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Complete set reagent for detecting lettuce mosaic virus

A technology of mosaic virus and lettuce, which is applied in the field of complete set of reagents for detection of lettuce mosaic virus, can solve the problems of low detection sensitivity, limited penetration rate, heavy workload, etc., and achieve the effect of high sensitivity, good specificity, and easy judgment

Inactive Publication Date: 2018-10-09
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection sensitivity of biological and serological detection methods is low, time-consuming and heavy workload; the observation of virus particle characteristics requires the use of electron microscopes, and the high cost and low penetration rate of electron microscopes limit the development of this technology; currently the most used It is a molecular detection technology, mainly including RT-PCR, fluorescent quantitative PCR and digital PCR, etc. These molecular detection technologies need to go through steps such as pre-denaturation, denaturation, annealing and extension, and each step requires a thermal cycler to ensure its temperature special requirements and the whole process takes a long time

Method used

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  • Complete set reagent for detecting lettuce mosaic virus
  • Complete set reagent for detecting lettuce mosaic virus
  • Complete set reagent for detecting lettuce mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of the complete set of reagents that detect lettuce mosaic virus

[0035] This embodiment provides a set of reagents for detecting lettuce mosaic virus, the set of reagents is composed of a primer pair named P1 and a single-stranded DNA named A1;

[0036] P1 consists of two single-stranded DNAs shown in sequence 1 (upstream primer sequence) and sequence 2 (downstream primer sequence) in the sequence table. During RT-RPA amplification, the RNA of lettuce mosaic virus can be used as a template to amplify the sequence with a size of 272bp DNA fragment;

[0037] The sequence of A1 is sequence 3 in the sequence list, and a DNA fragment with a sequence size of 428 bp can be amplified from the internal standard plasmid during RT-RPA amplification, and the sequence of the DNA fragment is sequence 3 in the sequence list.

[0038] The three single-stranded DNAs in the set of reagents are packaged independently, and the molar numbers of each single-s...

Embodiment 2

[0039] Embodiment 2, establishment of the method for detecting lettuce mosaic virus

[0040] The method steps of detection lettuce mosaic virus are as follows:

[0041] 1. Extraction of RNA

[0042] The total RNA of the samples to be tested was extracted according to the instruction manual of the Plant Total RNA Extraction Kit (Cat. No. DP432) from Tiangen Biotechnology Co., Ltd.; the obtained RNA was stored at -20°C for later use.

[0043] 2. RT-RPA amplification

[0044] The obtained RNA is used as a template, and the kit of reagents of Example 1 is used to carry out RT-RPA amplification to obtain the RPA amplification product, and a negative control (template is healthy lettuce virus RNA) and a blank control (template is ultrapure water) are set simultaneously.

[0045] The preparation method of RT-RPA amplification system (50 μL) is as follows: add 29.5 μL of rehydration buffer (Rehydration Buffer), 1 μL of each upstream primer and downstream primer (primer concentration ...

Embodiment 3

[0052] Embodiment 3, detect the specificity of the complete set of reagents of lettuce mosaic virus

[0053] The virus materials for testing are as follows: Arabidopsis mosaic virus; cucumber mosaic virus; tobacco ringspot virus; tomato ringspot virus; potato virus A; potato virus Y; zucchini mosaic virus; lettuce mosaic virus; a total of 8 kinds of RNA Viruses, 8 kinds of viruses are shown in Table 1. The above-mentioned test materials are all stored in the Chinese Academy of Inspection and Quarantine, and a negative control (the template is healthy lettuce RNA) and a blank control (the template is water).

[0054] Table 1. Tested viruses

[0055]

[0056] 8 kinds of test viruses in the above-mentioned embodiment 3 are respectively recorded in the following documents:

[0057] Arabidopsis mosaic virus: Development and detection application of monoclonal antibody against Arabidopsis mosaic virus. Phytosanitary, 2011, 25(6): 18-20.

[0058] Cucumber mosaic virus: Analysis...

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Abstract

The invention discloses a complete set reagent for detecting a lettuce mosaic virus. The complete set reagent for detecting the lettuce mosaic virus is composed of a primer pair named as P1 and DNA named as A1, wherein the P1 is composed of two single stranded DNAs shown as a sequence 1 and a sequence 2 in a sequence table; the A1 is DNA containing a recognition sequence of the P1. Experiments prove that the complete set reagent for detecting the lettuce mosaic virus is capable of effectively avoiding screening false negative while ensuring efficient amplification of target sequences, is excellent in specificity, sensitive, accurate, simple and rapid, and has a guiding significance on inspection and quarantine of inbound and outbound related plants and products.

Description

technical field [0001] The invention relates to a set of reagents for detecting lettuce mosaic virus in the field of biotechnology. Background technique [0002] Lettuce (Lactuca sativa L.) is a plant of the genus Lactuca in the Asteraceae family and is one of the most common vegetables. High-quality lettuce is rich in inorganic salts, vitamins, niacin and trace elements. Inorganic salts in lettuce help regulate the balance of salt in the body. Niacin can help diabetics improve sugar metabolism. Vitamins and trace elements can However, the occurrence of lettuce mosaic virus disease can seriously affect the quality of lettuce, resulting in a decrease in the nutritional value of lettuce. [0003] Lettuce mosaic virus disease is caused by lettuce mosaic virus (LMV) infection. After lettuce is infected with LMV under natural conditions, the edge of the leaves will appear irregular and shrunk, and then appear light green or light yellow. Mottled or mosaic symptoms; when the dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2521/107
Inventor 张永江向均
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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