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Method for detecting tomato spotted wilf virus

A technology of tomato spotted wilt virus and sequencing primers, which is applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc. It can solve the problems of cumbersome operation, false positive detection and false positive primers, etc., and achieve high sensitivity And the effect of high precision, easy operation and small sample volume

Active Publication Date: 2014-12-10
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection and confirmation methods used for TSWV generally use serology, biological hosts, and morphological tests to determine whether plants have plant viruses. These methods are time-consuming and cumbersome to operate, and cannot meet the needs of disease control. experience and professional background knowledge, which is highly subjective; when using PCR technology for confirmation, if the specificity of the primers is not strong, false positives may occur; due to the strong variability of tomato spotted wilt virus, the degree of base difference between different geographical strains Larger, the reported primers are prone to false positives due to site variation, therefore, more conservative detection primers or detection methods that reach the SNP level are required

Method used

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  • Method for detecting tomato spotted wilf virus
  • Method for detecting tomato spotted wilf virus
  • Method for detecting tomato spotted wilf virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Detect whether TSWV is contained in field samples

[0027] 1. Design specific primer sequences

[0028] Log in to the National Center for Bioinformatics (NCBI) of the United States through the Internet to query and retrieve the published N gene nucleotide sequence of TSWV, which does not contain unknown base component sequences. Use the Editseq and MegAlign software in the DNAStar 7.1 software package to edit the N gene nucleotide sequences of the selected samples, compare them one by one, and use the default parameters of the Clustal W Method (software) to perform homology on the selected N nucleotide sequences Sexual comparison to find the specific nucleotide sequence (target detection sequence) that characterizes the virus.

[0029] The design of PCR amplification primers and sequencing primers can be carried out by Assay Design SW software, and a set of primers with higher scores is used for experiments. According to the homology analysis results of DNAS...

Embodiment 2

[0046] Example 2: Detect whether TSWV is contained in imported tomato seeds

[0047] 1. Design specific primer sequences

[0048] With embodiment 1.

[0049] 2. Extract the RNA of the sample to be tested:

[0050] With embodiment 1.

[0051] 3. RT-PCR amplification with TSWV N primers

[0052] With embodiment 1.

[0053] 4. Perform agarose gel electrophoresis:

[0054] Perform agarose gel electrophoresis on the PCR product, take 2g of agarose, heat it in 100mL of electrophoresis buffer, fully dissolve, add ethidium bromide stock solution to a final concentration of 0.5μg / mL, make gel, add to the electrophoresis tank Electrophoresis buffer, so that the liquid level is just above the gel; mix 3μL ~ 6μL of PCR amplification products with an appropriate amount of sample buffer, and apply the sample; 9V / cm constant voltage electrophoresis until the bromophenol blue indicator migrates to the gel The middle part; the electrophoresis results were observed under the ultraviolet d...

Embodiment 3

[0056] Embodiment 3: Detect whether TSWV gene is contained in different virus samples

[0057] Using the pyrosequencing confirmation technology of the present invention, according to the detection steps, the tobacco ringspot virus (TRSV) isolated from imported Japanese stevia, the tomato black ring virus (TBRV) isolated from Italian cucumber seeds, and the cucumber isolated from Chilean watermelon seeds Mosaic virus (CMV) and tomato mosaic virus (ToMV), tomato yellow leaf curl virus (ToYCLV) isolated from tomato planting areas in Qingdao, and tomato spotted wilt virus-Impatiens necrotic spot virus (INSV) and Eight strains with similar genetic background to TSWV, including iris yellow spot virus (IYSV), were detected, and no amplified fragments and corresponding sequences specific to the TSWV-N gene were detected. Thereby proving that false positive results will not occur when detecting non-TSWV viruses.

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Abstract

The invention discloses a method for detecting a tomato spotted wilf virus according to a pyrosequencing technology. The method comprises the following steps: firstly, designing a specific primer and a pyrosequencing primer according to the gene N of the tomato spotted wilf virus; secondly, extracting ribonucleic acid of a sample to be detected, and performing polymerase chain reaction (PCR) amplification by the specific primer of the gene N; thirdly, detecting an amplification product by agarose gel electrophoresis, preparing a pyrosequencing single-stranded template if the length of a primer amplification fragment is 143 bp, producing pyrosequencing reaction, and finally judging whether the sample contains the tomato spotted wilf virus or not according to a PCR amplification result and a pyrosequencing result. The method is suitable for quickly detecting and confirming the tomato spotted wilf virus, and can be widely applied to epidemic situation monitoring in agricultural production and environment and confirmation of the tomato spotted wilf virus in import and export trade; the operation is very easy and convenient, and the required sample amount is small.

Description

technical field [0001] The invention belongs to the technical field of detection of plant pathogens, and in particular relates to a method for detecting tomato spotted wilt virus. 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. TSWV is the most important virus in the TosPovirus genus. It is widely distributed in temperate, subtropical and tropical regions around the world, infecting more than 900 monocotyledonous and dicotyledonous plants in 82 families, and endangering vegetables, flowers and various food crops such as tomato, Peppers, potatoes, peanuts, geba, peas, tobacco, chrysanthemums, anemones, zinnias, gloxinia, dahlias and cyclamen have caused serious losses to many commercial crops and garden plants. According to reports, the disease has caused 80% loss of peanuts and 50%-90% death of Geba, and in European r...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/70C12Q1/701C12Q2531/113C12Q2565/125C12Q2565/301
Inventor 吴兴海陈长法封立平魏晓棠冯黎霞张京宣王简
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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