烟草番茄斑萎病毒RT-LAMP检测体系的建立及优化

doi:10.13304/j.nykjdb.2023.0238

中国农业科技导报 ›› 2024, Vol. 26 ›› Issue (8): 140-150.DOI: 10.13304/j.nykjdb.2023.0238

• 动植物健康 • 上一篇

烟草番茄斑萎病毒RT-LAMP检测体系的建立及优化

张俊蕾¹^,²(), 盖晓彤², 赵正婷³, 刘弟⁴, 王金凤¹, 姜宁²(), 刘雅婷¹^,⁴()

^1.云南农业大学植物保护学院, 昆明 650201
^2.云南省烟草农业科学研究院, 昆明 650021
^3.云南农业大学农学与生物技术学院, 昆明 650201
^4.云南农业大学烟草学院, 昆明 650201

收稿日期:2023-03-29 接受日期:2023-05-24 出版日期:2024-08-15 发布日期:2024-08-12
通讯作者: 姜宁,刘雅婷
作者简介:张俊蕾 E-mail：jleiZHANGg@163.com
基金资助:
云南省烟草公司科技计划项目(2021530000242031);云南省科技厅基础研究专项(202101AU070129);国家重点研发计划项目(2021YFD1400200);国家自然科学基金项目(32260681)

Establishment and Optimization of RT-LAMP Assay System for Tobacco Tomato Spotted Wilt Virus

Junlei ZHANG¹^,²(), Xiaotong GE², Zhengting ZHAO³, Di LIU⁴, Jinfeng WANG¹, Ning JIANG²(), Yating LIU¹^,⁴()

^1.School of Plant Protection，Yunnan Agricultural University，Kunming 650201，China
^2.Yunnan Academy of Tobacco Agricultural Sciences，Kunming 650021，China
^3.School of Agriculture and Biotechnology，Yunnan Agricultural University，Kunming 650201，China
^4.School of Tobacco Science，Yunnan Agricultural University，Kunming 650201，China

Received:2023-03-29 Accepted:2023-05-24 Online:2024-08-15 Published:2024-08-12
Contact: Ning JIANG,Yating LIU

摘要/Abstract

摘要：

为快速检测烟草番茄斑萎病毒（tomato spotted wilt virus，TSWV），基于TSWV核外壳蛋白（nucleocapsid protein，NP）保守序列设计5组引物进行筛选，采用单一变量法对反应温度、时间及反应体系中dNTPs、Mg²⁺、甜菜碱的含量和内外引物比等参数进行优化，建立烟草TSWV反转录环介导等温扩增（loop-mediated isothermal amplification，RT-LAMP）检测体系，并采用RT-PCR（revers transcription-polymerase chain reaction）检测方法进行平行比对试验，验证优化后RT-LAMP的特异性、灵敏性及实用性。结果表明，烟草TSWV RT-LAMP检测体系最佳引物组为TS-N-4，在25 μL的反应体系中各组分最佳加入量为缓冲液2.5 μL、100 mmol·L^-1 MgSO₄ 0.5 μL、10 mmol·L^-1 dNTPs 0.5 μL、10 mmol·L^-1 FIP/BIP 1.5 μL、10 mmol·L^-1 F3/B3 0.5 μL、10 mmol·L^-1 LF/LB 1.5 μL、5 mmol·L^-1 Betaine 6 μL、Bst 2.0 WarmStar DNA Polymerase（8 000 U·mL^-1） 0.5 μL、M-MLV 酶（10 000 U·mL^-1） 0.125 μL、RNA （≥64.7 fg）1 μL，DEPC H₂O补至25 μL；最佳反应温度和时间分别为58 ℃、60 min。优化后的RT-LAMP灵敏度是RT-PCR的1 000倍，且田间样品检测结果与RT-PCR相符。建立的RT-LAMP方法特异性强、灵敏度高、操作简单，对于烟草TSWV的检测及监控有重要意义。

