甘薯褪绿矮化病毒西非株系RT-RPA-LFD检测方法的建立与应用

doi:10.3864/j.issn.0578-1752.2024.14.007

摘要/Abstract

摘要：

【目的】利用逆转录酶重组酶聚合酶扩增（reverse transcriptase recombinase polymerase amplification，RT-RPA）结合侧向流层析（lateral flow dipstick，LFD）试纸条技术，建立甘薯褪绿矮化病毒（sweet potato chlorotic stunt virus，SPCSV）西非株系（west African strain，WA）的RT-RPA-LFD检测方法。【方法】根据SPCSV-WA外壳蛋白基因和热激蛋白基因的保守序列设计并筛选扩增效果好、特异性强的引物和探针。然后对引物和探针的浓度、扩增体系、温度、反应时间等条件进行优化，建立SPCSV-WA的RT-RPA-LFD检测方法。利用该方法对SPCSV-EA、甘薯羽状斑驳病毒（sweet potato feathery mottle virus，SPFMV）、甘薯潜隐病毒（sweet potato latent virus，SPLV）、甘薯G病毒（sweet potato virus G，SPVG）等常见的甘薯病毒进行检测，验证方法的特异性；将感染SPCSV-WA甘薯叶片样品总RNA进行10倍梯度稀释，分别采用RT-PCR和RT-RPA-LFD进行检测，比较RT-PCR和RT-RPA-LFD方法的灵敏度；对采自田间的甘薯样品和试管苗样品进行RT-RPA、RT-RPA-LFD和RT-PCR检测，验证该方法的实用性。【结果】建立了SPCSV-WA的RT-RPA-LFD检测方法，最适引物为CSV357F/R，探针CSV-CP-Probe（47 bp），引物和探针工作浓度分别为0.2和0.06 μmol·L^-1，温度为42 ℃，时间5 min。该方法可对SPCSV-WA进行特异性检测，与甘薯上其他常见病毒无交叉反应。RT-RPA-LFD最低可检测到总RNA稀释至10^-4溶液，而RT-PCR最低检测到总RNA稀释至10^-3溶液，RT-RPA-LFD灵敏度是RT-PCR的10倍。田间采集的22份甘薯样品经RT-PCR、RT-RPA和RT-RPA-LFD检测，均检出11份阳性样品，3种方法检测结果一致。28份试管苗样品的检测结果表明，RT-PCR和RT-RPA-LFD检测结果一致，均检出5份阳性样品。【结论】建立了SPCSV-WA的RT-RPA-LFD检测方法，该方法具有快速、简便、特异、灵敏、可视的特点，既可用于甘薯脱毒试管苗样品的病毒检测，也适用于基层单位田间甘薯病毒样品的现场快速检测。

关键词: 甘薯褪绿矮化病毒西非株系, 逆转录酶重组酶聚合酶扩增, 侧向流层析, 快速检测, 甘薯

Abstract:

【Objective】 This study aims to establish a novel technique for detecting the west African strain of sweet potato chlorotic stunt virus (SPCSV-WA) by combining reverse transcriptase recombinase polymerase amplification and lateral flow dipstick (RT-RPA-LFD).【Method】 Primers and probes with good amplification results and strong specificity were designed and screened on the basis of the conserved sequences of the SPCSV-WA coat protein gene and heat shock protein gene. Then, the conditions such as primer and probe concentrations, amplification system, temperature, and reaction time were optimized to detect SPCSV-WA using RT-RPA-LFD. This method was used to detect common viruses on sweet potato such as the east African strain of sweet potato chlorotic stunt virus (SPCSV-EA), sweet potato feathery mottle virus (SPFMV), sweet potato latent virus (SPLV) and sweet potato virus G (SPVG) to verify the specificity. Total RNA from SPCSV-infected sweet potato leaves was diluted in a 10-fold gradient, and RT-PCR and RT-RPA-LFD were performed to compare the sensitivity of the two methods. Field sweet potato samples and test tube seedling samples were subjected to RT-RPA, RT-RPA-LFD, and RT-PCR detection to validate the practicality of the method.【Result】 The RT-RPA-LFD detection method for SPCSV-WA was established using the optimal primer CSV357F/R and the CSV-CP-Probe (47 bp). The working concentrations of the primer and probe were 0.2 and 0.06 μmol·L^-1, respectively. The reaction conditions were set to 42 ℃ for 5 min. This method could specifically detect SPCSV-WA and had no cross-reaction with other common viruses on sweet potato. The RT-RPA-LFD could detect viruses up to 10^-4dilution, whereas RT-PCR could only detect up to 10^-3 dilution, making the sensitivity of RT-RPA-LFD 10 times higher than that of RT-PCR. Of the 22 field-gathered sweet potato samples tested by RT-PCR, RT-RPA, and RT-RPA-LFD, 11 positive samples were consistently found across the three methods. The testing results of 28 test tube seedling samples showed that RT-PCR and RT-RPA-LFD consistently detected five positive samples.【Conclusion】 The RT-RPA-LFD detection method for SPCSV-WA has been established and it is characterized by its speed, simplicity, specificity, sensitivity, and visibility. This method can be used for testing virus-free test tube seedling samples of sweet potato as well as for on-site rapid detection of field sweet potato samples in basic level units.

