马铃薯病毒病实时荧光定量PCR检测技术

doi:10.6048/j.issn.1001-4330.2024.10.016

摘要/Abstract

摘要：

【目的】 鉴定马铃薯主要病毒病病原,为马铃薯病毒病的科学防治提供科学依据。【方法】 根据 GenBank 数据库注册序列设计马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)的检测引物,完成引物浓度及系统优化。【结果】 建立3种马铃薯病毒病的实时荧光定量PCR检测技术体系(real-time fluorescent quantitative RT-PCR,RT-qPCR),其标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为99.42%以上,扩增效率均为84.48%以上,可快速、准确检测出3种马铃薯病毒。【结论】 实时荧光定量PCR检测技术是一种高灵敏度、特异性强的检测方法,可实时监测马铃薯生产中病毒病的感染情况。

关键词: 马铃薯; 病毒病; 实时荧光定量PCR

Abstract:

【Objective】 Potato virus disease is one of the major diseases affecting the degradation of varieties and the decline of yields, and it is necessary to characterize the major virus pathogens of potato in the production process, so as to provide a scientific basis for the scientific prevention and control of potato virus diseases. 【Methods】 The primers for potato virus Y (PVY), potato virus S (PVS) and potato leaf roll virus (PLRV) were designed according to the sequences registered in the GenBank database, and the primer concentration and system optimization were completed. 【Results】 A real-time fluorescent quantitative RT-PCR (RT-qPCR) system was established for the detection of three potato virus diseases, and the standard curve showed a good linear relationship between the cycling threshold and the template concentration, with a correlation coefficient of 99.42%, and an amplification efficiency of 84.48%. The amplification efficiencies were all 84.48%, which could rapidly and accurately detect the three potato viruses. 【Conclusion】 Real-time fluorescence quantitative PCR is a highly sensitive and specific detection method, which can be used to monitor the infection of virus diseases in potato production in real time.

Key words: potato; virus disease; real-time fluorescence quantitative PCR

中图分类号:

S436.3

杨茹薇, 刘易, 古丽米拉·热合木土拉, 孙慧, 江应红. 马铃薯病毒病实时荧光定量PCR检测技术[J]. 新疆农业科学, 2024, 61(10): 2484-2490.

YANG Ruwei, LIU Yi, Gulimila Rehemutula, SUN Hui, JIANG Yinghong. Research on real-time fluorescence quantitative PCR detection of potato virus disease[J]. Xinjiang Agricultural Sciences, 2024, 61(10): 2484-2490.

图/表 10

表1 病毒引物序列

Tab.1 Virus primer sequences

病毒种类 Virus type	序列(5'-3') Sequence(5'-3')	Tm(℃)
PVY	F:TGAAAATGGAACCTCGCC	89.3
	R:AATGTGCCATGATTTGCC
PVS	F:TCGTBTGGAATTACATGCTG	90.6
	R:ATCAAATGTGTCAAAGCGG
PLRV	F:AAGAAGGCAATCCCTTCG	86.7
	R:ATGTCTCGCTTGAGCCTC

图1 RNA凝胶电泳结果

Fig.1 RNA gel electrophoresis results

图2 实时荧光定量PCR扩增曲线

Fig.2 Real-time fluorescence quantitative PCR amplification curve

图3 实时荧光定量PCR溶解曲线

Fig.3 Real-time fluorescence quantitative PCR lysis curve

图4 三种马铃薯病毒引物特异性检测

Fig.4 Specificity of three potato virus primer assays

图5 PVY、PVS、PLRV实时荧光定量PCR标准曲线

Fig.5 Standard curve of real-time fluorescence quantitative PCR for PVY, PVS, PLRV

表2 PVY、PVS、PLRV实时荧光定量PCR标准曲线

Tab.2 Standard curves of real-time fluorescence quantitative PCR for PVY, PVS, PLRV

