摘要
Pepino mosaic virus(PepMV)causes severe disease in tomato and other Solanaceous crops around globe.To effectively study and manage this viral disease,researchers need new,sensitive,and high-throughput approaches for viral detection.In this study,we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies(mAbs).A panel of highly specific and sensitive murine mAbs(15B2,8H6,23D11,20D9,3A6,and 8E3)could be produced through cell fusion,antibody selection,and cell cloning.Using the mAbs as the detection antibodies,we established double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA),Dot-ELISA,and Tissue print-ELISA for detecting PepMV infection in tomato plants.Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1310720 and 1:20480(weight/volume ratio(w/v),g/mL),respectively.Among the three methods developed,the Tissue print-ELISA was found to be the most practical detection technique.Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction(RT-PCR)and DNA sequencing,dem on strati ng all three serological methods are reliable and effective for monitoring PepMV.An ti-PepMV mAbs and the newly developed DAS-ELISA,Dot-ELISA,and Tissue print-ELISA can benefit PepMV detection and field epidemiological study,and management of this viral disease,which is already widespread in tomato plants in Yunnan Province of China.
目的:建立基于单克隆抗体的检测番茄等植物中凤果花叶病毒(PepMV)的血清学方法,为PepMV的田间调查和诊断及其科学防控提供快速实用的检测技术。创新点:首次制备了抗PepMV的高度特异和灵敏的单克隆抗体,并利用制备的单抗建立了3种能特异且灵敏地检测PepMV的血清学方法。方法:以差速离心方法提纯的PepMV粒子作为免疫原免疫BALB/c小鼠,通过杂交瘤技术获得了能稳定传代并分泌PepMV单克隆抗体的杂交瘤细胞株;杂交瘤细胞注射到小鼠腹腔获得单克隆抗体腹水,并以制备单抗为核心,根据血清学原理建立检测植物中PepMV的双抗夹心酶联免疫吸附试验(DAS-ELISA)、斑点酶联免疫吸附试验(Dot-ELISA)和组织印迹酶联免疫吸附试验(Tissue print-ELISA)三种血清学检测方法;利用田间番茄样品分析建立的血清学方法检测PepMV的有效性。结论:利用杂交瘤技术获得了6株能分泌高度特异灵敏PepMV单克隆抗体的杂交瘤细胞株,以分泌的单抗为核心建立了检测植株中PepMV的DASELISA,Dot-ELISA和Tissue print-ELISA三种高度灵敏的血清学新技术。三种建立的血清学技术检测感染PepMV的番茄植株均呈强阳性反应,而检测健康番茄及感染其他5种植物病毒的植株呈阴性反应,且DAS-ELISA和Dot-ELISA血清学技术检测番茄病叶粗提液的灵敏度分别达到1:1310720和1:20480倍稀释(质量体积比,g/mL)。田间样品检测结果发现,建立的血清学技术的检测结果与反转录聚合酶链反应(RT-PCR)的检测结果一致,表明建立的血清学方法可有效地用于植物中PepMV的检测。同时,本研究首次发现PepMV己在我国云南番茄作物上发生流行。PepMV单克隆抗体的制备及其灵敏血清学检测方法的建立有益于PepMV的田间调查和诊断及其科学防控。
基金
National Key R&D Program of China(Nos.2019YFD1001800 and 2017YFD0201604)
the National Natural Science Foundation of China(Nos.31772125 and 31972234)。
