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Tomato spotted wilf virus molecule standard sample and preparation method thereof

A technology for tomato spotted wilt virus and standard sample, which is applied in the field of tomato spotted wilt virus standard sample and its preparation, and achieves the effects of high stability, guaranteed quality control and good uniformity

Inactive Publication Date: 2016-08-17
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on the standard sample of this plant virus at home and abroad.

Method used

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  • Tomato spotted wilf virus molecule standard sample and preparation method thereof
  • Tomato spotted wilf virus molecule standard sample and preparation method thereof
  • Tomato spotted wilf virus molecule standard sample and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The preparation of embodiment 1 tomato spotted wilt virus molecular standard sample

[0026] (1) Collect plant tissues infected with tomato spotted wilt virus, and grind with liquid nitrogen;

[0027] Collect 0.1g of plant tissue infected with tomato spotted wilt virus and grind it into powder with liquid nitrogen, quickly transfer the ground material into a 1.5mL centrifuge tube, add 1mL Trizol Reagent, mix by inverting, 2℃~8℃, 12000g, centrifuge for 10min.

[0028] (2) Extract viral RNA from the plant tissue obtained in step (1);

[0029] Take the supernatant, keep it at 15°C-30°C for 5min; add 0.2mL chloroform, shake vigorously by hand (do not vortex) for 15s. 15℃~30℃, stand for 2min~3min; 2℃~8℃, 12000g, centrifuge for 15min. Carefully pipette approximately 600 μL of the upper aqueous phase without disturbing the middle and lower phases. Add 500 μL of isopropanol to mix the supernatant, and place it at 15°C to 30°C for 10 minutes. 2℃~8℃, centrifuge at 12000g for ...

Embodiment 2

[0050] The homogeneity experiment of embodiment 2 tomato spotted wilt virus molecular standard sample

[0051] The homogeneity determination of the tomato spotted wilt virus molecular standard sample is that the TSWV molecular standard sample prepared by the method of Example 1 is randomly divided into two sample groups, i.e. sample group A and sample group B, and each sample group has 15 standard samples. Samples, each sample is subjected to PCR amplification according to the following reaction conditions, and the concentration of PCR products (ng / μL) is measured. By comparing the concentration changes of PCR products, the uniformity of standard samples is evaluated according to statistical methods.

[0052] PCR reaction system: Add 1 μL of molecular standard sample of tomato spotted wilt virus (containing 1×10 -2 μg DNA), 2×PCR buffer (10-fold polymerase chain reaction mixture) 12.5 μL, 10 pmol / μL of 0.5 μL each of SEQ ID NO:3 and SEQ ID NO:4, and DEPC water to make up the v...

Embodiment 3

[0062] Stability experiment of embodiment 3 tomato spotted wilt virus molecular standard sample

[0063] The tomato spotted wilt virus molecular standard sample prepared by the method of embodiment 1 is randomly grouped, and each sample is carried out PCR amplification according to the PCR reaction system described in embodiment 2 and PCR reaction conditions, and the concentration of PCR product (ng / μL) is measured , by comparing the concentration changes of PCR products, evaluate the stability of standard samples according to statistical methods.

[0064] Two types of stability testing are employed:

[0065] One is the stability test at -20°C. 24 molecular standard samples of tomato spotted wilt virus were randomly selected, and 2 samples were tested every month for 12 consecutive months. The results are shown in Table 4.

[0066] The other is a stability test at a higher temperature of 20°C. 12 molecular standard samples of tomato spotted wilt virus were randomly selected, ...

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Abstract

The invention provides a tomato spotted wilf virus molecule standard sample and a preparation method thereof. The preparation method specifically comprises raw material selection of TSWV, preparation of a standard sample, uniformity and stability check, qualitative detection, value fixing and the like. The method for preparing the standard sample comprises the following steps: extracting virus RNA from TSWV virus solution, performing RT-PCR reaction amplification, purifying target gene fragments, connecting and transforming target fragments, and recovering plasmid. The tomato spotted wilf virus molecule standard sample disclosed by the invention is high in stability and uniformity, and suitable for rapid detection and confirmation of the tomato spotted wilf virus, provides a standard sample for detection research, pesticide research and application research of the tomato spotted wilf virus and can be widely applied to epidemic surveillance in agricultural production and environments and monitoring and detection of viruses in import and export trade. By using the standard sample disclosed by the invention, comparison of different laboratory results can be realized, so that the quality control of the laboratory is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of virus standard samples for detection and preparation methods thereof, and in particular relates to tomato spotted wilt virus standard samples and preparation methods thereof. Background technique [0002] In recent years, Tomato spotted wilt virus (TSWV) has been listed as one of the ten most harmful plant viruses in the world due to its wide host range and huge economic losses. In the 1960s to 1980s, the virus was prevalent in tobacco and tomato in Europe, America and Africa, with an annual incidence rate of 20% to 50%, causing losses of up to several billion dollars. In Hawaii, Brazil, Italy and South Africa, the prevalence of TSWV in the 1980s and 1990s led to near extinction of crops such as tomato and lettuce. In recent years, Tomato spotted wilt virus, especially TSWV, has become an important factor causing great economic losses of various commercial crops and ornamental plants all over the world. ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/70C12Q1/68C12R1/94
CPCC12Q1/6806C12Q1/701C12Q2600/166C12Q2531/113
Inventor 吴兴海陈长法李明哲王有福文朝慧栗智平李建勇宋涛王英超
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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