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Lotus latent virus double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit and preparation and detection methods

A latent virus and immune detection technology, applied in the field of biotechnology products for plant virus detection, can solve detection and other problems, achieve high sensitivity, facilitate large-scale commercial promotion, and good specificity

Active Publication Date: 2019-01-18
YANGZHOU UNIV
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  • Application Information

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Problems solved by technology

Therefore, it is imminent to develop rapid detection and identification technology for LLV. At present, LLV is mainly detected by RT-PCR method. Since there is no LLV-specific antiserum, enzyme-linked immunosorbent assay (ELISA) cannot be used for detection.

Method used

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  • Lotus latent virus double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit and preparation and detection methods
  • Lotus latent virus double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit and preparation and detection methods
  • Lotus latent virus double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit and preparation and detection methods

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Embodiment Construction

[0048] The present invention will be further described below in combination with specific embodiments and accompanying drawings.

[0049] (1) Prokaryotic expression and purification of LLV coat protein

[0050] ①Total RNA extraction (polysaccharide and polyphenol plant tissue lysis method)

[0051] ②Primer design

[0052] According to the obtained lotus root latent virus LLV-S2-2 isolate sequence, design and synthesize specific primers in amplifying LLV coat protein nucleotide sequence: LLVCPF (SEQ ID No.1) and LLVCPR (SEQ ID No.2 ); Respectively introduce restriction sites Nco I and Sac I 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.

[0053] ③RT-PCR

[0054] The total RNA of lotus root leaves was used as a template, and LLVCPR primers were used to synthesize cDNA by reverse transcriptase M-MLV. The reverse transcription system is shown in Table 1.

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Abstract

The invention discloses a lotus latent virus double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit and preparation and detection methods. The kit comprises an ELISA micro-pore plate,an enzyme-labeled antibody, a buffer solution, a color-developing solution and a stop solution, the micro-pore plate is coated by an LLV specific polyclonal antibody, and the enzyme-labeled antibody is an LLV specific polyclonal antibody marked by horseradish peroxidase. According to the kit, the adopted immunogen is an LLV coat protein antigen expressed by genetic engineering. The invention alsodiscloses a detection method of a lotus latent virus. According to the kit and the methods, a rapid, sensitive and simple method is provided for detection of the lotus latent virus on the basis of serology, the methods can be economic and practical, and an effective detection method is provided for prevention and control of lotus latency in lotus production and preparation of virus-free seedlingsthereof.

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 latent viruses in lotus root, and the invention also relates to the kit. Preparation method and detection method. Background technique [0002] "Stiff lotus root" is a kind of important lotus root disease that widely occurs in major lotus root producing areas in the world. The main symptom is brown spots on the surface of the lotus root. With the aggravation of the disease, the brown spots gradually develop into brown or black. Brown streaks, and deep below the epidermis, the disease occurs widely in lotus root cultivation areas in Jiangsu, seriously restricting the sustainable and stable development of lotus root industry. [0003] Lotus latent virus (LLV) belongs to the genus Potyvirus in the family Potyviridae. The virus genome is about ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 贺振陈雯陈春峰刘娴张坤李良俊
Owner YANGZHOU UNIV
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