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

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

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。

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.

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