Pepper vein mottle virus multi-gene joint detection and identification method

A mottle virus, combined detection technology, used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc.

Pending Publication Date: 2020-10-23
FUJIAN AGRI & FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no research report on the application of multi-gene combined detection method to detect ChiVMV

Method used

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  • Pepper vein mottle virus multi-gene joint detection and identification method
  • Pepper vein mottle virus multi-gene joint detection and identification method
  • Pepper vein mottle virus multi-gene joint detection and identification method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Serology and routine RT-PCR detection of Indian import pepper sample of embodiment 1

[0031] Weigh 0.1 g of each pepper sample, perform DAS-ELISA detection according to the instructions of the virus kit, and measure its optical density (OD) at 405 nm with a multi-functional microplate reader. When the sample OD 405 nm and negative control OD 405 When the nm ratio is greater than 2, it is judged as positive. Wherein, in this embodiment, the sample material carrying ChiVMV is used as a positive control, healthy peppers are used as a negative control, and the extraction buffer is used as a blank control.

[0032] The positive samples of chili pepper infected with ChiVMV were extracted by Trizol reagent method, and the extraction method was carried out according to the kit instructions. Take 3 μL of total RNA, add 1 μL of random primers (Random Primer), and perform reverse transcription according to the operating instructions to obtain cDNA. PCR amplification uses 25 ...

Embodiment 2

[0034] Example 2 Establishment and optimization of multi-gene joint detection system

[0035] Using the cDNA of a ChiVMV-positive sample as a template, two primers of the same concentration were used to react with CP337-F / CP337-R and CI655-F / CI655-R, and a multi-gene joint detection based on ChiVMV CP and CI was preliminarily established system. 25 μL total reaction system, including 2 μL cDNA, 1 μL each of the two primer pairs of 10 μmol / L, 2×PCR Master Mix 12.5 μL, ddH 2 O 6.5 μL. 94 °C, 3 min; 94 °C, 30 s, 50 °C, 45 s, 72 °C, 1 min, a total of 35 cycles, 72 °C extension for 10 min. Take 5 μL of the PCR product for detection by 1.5% agarose gel electrophoresis, and observe the gel electrophoresis results.

[0036] According to the results of gel electrophoresis, the reaction system was partially optimized, and the annealing temperature of the reaction conditions was optimized according to the Tm values ​​of the two pairs of primers. A total of 6 settings were set at 46 °C...

Embodiment 3

[0041] Example 3 Multigene Joint Detection Specificity and Sensitivity Detection

[0042] Using ChiVMV, ChiRSV, CMV, PeVYV, and PVY virus positive samples as materials, RNA was extracted and tested according to the established multi-gene joint detection method, and a single gene was used for conventional RT-PCR detection to determine the specificity of the multi-gene joint detection method. sex.

[0043] After the reverse transcription, the pepper sample cDNA was diluted in a 10-fold gradient, and the original solution was diluted to 10 times in sequence. -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 , according to the established multi-gene joint detection method for detection, while using a single gene for conventional RT-PCR sensitivity detection, and comparing the two to determine the sensitivity of the multi-gene joint detection method.

[0044] The optimized multi-gene combined detection system can simultaneously amplify the target fragments of CP gene and CI gene ...

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Abstract

The invention provides a pepper vein mottle virus multi-gene joint detection and identification method, and belongs to the field of plant virus detection. The method adopts two primer pairs: CP337-F/CP337-R and CI655-F/CI655-R, and establishes a multi-gene combined detection system based on ChiVMV coat protein (CP) and cytoplasm inclusion body (CI). The method has good specificity, the sensitivityis equivalent to that of single RT-PCR and is 10<-4> orders of magnitudes, and good detection sensitivity is shown. The multi-gene joint detection system established by the invention can simultaneously detect CP and CI gene conserved region fragments of ChiVMV in one reaction, and can meet the requirements of rapid and accurate detection of capsicum vein mottle virus in port entry and exit and agricultural production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multi-gene combined detection and identification method for capsicum vein mottle virus. Background technique [0002] chili( Capsicum annuum L. ) is an annual or limited perennial herbaceous plant of the genus Capsicum in the family Solanaceae, native to the tropical regions of Central and South America, and widely cultivated worldwide. In my country, the pepper planting area is as high as 140 hm 2 More than 10% of all vegetable planting area in my country. Virus disease is a kind of important disease on pepper crops, which seriously affects the quality and yield of pepper crops. At present, there are more than 70 kinds of pepper viruses reported in succession all over the world, and at least 37 kinds have been found in our country, among which pepper vein mottle virus ( Chilli vein mottle virus, ChiVMV) is one of the important viruses on peppers. It spreads quickly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16
Inventor 高芳銮沈建国蔡伟杨宏凯陈细红杨晶文
Owner FUJIAN AGRI & FORESTRY UNIV
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