Highly sensitive serological approaches for Pepino mosaic virus detection
- PDF / 1,835,485 Bytes
- 12 Pages / 595.22 x 842 pts (A4) Page_size
- 78 Downloads / 245 Views
811
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) ISSN 1673-1581 (Print); ISSN 1862-1783 (Online) www.jzus.zju.edu.cn; www.springerlink.com E-mail: [email protected]
Highly sensitive serological approaches for Pepino mosaic virus detection* Wan-qin HE§1, Jia-yu WU§2, Yi-yi REN2, Xue-ping ZHOU1,3, Song-bai ZHANG4, Ya-juan QIAN1, Fang-fang LI†‡3, Jian-xiang WU†‡1 1 2
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
Department of Applied Biological Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China 3
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
4
Hunan Plant Protection Institute, Chinese Academy of Agricultural Sciences, Changsha 410125, China †
E-mail: [email protected]; [email protected]
Received May 17, 2020; Revision accepted July 30, 2020; Crosschecked Sept. 8, 2020
Abstract: 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 1:1 310 720 and 1:20 480 (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. Key words: Pepino mosaic virus; Monoclonal antibody; Serological method; Double antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA); Dot-ELISA; Tissue print-ELISA https://doi.org/10.1631/jzus.B2000255 CLC number: S41-30
1 Introduction ‡
Corresponding authors The two authors contributed equally to this w
Data Loading...
