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Peach latent mosaic viroid molecule standard sample and preparation method thereof

A standard sample and virus-like technology, applied in biochemical equipment and methods, DNA preparation, DNA / RNA fragments, etc., to achieve good uniformity, quality control, and high stability

Inactive Publication Date: 2016-09-21
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 viroid at home and abroad.

Method used

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  • Peach latent mosaic viroid molecule standard sample and preparation method thereof
  • Peach latent mosaic viroid molecule standard sample and preparation method thereof
  • Peach latent mosaic viroid molecule standard sample and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of Molecular Standard Sample of Peach Latent Mosaic Virus

[0024] (1) Extract viral RNA;

[0025] Collect 0.1g of plant tissue infected with peach latent mosaic virus and add liquid nitrogen to grind it into a powder, quickly transfer the ground material into a 1.5mL centrifuge tube, add 1mL Trizol Reagent, mix upside down, 2℃~8℃, 12000g, Centrifuge for 10 min. 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 10min. Remove the supernatant, add 1mL 75% ethanol to the precipitate, and wash; centrifuge at 7500g at 2°C to 8°C for 5min. Remove the supernatant, and after...

Embodiment 2

[0044] Example 2 Homogeneity Experiment of Peach Latent Mosaic Vioid Molecular Standard Sample

[0045] The homogeneity determination of peach latent mosaic viroid molecular standard sample is that the PLMVd molecular standard sample prepared as the method of embodiment 1 is randomly divided into two sample groups, i.e. sample group A and sample group B, each sample group has For 15 standard samples, each sample was subjected to PCR amplification according to the following reaction conditions, and the concentration of PCR products (ng / μL) was measured. By comparing the concentration changes of PCR products, the uniformity of standard samples was evaluated according to statistical methods.

[0046] PCR reaction system: Add 1 μL of peach latent mosaic virus molecular standard sample (containing 1×10 -2 μg DNA), 2×PCR buffer (10-fold polymerase chain reaction solution) 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 volume to 25 μL...

Embodiment 3

[0058] Example 3 Stability Experiment of Peach Latent Mosaic Vioid Molecular Standard Sample

[0059] The peach latent mosaic viroid 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 the PCR reaction condition, and measures PCR product concentration (ng / μL), by comparing the concentration changes of PCR products, the stability of standard samples was evaluated according to statistical methods.

[0060] Two types of stability testing are employed:

[0061] One is the stability test at -20°C. 24 peach latent mosaic virus molecular standard samples were randomly selected, and 2 samples were tested every month for 12 consecutive months. The results are shown in Table 4.

[0062] The other is the stability test at a higher temperature of 20°C. 12 molecular standard samples of peach latent mosaic virus were randomly selected, and 3...

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Abstract

The invention provides a peach latent mosaic viroid molecule standard sample and a preparation method thereof. The preparation method specifically comprises the processes of selecting raw materials of PLMVd, preparing a standard sample, checking uniformity and stability, carrying out qualitative detection, determining value and the like. The preparation method of the standard sample comprises the following steps: extracting virus RNA in a PLMVd virus solution, carrying out RT-PCR reaction, amplifying and purifying target gene fragments, connecting and transforming target fragments; and recycling plasmids. The peach latent mosaic viroid molecule standard sample is high in stability and high in uniformity, is suitable for rapid detection and determination of the peach latent mosaic viroid, provides the standard sample demand for detection research, pesticide research and application research of the peach latent mosaic viroid, and can be widely applied to disease surveillance and control in the agricultural production and environment and surveillance and detection of the peach latent mosaic viroid in import and export trade. Through application of the standard sample prepared by the method, different laboratory results can be compared, so that the quality control of the laboratories can be ensured.

Description

technical field [0001] The invention belongs to the technical field of virus standard products for detection and preparation methods thereof, and in particular relates to peach latent mosaic virus standard samples and preparation methods thereof. Background technique [0002] Peach latent mosaic viroid (PLMVd) belongs to the Avsunviroidae family and usually contains about 338 nucleotides without a central conserved region. It occurs in peach cultivation areas all over the world, and mainly infects stone fruit trees such as peaches, pears, plums, and apricots: PLMVd is a latent infection on many peach tree (Prunus persica) varieties, and the incubation period can reach 5 ~7 years, usually no obvious leaf symptoms, but the growth of poisonous peach trees is slow, the tree vigor declines, and the resistance decreases. After the infected variety is infected, white or yellow creamy mosaic leaves (Peach calico) or printed lesions, chlorotic spots (Peach blotch) are formed on the ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/70C12Q1/68
CPCC12Q1/70C12Q1/6806C12Q2600/166C12Q2531/113
Inventor 吴兴海张成标王振华李明哲邵秀玲张京宣王英超
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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