柑橘黄化脉明病毒RT-RPA检测方法的建立

doi:10.3864/j.issn.0578-1752.2021.15.009

摘要/Abstract

摘要：

【目的】利用逆转录重组酶聚合酶扩增技术（reverse transcription-recombinase polymerase amplification,RT-RPA）,结合侧向流层析（lateral flow dipstick,LFD）试纸条,建立一种快速简便、特异、灵敏、裸眼可视的柑橘黄化脉明病毒（citrus yellow vein clearing virus,CYVCV）检测新方法。【方法】根据CYVCV外壳蛋白基因的保守序列设计5对引物,通过样品检测筛选出扩增效果好、特异性强的引物对。将选出的引物进行标记物修饰,并设计对应的特异性探针,分别设置6个反应时间梯度（5、10、20、30、40、50 min）和8个反应温度梯度（37、38、39、40、41、42、43、44℃）,对RT-RPA反应条件进行优化,建立CYVCV的RT-RPA检测体系。分别以仅感染了CYVCV、柑橘叶斑驳病毒（citrus leaf blotch virus,CLBV）、柑橘衰退病毒（citrus tristeza virus,CTV）、柑橘碎叶病毒（citrus tatter leaf virus,CTLV）、柑橘裂皮病类病毒（citrus exocortis viroid,CEVd）、柑橘鳞皮病毒（citrus psorosis virus,CPV）、温州蜜柑萎缩病毒（satsuma dwarf virus,SDV）、柑橘黄龙病菌（Candidatus Liberibacter asiaticus,CLas）和柑橘溃疡病菌（Xanthomonas citri subsp. citri,Xcc）柑橘材料的总核酸为模板,采用所筛选的最佳引物、探针和反应条件进行检测,评价所建立RT-RPA检测体系的特异性。将感染了CYVCV的柑橘总RNA样品进行10倍梯度稀释,以原液和10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7稀释液作为模板,平行进行RT-PCR及RT-RPA的检测,评价所建立检测方法的灵敏度。随机采集来自田间9个不同品种柑橘的叶片,同时进行RT-RPA和RT-PCR检测,检验所建立RT-RPA检测方法的适用性。【结果】建立了CYVCV的RT-RPA检测体系：最佳引物为CY1-F/R,对应探针为CY1-Probe（47 bp）,最佳反应条件为39℃,30 min,特异扩增目的片段为177 bp,检测结果可通过侧向流层析试纸条直接判读。特异性检测结果显示,利用该检测体系仅对感染了CYVCV的样品检测结果为阳性,其余均为阴性;灵敏度检测结果显示,RT-RPA与RT-PCR方法均最低可检测到10^-4稀释液,两种方法检测灵敏度相当。随机采集的45株田间柑橘样品中,RT-PCR与RT-RPA均检测出37个阳性样品,检出率均为82.2%,表明该方法检测效果稳定可靠。【结论】建立了CYVCV的RT-RPA检测方法,该检测方法操作简单、反应快速,结果裸眼可视,适用于基层条件不足的实验室或者植检站现场快速检测。

关键词: 柑橘, 柑橘黄化脉明病毒, RT-RPA, 侧向流层析试纸条, 快速检测

Abstract:

【Objective】 The objective of this study is to establish a fast, simple, accurate and visualized with naked eyes new detection method for citrus yellow vein clearing virus (CYVCV) using reverse transcription-recombinase polymerase amplification (RT-RPA) combined with lateral flow dipstick (LFD).【Method】 Five pairs of primers were designed according to the conservative sequence of the coat protein gene of CYVCV. By detecting different samples, the pair of primers with the best amplification efficiency and specificity was selected. The selected primers were modified and its corresponding specific probe was designed. According setting 6 reaction gradient times (5, 10, 20, 30, 40 and 50 min) and 8 reaction gradient temperatures (37, 38, 39, 40, 41, 42, 43 and 44℃), the RT-RPA system for CYVCV detection was optimized. The specificity of the established RT-RPA was evaluated by detecting the samples infected with CYVCV, citrus leaf blotch virus (CLBV), citrus tristeza virus (CTV), citrus tatter leaf virus (CTLV), citrus exocortis viroid (CEVd), citrus psorosis virus (CPV), satsuma dwarf virus (SDV), Candidatus Liberibacter asiaticus (CLas) and Xanthomonas citri subsp. citri (Xcc), respectively. The citrus total RNA samples infected with CYVCV was diluted by 10 times. The original RNA solution and 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7 dilutions were used as templates for testing the sensitivity of RT-RPA, and the sensitivity was compared with RT-PCR. Leaves of different citrus varieties were randomly collected from the field. RT-RPA and RT-PCR were used at the same time to test the applicability of the established RT-RPA detection method.【Result】 A RT-RPA detection system for CYVCV was established, with primer pairs CY1-F/R and corresponding probe CY1 (47 bp). It could specifically amplify the target fragment of CYVCV with a size of 177 bp. The best reaction conditions were 39℃, 30 min. The result could be judged by the LFD test strip directly. In the specific test, only samples infected with CYVCV were positive, and the rest were negative. In the sensitivity detection, 10^-4 dilution was the lowest detection sensitivity of RT-RPA and RT-PCR. The sensitivity of the two methods was equivalent. Among the 45 field citrus samples taken randomly, 37 samples were positive by RT-PCR and RT-RPA, and the positive rate was both 82.2%, indicating that the RT-RPA method established in this study was stable and reliable.【Conclusion】 A RT-RPA detection method for CYVCV is established. The method is convenient, rapid, and visualized. It can be applied to on-site rapid detection for the labs with insufficient basic conditions or plant protection and quarantine station.

