Highly sensitive serological approaches forPepino mosaic virusdetection
Highly sensitive serological approaches forPepino mosaic virusdetection
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用于检测菠萝花叶病毒的高灵敏血清学方法
DOI：
10.1631/jzus.b2000255
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发表时间：
2020-10-01
影响因子：
5.1
通讯作者：
Wu, Jian-xiang
中科院分区：
文献类型：
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作者：
He, Wan-qin;Wu, Jia-yu;Wu, Jian-xiang
佩皮诺花叶病毒（PepMV）是地球仪上危害番茄等茄科作物的一种严重病害。为了有效地研究和管理这种病毒性疾病，研究人员需要新的，灵敏的，高通量的病毒检测方法。本研究从本氏烟（Nicotiana benthamiana）植株中分离纯化PepMV病毒颗粒，并将其免疫BALB/c小鼠，制备了分泌抗PepMV单克隆抗体（mAb）的杂交瘤细胞。通过细胞融合、抗体选择和细胞克隆，可以产生一组高度特异性和敏感性的鼠mAb（15 B2、8H 6、23 D11、20 D9、3A 6和8 E3）。以该单克隆抗体为检测抗体，建立了检测番茄PepMV感染的双抗体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）。灵敏度分析试验的结果数据表明，DAS-ELISA和Dot-ELISA在分别以1：1310720和1：20480（重量/体积比（w/v），g/mL）稀释时，可有效监测PepMV感染组织粗提物中的病毒。在所开发的三种方法中，组织印迹-ELISA被认为是最实用的检测技术。通过RT-PCR和DNA测序验证了所建立的血清学方法对田间样品的检测结果，证明了3种血清学方法对监测PepMV的可靠性和有效性。抗PepMV单克隆抗体和新开发的DAS-ELISA、Dot-ELISA和Tissue print-ELISA可用于PepMV的检测和田间流行病学研究，以及对已在云南省番茄上广泛流行的PepMV的防治。
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 infectedNicotiana benthamianaplants 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, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-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.