DAS-ELISA与RT-PCR检测马铃薯病毒结果比较与分析

Comparison and Analysis of DAS-ELISA and RT-PCR Results for Potato Virus Detection

摘要

马铃薯病毒病是制约马铃薯产业健康发展的重要因素之一，建立快速、准确的检测技术是病毒病防控的关键前提。比较分析双抗体夹心酶联免疫吸附测定（Double antibody sandwich-enzyme linked immunosorbent assay，DAS-ELISA）和反转录聚合酶链式反应（Reverse transcription-polymerase chain reaction，RT-PCR）两种方法在检测不同来源马铃薯材料病毒含量差异与适用性，为实现马铃薯产业不同场景样品病毒病的高效快速检测提供依据。采用DAS-ELISA和RT-PCR分别对23份田间显症马铃薯样品和30份微茎尖离体培养材料进行不同病毒[马铃薯Y病毒（Potato virus Y，PVY）、马铃薯S病毒（Potato virus S，PVS）、马铃薯X病毒（Potato virus X，PVX）、马铃薯M病毒（Potato virus M，PVM）和马铃薯卷叶病毒（Potato leafroll virus，PLRV）]检测，对比分析结果表明，大田显症样品，DAS-ELISA与RT-PCR检测结果一致性高；隐症离体培养材料，两次DAS-ELISA检测结果重复性较差，与RT-PCR结果的一致性分别为59.33%和80.66%。本研究认为DAS-ELISA方法适用于田间病毒显症样品的高通量初步筛查与鉴定，RT-PCR适用于脱毒苗、基础种薯等病毒隐症关键材料的精准检测。

Abstract

Potato viral diseases are one of the key factors limiting the healthy development of the potato industry. Establishing rapid and accurate detection techniques is an essential prerequisite for controlling these viral diseases. Comparative analysis of double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) in detecting viral content differences and applicability across potato materials from various sources provides a basis for achieving efficient and rapid detection of viral diseases in samples across different scenarios within the potato industry. DAS-ELISA and RT-PCR were applied to detect various viruses (Potato virus Y, Potato virus S, Potato virus X, Potato virus M, and Potato leafroll virus) in 23 field symptomatic potato samples and 30 micro-apical shoot tip in vitro cultures. Comparative analysis revealed that the results of DAS-ELISA and RT-PCR were highly consistent for symptomatic samples from fields; for latent in vitro cultures, DAS-ELISA showed poor repeatability in duplicate tests, with concordance rates of 59.33% and 80.66% against RT-PCR, respectively. This study concludes that DAS-ELISA is suitable for high-throughput preliminary screening and identification of field samples with visible virus symptoms, while RT-PCR is appropriate for precise detection of asymptomatic materials such as plantlets in vitro and foundation seed potatoes.

关键词

马铃薯 / 病毒 / DAS-ELISA / RT-PCR

Key words

potato / virus / DAS-ELISA / RT-PCR

参考文献

[1] 赵洁, 张清凤, 普春, 等. 昭通市马铃薯病毒病发生情况分析[J]. 安徽农业科学, 2023, 51(11): 114-116.

[2] 周艳玲, 刘学敏, 孟玉芹. 马铃薯Y病毒的检测技术[J]. 中国马铃薯, 2000, 14(2): 89-93.

[3] 孙琦. 马铃薯脱毒苗的RT-PCR检测技术研究[D]. 泰安: 山东农业大学, 2003.

[4] Clark M F, Adams A N. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses[J]. Journal of General Virology, 1977, 34(3): 475-483.

[5] 申宇, 高艳玲, 范国权, 等. DAS-ELISA与RT-PCR法检测马铃薯S病毒的比较[J]. 黑龙江农业科学, 2016(10): 58-60.

[6] Zhu Y J, Lim S T S, Schenck S, et al. RT-PCR and quantitative real-time RT-PCR detection of Sugarcane Yellow Leaf Virus (SCYLV) in symptomatic and asymptomatic plants of Hawaiian sugarcane cultivars and the correlation of SCYLV titre to yield[J]. European Journal of Plant Pathology, 2010, 127(2): 263-273.

[7] Malan S S. ELISA detection of Apple chlorotic leafspot virus (ACLSV) and Apple mosaic virus (ApMV) in comparison to RT-PCR detection and the determination of genetic variation of these virus species in South Africa[J]. Acta Horticulturae, 2020, 1297(65): 499-506.

[8] 庄木, 王晓武, 谢开云, 等. 一步RT-PCR和DAS-ELISA检测马铃薯X病毒的比较[C]// 中国植物病理学会. 第三次全国植物病毒和病毒防治学术研讨会论文集. 昆明, 2003: 87-88.

[9] 温玉洁, 赵文龙, 葛红霞. 采用DAS-ELISA和RT-PCR方法对马兜铃病毒病的研究[J]. 防护林科技, 2015(8): 36-39.

[10] 陈建军, 刘崇怀, 古勤生, 等. DAS-ELISA、RT-PCR和IC-RT-PCR检测葡萄卷叶病毒Ⅲ的比较研究[J]. 果树学报, 2003, 20(3): 173-177.

[11] 崔健, 崔雅琦, 刘智慧, 等. 酶联免疫法快速检测大批量马铃薯病毒[J]. 中南农业科技, 2023, 44(6): 132-134.

[12] 崔红红, 张幸媛, 田宇豪, 等. 湖南省马铃薯病毒病发生情况调查[J]. 植物保护, 2022, 48(1): 198-203.

[13] Peiman M, Xie C. Development and evaluation of a multiplex RT-PCR for detecting main viruses and a viroid of potato[J]. Acta Virologica, 2006, 50(2): 129-133.

[14] 张丽, 聂峰杰, 张国辉, 等. 3种检测方法在脱毒马铃薯种薯病毒检测中的应用及检测效果比较[J]. 西北农业学报, 2015, 24(7): 119-124.

[15] 吴丽萍. 马铃薯两种病毒的RT-PCR和ELISA检测技术的研究[D]. 兰州: 甘肃农业大学, 2006.

[16] Mahmoud S Y M. Detection of cucumber mosaic virus in some ornamental plants and elimination of nonspecific ELISA reactions[J]. Archives of Phytopathology and Plant Protection, 2011, 44: 1410-1424.

[17] Sikora E J, Andrianifahanana M, Murphy J F. Detection of cucumber mosaic cucumovirus in weed species: A cautionary report on nonspecific reactions in ELISA[J]. Canadian Journal of Plant Pathology, 1999, 21(4): 338-344.

[18] 程胜群, 吕文河, 高艳玲, 等. 马铃薯S病毒RT-qPCR通用检测体系的建立与应用[J]. 华北农学报, 2020, 35(6): 187-194.

[19] 李宗泽, 陆占军, 史云云, 等. 宁夏马铃薯脱毒种薯主要病毒病调查[J]. 作物杂志, 2024(6): 232-236.

[20] 杨茹薇, 邢斌德, 刘易, 等. 马铃薯病毒病病原研究[J]. 安徽农学通报, 2021, 27(9): 96-97, 146.

[21] 庞博. 马铃薯四种病毒多重PCR检测体系的建立[D]. 兰州: 甘肃农业大学, 2014.

[22] 王玥, 张永鹏, 马永强. 青海地区马铃薯主要病毒检测与分析[J]. 青海大学学报, 2024, 42(4): 26-32.

[23] 杨茹薇, 刘易, 李江涛, 等. 马铃薯病毒病病原RT-PCR分子鉴定[J]. 新疆农业科学, 2023, 60(12): 3032-3040.

基金资助

湖南省现代农业产业技术体系(HARS-02)