Research on real-time fluorescence quantitative PCR detection of potato virus disease

doi:10.6048/j.issn.1001-4330.2024.10.016

Abstract

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

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

CLC Number:

S436.3

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.

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.

Figures/Tables 10

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

Fig.1 RNA gel electrophoresis results

Fig.2 Real-time fluorescence quantitative PCR amplification curve

Fig.3 Real-time fluorescence quantitative PCR lysis curve

Fig.4 Specificity of three potato virus primer assays

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

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

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

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

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

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