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Kit for rapidly detecting tomato yellow leaf curl virus and application thereof

A technology of tomato yellowing curved leaves and a kit, which is applied in the biological field, can solve the problems of large interference from human factors, unsuitability for rapid detection, and low reliability of detection results, etc., and achieve the effect of high sensitivity

Active Publication Date: 2014-10-08
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biological detection methods and electron microscope observation methods are greatly disturbed by human factors, and the reliability of the detection results is low; serological detection is mainly ELISA method, but its sensitivity and specificity are not high, and it is not suitable for rapid detection

Method used

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  • Kit for rapidly detecting tomato yellow leaf curl virus and application thereof
  • Kit for rapidly detecting tomato yellow leaf curl virus and application thereof
  • Kit for rapidly detecting tomato yellow leaf curl virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The preparation of embodiment 1 kit

[0040] 1. Primer design

[0041] According to the multiple sequence comparison of the nucleotide sequences of the Chinese dominant strains of tomato yellow leaf curl virus registered on NCBI, the primers were designed. The primer sequences are as follows. For the verification of the primer effectiveness, see figure 1 :

[0042] Upstream primer (TY-V1): 5'—AGTACTATGTCGAAGCGWCCAG—3' (shown in SEQ ID NO.1, W=A / T)

[0043] Downstream primer (TY-V2): 5'-TCTAGATTAATTTKRTATTGAATCATAGA-3' (shown in SEQ ID NO.2, K=G / T, R=A / G)

[0044] The size of the amplified target fragment is 789bp.

[0045] 2. Preparation of extraction buffer and washing buffer

[0046] (1) Preparation of extraction buffer (100ml)

[0047]

[0048] Add 90ml ddH 2 O, adjust the pH to 7.8, dilute to 100ml, and store at room temperature.

[0049] (2) Preparation of washing buffer (100ml)

[0050]

[0051] Add 90ml ddH 2 O, adjust the pH to 7.4, dilute to 100m...

Embodiment 2

[0053] Example 2 Application of the kit of the present invention in the detection of tomato yellow leaf curl virus

[0054] 1. Sample material: Tomato samples known to be infected with tomato yellow leaf curl virus in 7 provinces and cities were used as test objects, and healthy tomato samples were used as negative controls.

[0055] 2. Method

[0056] (1) Sample processing

[0057] Take 10 mg of tomato diseased leaves in a 1.5 mL centrifuge tube, add 100 μL of extract buffer (prepared according to the method in Example 1) and grind thoroughly; then centrifuge at 12000 rpm for 5 min; take 50 μL of the supernatant and add it to a 0.2 mL PCR tube; 37 ° C Incubate in a water bath for 10 minutes to facilitate the adsorption of virus particles on the wall of the PCR tube; pour off the liquid in the PCR tube; wash the PCR tube twice with 100 μL of washing solution buffer (prepared according to the method in Example 1); fully dry the PCR tube ;

[0058] (2) PCR amplification

[0...

Embodiment 3

[0066] The sensitivity detection analysis (detection limit analysis) of embodiment 3 inventive method

[0067] Control group: take tomato leaves infected with TYLCV, extract DNA by conventional CTAB method, dilute 1:1, 1:10, 1:100, 1:1000, 1:5000, 1:10000 times with double distilled water, perform PCR Amplified, the product was subjected to 1.2% agarose gel electrophoresis, stained with EB, and photographed on the BioRad gel imaging system, such as image 3 as shown in a.

[0068] The method of the present invention: get the tomato leaf that infects TYLCV, get 50 μ L supernatant after extracting buffer treatment, with the supernatant after extracting buffer treatment of healthy tomato plant (not infected with TYLCV) leaf, carry out 1:1, 1 : 10, 1: 100, 1: 1000, 1: 5000, 1: 10000, 1: 20000 times of dilution, the supernatants of different dilution times were respectively incubated according to the method of embodiment 2, washed with buffer solution, PCR amplification The produ...

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Abstract

The invention discloses a kit for rapidly detecting tomato yellow leaf curl virus and application thereof. A pair of specific primers is designed according to the nucleotide sequence of a dominant strain of tomato yellow leaf curl virus in our country, and also an extraction buffer and a washing buffer for processing a sample are designed. By using the kit for rapid detection on tomato yellow leaf curl virus, a virus-inflected tomato sample does not need extraction of DNA, only a small amount of a disease leaf (about 10 mg) is needed, and the disease leaf only needs once PCR reaction after being processed by the extraction buffer and the washing buffer, so that identification on tomato yellow leaf curl virus is realized. Experiments prove that the method has relatively high sensitivity and the sensitivity reaches 1E4. Also, the method is capable of performing virus containing identification on virus-transmission vectors Q-biotype bemisia tabaci and B-biotype bemisia tabaci of tomato yellow leaf curl virus. Compared with traditionally biology and serology, the method is a simple, convenient, rapid, economic and sensitive method for detecting tomato yellow leaf curl virus.

Description

technical field [0001] The invention relates to a virus detection kit, in particular to a rapid detection kit for tomato yellow leaf curl virus and its application. The invention belongs to the field of biotechnology. Background technique [0002] Tomato yellow leaf curl virus (TYLCV) is a virus that seriously endangers tomato production. The virus is a single-stranded circular DNA virus with a genome size of 2.7-2.8kb and belongs to the Geminiviridae family. Begomovirus belongs to the genus Begomovirus, and its vectors are B and Q types of whitefly (Bemisia tabaci). Tomato yellow leaf curl virus disease can occur at all stages of tomato growth. After the plants are infected in the early growth and development stage, there are no obvious symptoms on the appearance of the plants. After that, the main symptoms are stagnant or slow plant growth, plant dwarfing, and obvious internodes. Shorter, thicker and smaller leaves, yellowing from the edge of the leaf to the vein area, wi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2565/125
Inventor 许向阳李景富王傲雪薛东齐姜景彬陈秀玲桑毅振
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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