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Sugarcane streak mosaic virus double-antibody sandwich enzyme-linked immunosorbent assay kit as well as preparation method and detection method of kit

A mosaic virus and immune detection technology, applied in the field of biotechnology products for plant virus detection, can solve problems such as inability to detect, and achieve the effects of simple operation, high sensitivity, and large-scale commercial promotion.

Inactive Publication Date: 2018-11-09
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is imminent to develop rapid detection and identification technology for SCSMV. At present, SCSMV is mainly detected by electron microscope technology and molecular biology technology. Since there is no specific antiserum for SCSMV, enzyme-linked immunosorbent assay (ELISA) cannot be used for detection.

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  • Sugarcane streak mosaic virus double-antibody sandwich enzyme-linked immunosorbent assay kit as well as preparation method and detection method of kit
  • Sugarcane streak mosaic virus double-antibody sandwich enzyme-linked immunosorbent assay kit as well as preparation method and detection method of kit
  • Sugarcane streak mosaic virus double-antibody sandwich enzyme-linked immunosorbent assay kit as well as preparation method and detection method of kit

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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 SCSMV coat protein

[0049] ①Total RNA extraction (polysaccharide and polyphenol plant tissue lysis method)

[0050] ②Primer design

[0051] According to the obtained SCSMV-M114 isolate sequence, design and synthesize specific primers in amplifying the SCSMV coat protein nucleotide sequence: SCSMCPF F (SEQ ID No.1) and SCSMCPR F (SEQ ID No.2); introduce respectively Restriction sites Nco I and Sac I were used to connect the prokaryotic expression recombinant vector pET-28a. The primers were purified by PAGE and sent to Shanghai Sangon Bioengineering Co., Ltd. for synthesis.

[0052] ③RT-PCR

[0053] Using the total RNA of sugarcane leaves as a template, using SCSMCPR primers, cDNA was synthesized by reverse transcriptase M-MLV. The reverse transcription system is shown in Table 1.

[0054] ...

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Abstract

The invention discloses a sugarcane streak mosaic virus double-antibody sandwich enzyme-linked immunosorbent assay kit as well as a preparation method and detection method of the kit. The kit containsa micro-hole plate for ELISA, an enzyme labeled antibody, a buffer solution, a developing solution and a stop solution, wherein the micro-hole plate is coated with an SCSMV specific polyclonal antibody, and the enzyme labeled antibody is horseradish peroxidase labeled SCSMV specific polyclonal antibody. An immunogen adopted in the kit is a genetically engineered expressed SCSMV coat protein antigen. The invention further discloses a detection method for sugarcane streak mosaic viruses. A rapid, sensitive, simple, convenient, economic and practical method is provided for the detection of the sugarcane streak mosaic viruses in the serological level, and an effective method is provided for the prevention and treatment of the sugarcane streak mosaic viruses in the production of sugarcane andthe preparation of virus-free seedlings of the sugarcane streak mosaic viruses.

Description

technical field [0001] The invention relates to the field of biotechnology products for plant virus detection, in particular to a double-antibody sandwich enzyme-linked immunoassay kit (DAS-ELISA) that can be used for rapid detection of sugarcane streak mosaic virus, and the invention also relates to the kit preparation method and detection method. Background technique [0002] Sugarcane mosaic disease (SMD) is an important disease of sugarcane that widely occurs in major sugarcane producing areas in the world. The main symptoms are yellow and green, irregular mosaic, stripes or mottled leaves. , the leaf color is lighter, the growth potential is poor, and the plants are dwarfed, which can generally cause 10-40% yield loss and sugar content drop. This disease is widespread in the sugarcane cultivation areas of Yunnan and Guangxi provinces and the national sugarcane germplasm resource garden occurred, seriously restricting the sustainable and stable development of the sugar ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 贺振陈春峰陈雯甘海峰张坤
Owner YANGZHOU UNIV
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