李矮缩病毒重组酶聚合酶扩增—侧流层析试纸条检测方法的建立

doi:10.16420/j.issn.0513-353x.2020-0174

摘要/Abstract

摘要：

根据李矮缩病毒（prune dwarf virus,PDV）外壳蛋白基因序列的保守区设计特异性引物和探针,建立了PDV的重组酶聚合酶扩增—侧流层析试纸条（recombinase polymerase amplification combined with lateral flow dipstick,RPA-LFD）检测方法。该方法在恒温39 ℃下反应20 min,具有标记的扩增子可达到检测水平。扩增子检测室（amplicon detection champer）中的侧流层析试纸条在20 min内呈现可视化检测结果。该方法与侵染樱桃的另外6种病毒（CGRMV、PNRSV、LchV-1、PBNSPaV、CVA和CNRMV）无交叉反应。灵敏度试验表明,用RPA-LFD方法检测PDV的灵敏性是PCR方法的10倍。用该方法检测13个田间样品,其检测结果与PCR检测结果一致。RPA-LFD法具有操作简单、快速、灵敏度高、特异性强等特点,适合对PDV样品的快速检测与鉴定。

关键词: 李矮缩病毒, 樱桃, 重组酶聚合酶扩增—侧流层析试纸条, 检测

Abstract:

Recombinase polymerase amplification combined with lateral flow dipstick（RPA-LFD）method was developed to detect prune dwarf virus（PDV）in cherry,using the specific primers and probe based on the conserved coat protein gene sequences of PDV. The RPA method could be conducted under isothermal conditions optimized to be 39 ℃ for 20 min,and the terminally labeled amplicon can reach the detection level. The detection results could be visually inspected with lateral flow dipstick located in amplicon detection champer within 20 min. The established method was specific and no cross-reactivity was detected with other six cherry-infecting viruses（CGRMV,PNRSV,LchV-1,PBNSPaV,CVA and CNRMV）. The sensitivity test showed that the RPA was about 10-fold sensitive than that of PCR. This method was verified effectively by testing 13 field-collected samples,and the results were consistent with those detected by PCR method. The RPA-LFD detection technique developed in this study is a simple,rapid,sensitive and specific method,which could be applicable for rapid and accurate detection and identification of PDV.

Key words: prune dwarf virus, cherry, recombinase polymerase amplification combined with lateral flow dipstick（RPA-LFD）, detection

中图分类号:

陈玲, 闫国华, 张晓明, 周宇, 王晶, 段续伟, 李彦林, 张开春. 李矮缩病毒重组酶聚合酶扩增—侧流层析试纸条检测方法的建立[J]. 园艺学报, 2021, 48(1): 183-192.

CHEN Ling, YAN Guohua, ZHANG Xiaoming, ZHOU Yu, WANG Jing, DUAN Xuwei, LI Yanlin, ZHANG Kaichun. Establishment of Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick for Detection of Prune Dwarf Virus[J]. Acta Horticulturae Sinica, 2021, 48(1): 183-192.

