Molecular detection primer specific for phytophthora sojae and application thereof

A technology for molecular detection and Phytophthora sojae, applied in the biological field, can solve the problems of distinguishing, unsuitable for quantitative detection methods, etc., and achieve the effect of high specificity and sensitivity

Active Publication Date: 2014-02-12
PLANT PROTECTION & QUALITY & SAFETY OF AGRI PRODS INST ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the rapid development of modern molecular biology and the continuous publication of the genome sequences of various species, it was found that the ITS sequences of many different species have a high similarity, and it is difficult to distinguish these species using ITS sequences.
Moreover, because ITS is multi-copy in the genome, it is not suitable for the development of quantitative detection methods

Method used

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  • Molecular detection primer specific for phytophthora sojae and application thereof
  • Molecular detection primer specific for phytophthora sojae and application thereof
  • Molecular detection primer specific for phytophthora sojae and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Detection results of soybean susceptible plants:

[0029] 1) DNA extraction of diseased plants: Disinfect soybean leaves or rhizomes with water-soaked lesions with 70% alcohol, grind them with liquid nitrogen, take a small amount of powder, and use CTAB method to extract genome. Alkaline lysis can also be used to quickly extract DNA. The method is as follows: Take a section of diseased plant tissue, add 10 μl 0.5 M NaOH per mg of tissue, grind it thoroughly, transfer it to a 1.5 ml EP tube, centrifuge at 12,000 rpm for 5 minutes, and take 1 μl was directly used for PCR amplification.

[0030] 2) Ordinary PCR amplification verification: take 1 μl of DNA solution as a reaction template, use the Phytophthora soybean specific base sequence PsYkt6-F / PsYkt6-R as primers, and perform PCR amplification with the reagents and dosages mentioned above, Take 10 μl of the amplified product for gel electrophoresis detection. The results show that the diseased plants can amplify spe...

Embodiment 2

[0033] The detection results of residual Phytophthora soybean oospores in the soil:

[0034] 1) Extraction of microbial DNA in soil: Each soil sample was dried and crushed, then 0.5g of soil sample was weighed, and DNA was extracted using FastDNA SPIN kit (Q-Biogene Ltd, USA).

[0035] 2) Ordinary PCR amplification verification: Take 1 μL of DNA solution as a reaction template, use the PsYkt6-F / PsYkt6-R primers specific to Phytophthora sojae and the Ykt6 Phytophthora general primers Ykt6F / Ykt6R in a complete set of the present invention. PCR was used to amplify, and 10 μL of the amplified product was used for gel electrophoresis detection. The results showed that among the 14 soils collected from soybean fields in different regions, 11 could detect the target band, indicating that there were eggs of Phytophthora sojae. Spores, the artificial inoculation of the soil containing Phytophthora sojae oospores can also amplify the target band, but no band is amplified in the negat...

Embodiment 3

[0038] Detection results of zoospores in water sources polluted by Phytophthora sojae:

[0039] 1) Zoospore enrichment and DNA extraction: Phytophthora sojae can form sporangia and release a large number of zoospores in an environment with a water film, which is an important way for re-infection. Take 500 mL of water source contaminated by Phytophthora sojae, centrifuge it at 5000 g for 20 min, pour off the supernatant, suspend the precipitated zoospores with 100 μL of water, transfer to a 1.5 mL centrifuge tube, add 0.05 g of quartz sand, and vortex for 10 seconds After centrifugation at 2000rpm for 5 minutes, the supernatant was taken for PCR amplification.

[0040] 2) Ordinary PCR amplification verification: take 1 μL zoospore supernatant as a reaction template, and use the PsYkt6-F / PsYkt6-R primers specific to Phytophthora sojae and Ykt6 Phytophthora general primers Ykt6F / Ykt6R of the present invention as a complete set Using nested PCR for amplification, 10 μL of the a...

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Abstract

The invention designs a molecular detection primer special for phytophthora sojae, which adopts R-SNARE protein coding gene Ykt6 needed by cell survival as a target. The primer has high sensitivity and is matched with a universal primer Ykt6F/Ykt6R of Ykt6 phytophthora; a nested PCR method is adopted, so that the sensitivity can be improved by 1000 times and trace phytophthora sojae can be detected; according to the method, specificity and sensitivity are high and hypha, oospore and zoospore of the phytophthora sojae can be detected. Simultaneously, the special for the phytophthora sojae is utilized for developing the detection method for real-time quantitative PCR and a linear regression equation, the amount of infected phytophthora sojae in soybean can be accurately and quantitatively detected and the content of the oospore of the residual phytophthora sojae in the soil can be detected so as to provide forceful data for prediction and forecast of diseases and provide technical support for entry and exit quarantine.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically relates to a molecular detection primer for Phytophthora soybean specificity and application thereof. Background technique [0002] Soybean is an important economic crop in the world, Phytophthora sojae ( Phytophthora sojae ) root rot, which infects soybeans and causes sudden death of seedlings and adult plants, is one of the main diseases in soybean production in the world, and it brings huge economic losses to the soybean industry in the world every year. Phytophthora sojae can infect soybeans at various stages of soybean growth, and the disease is particularly serious in wet and rainy seasons. Because Phytophthora sojae reproduces sexually in a homologous manner, a large number of oospores can be produced on diseased plants. Plants The oospores left in the soil after harvesting become the primary infection source of Phytophthora sojae, and it is reported that the oospores of Phytoph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12Q1/04C12N15/11G01N21/64
CPCC12Q1/04C12Q1/6851C12Q1/686C12Q2545/114C12Q2561/113C12Q2531/113
Inventor 赵伟戚仁德汪涛
Owner PLANT PROTECTION & QUALITY & SAFETY OF AGRI PRODS INST ANHUI ACAD OF AGRI SCI
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