μL⁻¹、100 fg·μL⁻¹、10 fg·μL⁻¹、1 fg·μL⁻¹。

Figure 3. Comparison on sensitivity of RPA-based and PCR detections of SPLCV

Note: M: DL2 000DNA marker; 1-8: DNA concentrations of SPLCV at 10 ng·μL⁻¹, 1 ng·μL⁻¹, 100 pg·μL⁻¹, 10 pg·μL⁻¹, 1 pg·μL⁻¹, 100 fg·μL⁻¹, 10 fg·μL⁻¹, and 1 fg·μL⁻¹.

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表 1 RPA检测用引物

Table 1. Nucleotide sequences of RPA primers used

用途 Usage	引物名称 Primer name	序列（5′-3′） Sequence	片段大小 Length/bp
RPA	SPLCV（RPA）F	AGGCTGAACTTCGAGACAGCTATCGTGCCCTAC	464
RPA	SPLCV（RPA）R	AAGACCTGCATTCTATCCCTCAGATCCATTCGGAT	464
PCR	SPLCV（PCR）F	GGAACAGGCATTAGTTAGGA	161
PCR	SPLCV（PCR）R	TGCAACGCAGAGTCTGATAT	161

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参考文献(16)

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