Specific Molecular Detection Primers for Soybean Gray Spot and Its Application

A technology for gray spot disease and soybean, which is applied in the field of specific molecular detection primers for soybean gray leaf spot bacteria, can solve the problems of high similarity, inability to design specific detection primers, etc., and achieves strong specificity, simple and fast method, and high sensitivity. Effect

Active Publication Date: 2020-12-01
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the ITS sequences of some fungi, including C. sojae, have high interspecies similarity, and it is impossible to design specific detection primers

Method used

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  • Specific Molecular Detection Primers for Soybean Gray Spot and Its Application
  • Specific Molecular Detection Primers for Soybean Gray Spot and Its Application
  • Specific Molecular Detection Primers for Soybean Gray Spot and Its Application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Establishment of a specific molecular detection method for Soybean gray spot fungus

[0022] 1. Primer Design

[0023] The most commonly used ITS sequences in the molecular detection of plant pathogenic fungi have high similarities among some fungal species, and it is difficult to directly use them for accurate species identification. In the present invention, through the multiple alignment analysis of ITS, β-tublin, Actin, Cytb, EF-1α and other gene sequences of Soybean gray spot and other plant pathogenic fungi, multiple soybean gray spots are finally found in the β-tublin gene sequence Pathogen-specific loci, and based on this, specific detection primers for Soybean gray spot were designed and screened. The β-tublin gene sequences of Cercospora sojina and other fungi of the genus Cercospora were downloaded from the GenBank database, and multiple alignment analysis was performed using MEGA 5.1 software, and molecular detection primers were designed for the specific s...

Embodiment 2

[0034] Specific Validation of Primers for Detection of Gray Spot of Soybean

[0035] 1. Extraction of genomic DNA from the sample to be tested

[0036] Genomic DNA of soybean sclerotinia, soybean purple spot, soybean Phytophthora root rot, soybean scab and soybean blight were extracted by the above-mentioned SDS method.

[0037] 2. Specific detection of primers for detection of gray spot disease of soybean

[0038]PCR amplification was carried out using the genomic DNA of the above-mentioned tested fungi as templates. The PCR reaction system contains: 2.5 μL 10×PCR buffer, 1.5 μL Mg 2+ (25mM), 0.5μL dNTP (each 10mM), 2.5U Taq DNA polymerase, 1.0μL each of upstream primer TF1 (10μM) and downstream primer TR1 (10μM), 1.5μL DNA template, ddH 2 O 16.5 μL. The PCR reaction program was: 94°C for 3min; 35 cycles of 94°C for 30s, 57°C for 30s, 72°C for 1min; 72°C for 10min. 5.0 μL of the PCR product was electrophoresed on a 1.5% (m / v) agarose gel, stained with ethidium bromide, a...

Embodiment 3

[0042] Sensitivity detection of detection primers for soybean gray spot bacterium

[0043] 1. Extraction of Genomic DNA of Soybean Gray Spot

[0044] Genomic DNA of Soybean gray spot was extracted by the above-mentioned SDS method

[0045] 2. DNA concentration determination and dilution

[0046] Use a NanoDrop ultra-micro spectrophotometer to measure the concentration of the above-mentioned genomic DNA, and use ddH 2 Oy was serially diluted to 100ng / μL, 10ng / μL, 1ng / μL, 10 -1 ng / μL, 10 -2 ng / μL and 10 -3 ng / μL.

[0047] 3. Perform PCR amplification using the above-mentioned different concentrations of DNA as templates. The PCR reaction system contains: 2.5 μL 10×PCR buffer, 1.5 μL Mg 2+ (25mM), 0.5μL dNTP (each 10mM), 2.5U Taq DNA polymerase, 1.0μL each of upstream primer TF1 (10μM) and downstream primer TR1 (10μM), 1.5μL DNA template, ddH 2 O 16.5 μL. The PCR reaction program was: 94°C for 3min; 35 cycles of 94°C for 30s, 57°C for 30s, 72°C for 1min; 72°C for 10min. ...

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Abstract

The present invention relates to a cercospora edulis specific molecular detection primer and application thereof, the cercospora edulis specific molecular detection primer includes an upstream primerand a downstream primer as shown in sequence tables SEQ ID No.1-2. The cercospora edulis specific molecular detection primer pair can amplify specifically in cercospora edulis to obtain a target product of 769 bp. The application comprises application of the cercospora edulis specific molecular detection primer in detection of soybean Cercospora sojina Hara and preparation of a detection kit for detecting the soybean Cercospora sojina Hara. The primer has high specificity and can be used for distinguishing soybean sclerotinia blight, Soybean Phytophthora Root, Soybean purple blotch, soybean gibberellic disease, soybean wilt and other soybean common disease pathogenic bacteria, and has high sensitivity, the detection sensitivity of cercospora edulis genomic DNA is up to 1ng / mu L; the usemethod is simple and quick, and cloning, sequencing, enzyme digestion and other operation are not needed for PCR products.

Description

technical field [0001] The invention relates to a specific molecular detection primer for soybean gray spot bacterium and application thereof. Background technique [0002] Soybean is a major source of protein in human food and livestock feed, and is also an important oil crop that is widely grown worldwide. In my country, soybean has become the fifth largest crop after rice, wheat, corn and cotton, and occupies an important position in my country's agricultural production. Soybean gray spot is a fungal disease caused by the infection of Cercospora sojina Hara. It is a serious hazard to soybean production. In the year when the disease is prevalent, it can generally cause a 10%-30% reduction in production, and in severe cases it can reduce production by 50%. %, but also seriously affect the quality of soybeans. The fungus overwinters on the diseased residue and seeds with mycelium and conidia, and becomes the source of primary infection in the following year. After the init...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6895
Inventor 宋爽张俊华陈宇飞
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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