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A kind of apple mosaic virus detection kit and preparation method thereof

An apple mosaic virus, detection kit technology, applied in biochemical equipment and methods, microorganism-based methods, and microorganism determination/inspection, etc., can solve problems such as operator poisoning, and achieve high sensitivity and low detection limit. , bright color effect

Inactive Publication Date: 2018-08-03
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is prone to non-specific amplification and false positive results, and the olfactory ethidium used in electrophoresis is toxic to the operator

Method used

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  • A kind of apple mosaic virus detection kit and preparation method thereof
  • A kind of apple mosaic virus detection kit and preparation method thereof
  • A kind of apple mosaic virus detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0042] ⑤Preparation of 20×SSC

[0043] The preparation method of 20×SSC: Weigh 8.76g NaCl and 4.41g trisodium citrate, add 45mL deionized water, and then adjust the pH value to 7.0 with dilute hydrochloric acid.

[0044] ⑥Preparation of 1×Exo III buffer

[0045] The preparation method of 1×Exo III buffer: 10×Exo III buffer (purchased from Bao Biological Engineering (Dalian) Co., Ltd., 500mM Tris-HCl, pH 8.0, 50mM MgCl 2 , 10mM DTT) was diluted 10 times, that is, 5μL of 10×ExoIII buffer was added to 45μL of water to obtain 1×ExoIII buffer, which consisted of 50mM Tris-HCl, pH 8.0, 5mM MgCl 2 , 1 mM DTT.

[0046] The DNA sequence used in the test in the examples of the present invention is as follows, synthesized by Takara Company and purified by HPLC. All solutions were prepared with ultrapure water (resistance of 18.2MΩ.cm).

[0047] ①Specific DNA probe 1

[0048] AuNPs-SH-5'-AAAAAAACGAAGTCATCAAAAAAA-3'

[0049] ② DNA probe 2

[0050] 5'-AGAGTTGTGTTTGGAGGA-3'

[0051] ...

Embodiment 1

[0059] A kind of apple virus detection test strip of the present invention, as figure 1 As shown, it consists of a strip bottom plate, sample pads overlapped sequentially on the strip bottom plate, a gold label conjugate pad coated with specific DNA probe 1, a specific coated nitrocellulose membrane and an absorbent pad. One end of DNA probe 1 is combined with nano gold, and the nitrocellulose membrane is covered with a linear detection line T line coated with DNA probe 2 and a linear quality control line C line coated with DNA probe 3, such as figure 1 As shown, a large number of single-stranded DNAs (sequences 4 and 2*) were obtained through the circular amplification principle of targeted priming and ExoIII cleavage in the apple virus liquid to be tested, and the single-stranded DNAs were compatible with sequence 4* and DNA Probe 2 is complementary.

[0060] 1. Preparation of sample pad

[0061] A 0.01 mol / L Tris-HCl buffer solution was prepared, wherein the Tris-HCl buff...

Embodiment 2

[0073] 1. The preparation of the sample pad, the preparation of the gold-labeled conjugate pad coated with specific DNA probes, and the preparation of the specific coated nitrocellulose membrane are the same as in Example 2

[0074] 2. Collect samples of apple mosaic virus-positive plants and virus-free plants preserved by Yantai Academy of Agricultural Sciences, and use liquid nitrogen quick-frozen grinding and plant RNA extraction kits to extract total RNA from apple leaves.

[0075] 3. Preparation of tested single-stranded DNA

[0076] Convert 10 μmol / L structure to 5'-TCCTCCAAACACAACTCTTGATGACTTCGTAGAGTT

[0077] The hairpin DNA of GTGTTTGGAGGAGCTTCCCACTGCCAACCT-3' was heated at 95°C for 10 minutes, cooled naturally, and the actual apple virus liquid was heated at 95°C for 10 minutes, and immediately placed in an ice bath, and then 2.5 μL of 500 nM hairpin DNA was hybridized with 2.5 μL of the apple virus liquid extracted above to be tested. 45 μL of 1×ExoⅢ buffer was rea...

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Abstract

The invention relates to an apple virus detection test strip and a preparation method thereof. The test strip is characterized in that: target initiated and Exonuclease III assisted amplification strategy is employed for detection, a lot of single stranded DNA produced after apple virus initiation and Exonuclease III cutting circulation can be taken as a to-be-tested solution on a sample pad, a part of the sequence of the single stranded DNA forms complementary pairing with a DNA probe 1 on a glass fiber combined pad, and the other part of the sequence forms complementary pairing with a DNA probe 2 on a nitrocellulose membrane; and the DNA of a DNA probe 1-nanogold compound forms complementary pairing with a DNA probe 3. The preparation method consists of: conducting lapping molding on the prepared sample pad, the specifically coated glass fiber combined pad, the specifically coated nitrocellulose membrane and a water absorbing pad in order, sticking the lapped product on a base plate, and cutting the product into 3-5mm wide thin strips, thus obtaining the finished product. The test strip has the advantages of high sensitive and high selective detection of apple viruses.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, in particular to a method for detecting apple viruses. Background technique [0002] Apples are native to China and are the largest fruit variety in China. They have been cultivated in my country for more than 3,000 years. At present, 25 provinces in my country have apple production and cultivation, and there is the largest contiguous apple orchard in the world, and its planting area and output both rank first in the world. The export volume of fresh apples in my country increased by more than three times from 2000 to 2010, and it has become the world's largest producer and exporter of concentrated apple juice. The annual output and export volume both account for about 60% of the global total, and the annual export value exceeds 1 billion Dollar. In recent years, with the vigorous development of the apple industry, apple virus disease has become an important factor that seriously r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6834C12R1/94
Inventor 兰伟纪秀翠徐慧
Owner LUDONG UNIVERSITY