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Primer, probe and kit for detecting soybean damping-off disease based on recombinase polymerase amplification method

A recombinase polymerase and kit technology, applied in the biological field, can solve the problems of long cycle, poor specificity and low sensitivity of detection methods, and achieve the effects of high accuracy, convenient operation and simple requirements.

Active Publication Date: 2019-01-18
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] Aiming at the problems of long period, poor specificity and low sensitivity of the biological detection method of Pythium melon and fruit in the prior art, the purpose of the present invention is to provide a method based on the amplification method of recombinase polymerase to detect Pythium melon and fruit caused by Pythium melon and fruit. Primers, probes and kits for soybean damping-off

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  • Primer, probe and kit for detecting soybean damping-off disease based on recombinase polymerase amplification method
  • Primer, probe and kit for detecting soybean damping-off disease based on recombinase polymerase amplification method

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Effect test

Embodiment 1

[0027] Embodiment 1: Utilize RPA method to detect Pythium melon and fruit

[0028] RPA primers and probes for detecting Pythium melon and fruit: the forward primer F-primer is shown in SEQ ID NO.1, the reverse internal primer R-primer is shown in SEQ ID NO.2, and the probe Probe is shown in SEQ ID NO.1 Shown in ID NO.3.

[0029] The total volume of the RPA kit for detecting Pythium melon and fruit is 50 μL, the concentration of each reagent is: 29.5 μL RehydrationBuffer, 8.2 μL DEPC-treated water, and the final concentration of forward primer and reverse primer is 420nmol L -1 , the final concentration of the probe was 120 nmol L -1 , the final concentration of MgAC was 14mmol L -1 , 5 μL DNA template.

[0030] RPA detection method: extract the DNA of the microorganism to be tested, take 5 μL of the DNA solution as a reaction template, add 45 μL of the detection solution in the kit for RPA, and the RPA reaction procedure is: 39 ° C reaction amplification for 30 minutes, and...

Embodiment 2

[0032] Embodiment 2: the sensitivity test of Pythium melon and fruit RPA reaction

[0033]In order to determine the sensitivity of the RPA detection method, measure the concentration of the extracted Pythium melon and fruit DNA with a spectrophotometer and perform a 10-fold dilution. Set the DNA concentration range to 37.5ng-3.75fg, and take the diluted DNA of each concentration to dilute 5 μL of the solution was used as a template, and 45 μL of the kit solution was added to carry out the RPA reaction. The reaction procedure was: reaction and amplification at 39°C for 30 minutes. The results show that: as the RPA reaction continues, the fluorescence values ​​of different DNA concentrations are increasing with time, and the fluorescence intensity decreases with the decrease of the amount of DNA template, indicating that when the DNA concentration is 3.75fg, it is enough detected by the RPA method ( figure 2 ).

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Abstract

A primer, probe and kit for detecting soybean damping-off disease based on recombinase polymerase amplification method are disclosed. The primer pair comprises forward primer F as shown in SEQ ID NO.Primer and reverse primer R as shown in SEQ ID NO. 2- Primer; The sequence of the probe Probe is shown in SEQ ID NO. 3. Compared with the traditional detection technology for identifying melon and fruit Pythium according to the morphological characteristics, the invention has higher accuracy, sensitivity and effectiveness, is convenient to operate and has good practicability, provides a new technical platform for the detection of melon and fruit Pythium, and can be used for the high sensitivity and rapid detection of soybean damping-off caused by melon and fruit Pythium.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a probe and a kit for detecting soybean damping-off disease based on a recombinase polymerase amplification method. Background technique [0002] Soybean damping-off is a common disease in soybean production, and its main pathogen is Pythium melon and fruit. The pathogen mainly infects the stem base of soybean seedlings. Water-soaked streaks appear on the young stems near the surface at the beginning of the disease, and then the diseased part becomes soft and constricted, turning dark brown, and the diseased seedlings quickly fold and die. Irregular brown spots appear at the beginning after the root is damaged, which seriously causes root rot, and the aboveground stems and leaves wilt or yellow. At present, there are no effective control measures for soybean damping-off disease. Strengthening quarantine and preventing the spread of pathogenic bacteria are the m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107C12Q2521/507C12Q2521/101
Inventor 窦道龙沈丹宇景茂峰张正光赵媛媛于佳逯欣宇
Owner NANJING AGRICULTURAL UNIVERSITY
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