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Broad bean latent virus M double-antibody sandwich enzyme-linked immunoassay kit and preparation and detection methods thereof

A latent virus and immune detection technology is applied in the field of biotechnology products for plant virus detection, which can solve the problems of no VCV-M specific antiserum and undetectability, and achieve large-scale commercial promotion, simple operation, and good performance. The effect of applying the foreground

Pending Publication Date: 2020-12-01
YANGZHOU UNIV
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Problems solved by technology

Therefore, it is imminent to develop a rapid detection and identification technology for VCV-M. At present, there is no further experimental research on the basic biological characteristics of VCV-M. In addition, there is no VCV-M specific antiserum, and it is impossible to use enzyme-linked Immunosorbent assay (ELISA) detection

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  • Broad bean latent virus M double-antibody sandwich enzyme-linked immunoassay kit and preparation and detection methods thereof
  • Broad bean latent virus M double-antibody sandwich enzyme-linked immunoassay kit and preparation and detection methods thereof
  • Broad bean latent virus M double-antibody sandwich enzyme-linked immunoassay kit and preparation and detection methods thereof

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Embodiment Construction

[0047] The present invention will be further described below in combination with specific embodiments and accompanying drawings.

[0048] (1) Prokaryotic expression and purification of VCV-M coat protein

[0049] ①Total RNA extraction (Trizol method)

[0050] ②Primer design

[0051] According to the obtained VCV-M isolate sequence, design and synthesize specific primers in amplifying the VCV-M coat protein nucleotide sequence: VCV-M / CP / F (SEQ ID No.1) and VCV-M / CP / R (SEQ ID No.2); restriction sites NcoI and NotI were respectively introduced to connect the prokaryotic expression recombinant vector pET-28a. The primers were purified by PAGE and sent to Nanjing Qingke Biotechnology Co., Ltd. for synthesis.

[0052] ③RT-PCR

[0053] The total RNA of faba bean leaves was used as a template, and VCVMCPR primer was used to synthesize cDNA by reverse transcriptase M-MLV. The reverse transcription system is shown in Table 1.

[0054] Table 2 Reverse transcription system

[0055]...

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Abstract

The invention discloses a broad bean latent virus M double-antibody sandwich enzyme-linked immunoassay kit as well as a preparation method and a detection method thereof. The test kit comprises a microwell plate for ELISA, an enzyme-labeled antibody, a buffer solution, a developing solution and a stop solution, the microwell plate is coated with a VCV-M specific polyclonal antibody with an amino acid sequence shown as SEQ ID No.3, and the enzyme-labeled antibody is an alkaline phosphatase labeled VCV-M specific polyclonal antibody. The immunogen adopted by the invention is a VCV-M coat proteinantigen expressed by genetic engineering, a rapid, sensitive, simple and convenient method is provided for VCV-M detection on the serological level, the method is economical and practical, and a certain effective method is provided for subsequent broad bean field planting and virus disease prevention and control.

Description

technical field [0001] The invention belongs to the technical field of biotechnology products for plant virus detection, and in particular relates to a broad bean latent virus M double-antibody sandwich enzyme-linked immunoassay kit and a preparation and detection method. Background technique [0002] Vicia cryptic virus M (VCV-M) was first reported in Hangzhou, Zhejiang Province, China in 2009. VCV-M is mainly transmitted by seeds or by cell mitosis, and there is no obvious symptom after the plant is infected, but it often infects plants in cooperation with other viruses, showing more serious symptoms. [0003] VCV-M belongs to the family Amalgaviridae, a member of the genus Amalgavirus. The virus genome is about 3.4kb, and the VCV-M genome consists of two double-stranded RNA fragments. The positive strand of the RNA contains two partially overlapping ORFs, ORF2 translocated by ribosomes by +1. The protein encoded by ORF2 contains an RNA-dependent RNA polymerase conserve...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/535C07K16/10
CPCG01N33/56983G01N33/54306G01N33/535C07K16/10G01N2333/08
Inventor 张坤徐红梅陈佳欢贺振庄新建甘海峰
Owner YANGZHOU UNIV
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