Primer for detecting potato ralstonia solancearum and PCR detection method adopted by primer
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A technology of R. solanacearum and potato, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of narrow detection range, low detection efficiency, and poor sensitivity, and achieve high detection efficiency , Wide range of detection, good detection specificity
Inactive Publication Date: 2016-11-16
黑龙江省农业科学院植物脱毒苗木研究所
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[0006] The purpose of the invention is to solve the problems that the existing method has narrow detection range, poor specificity, poor sensitivity and low detection efficiency to the pathogenic bacteria of potato bacterial wilt
Method used
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specific Embodiment approach 1
[0028] Specific embodiment one: a kind of primer of detection potato solanacearum of the present embodiment, this primer pair is as follows:
[0029] QK5-1: 5'-GCTAATACCGCATACGAC-3'
[0030] QK3-1: 5'-GAGCGTCAGTGTTATCCC-3'.
specific Embodiment approach 2
[0031] Specific embodiment two: a kind of PCR method of detecting potato solanacear of the present embodiment, it is to carry out according to the following steps:
[0032] 1. Extract the genomic DNA of the sample to be tested;
[0033] Two, with the primer of claim 1 as detection primer, the DNA that step 1 extracts is as template, carries out PCR amplification reaction;
[0034] 3. PCR products were subjected to 1% agarose gel electrophoresis;
[0035] 4. Analyze the amplified slice: there is an amplified band at the 591bp position, indicating that the sample is positive, and the sample to be detected contains R. solanacearum.
specific Embodiment approach 3
[0036] Specific embodiment three: the difference between this embodiment and specific embodiment two is: the PCR system of PCR amplification is as follows:
[0037]
[0038] PCR amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 50-63°C for 40 s, extension at 72°C for 100 s, 35 cycles, extension at 72°C for 10 min, and storage at 4°C. Others are the same as in the second embodiment.
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Abstract
The invention provides a primer for detecting potato ralstonia solancearum and a PCR detection method adopted by the primer, relating to a primer and a PCR detection method adopted by the primer, and aiming at solving the problems that when the existing methods are adopted for detecting pathogenic bacteria of potato ralstonia solancearum, the detection range is narrow, the specificity is poor, the sensitivity is poor, and the detection efficiency is low. The primer has the sequence as follows: QK5-1: 5'-GCTAATACCGCATACGAC-3'; QK3-1: 5'-GAGCGTCAGTGTTATCCC-3'. The PCR detection method comprises the following steps: extracting the total genomic DNA, carrying out PCR detection, verifying the detection primer, and if an amplification band appears at the 591bp position, indicating that the sample is positive. According to the primer and the PCR detection method, the detection sensitivity can achieve 1pg of template DNA, the detection efficiency is high, the detected sample can be potato plant DNA and tuber DNA, even PCR detection can be carried out directly by taking bacterial liquid as a template.
Description
technical field [0001] The invention relates to a detection primer and a PCR detection method thereof. Background technique [0002] Potato bacterial wilt, also known as bacterial wilt. In terms of occurrence area or hazard, bacterial wilt is a common and frequently-occurring disease second only to late blight among potato diseases, and it is a major bacterial disease worldwide. Bacterial wilt has a wide range of distribution, mainly in tropical and subtropical regions with warm and humid climates and abundant rainfall. Because bacterial wilt is difficult to control, it has no immune antigen and can be transmitted through soil. Once bacterial wilt occurs, the yield of the diseased field will be greatly reduced, and the yield loss of severe disease will reach about 80%. [0003] The disease is caused by a bacterial infection called Ralstonia solanacearum. This bacterium is a plant pathogenic bacterium that parasitizes vascular bundles. After invading the vascular bundles, ...
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