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Method for rapidly detecting prunus necrotic ring-spot virus of sweet cherries

A ringspot virus and sweet cherry technology, applied in the direction of microbe-based methods, biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the research of non-cherry disease detection test strips and detection methods, and limit test strips and development, long experimental cycle and other problems, to achieve the effect of short color development time, reduce the preparation cycle, and improve the detection sensitivity

Pending Publication Date: 2020-07-14
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional detection test strips are immunological test strips based on the specific combination of antigens and antibodies, which require tedious and long experimental cycles to prepare monoclonal antibodies, which limits the research and development of test strips
Therefore, there is no cherry disease detection test strip and detection method at present

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A test strip for rapid detection of Prunus necrotic ringspot virus of the present embodiment consists of a sample pad, a gold label binding pad coated with a nano-gold-Probe1 complex, a nitrocellulose membrane, an absorbent pad and an adhesive bottom plate Composition, sample pad, gold standard bonding pad, nitrocellulose membrane with detection line Probe2 and quality control line Probe3, and water-absorbent pads are glued to the adhesive bottom plate in sequence, and there should be 2-3mm overlap between each part. Real, dry storage under 4 ℃, the preparation process of described test strip is as follows:

[0039] 1. Design of DNA fragments and probes

[0040] Through the 95 PNRSV virus nucleic acid sequences found in the NCBI database, the highly specific conserved region was found, and the detection sequence was determined, and based on this, hairpin DNA, nano-gold Probe1, Probe2 and Probe3 were designed. Among them, the 5' end of Probe1 is marked with a sulfhydryl...

Embodiment 2

[0059] A test strip for rapid detection of Prunus necrotic ringspot virus of the present embodiment consists of a sample pad, a gold label binding pad coated with a nano-gold-Probe1 complex, a nitrocellulose membrane, an absorbent pad and an adhesive bottom plate Composition, sample pad, gold standard bonding pad, nitrocellulose membrane with detection line Probe2 and quality control line Probe3, and water-absorbent pads are glued to the adhesive bottom plate in sequence, and there should be 2-3mm overlap between each part. Real, dry storage under 4 ℃, the preparation process of described test strip is as follows:

[0060] 1. Design of DNA fragments and probes

[0061] Through the 95 PNRSV virus nucleic acid sequences found in the NCBI database, the highly specific conserved region was found, and the detection sequence was determined, and based on this, hairpin DNA, nano-gold Probe1, Probe2 and Probe3 were designed. Among them, the 5' end of Probe1 is marked with a sulfhydryl...

Embodiment 3

[0080] A kind of detection method of rapid detection Prunus necrotic ringspot virus of the present embodiment comprises the following steps:

[0081] 1. Sample preparation

[0082] Take the leaves of PNRSV-positive cherry plants and virus-free tissue-cultured seedlings (negative control), rinse them with tap water, and then wash them with distilled water. For RNA, 1 μg of total RNA was treated with DNase I to remove residual DNA, heated at 95°C for 5 min to inactivate the enzyme, cooled on ice, and cDNA was obtained by reverse transcription using a cDNA first-strand synthesis kit.

[0083] 2. ExoⅢ digestion amplification

[0084]Measure the concentration of the cDNA prepared in the above steps, dilute to 100ng / mL, take 10 μL, add 5 μL 10×hybridization buffer (3M NaCl, 0.1% SDS, 0.1M PB, pH7.0), heat at 95°C for 5min, and then quickly set aside In an ice-water bath, make it form a free linear state. Add 5 μL of 10 μM hairpin-type DNA solution into 10 μL double-distilled wate...

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Abstract

The invention relates to the technical field of plant virus detection, and in particular relates to a method for detecting prunus necrotic ring-spot virus of sweet cherries. The method comprises the following steps of: (1), designing and synthesizing a DNA fragment and a probe; (2), preparing nanogold; (3), preparing a nanogold-probe 1 compound; (4), assembling a test strip; (5), preparing a sample; (6), performing exonuclease III signal amplification; and (7), detecting the test strip. According to the method in the invention, the test strip is prepared by adoption of a nucleic acid detectionprobe; an antibody is unnecessary to prepare; the preparation process is simple; the cost is low; the probe is designed for a highly specific conserved region of the prunus necrotic ring-spot virus of the sweet cherries; the detection effect is good; the specificity is high; false positive is difficultly generated; signal amplification is carried out by utilization of cyclic enzyme digestion of exonuclease III; the detection sensitivity is increased; furthermore, a professional staff and a special instrument are unnecessary; carrying and detection are convenient; a result can be measured by naked eyes; the colour development time is short; and the colour development time is only 10 min.

Description

technical field [0001] The invention relates to the technical field of plant virus detection, in particular to a method for detecting Prunus necrotic ringspot virus. Background technique [0002] Sweet cherry (Cerasus avium (L.) Moench.), also known as big cherry, is a plant of the subgenus Prunus in the Rosaceae family, and is the main species of cherry in my country at present. Sweet cherry originated in Europe and West Asia, has a long history of cultivation, and has many cultivars and a wide range of distribution. Sweet cherries were introduced into my country around 1871 and are currently mainly planted in the Bohai Bay area. Because of its bright color, crystal clear, sweet and delicious taste, and rich nutrition, it enjoys the reputation of "treasures among fruits", has extremely high nutritional value and commercial value, and is favored by consumers. [0003] With the continuous development of my country's sweet cherry industry, viral diseases are becoming more an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/682C12R1/93
CPCC12Q1/701C12Q1/682C12Q2525/301C12Q2521/319C12Q2565/625C12Q2563/155C12Q2563/137
Inventor 王磊宿红艳吴凡林朱冬姿王雪辉王先钰
Owner LUDONG UNIVERSITY
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