Key words: west African strain of sweet potato chlorotic stunt virus (SPCSV-WA), reverse transcriptase recombinase polymerase amplification (RT-RPA), lateral flow dipstick (LFD), rapid detection, sweet potato

王永江, 乔奇, 王爽, 赵付枚, 田雨婷, 张德胜, 张振臣. 甘薯褪绿矮化病毒西非株系RT-RPA-LFD检测方法的建立与应用[J]. 中国农业科学, 2024, 57(14): 2781-2790.

WANG YongJiang, QIAO Qi, WANG Shuang, ZHAO FuMei, TIAN YuTing, ZHANG DeSheng, ZHANG ZhenChen. Establishment and Application of RT-RPA-LFD Detection Method for Sweet Potato Chlorotic Stunt Virus WA Strain[J]. Scientia Agricultura Sinica, 2024, 57(14): 2781-2790.

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体系 System	编号 Number	引物名称 Primer name	引物和探针序列 Primer and probe sequence (5′-3′)	大小 Size (bp)	来源 <BOLD>S</BOLD>ource
RT-RPA	C1	CSV170F	ATGGCTGATAGCACTAAAGTCGAAGAGAAGAAC	170	本试验This study
	C1	CSV170R	GTAAGAATACCACTRGTTARCTCCATATCTC	170	本试验This study
	C2	CSV216F	GRCTTCCAACTATACTCTTTCCCGT	216	本试验This study
	C2	CSV216R	GATATTATGTCAAAGAGTCAAGAGGATG	216	本试验This study
	C3	CSV237F	GGCRTGGGCAAATCAGAGTACGTCCGAAAAGAAT	237	本试验This study
	C3	CSV237R	TTTCGCCTGYAAATGGTAATCGGGTTTAACAATAC	237	本试验This study
	C4	CSV253F	TTTGGGAAGACGAGATATGGAGYTAACTAGTGGT	253	本试验This study
	C4	CSV253R	TTTCGGACGTACTCTGATTTGCCCATGCCTGW	253	本试验This study
	C5	SCV259F	YGGCTAGTTTGGGAAGACGAGATATGGAGYTAA	259	本试验This study
	C5	CSV259R	TCGGACGTACTCTGATTTGCCCAYGCCTGW	259	本试验This study
	C6	CSV357F	[6-Fam]-TTTGGGAAGACGAGATATGGAGYTAACYAGTG	357	本试验This study
		CSV357R	Biotin-GGCATGTTAGRTTGAATATGGTTTATGAAGTTGT
		CSV-CP-Probe	[6-Fam]-CAGATATTATGTCAAAGAGTCAAGAGGAT[THF] AGTTTAAGCGTCATATG-C3Spacer
	C7	CSV405F	CGGRTCGGARTTTACYCCMACGTGTYGTCYRTYA	405	本试验This study
	C7	CSV405R	CCKAACGATTCACATACAGACTTCATAAACATYC	405	本试验This study
	C8	CSV313F	WCAGGCRTGGGCAAATCAGAGTACGTCCGAAAA	313	本试验This study
	C8	CSV313R	CTGCGGTTGTCGCCAAATCATTACCTCTCATG	313	本试验This study
RT-PCR		CP-F	ATGGCTGATAGCACTAAAGTCGA	774	文献[7] Reference [7]
		CP-R	TCAACAGTGAAGACCTGTTCCAG	774	文献[7] Reference [7]
		SPCSV-EA-SPSP1-T7	TAATACGACTCACTATAATGRMTACTGRCAAAGTAAACGATG	791	文献[10] Reference [10]
		SPCSV-EA-SPSP4	TCAACAGTGAAGACCRGYACCRGTCAA
		SPVG-P5-T7	TAATACGACTCACTATAACTGAATTCTCYRCTGAAGARAYATAYGATGC	1017	文献[28] Reference [28]
		SPVG-P3	ACTAAGCTTTTACTGCACACCCCTCATACC	1017	文献[28] Reference [28]
		SPLV-P5-T7	TAATACGACTCACTATAGCCGAY GAAACCATCMTCGAT	1093	文献[29] Reference [29]
		SPLV-P3	GTCTC Y GGTATAAGACG	1093	文献[29] Reference [29]

编号 Number	引物和探针反应浓度 Concentration of primer and probe (μmol·L^-1)	镁离子浓度 Concentration of Mg²⁺(μmol·L^-1)
A	CSV357F (2×10^-1)/CSV357R (2×10^-1)+CSV-CP-Probe (6×10^-2)	2.10×10⁴
B	CSV357F-fam (2×10^-1)/CSV357R (2×10^-1)	2.10×10⁴
C	CSV357F (4×10^-1)/CSV357R (4×10^-1)+CSV-CP-Probe (8×10^-2)	1.68×10⁴
D	CSV357F-fam (2×10^-1)/CSV357R (2×10^-1)	1.68×10⁴
E	CSV357F (2×10^-1)/CSV357R (2×10^-1)+CSV-CP-Probe (6×10^-2)	1.68×10⁴
F	CSV357F (1.6×10^-1)/CSV357R (1.6×10^-1)+CSV-CP-Probe (6×10^-2)	1.68×10⁴