Key words: citrus, citrus yellow vein clearing virus (CYVCV), RT-RPA, lateral flow dipstick test strip, rapid detection

马志敏,许建建,段玉,王春庆,苏越,张琦,宾羽,周常勇,宋震. 柑橘黄化脉明病毒RT-RPA检测方法的建立[J]. 中国农业科学, 2021, 54(15): 3241-3249.

MA ZhiMin,XU JianJian,DUAN Yu,WANG ChunQing,SU Yue,ZHANG Qi,BIN Yu,ZHOU ChangYong,SONG Zhen. Establishment of RT-RPA for Citrus Yellow Vein Clearing Virus (CYVCV) Detection[J]. Scientia Agricultura Sinica, 2021, 54(15): 3241-3249.

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https://www.chinaagrisci.com/CN/Y2021/V54/I15/3241

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体系 System	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	引物序列位置 Primer sequence position
RT-RPA	CY1-F	GTCACTAACT CCATAGCTAA CCCTAAACAGAC	6561-6737
	CY1-R	[biotin]-GTTGAGATCTTCGTTCGCTCCAATAATATCAG	6561-6737
	CY1-Probe	[6-FAM]CT ATCGGGAAAG CTTGGGTCCG CATCTTG[THF]AC ATCGATCCTG CCAAC-C3 Spacer	6598-6644
	CY2-F	AAAACCCACA ACAATGGGGT CTCTCCACCT GCC	6462-6652
	CY2-R	AAGAACACGTTGGCAGGATCGATGTTCAAGATGCG	6462-6652
	CY3-F	GTCTACCGACGACAACAAGGGCAAACAACC	6287-6435
	CY3-R	TACGCTTTTCGATGGGCTCTTGGTTTTCCT	6287-6435
	CY4-F	AACAAGGGCAAACAACCACTTCACCCGACA	6300-6435
	CY4-R	TACGCTTTTCGATGGGCTCTTGGTTTTCCT	6300-6435
	CY5-F	GCCCTAACGACACGACCCCGAAACCTATCC	6337-6439
	CY5-R	GTGATACGCTTTTCGATGGGCTCTTGGTTT	6337-6439
RT-PCR	614F	TACCGCAGCTATCCATTTCC	6174-6785
RT-PCR	614R	GCAGAAATCCCGAACCACTA	6174-6785

品种 Variety	样品株数 Number of samples	阳性样品数（阳性率） Positive number (Positive rate)
品种 Variety	样品株数 Number of samples	RT-RPA	RT-PCR
尤力克柠檬 Eureka lemon	5	5 (100%)	5 (100%)
纽荷尔脐橙 Newhall navel orange	5	5 (100%)	5 (100%)
塔罗科血橙 Tarocco	5	5 (100%)	5 (100%)
冰糖橘 Bingtangju	5	5 (100%)	5 (100%)
默科特橘 Murcott angerine	5	5 (100%)	5 (100%)
沙田柚 Shatianyou	5	2 (40%)	2 (40%)
强德勒柚 Chandler pummelo	5	0 (0)	0 (0)
沃柑 Orah	5	5 (100%)	5 (100%)
爱媛38 Ehime 38 hybrid citrus	5	5 (100%)	5 (100%)
总计 Total	45	37 (82.2%)	37 (82.2%)