图/表 4

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引物来源 Primer source	参考基因组 Reference genome	产物/bp Product	引物位置/nt Location	引物名称 Primer name	引物序列（5′-3′） Primer sequence
本实验室设计	NC_008038	130	1 526 ~ 1 558	RPA-PDV-F	CGTTACTGCCTTGATGTTTCTAATGGTGTCTAC
Design by our Lab			1 680 ~ 1 651	RPA-PDV-R	Biotin-CCAACGGTCATACCAGTAGGAGCAAGAATT
			1 551 ~ 1 597	RPA-PDV- Probe	FAM-GTGTCTACGGACTCATTAAAGGTTTCGATGTG(THF)AATGCGCCTGTGGCG -PO₄
宗晓娟等, 2012	NC_008038	304	1 341 ~ 1 360	PDV-F	CGAAGTCTATTTCCGAGTGG
			1 644 ~ 1 625	PDV-R	CCACTGGCTTGTTTCGCTGT
Bajet et al.,2008	Y10237.1	300	7 090 ~ 7 109	LChV-1-F	GGTTGTCCTCGGTTGATTAC
			7 389 ~ 7 370	LChV-1-R	GGCTTGGTTCCATACATCTC
侯义龙等, 2005	NC_004364	455	1 185 ~ 1 209	PNRSV-F	ACGCGCAAAAGTGTCGAAATCTAAA
			1 639 ~ 1 615	PNRSV-R	TGGTCCCACTCAGAGCTCAACAAAG
Villamor & Eastwell,2013	KY178277	685	7 468 ~ 7 486	CGRMV-F	CTAAGTCCTCCAACCCTCA
			8 152 ~ 8 133	NGRRM-TL	ATNGGTTGAATTTGGCCAGT
Villamor & Eastwell,2013	KR820549	658	7 559 ~ 7 579	CNRMV-F	CTTCAAGAARGAGYGATATTG
			8 216 ~ 8 197	NGRRM-TL	ATNGGTTGAATTTGGCCAGT
陈玲等,2020	EF546442	804	2 828 ~ 2 847	PBNSPaV-F	GAGACTCGCGATGGACAGAG
			3 631 ~ 3 612	PBNSPaV-R	GGCAATTTCCACAACAGCGT
	KT285841	567	6 170 ~ 6 190	CVA-F	AGCCAGAAGGTATCATGCCAG
			6 718 ~ 6 736	CVA-R	ATGACATGCCTGCTGGGAG
	XM_021949634	361	958 ~ 977	Internal Control-F	TGCGGTCAAAGCTTCTCAGT
			1 318 ~ 1 299	Internal Control-R	AACATTTGCGACGACGACCC

引物来源 Primer source	参考基因组 Reference genome	产物/bp Product	引物位置/nt Location	引物名称 Primer name	引物序列（5′-3′） Primer sequence
本实验室设计	NC_008038	130	1 526 ~ 1 558	RPA-PDV-F	CGTTACTGCCTTGATGTTTCTAATGGTGTCTAC
Design by our Lab			1 680 ~ 1 651	RPA-PDV-R	Biotin-CCAACGGTCATACCAGTAGGAGCAAGAATT
			1 551 ~ 1 597	RPA-PDV- Probe	FAM-GTGTCTACGGACTCATTAAAGGTTTCGATGTG(THF)AATGCGCCTGTGGCG -PO₄
宗晓娟等, 2012	NC_008038	304	1 341 ~ 1 360	PDV-F	CGAAGTCTATTTCCGAGTGG
			1 644 ~ 1 625	PDV-R	CCACTGGCTTGTTTCGCTGT
Bajet et al.,2008	Y10237.1	300	7 090 ~ 7 109	LChV-1-F	GGTTGTCCTCGGTTGATTAC
			7 389 ~ 7 370	LChV-1-R	GGCTTGGTTCCATACATCTC
侯义龙等, 2005	NC_004364	455	1 185 ~ 1 209	PNRSV-F	ACGCGCAAAAGTGTCGAAATCTAAA
			1 639 ~ 1 615	PNRSV-R	TGGTCCCACTCAGAGCTCAACAAAG
Villamor & Eastwell,2013	KY178277	685	7 468 ~ 7 486	CGRMV-F	CTAAGTCCTCCAACCCTCA
			8 152 ~ 8 133	NGRRM-TL	ATNGGTTGAATTTGGCCAGT
Villamor & Eastwell,2013	KR820549	658	7 559 ~ 7 579	CNRMV-F	CTTCAAGAARGAGYGATATTG
			8 216 ~ 8 197	NGRRM-TL	ATNGGTTGAATTTGGCCAGT
陈玲等,2020	EF546442	804	2 828 ~ 2 847	PBNSPaV-F	GAGACTCGCGATGGACAGAG
			3 631 ~ 3 612	PBNSPaV-R	GGCAATTTCCACAACAGCGT
	KT285841	567	6 170 ~ 6 190	CVA-F	AGCCAGAAGGTATCATGCCAG
			6 718 ~ 6 736	CVA-R	ATGACATGCCTGCTGGGAG
	XM_021949634	361	958 ~ 977	Internal Control-F	TGCGGTCAAAGCTTCTCAGT
			1 318 ~ 1 299	Internal Control-R	AACATTTGCGACGACGACCC