引物 Primer	标准曲线方程 Standard curve equation	R²	扩增效率 Amplification efficiency(%)	斜率 Slope	Y轴截距 Y-Intercept
PVY	Y=-3.57X+37.05	0.994 2	90.59	3.57	37.05
PVS	Y=-3.76X+39.93	0.996 4	84.48	3.76	39.93
PLRV	Y=-3.72X+40.16	0.998 2	85.7	3.72	40.16

图6 不同患病植株PVY基因相对表达量

Fig.6 Relative expression of PVY gene in different diseased plants

图7 不同患病植株PVS基因相对表达量

Fig.7 Relative expression of PVS gene in different diseased plants

图8 不同患病植株PLRV基因相对表达量

Fig.8 Relative expression of PLRV gene in different diseased plants

参考文献 21

[1]	SALAZARL F. 马铃薯病毒及其防治[M].阎文昭, 译. 北京: 中国农业科学技术出版社, 2000.
	Salazarl F. Potato virus and its control[M]. YAN Wenzhao, Translator. Beijing: China Agricultural Science and Technology Press, 2000.
[2]	吴丽萍, 王蒂, 司怀军, 等. 马铃薯S病毒的RT-PCR检测[J]. 中国马铃薯, 2006, 20(4): 200-203.
	WU Liping, WANG Di, SI Huaijun, et al. Detection of potato S virus by RT-PCR[J]. Chinese Potato, 2006, 20(4): 200-203.
[3]	李华伟, 罗文彬, 纪荣昌, 等. 福建省马铃薯主要病虫害种类调查及防控技术[J]. 福建农业科技, 2014,(12): 59-62.
	LI Huawei, LUO Wenbin, JI Rongchang, et al. Main kinds of pests and diseases of potato and the control technology in Fujian Province[J]. Fujian Agricultural Science and Technology, 2014,(12): 59-62.
[4]	薛超. 植物病毒检测技术研究进展分析[J]. 农业与技术, 2020, 40(16): 48-50.
	XUE Chao. Research progress analysis of plant virus detection technology[J]. Agriculture and Technology, 2020, 40(16): 48-50.
[5]	Lacomme C, Jacquot E. General characteristics of potato virus Y (PVY) and its impact on potato production: an overview[M]//Lacomme C, Glais L, Bellstedt D, et al. Potato virus Y: biodiversity, pathogenicity, epidemiology and management. Cham: Springer, 2017: 1-19.
[6]	Mancini V, Murolo S, Romanazzi G. Diagnostic methods for detecting fungal pathogens on vegetable seeds[J]. Plant Pathology, 2016, 65(5): 691-703.
[7]	尚晓楠, 吴蓓蕾. 马铃薯X病毒荧光定量PCR检测体系的建立及应用[J]. 植物保护, 2016, 42(3): 165-169.
	SHANG Xiaonan, WU Beilei. Development and application of a real-time quantitative PCR system for detection of Potato virus X[J]. Plant Protection, 2016, 42(3): 165-169.
[8]	秦子禹, 孙建设, 王娜, 等. 苹果茎痘病毒TaqMan探针实时荧光定量RT-PCR检测方法的建立[J]. 园艺学报, 2015, 42(7): 1400-1408.
	QIN Ziyu, SUN Jianshe, WANG Na, et al. Development of a TaqMan Real-time RT-PCR Method for Detecting Apple stem pitting virus(ASPV)[J]. Acta Horticulturae Sinica, 2015, 42(7): 1400-1408. DOI
[9]	周俊, 范旭东, 董雅凤, 等. 葡萄扇叶病毒实时荧光定量RT-PCR检测方法的建立及应用[J]. 园艺学报, 2016, 43(3): 538-548. DOI
	ZHOU Jun, FAN Xudong, DONG Yafeng, et al. Development and Application of a Quantitative RT-PCR Approach for Quantification of Grapevine fanleaf virus[J]. Acta Horticulturae Sinica, 2016, 43(3): 538-548. DOI
