重组酶聚合酶扩增技术在植物病毒检测中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0453

摘要/Abstract

摘要：

随着分子生物学技术的发展,多种核酸等温扩增技术逐渐被开发出来。其中,重组酶聚合酶扩增（recombinase polymerase amplification,RPA）作为一种快速、灵敏的检测技术具有很大的优势。目前,RPA已应用于转基因生物、各类病原物及食品安全检测等多个领域,并作为新兴技术在植物病毒检测领域中快速发展。RPA技术只需一对引物,在恒温条件下（37-42℃）只需30 min左右即可完成反应,具有较高的灵敏度与特异性。因此,该技术正迅速成为一种能够用于条件有限的实验室或现场植物病毒检测的手段。本文介绍了RPA技术的检测原理、引物设计和应用方式,综述了其在植物病毒检测中的最新研究进展及存在的问题,为RPA技术在植物病毒检测中的应用提供参考。

关键词: 重组酶聚合酶扩增, 植物病毒, 检测, 应用

Abstract:

With the development of molecular biology technology,a number of nucleic acid isothermal amplification technologies have been developed gradually. Recombinase polymerase amplification（RPA）,as a rapid and sensitive detection technique,has great advantages. At present,RPA has been applied in many fields on detections,such as genetically modified organism,a variety of pathogens and food safety. As an emerging technology,RPA is developing rapidly in plant virus detection. The RPA technique requires only a pair of primers and is finished in only 30 min at constant temperature（37-42^oC）,which has high sensitivity and specificity. As a result,the RPA technique is rapidly becoming a very effective tool for detection of plant viruses in under-equipped laboratories or in on-site facilities. In this review,the detection principle,primer design and application for RPA are introduced,and the recent research progress and existing problems of RPA in plant virus detection is reviewed,aiming to provide references for the application of RPA in te detection of plant detection.

Key words: recombinase polymerase amplification, plant virus, detection, application

罗雪琮, 安梦楠, 吴元华, 夏子豪. 重组酶聚合酶扩增技术在植物病毒检测中的应用[J]. 生物技术通报, 2022, 38(2): 269-280.

LUO Xue-cong, AN Meng-nan, WU Yuan-hua, XIA Zi-hao. Applications of Recombinase Polymerase Amplification in Plant Virus Detection[J]. Biotechnology Bulletin, 2022, 38(2): 269-280.

图/表 3

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[78]	Hammond RW, Zhang SL. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification[J]. J Virol Methods, 2016, 236:62-67. doi: 10.1016/j.jviromet.2016.06.013 URL