关键词: 番茄斑萎病毒, NP基因, 反转录-环介导等温扩增, 快速检测

Abstract:

To rapidly detect tobacco tomato spotted wilt virus （TSWV）， 5 sets of primers were designed based on the conserved sequence of TSWV nucleocapsid protein （NP） for screening， and parameters such as reaction temperature and time， volumetric molar concentrations of dNTPs， Mg²⁺， betaine， and internal and external primer ratios in the reaction system were optimized using the univariate method to establish the tobacco TSWV reverse transcription loop-mediated isothermal amplification（RT-LAMP）?， the specificity， sensitivity， and practicality of the optimized RT-LAMP were verified by parallel comparison tests using the revers transcription-polymerase chain reaction（RT-PCR） assay. The results showed that the best primer set for the tobacco TSWV RT-LAMP assay was TS-N-4， and the optimal addition of each component in the 25 μL reaction system were 2.5 μL of buffer， 0.5 μL of MgSO₄ （100 mmol·L^-1）， 0.5 μL of dNTPs （10 mmol·L^-1）， 1.5 μL of FIP/BIP （10 mmol·L^-1）， 0.5 μL of F3 /B3 （10 mmol·L^-1）， 1.5 μL LF/LB （10 mmol·L^-1）， 6 μL of Betaine （5 mmol·L^-1）， 0.5 μL of Bst 2.0 WarmStar DNA Polymerase （8 000 U·mL^-1），0.125 μL of M-MLV enzyme （10 000 U·mL^-1）， 0.125 μL of RNA （≥64.7 fg）， and added DEPC H₂O to 25 μL. The optimum reaction temperature and time were 58 ℃ and 60 min， respectively. The optimized RT-LAMP was 1 000 times more sensitive than RT-PCR， and the results of field samples were consistent with RT-PCR. The RT-LAMP method established in this study was specific， sensitive， and simple to operate， which was important for the detection and monitoring of TSWV in tobacco.

Key words: tomato spotted wilt virus, NP gene, reverse transcription-loop-mediated isothermal amplification, rapid detection

中图分类号:

S435.72

张俊蕾, 盖晓彤, 赵正婷, 刘弟, 王金凤, 姜宁, 刘雅婷. 烟草番茄斑萎病毒RT-LAMP检测体系的建立及优化[J]. 中国农业科技导报, 2024, 26(8): 140-150.

Junlei ZHANG, Xiaotong GE, Zhengting ZHAO, Di LIU, Jinfeng WANG, Ning JIANG, Yating LIU. Establishment and Optimization of RT-LAMP Assay System for Tobacco Tomato Spotted Wilt Virus[J]. Journal of Agricultural Science and Technology, 2024, 26(8): 140-150.

