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Universal primer for identifying pathogenic species of potato scabs and detection method

A technology for potato scab and scab bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of low identification efficiency, poor accuracy, and low identification resolution, and achieve identification The effect of high efficiency, good accuracy and high resolution

Active Publication Date: 2017-07-18
黑龙江省农业科学院植物脱毒苗木研究所
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem of low identification resolution, low identification efficiency and poor accuracy of the pathogenic bacteria of potato scab

Method used

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  • Universal primer for identifying pathogenic species of potato scabs and detection method
  • Universal primer for identifying pathogenic species of potato scabs and detection method
  • Universal primer for identifying pathogenic species of potato scabs and detection method

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Experimental program
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specific Embodiment approach 1

[0025] Specific embodiment one: a kind of general primer for identification of potato scab pathogenic bacteria species of the present embodiment, the primer pair is as follows:

[0026] gyrBF2: 5'-TCTGCACGGYGTSGGYGTCTC-3'

[0027] gyrBR2: 5'-TTGCGGGAGTTGAGGTAC-3'.

[0028] The primer sequences of this embodiment are shown in Sequence Listing Seq ID No: 8 and Sequence Listing Seq ID No: 9.

specific Embodiment approach 2

[0029] Specific embodiment two: a kind of detection method for identifying potato scab pathogen species of the present embodiment, it is carried out according to the following steps:

[0030] 1. Extract the genomic DNA of the sample to be tested;

[0031] 2. Use the primers of the specific embodiment 1 as identification primers, and use the DNA extracted in step 1 as a template to carry out PCR amplification reaction;

[0032] 3. The PCR products were electrophoresed on a 1% agarose gel;

[0033] 4. Analyze the amplified fragment: there is an amplified band at the 400bp position, indicating that the PCR product of the sample can be sequenced, and then sequenced;

[0034] 5. Compare the sequenced sequence with the data in the gene database, and after the alignment is successful, the detection for identifying the species of potato scab pathogens is completed.

specific Embodiment approach 3

[0035] Embodiment 3: The difference between this embodiment and Embodiment 2 is that the PCR system for PCR amplification is as follows:

[0036]

[0037] PCR amplification conditions: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 45 s, annealing at 50-65 °C for 45 s, extension at 72 °C for 90 s, 35 cycles, extension at 72 °C for 10 min, and storage at 4 °C.

[0038] Others are the same as in the second embodiment.

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Abstract

The invention provides a universal primer for identifying pathogenic species of potato scabs and a detection method, which relates to a detection primer and a PCR (polymerase chain reaction) amplification method. The invention aims at solving the problems of low identification resolution ratio, low identification efficiency and poor identification accuracy of the pathogenic species of the potato scabs. The sequences of the primer are gyrBF2:5'-TCTGCACGGYGTSGGYGTCTC-3' and gyrBR2:5'-TTGCGGGAGTTGAGGTAC-3'. The detection method comprises the following steps: extracting genomic DNA of a bacterial strain, performing PCR amplification, sequencing products with fragment size of 400bp, comparing a sequencing result and a gene database and finally determining the pathogenic spices. The universal primer has comparatively high identification resolution ratio between spices, high identification efficiency and high repeatability. A detected sample can be pure bacterial strain DNA, and potato tuber tissue DNA after being infected by a single pathogenic bacterium serves as a template for PCR amplification and sequencing.

Description

technical field [0001] The present invention relates to a detection primer and a PCR amplification method thereof. Background technique [0002] Potato common scab, a soil-borne disease that affects the appearance, grade and quality of potatoes, has a wide distribution and is a serious world-class disease. Potato scab mainly damages tubers. The pathogen invades from the lenticels, and brown spots appear on the epidermis of the tuber at the initial stage, which gradually expands later. The severity of the disease varies with the variety, plot, and year, and the size and depth of the lesions also vary with the type of pathogenic bacteria, the degree of susceptibility of the variety, and the environmental conditions. In severe cases, the lesions are contiguous, which seriously reduces the appearance quality of tubers and affects their commercial value and processing value. Economic losses from scab in Canada range from $90-102 per hectare, and national economic losses from sc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/465
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 魏琪闵凡祥王晓丹高云飞杨帅董学志王文重张抒吕典秋宿飞飞张静华李辉
Owner 黑龙江省农业科学院植物脱毒苗木研究所
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