病毒 Virus	英文全称 Full name in English	缩写 Acronym	属 Genus	方法 Method	模板 Template	灵敏度 Sensitivity	参考文献 Reference
苹果茎沟病毒	Apple stem grooving virus	ASGV	Capillovirus	RT-RPA	纯化的RNA	4.7 ng	[39]
苹果茎痘病毒	Apple stem pitting virus	ASPV	Foveavirus	RT-RPA	CP体外转录物	10^-4稀释度	[40]
迈索尔香蕉线条病毒	Banana streak Mysore virus	BSMYV	Badnavirus	RT-RPA	纯化的RNA	10^-2稀释度	[41]
					叶片粗提物	10^-2稀释度
大麦黄矮病毒	Barley yellow dwarf virus	BYDV	Luteovirus	RT-RPA	CP体外转录物	10^-7稀释度	[42]
菜豆荚斑驳病毒	Bean pod mottle virus	BPMV	Comovirus	RT-RPA	总RNA	10^-4稀释度	[43]
				IAC-RT-RPA	总RNA	0.05 ng	[44]
樱桃病毒A	Cherry virus A	CVA	Capillovirus	RT-RPA-LFS	总RNA	10^-4稀释度	[45]
辣椒脉斑驳病毒	Chilli veinal mottle virus	ChiVMV	Potyvirus	RT-RPA	总RNA	10 fg	[46]
柑橘衰退病毒	Citrus tristeza virus	CTV	Closterovirus	RT-RPA-AuNP-EIS	CTV-p20 gene	1 pg	[47]
				RT-RPA-LFICA	总RNA	3.77×10⁵拷贝	[48]
					RNA体外转录物	6.288×10⁸拷贝
黄瓜花叶病毒	Cucumber mosaic virus	CMV	Cucumovirus	RT-exoRPA	纯化的RNA	3 pg	[27]
					叶片粗提物	10^-4稀释度
					纯化的RNA	3 pg
					叶片粗提物	10^-5稀释度
黄瓜绿斑驳花叶病毒	Cucumber green mottle mosaic virus	CGMMV	Tobamovirus	RT-RPA	总RNA	10^-6稀释度	[49]
				RT-RPA	总RNA	0.5 pg	[50]
南瓜黄矮化失调病毒	Cucurbit yellow stunting disorder virus	CYSDV	Crinivirus	RT-exoRPA	纯化的总RNA	10拷贝	[51]
葡萄卷叶伴随病毒2号	Grapevine leafroll-associated virus 2	GLRaV-2	Ampelovirus	RT-RPA	总RNA	10^-4稀释度	[52]
葡萄卷叶伴随病毒3号	Grapevine leafroll-associated virus 3	GLRaV-3	Ampelovirus	RT-RPA	总RNA	10^-3稀释度	[15]
莴苣花叶病毒	Lettuce mosaic virus	LMV	Potyvirus	RT-RPA	总RNA	10^-2稀释度	[53]
小樱桃病毒2号	Little cherry virus 2	LChV2	Ampelovirus	RT-RPA-LFS	叶片粗提物	10^-2稀释度	[54]
					纯化的RNA	0.1 ng
莲藕潜隐病毒	Lotus latent virus	LLV	Potyvirus	RT-RPA	质粒	7x10³拷贝	[55]
玉米褪绿斑驳病毒	Maize chlorotic mottle virus	MCMV	Machlomovirus	RT-RPA	总RNA	2.3×10^-6 μg	[56]
					总RNA	10^-4稀释度	[57]
胡椒黄斑驳病毒	Pepper yellow mottle virus	PYMoV	Badnavirus	RPA	总DNA	10^-5稀释度	[58]
					粗提物	10^-3稀释度
				RT-RPA	总RNA	10^-5稀释度
李痘病毒	Plum pox virus	PPV	Potyvirus	RT-RPA-LFS	叶片粗提物	10^-4稀释度	[59]
					RNA体外转录物	1 fg
				real-time RT-RPA	叶片粗提物	10^-4稀释度