图/表 11

表 1 RT-LAMP和RT-PCR特异性检测TSWV引物

Table 1 Specific primers for RT-LAMP and RT-PCR for TSWV

引物 Primer		序列 Sequence（5’-3’）
TS-N-1	F3	ACTTCCTTTAGCATTAGGATTG
	B3	AGCTTTGAGATGATCAGTGT
	FIP	GTGCTAAAAAGCAAAGCATTTGAAA-GGAGCTAAGTATAGCAGCATAC
	BIP	TAAGGCTTCCCTGGTGTCAT-TGTCTTGGCTATATATCAGGATG
	LF	GAAGATCAGGTGAAGAAAGGGAAA
	LB	ACTTCTTTGGATCGATCCCGA
TS-N-2	F3	ACTTCCTTTAGCATTAGGATTG
	B3	AGCTTTGAGATGATCAGTGT
	FIP	GTGCTAAAAAGCAAAGCATTTGAAA-GGAGCTAAGTATAGCAGCATA
	BIP	TAAGGCTTCCCTGGTGTCATA-TGTCTTGGCTATATATCAGGATG
	LF	GATCAGGTGAAGAAAGGGAAAGAG
	LB	TTTGGATCGATCCCGAGGTCT
TS-N-3	F3	TCTTCACCTGATCTTCATTCA
	B3	TCTGTGAGGCTTGCCATA
	FIP	CGGGATCGATCCAAAGAAGTATGAC-CTTTTTAGCACAGTGCAAAC
	BIP	GCATCCTGATATATAGCCAAGACAA-TGCTGGGAGGTAGCTTAC
	LF	ACCAGGGAAGCCTTAGGAAAA
	LB	CATCTCAAAGCTATCAACTGAAGCA
TS-N-4	F3	ACTTCCTTTAGCATTAGGATTG
	B3	AGCTTTGAGATGATCAGTGT
	FIP	GTGCTAAAAAGCAAAGCATTTGAAA-GGAGCTAAGTATAGCAGCATA
	BIP	TAAGGCTTCCCTGGTGTCAT-TGTCTTGGCTATATATCAGGATG
	LF	GATCAGGTGAAGAAAGGGAAAGAG
	LB	ACTTCTTTGGATCGATCCCGA
TS-N-5	F3	ACTTCCTTTAGCATTAGGATTG
	B3	GATAGCTTTGAGATGATCAGTG
	FIP	GTGCTAAAAAGCAAAGCATTTGAAA-GGAGCTAAGTATAGCAGCATA
	BIP	TAAGGCTTCCCTGGTGTCAT-TTGTCTTGGCTATATATCAGGATG
	LF	GATCAGGTGAAGAAAGGGAAAGAG
	LB	ACTTCTTTGGATCGATCCCGAG
RT-PCR	TSWV-F	CTGCACAATCCCAAGACA
RT-PCR	TSWV-R	GCTAAGAGATTGAGRAATGGTATAATAGATTC

图1 TSWV不同RT-LAMP引物扩增结果A：凝胶电泳图；B：荧光可视化结果；M—2 000 DNA marker；1、3、5、7、9—阴性对照；2、4、6、8、10—TSWV的RT-LAMP引物TS-N-1~TS-N-5

Fig. 1 Amplification results of TSWV with different RT-LAMP primersA： Gel electrophoresis； B： Fluorescence visualization results； M—2 000 DNA marker； 1， 3， 5， 7， 9—Negative control； 2， 4， 6， 8， 10—RT-LAMP primers TS-N-1~TS-N-5 of TSWV

图2 TSWV RT-LAMP反应温度优化A：凝胶电泳；B：荧光可视化结果；M—2 000 DNA marker；1—阴性对照；2~7—6个反应温度58、60、62、64、66、68 ℃

Fig. 2 TSWV RT-LAMP reaction temperature optimizationA： Gel electrophoresis； B： Fluorescence visualization results： M—2 000 DNA marker； 1—Negative control； 2~7—6 reaction time gradients 58， 60， 62， 64， 66， 68℃

图3 TSWV RT-LAMP反应时间的优化A：凝胶电泳；B：荧光可视化结果；M—2 000 DNA marker；1—阴性对照；2~7—6个反应温时间30、40、50、60、70、80 min

Fig. 3 Optimization of the TSWV RT-LAMP reaction timesA： Gel electrophoresis； B： Fluorescence visualization results： M—2 000 DNA marker； 1—Negative control； 2~7—6 reaction time gradient 30，40，50，60，70，80 min

图4 TSWV RT-LAMP甜菜碱含量的优化A：凝胶电泳；B：荧光可视化结果；M—2 000 DNA marker；1—阴性对照；2~8—7个反应含量0.0、0.6、0.8、1.0、1.2、1.4、1.6 mmol·L-1

Fig. 4 Optimization of betaine content of TSWV RT-LAMPA： Gel electrophoresis； B： Fluorescence visualization results； M—2 000 DNA marker； 1—Negative control； 2~8—7 reaction contents 0.0， 0.6， 0.8， 1.0， 1.2， 1.4， 1.6 mmol·L-1

