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Molecular detection primer and quick detection method for thielaviopsis basicola

A black rot pathogen and molecular detection technology, which is applied in the field of agricultural biology, can solve the problems of differentiating pathogens from closely related species, low sensitivity, and difficulty in early diagnosis of tobacco root black rot, and achieves good practicability, high sensitivity, and specificity. strong effect

Pending Publication Date: 2016-01-27
云南省烟草公司大理州公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the existing difficulty in early diagnosis and low sensitivity of tobacco root black rot, which makes it difficult to provide timely and accurate scientific basis for prevention and control, and the difference in rDNA-ITS sequence in existing molecular detection is very small, it is difficult to design primers targeting ITS. To solve the technical problem of distinguishing some pathogenic bacteria of closely related species, the present invention provides a highly sensitive, early-diagnosed tobacco root black rot pathogen, specific molecular detection primers, accurate results, and easy-to-operate tobacco root black rot bacteria Rapid Molecular Assays

Method used

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  • Molecular detection primer and quick detection method for thielaviopsis basicola
  • Molecular detection primer and quick detection method for thielaviopsis basicola
  • Molecular detection primer and quick detection method for thielaviopsis basicola

Examples

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Embodiment 1

[0028] Embodiment 1 PCR primer design of the present invention and the specific amplification of primers to tobacco root black rot bacteria

[0029] 1. Primer design and synthesis

[0030] Download the β-tubulin gene sequences of Tobacco root rot and other species from GenBank, and use ClustalW software for multiple sequence alignment and analysis to find the specific sequence of tobacco root rot β-tubulin gene; use Primers3 software for primers Design, designed tobacco root black rot pathogen molecular detection primer is made up of upstream primer Tbas-tubF and downstream primer Tbas-tubR, the base sequence of described upstream primer Tbas-tubF is as shown in SEQIDNO: 1, and described downstream primer Tbas The base sequence of -tubR is shown as SEQ ID NO: 2, and the expected fragment size for the specific amplification of Tobacco root rot is 254bp. The designed primers were synthesized by Shanghai Invitrogen Company.

[0031] 2. Test materials: tobacco root black rot (Th...

Embodiment 2

[0045] Embodiment 2 Sensitivity detection of primers of the present invention to tobacco root black rot

[0046] 1. Dilution of DNA concentration: After measuring the concentration of the extracted tobacco root black rot genomic DNA by a ThermoNanoDrop spectrophotometer, the genomic DNA concentration was 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg. spare.

[0047] 2. with the sensitivity detection of upstream primer Tbas-tubF of the present invention and downstream primer Tbas-tubR to tobacco root black rot bacterium

[0048] The upstream primer Tbas-tubF and the downstream primer Tbas-tubR were used to perform PCR amplification on 10-fold serially diluted genomic DNA of Tobacco root black rot, respectively. The reaction system and PCR amplification procedure of PCR amplification were the same as in Example 1.

[0049] 3. Detection results: After the PCR, 1.5% agarose gel electrophoresis was performed to detect the amplified products. Such as figure 2 It ...

Embodiment 3

[0050] Early diagnosis of root black rot fungus in the pathogenic tissue of tobacco in embodiment 3

[0051] 1. Preparation of Tobacco Root Black Rot Disease-Like Tissues

[0052] Make the concentration approximately 1 x 10 5 The conidia suspension of cfu / ml of tobacco root black rot fungus or the hyphae block of tobacco root black rot fungus cultured for 1 week by cutting out PDA with a scalpel was inoculated in the rhizome of tobacco variety Honghua Dajinyuan grown for 6 weeks, 22~ Moisturize in an artificial climate box at 23°C. Tobacco rhizomes 6h, 12h, 24h, 48h, 72h and 5d after inoculation were taken as disease-like tissues for DNA extraction.

[0053] 2. Rapid DNA extraction and PCR amplification of diseased tobacco tissue

[0054] DNA was extracted from the tobacco-infected diseased stems or diseased root tissues of tobacco root black rot infection for 6h, 12h, 24h, 48h, 72h and 5d according to the NaOH rapid lysis method, as follows:

[0055]Tobacco diseased stems...

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Abstract

The invention discloses a molecular detection primer and a quick detection method for thielaviopsis basicola. The molecular detection primer and the quick detection method have the advantages that the specific primer which is high in detection sensitivity and specificity and can diagnose black roots of tobaccos in fields at early stages and monitor and identify germs is designed according to multiple comparative analysis on beta-tubulin gene sequences of the thielaviopsis basicola and other species, the detection sensitivity of the molecular primer can reach 10 pg, the tobacco infection thielaviopsis basicola can be detected within 24 hours, and the lengths of fragments of amplification products are 254 bp; the timely and accurate detection method and scientific evidence can be provided for identifying the thielaviopsis basicola, monitoring the thielaviopsis basicola at early stages and timely preventing and treating the thielaviopsis basicola.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology. It specifically relates to the rapid and sensitive molecular detection of tobacco root black rot, and can be used for the early diagnosis of tobacco root black rot and the detection and identification of pathogens. Background technique [0002] Tobacco root black rot is an important soil-borne disease in tobacco production caused by Thielaviopsis basicola (Berk. et Br.) . It spreads all over the world's major tobacco-producing areas and causes significant losses in tobacco production. The pathogen can infect and damage a variety of plants, such as tobacco, cotton, beans, peanuts, citrus and various ornamental plants. Tobacco root black rot is a worldwide disease, which occurs heavily in my country's Yunnan, Guizhou, Hubei and other provinces, and the incidence rate of severe plots can reach more than 30%. Shandong, Henan, Anhui, Jilin, Fujian and other provinces also have occurrences. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895
Inventor 赵志坚户艳霞曹继芬杨明英王新中徐发华周丽凤
Owner 云南省烟草公司大理州公司