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PVY single virus colloidal gold rapid test strip

A technology of colloidal gold and colloidal gold pad, applied in the field of pathogen detection, can solve the problems of being expensive and not suitable for the detection of smoke seedlings, and achieve the effect of simple operation, easy detection of a large number of samples, and strong innovation

Active Publication Date: 2017-03-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Foreign commercial test strips are very expensive, the price is 50-60 yuan / sheet, which is not suitable for the detection of a large number of tobacco seedlings in production

Method used

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  • PVY single virus colloidal gold rapid test strip
  • PVY single virus colloidal gold rapid test strip
  • PVY single virus colloidal gold rapid test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The isolation of PVY virus strain of embodiment 1 Guangdong smoking area

[0034]1. From 2012 to 2014, from Nanxiong, Shixing, Ruyuan, and Lechang in Shaoguan City, Guangdong Province, Lianzhou in Qingyuan City, Wuhua, Jiaoling, Dapu and Meixian in Meizhou City, etc. 29 Sampling was carried out on tobacco plants exhibiting common mosaic disease in tobacco fields in 3 towns and 33 villages. The sampling points covered all smoking areas in Guangdong Province. A total of 72 samples. Separate and identify them by biology and ELISA (enzyme-linked immunosorbent assay).

[0035] A total of 49 PVY isolates were isolated, all of which have been verified by ELISA. The results of biological identification showed that the collected PVY isolates belonged to common strains.

[0036] 2. Amplifying the entire CP (coat protein) gene of the PVY virus. After the gel recovery of the PCR product, it was ligated with the pMD-18T vector and transformed into Escherichia coli E.coli DH5α. ...

Embodiment 2

[0040] Example 2 Preparation of CP gene prokaryotic expression specific protein

[0041] 1. Prepare CP-P-GD prokaryotic expression protein, construct CP-P-GD on the pET30a-GST vector, transform BL21 strain, carry out prokaryotic expression, and purify the expressed protein.

[0042] The relevant information is summarized in Table 1 below.

[0043] Table 1

[0044]

[0045] 2. The specific method is as follows.

[0046] (1) Target gene sequence: according to the codon-optimized sequence of Escherichia coli, enzyme cutting sites EcoR I and Xho I are added at both ends. Synthesize the target gene into pUC57 vector.

[0047] (2) Connect the target gene to the pET30a-GST vector by enzyme digestion and ligation. The schematic diagram of the constructed recombinant vector is shown in the attached figure 1 shown.

[0048] Digestion of gene fragments: 43 μl recombinant plasmid, 1 μl EcoR I, 1 μl Xho I, 5 μl 10×Buffer, react overnight at 37°C. (Agarose Gel DNA Recovery Kit, BPI...

Embodiment 3

[0081] Example 3 Construction of hybridoma cell lines expressing PVY virus-specific monoclonal antibody

[0082] The CP gene obtained in Example 2 was used to prokaryotically express a specific protein, and the expressed protein was used to immunize mice; a fusion test was performed to obtain a positive hybridoma cell line producing a specific monoclonal antibody to PVY virus.

[0083] 1. The specific method is shown in Table 2.

[0084] Table 2

[0085]

[0086]

[0087] 2. The specific method is as follows:

[0088] (1) immunity

[0089] 1) With "PVY", according to the amount of 60ug protein / mouse, 4 SPF BALB / c female mice were subcutaneously immunized for the first time, and the numbers were: 1, 2, 3, 4.

[0090] 2) Two weeks after the initial immunization, the subcutaneous booster immunization was given for the first time, with an immune dose of 30ug protein per mouse.

[0091] 3) Two weeks after the first booster immunization, a second subcutaneous booster immun...

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Abstract

The invention discloses a PVY single virus colloidal gold rapid test strip which is composed of a sample pad, a colloidal gold pad, an NC membrane, water absorption filter paper and a backing; the colloidal gold pad is coated with a colloidal gold-labeled specific PVY antibody, and the antibody is secreted from a hybridoma cell strain BALBc-15-8; the NC membrane is provided with a test line and a control line, the test line is coated with a specific PVY antigen, and the control line is coated with a colloidal gold-labeled secondary antibody. The test strip is rapid to operate, sensitive, accurate, low in cost and easy and convenient to operate for testing PVY in Guangdong tobacco-growing areas, achieves simultaneous multi-sample diagnosis in one-time sampling, is very suitable for conducting site primary screening on a large scale of samples and has an application value in practical production and a good application and popularization prospect.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection. More specifically, it relates to a PVY single virus colloidal gold rapid detection test strip. Background technique [0002] Potato virus Y (PVY) is one of the main viruses infecting tobacco. The viral diseases it causes cause huge economic losses to the tobacco industry every year. Rapid and accurate qualitative detection of related viruses is one of the important means to prevent and control diseases and reduce losses in production. [0003] In scientific research, the detection methods of plant viruses mainly include biological detection methods (discrimination of symptom types, identification of hosts, etc.), electron microscope technology, serological detection methods (enzyme linked immunosorbent assay (ELISA), etc.) and molecular Biological detection methods (including PCR technology, nucleic acid probe technology, etc.), etc. Although the isolation of virus by electron micr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 阮小蕾
Owner SOUTH CHINA AGRI UNIV
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