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Colloidal gold immune test strip for rapid detection of Papaya ringspot virus

A technology of colloidal gold test paper and ringspot virus, which is applied in the field of diagnosis of plant disease pathogens, can solve the problems of slow identification speed, difficulty in accurately judging symptoms, and difficulty in judging, and achieves accurate and reliable results, low production costs, and easy popularization and use.

Inactive Publication Date: 2013-11-27
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although traditional biological methods for identifying viruses are simple to operate and do not require special equipment, they have the following disadvantages: (1) A series of identification hosts are required for identification, and non-professionals often face difficulties in accurately judging the symptoms that appear question
(2) In actual identification, multiple viruses often infect the host together, causing great changes in symptoms and making it difficult to judge; (3) The identification speed is slow and the cycle is long
The time required to identify the symptoms of the host after inoculation is generally more than 3 days, and some even as long as 20 days; (4) The identification results are easily affected by the season and the surrounding environment, and a relatively independent space is required to avoid the spread of the virus. Cross-infection; (5) Some viruses do not have a suitable identification host and cannot be identified by traditional biological methods
Although enzyme-linked immunosorbent assay and molecular biology methods for virus detection have the advantages of strong detection specificity, high sensitivity, and shorter detection time than traditional biological methods, there are at least the following shortcomings: (1) Both methods require Special instruments and equipment, and the operators generally have to undergo special training, so that these two methods of detecting viruses can only be completed in the laboratory, which is not conducive to the promotion and use in grassroots units; (2) Comparing with traditional biological methods Compared with other methods, although the detection time has been greatly shortened, the time required is at least several hours, which is still not suitable for rapid detection in the field; (3) the cost of detection is relatively high, especially for molecular biology detection methods. Requires expensive reagents

Method used

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  • Colloidal gold immune test strip for rapid detection of Papaya ringspot virus
  • Colloidal gold immune test strip for rapid detection of Papaya ringspot virus
  • Colloidal gold immune test strip for rapid detection of Papaya ringspot virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] A papaya ringspot virus immune colloidal gold test strip is characterized in that the preparation method comprises the following steps:

[0030] 1) The PRSV-Vb strain was rubbed inoculated on the systemically infected host zucchini, and the zucchini leaves with typical symptoms were used as materials for purifying PRSV; 200 g of diseased leaves were taken in a mortar, and liquid nitrogen was added until the diseased leaves were covered. Quickly grind the diseased leaves into powder with a grinding rod, transfer the powder to a beaker, add 500 mL of phosphate buffer A pre-cooled at 4°C; the concentration of the phosphate buffer A is 0.5M, pH7.2, and additionally 0.1 M EDTA disodium, 1 M urea, 0.3% w / v Na 2 SO 3 , 0.5% w / v tritonX-100; stir at 4°C for 1 h to obtain a suspension buffer, add 300 mL of CCl 4 with CHCl 3 equal volume mixture, stirred at 4°C for 30 min, 3000 × g Centrifuge for 15 min, take the upper layer solution, and pass the upper layer solution through...

Embodiment 2

[0040] Detection of PRSV infection dynamics on zucchini with colloidal gold immunoassay strips

[0041] Take the zucchini seedlings cultivated in the insect-proof greenhouse, sprinkle a small amount of carborundum evenly on one of the tender leaves, dip the finger to pick up 10 μg / mL purified PRSV solution, rub and inoculate on the leaf surface sprinkled with carborundum, and inoculate with distilled water after about 1 min. Rinse the carborundum on the inoculated leaves, and place the inoculated zucchini in an insect-proof greenhouse for cultivation. On the 3rd, 5th, 7th, 9th, 11th, 13th and 15th day after inoculation, take 0.5 g samples from the inoculated leaves, add 5 mL of 0.01 M phosphate buffer with pH 7.0, grind thoroughly, and centrifuge at 5000 g for 1 min , the supernatant is the sample to be tested. Take 200 μL of the sample to be tested and drop them on the sample pad of the test strip prepared in Example 1. After 15 minutes, judge the detection result according ...

Embodiment 3

[0054] Fifty samples of suspected papaya ringspot virus disease were collected from papaya planting areas in different areas of Fujian Province, 0.5 g of each sample was weighed, and 5 mL of 0.01 M phosphate buffered saline with pH 7.0 was added, fully ground, Centrifuge at 5000 g for 1 min, and the supernatant is the sample solution to be tested. Insert the test strip prepared in Example 1 into the sample solution to be tested, and judge the test result after 10 minutes according to the color development of the detection line and the quality control line on the test strip. The results showed that 46 of the 50 samples were positive and 4 were negative; at the same time, the 50 samples were detected by indirect ELISA method, and the detection results were 47 positive and 3 negative. Comparing the results obtained by the two detection methods, it can be seen that the accuracy and reliability of the test strip detection results are close to the ELISA detection method.

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Abstract

The invention relates to preparation and application of an immune colloidal gold test strip for rapid detection of Papaya ringspot virus (PRSV). The preparation method includes: taking PRSV-Vb bred on summer squashes as a material, adopting purified PRSV as an antigen for preparation of a polyclonal antibody, and labelling the polyclonal antibody by colloidal gold to form a gold-labeled antibody, coating the detection line and quality control line of a nitrocellulose membrane respectively with the polyclonal antibody and goat anti-rabbit IgG, coating a combination pad prepared from a glass cellulose membrane with the gold-labeled antibody, then sticking the nitrocellulose membrane coated by the polyclonal antibody and the goat anti-rabbit IgG on a PVC base plate, finally sticking the gold-labeled combination pad and a sample pad prepared from glass cellulose at the detection line end in order, and sticking absorbent paper at the quality control line end, thus obtaining the PRSV colloidal gold immune test strip. The colloidal gold immune test strip provided in the invention has the advantages of simple operation, fast detection speed, no need of special equipment, accurate and reliable detection result, and low cost, and can be used for early diagnosis of field diseased plants.

Description

technical field [0001] The invention belongs to the technical field of plant disease pathogen diagnosis, and relates to a rapid diagnosis method of plant viruses, in particular to the preparation and application of an immune colloidal gold test strip for detecting papaya ringspot virus. Background technique [0002] Papaya ringspot virus (PRSV) is an important member of Potavirus genus and one of the important viruses that cause papaya virus disease. The virus was first reported in the United States in the 1940s. my country introduced papaya for planting and production in the early 1950s, and the virus was found on papaya in 1959, and PRSV was widely prevalent in my country after 1962. Once papaya plants are infected by PRSV, the mortality rate can be as high as 90%, which makes papaya production suffer a devastating blow. In 2000, thousands of acres of papayas planted in Hainan Province were almost completely destroyed due to PRSV infection. Due to the severe damage caus...

Claims

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Application Information

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IPC IPC(8): G01N33/569
Inventor 陈启建叶通欧阳明安林诗发谭庆伟
Owner FUJIAN AGRI & FORESTRY UNIV
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