[10]	Chen H M, Zhou Y, Wang X F, et al. Detection of Citrus yellow vein clearing virus by Quantitative Real-time RT-PCR[J]. Horticultural Plant Journal, 2016, 2(4): 188-192.
[11]	Babujee L, Witherell R A, Mikami K, et al. Optimization of an isothermal recombinase polymerase amplification method for real-time detection of Potato virus Y O and N types in potato[J]. Journal of Virological Methods, 2019, 267: 16-21.
[12]	Cheng J H, Jiang Y H, Rao P B, et al. Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen[J]. Archives of Virology, 2013, 158(2): 379-386. DOI PMID
[13]	Agindotan B O, Shiel P J, Berger P H. Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan© real-time RT-PCR[J]. Journal of Virological Methods, 2007, 142(1/2): 1-9.
[14]	王玉倩, 薛秀花. 实时荧光定量PCR技术研究进展及其应用[J]. 生物学通报, 2016, 51(2): 1-6.
	WANG Yuqian, XUE Xiuhua. Research progress and application of real-time fluorescence quantitative PCR technology[J]. Bulletin of Biology, 2016, 51(2): 1-6.
[15]	程群, 朱云芬, 沈艳芬, 等. 马铃薯S病毒RT-PCR检测技术的研究[J]. 氨基酸和生物资源, 2010, 32(4): 8-11.
	CHENG Qun, ZHU Yunfen, SHEN Yanfen, et al. Study on detection of potato virus S by RT-PCR[J]. Amino Acids & Biotic Resources, 2010, 32(4): 8-11.
[16]	张威, 白艳菊, 文景芝, 等. 马铃薯六种主要病毒通用RT-PCR检测体系的建立[J]. 中国马铃薯, 2015, 29(4): 222-227.
	ZHANG Wei, BAI Yanju, WEN Jingzhi, et al. Establishment of universal RT-PCR system for detection of six major potato viruses[J]. Chinese Potato Journal, 2015, 29(4): 222-227.
[17]	姜立慧. 四川省6种主要马铃薯病毒的发生及其多重RT-PCR检测体系的建立[D]. 雅安: 四川农业大学, 2016.
	JIANG Lihui. The Incidence of Six Kinds of Main Potato Viruses and the Establishment of Multiplex RT-PCR Detection System in Sichuan[D]. Yaan: Sichuan Agricultural University, 2016.
[18]	聂峰杰, 詹红, 张丽, 等. RT-PCR技术对宁夏马铃薯脱毒种薯病毒检测的研究[J]. 植物保护, 2016, 42(5): 188-193.
	NIE Fengjie, ZHAN Hong, ZHANG Li, et al. Detection of virus on virus-free potatoes in Ningxia by RT-PCR[J]. Plant Protection, 2016, 42(5): 188-193.
[19]	Kogovšek P, Gow L, Pompe-Novak M, et al. Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates[J]. Journal of Virological Methods, 2008, 149(1): 1-11. DOI PMID
[20]	陈兆贵, 叶新友, 邢澍祺, 等. 马铃薯卷叶病毒实时荧光定量PCR检测技术研究[J]. 湖南农业科学, 2018, 55(9): 9-12.
	CHEN Zhaogui, YE Xinyou, XING Shuqi, et al. Study on real time fluorescence quantitative PCR detection technology of potato leaf roll virus[J]. Hunan Agricultural Sciences, 2018, 55(9): 9-12.
[21]	张永江, 辛言言, 李桂芬, 等. 实时荧光RT-PCR方法检测马铃薯V病毒[J]. 江苏农业科学, 2013, 41(6): 36-38.
	ZHANG Yongjiang, XIN Yanyan, LI Guifen, et al. Detection of potato V virus by real-time fluorescence RT-PCR[J]. Jiangsu Agricultural Sciences, 2013, 41(6): 36-38.