图5 TSWV RT-LAMP dNTPs反应含量的优化A：凝胶电泳；B：荧光可视化结果；M—2 000 DNA marker；1—阴性对照；2~8—7个反应含量0.0、0.2、0.6、1.0、1.4、1.8、2.2 mmol·L-1

Fig. 5 Optimization of TSWV RT-LAMP dNTPs reaction contentA： Gel electrophoresis； B： Fluorescence visualization results； M—2 000 DNA marker； 1—Negative control； 2~8—7 reaction contents 0.0， 0.2， 0.6， 1.0， 1.4， 1.8， 2.2 mmol·L-1

图6 TSWV RT-LAMP Mg2+反应含量的优化A：凝胶电泳；B：荧光可视化结果；M—2 000 DNA marker；1—阴性对照；2~7—6个反应含量2、4、6、8、10、12 mmol·L-1

Fig. 6 Optimization of TSWV RT-LAMP Mg2+ reaction contentA： Gel electrophoresis； B： Fluorescence visualization results； M—2 000 DNA marker； 1—Negative control； 2~7—6 reaction contents 2， 4， 6， 8， 10， 12 mmol·L-1

图7 TSWV RT-LAMP内外引物用量比的优化A：凝胶电泳；B：荧光可视化结果；M—2 000 DNA marker；1—阴性对照；2~9—8个引物用量比梯度1∶1~8∶1

Fig. 7 Optimization of inside and outside primers content ratio of TSWV RT-LAMPA： Gel electrophoresis； B： Fluorescence visualization results；M—2 000 DNA marker； 1—Negative control； 2~9—8 primer ratio gradient 1∶1~8∶1

图8 TSWV RT-LAMP灵敏度检测A：RT-LAMP凝胶电泳；1—阴性对照；2~8—7个RNA的含量642.7×100 ~ 642.7×10-6 ng·μL-1；B：RT-PCR凝胶电泳图；1~7—7个RNA的含量642.7×100~642.7×10-6 ng·μL-1；8—阴性对照；M—2 000 DNA marker；C：荧光可视化；1—阴性对照；2~8—7个RNA的含量642.7×100~642.7×10-6 ng·μL-1

Fig. 8 TSWV RT-LAMP sensitivity detectionA： Gel electrophoresis diagram； 1—Negative control； 2~8—Seven RNA reaction contents of 642.7×100~642.7×10-6 ng·μL-1；B： Gel electrophoresis diagram； 1~7—Seven RNA reaction contents of 642.7×100~642.7×10-6 ng·μL-1；8—Negative control；M—2 000 DNA marker； C： Fluorescence visualization results； 1—Negative control； 2~8—Seven RNA reaction contents of 642.7×100~642.7×10-6 ng·μL-1

图9 TSWV RT-LAMP特异性检测A：凝胶电泳图；B：荧光可视化结果；M—2000 DNA marker；1—阴性对照；2~10—TSWV、TMV、CMV、ToBRFV、HCRV、TZSV、TVBMV、ChiVMV、PVY的RNA

Fig. 9 TSWV RT-LAMP specificity detectionA： Gel electrophoresis； B： Fluorescence visualization results； M—2 000 DNA marker； 1—Negative control； 2~10—RNA for TSWV， TMV， CMV， ToBRFV， HCRV， TZSV， TVBMV， ChiVMV and PVY

图10 TSWV RT-LAMP和RT-PCR田间样品检测结果A：凝胶电泳；B：荧光可视化结果；M—2 000 DNA marker；1—阴性对照；2—阳性对照；3~12—田间烟草叶片样品

Fig. 10 Results of TSWV RT-LAMP and RT-PCR field samplesA： Gel electrophoresis； B： Fluorescence visualization results； M—2 000 DNA marker； 1—Negative control； 2—Positive control； 3~12—Field tobacco leaf samples

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烟草番茄斑萎病毒RT-LAMP检测体系的建立及优化

Establishment and Optimization of RT-LAMP Assay System for Tobacco Tomato Spotted Wilt Virus

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