					RNA体外转录物	16 fg
				RT-RPA	总RNA	1.06 ng	[60]
马铃薯Y病毒	Potato virus Y	PVY	Potyvirus	RT-RPA	纯化的总RNA	10^-2稀释度	[61]
					叶片粗提物	10^-2稀释度
				real-time RT-RPA	纯化的总RNA	10^-3稀释度
李矮缩病毒	Prune dwarf virus	PDV	Ilarvirus	RPA-LFD	总RNA	170 fg	[20]
水稻黑条矮缩病毒	Rice black-streaked dwarf virus	RBSDV	Fijivirus	RT-RPA-LFS	总RNA	10^-1稀释度	[62]
玫瑰丛簇病毒	Rose rosette virus	RRV	Emaravirus	RT-exoRPA	RNA体外转录物	1 fg	[28]
				RT-RPA	RNA体外转录物	1 fg	[63]
南方菜豆花叶病毒	Southern bean mosaic virus	SBMV	Sobemovirus	RT-RPA	总RNA	10^-3稀释度	[64]
				IAC-RT-RPA	总RNA	0.05 ng	[44]
大豆花叶病毒	Soybean mosaic virus	SMV	Potyvirus	RT-RPA	总RNA	10^-4稀释度	[65]
				IAC-RT-RPA	总RNA	0.05 ng	[44]
番茄褪绿病毒	Tomato chlorosis virus	ToCV	Crinivirus	RT-RPA	总RNA	3 ng	[66]
番茄斑萎病毒	Tomato spotted wilt virus	TSWV	Tospovirus	RT-RPA	总RNA	10^-5稀释度	[67]
淮山药温和花叶病毒	Yam mild mosaic virus	YMMV	Potyvirus	Real-time RT-RPA	叶片粗提物	10^-1稀释度	[68]
薯蓣花叶病毒	Yam mosaic virus	YMV	Potyvirus	RT-exoRPA	纯化的RNA	14 pg	[69]
				Real-time RT-RPA	叶片粗提物	10^-3稀释度	[68]
香蕉束顶病毒	Banana bunchy top virus	BBTV	Babuvirus	RPA	基因组DNA	10^-6稀释度	[70]
					叶片粗提物	10^-6稀释度
菜豆金色黄花叶病毒	Bean golden yellow mosaic virus	BGYMV	Begomovirus	RPA	叶片粗提物	10^-3稀释度	[71]
柑橘黄花叶病毒	Citrus yellow mosaic virus	CYMV	Badnavirus	RPA	叶片粗提物	10^-5稀释度	[72]
				IC-RPA	叶片粗提物	-
紫云英矮缩病毒	Milk vetch dwarf virus	MDV	Nanovirus	RPA-LFS	质粒	10拷贝	[24]
				RPA	质粒	10拷贝
番茄斑驳病毒	Tomato mottle virus	ToMoV	Begomovirus	RPA	叶片粗提物	10^-3稀释度	[44]
番茄黄化曲叶病毒	Tomato yellow leaf curl virus	TYLCV	Begomovirus	RPA	总DNA	50 pg	[73]
				RPA-AuNP	总DNA	1拷贝	[31]
				RPA	质粒	9.6 pg	[71]
					粗提物	10^-3稀释度
啤酒花矮化类病毒	Hop stunt viroid	HSVd	Hostuviroid	RT-RPA-LFS	总RNA	10 ng	[74]
					RNA体外转录物	2×10⁹拷贝
				RT-RPA	基因组RNA	-	[75]
桃潜隐花叶类病毒	Peach latent mosaic viroid	PLMVd	Pelamoviroid	RT-RPA	总RNA	10^-7稀释度	[76]
马铃薯纺锤块茎类病毒	Potato spindle tuber viroid	PSTVd	Pospiviroid	RT-RPA-LFA	RNA体外转录物	10⁶拷贝	[77]
					总RNA	10^-7稀释度
番茄褪绿矮缩类病毒	Tomato chlorotic dwarf viroid	TCDVd	Pospiviroid	RT-RPA-LFS	RNA体外转录物	1 pg	[78]
					叶片粗提物	25倍稀释
					种子粗提物	10^-1稀释度

