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Rapid detection method of molecular variant of citrus tatter leaf virus

A technology for leaf-shattering virus and detection methods, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., which can solve the problems of complex staining procedures, difficulty in defining the type of variation, and long electrophoresis time.

Inactive Publication Date: 2009-10-21
SOUTHWEST UNIVERSITY +1
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AI Technical Summary

Problems solved by technology

It is difficult to define the type of variation by this method, and a single standard isolate is required, so the repeatability is poor, and there may be differences between the results of different laboratories, and the electrophoresis time of this method is long, and the later staining procedure is more complicated. need extensive experience
[0006] So far, there is no report on a method suitable for high-throughput rapid detection of molecular mutations of citrus leaf scraper virus

Method used

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  • Rapid detection method of molecular variant of citrus tatter leaf virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Nucleic acid extraction

[0027] Weigh 5 mg of virus host skin or leaf into a sterile centrifuge tube, grind in liquid nitrogen, add 60 μL of TES buffer solution and 60 μL of saturated phenol:chloroform:isoamyl alcohol (25:24:1), and mix well; Water bath at 70°C for 5 to 10 minutes; centrifuge at 13,000 rpm for 5 minutes at room temperature, absorb 40 μL of the supernatant and add it to a microcolumn made of Sephadex G-50-80, place in a new sterile centrifuge tube, centrifuge at 5,000 rpm for 4 minutes, and collect eluent.

Embodiment 2

[0028] Example 2: Reverse transcription polymerase chain amplification

[0029] Design specific primers CTLVR: 5′-AGAGTGGACA AACTCTAGAC-3′, CTLVF: 5′-CCCTCTCAGC TAGAATTGAA-3′ to amplify the 889bp nucleic acid sequence including the coat protein gene of citrus leaf scraper virus, reverse transcription polymerase chain Amplification was carried out in a PCR machine, and the total volume of the reaction system used was 10 μL, including: 5 μL of 2γPCR buffer, 1.25 μL of 10 μmol / μL primers, 0.3 μL of 50 mmol / L MgSO4, 0.2 μL of 5 U / μL RT / Taq enzyme (Invitrogen), 1 μL of nucleic acid mixture extracted according to the above method, 1.5 μL of ultrapure water; the reaction conditions are: 42°C, 30 minutes; 94°C, 2 minutes; 94°C for 20s, 54.5°C for 20s, 72°C for 45s, 40 cycles; 72 °C for 10 minutes.

Embodiment 3

[0030] Example 3: Restriction enzyme digestion

[0031] After reverse transcription polymerase chain amplification, directly add 10 μL of the following mixture for Hinf I digestion reaction: ultrapure water 6.25 μL; buffer B 2.5 μL; BSA (10 μg / μL) 0.25 μL; Hinf I (10U / μL ) 1 μL; after mixing thoroughly, bathe in water at 37°C for 1 hour.

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Abstract

The invention relates to a rapid detection method for diagnosing molecular variant of citrus tatter leaf virus (CTLV), comprising the steps of: designing a specific primer to perform a reverse transcription polymerase chain reaction augmentation (RT-PCR) on the nucleic acid sequences in a special area of CTLV genome, and using Taq I, Mse I, Hinf I or other restriction enzyme to digest the RT-PCR product, and then diagnosing the molecular variant of CTLV according to an electrophoretogram. The method of the invention is characterized by high speed, low cost, good repeatability, high stability and strong operability and the like, and is suitable for large-scale and high-flux sample analysis, and is applicable to various uses such as field sample detection, CTLV epidemic monitoring and detoxication plantlet identification.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a rapid detection method for the molecular variation of citrus broken leaf virus. Background technique [0002] Citrus tatter leaf virus (CTLV) caused by citrus tatter leaf virus (CTLV) is one of the important diseases threatening the production of citrus, which can lead to about 25% yield reduction of citrus with trifoliate as rootstock. Citrus leaf shred virus has occurred in many countries such as the United States, Japan, South Africa, Australia and China, especially in Japan and China. In some areas of Zhejiang, Hunan, Fujian and Guangxi provinces, citrus broken leaf virus has caused relatively serious damage. Citrus Leaf Broken Virus is an RNA virus, which is prone to recombination and mutation. Once a highly pathogenic mutant strain becomes popular, it will bring immeasurable losses to the citrus industry. [0003] So far, the identification of indicator plants i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 宋震周常勇李中安
Owner SOUTHWEST UNIVERSITY
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