病毒 Virus	英文全称 Full name in English	缩写 Acronym	属 Genus	方法 Method	模板 Template	灵敏度 Sensitivity	参考文献 Reference
苹果茎沟病毒	Apple stem grooving virus	ASGV	Capillovirus	RT-RPA	纯化的RNA	4.7 ng	[39]
苹果茎痘病毒	Apple stem pitting virus	ASPV	Foveavirus	RT-RPA	CP体外转录物	10^-4稀释度	[40]
迈索尔香蕉线条病毒	Banana streak Mysore virus	BSMYV	Badnavirus	RT-RPA	纯化的RNA	10^-2稀释度	[41]
					叶片粗提物	10^-2稀释度
大麦黄矮病毒	Barley yellow dwarf virus	BYDV	Luteovirus	RT-RPA	CP体外转录物	10^-7稀释度	[42]
菜豆荚斑驳病毒	Bean pod mottle virus	BPMV	Comovirus	RT-RPA	总RNA	10^-4稀释度	[43]
				IAC-RT-RPA	总RNA	0.05 ng	[44]
樱桃病毒A	Cherry virus A	CVA	Capillovirus	RT-RPA-LFS	总RNA	10^-4稀释度	[45]
辣椒脉斑驳病毒	Chilli veinal mottle virus	ChiVMV	Potyvirus	RT-RPA	总RNA	10 fg	[46]
柑橘衰退病毒	Citrus tristeza virus	CTV	Closterovirus	RT-RPA-AuNP-EIS	CTV-p20 gene	1 pg	[47]
				RT-RPA-LFICA	总RNA	3.77×10⁵拷贝	[48]
					RNA体外转录物	6.288×10⁸拷贝
黄瓜花叶病毒	Cucumber mosaic virus	CMV	Cucumovirus	RT-exoRPA	纯化的RNA	3 pg	[27]
					叶片粗提物	10^-4稀释度
					纯化的RNA	3 pg
					叶片粗提物	10^-5稀释度
黄瓜绿斑驳花叶病毒	Cucumber green mottle mosaic virus	CGMMV	Tobamovirus	RT-RPA	总RNA	10^-6稀释度	[49]
				RT-RPA	总RNA	0.5 pg	[50]
南瓜黄矮化失调病毒	Cucurbit yellow stunting disorder virus	CYSDV	Crinivirus	RT-exoRPA	纯化的总RNA	10拷贝	[51]
葡萄卷叶伴随病毒2号	Grapevine leafroll-associated virus 2	GLRaV-2	Ampelovirus	RT-RPA	总RNA	10^-4稀释度	[52]
葡萄卷叶伴随病毒3号	Grapevine leafroll-associated virus 3	GLRaV-3	Ampelovirus	RT-RPA	总RNA	10^-3稀释度	[15]
莴苣花叶病毒	Lettuce mosaic virus	LMV	Potyvirus	RT-RPA	总RNA	10^-2稀释度	[53]
小樱桃病毒2号	Little cherry virus 2	LChV2	Ampelovirus	RT-RPA-LFS	叶片粗提物	10^-2稀释度	[54]
					纯化的RNA	0.1 ng
莲藕潜隐病毒	Lotus latent virus	LLV	Potyvirus	RT-RPA	质粒	7x10³拷贝	[55]
玉米褪绿斑驳病毒	Maize chlorotic mottle virus	MCMV	Machlomovirus	RT-RPA	总RNA	2.3×10^-6 μg	[56]
					总RNA	10^-4稀释度	[57]
胡椒黄斑驳病毒	Pepper yellow mottle virus	PYMoV	Badnavirus	RPA	总DNA	10^-5稀释度	[58]
					粗提物	10^-3稀释度
				RT-RPA	总RNA	10^-5稀释度
李痘病毒	Plum pox virus	PPV	Potyvirus	RT-RPA-LFS	叶片粗提物	10^-4稀释度	[59]
					RNA体外转录物	1 fg
				real-time RT-RPA	叶片粗提物	10^-4稀释度
					RNA体外转录物	16 fg
				RT-RPA	总RNA	1.06 ng	[60]
马铃薯Y病毒	Potato virus Y	PVY	Potyvirus	RT-RPA	纯化的总RNA	10^-2稀释度	[61]
					叶片粗提物	10^-2稀释度
				real-time RT-RPA	纯化的总RNA	10^-3稀释度
李矮缩病毒	Prune dwarf virus	PDV	Ilarvirus	RPA-LFD	总RNA	170 fg	[20]
水稻黑条矮缩病毒	Rice black-streaked dwarf virus	RBSDV	Fijivirus	RT-RPA-LFS	总RNA	10^-1稀释度	[62]
玫瑰丛簇病毒	Rose rosette virus	RRV	Emaravirus	RT-exoRPA	RNA体外转录物	1 fg	[28]
				RT-RPA	RNA体外转录物	1 fg	[63]
南方菜豆花叶病毒	Southern bean mosaic virus	SBMV	Sobemovirus	RT-RPA	总RNA	10^-3稀释度	[64]
				IAC-RT-RPA	总RNA	0.05 ng	[44]
大豆花叶病毒	Soybean mosaic virus	SMV	Potyvirus	RT-RPA	总RNA	10^-4稀释度	[65]
				IAC-RT-RPA	总RNA	0.05 ng	[44]
番茄褪绿病毒	Tomato chlorosis virus	ToCV	Crinivirus	RT-RPA	总RNA	3 ng	[66]
番茄斑萎病毒	Tomato spotted wilt virus	TSWV	Tospovirus	RT-RPA	总RNA	10^-5稀释度	[67]
淮山药温和花叶病毒	Yam mild mosaic virus	YMMV	Potyvirus	Real-time RT-RPA	叶片粗提物	10^-1稀释度	[68]
薯蓣花叶病毒	Yam mosaic virus	YMV	Potyvirus	RT-exoRPA	纯化的RNA	14 pg	[69]
				Real-time RT-RPA	叶片粗提物	10^-3稀释度	[68]
香蕉束顶病毒	Banana bunchy top virus	BBTV	Babuvirus	RPA	基因组DNA	10^-6稀释度	[70]
					叶片粗提物	10^-6稀释度
菜豆金色黄花叶病毒	Bean golden yellow mosaic virus	BGYMV	Begomovirus	RPA	叶片粗提物	10^-3稀释度	[71]
柑橘黄花叶病毒	Citrus yellow mosaic virus	CYMV	Badnavirus	RPA	叶片粗提物	10^-5稀释度	[72]
				IC-RPA	叶片粗提物	-
紫云英矮缩病毒	Milk vetch dwarf virus	MDV	Nanovirus	RPA-LFS	质粒	10拷贝	[24]
				RPA	质粒	10拷贝
番茄斑驳病毒	Tomato mottle virus	ToMoV	Begomovirus	RPA	叶片粗提物	10^-3稀释度	[44]
番茄黄化曲叶病毒	Tomato yellow leaf curl virus	TYLCV	Begomovirus	RPA	总DNA	50 pg	[73]
				RPA-AuNP	总DNA	1拷贝	[31]
				RPA	质粒	9.6 pg	[71]
					粗提物	10^-3稀释度
啤酒花矮化类病毒	Hop stunt viroid	HSVd	Hostuviroid	RT-RPA-LFS	总RNA	10 ng	[74]
					RNA体外转录物	2×10⁹拷贝
				RT-RPA	基因组RNA	-	[75]
桃潜隐花叶类病毒	Peach latent mosaic viroid	PLMVd	Pelamoviroid	RT-RPA	总RNA	10^-7稀释度	[76]
马铃薯纺锤块茎类病毒	Potato spindle tuber viroid	PSTVd	Pospiviroid	RT-RPA-LFA	RNA体外转录物	10⁶拷贝	[77]
					总RNA	10^-7稀释度
番茄褪绿矮缩类病毒	Tomato chlorotic dwarf viroid	TCDVd	Pospiviroid	RT-RPA-LFS	RNA体外转录物	1 pg	[78]
					叶片粗提物	25倍稀释
					种子粗提物	10^-1稀释度