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Detection primer set, detection kit and detection method for rapidly detecting Ralstonia solanacearum

A technology for detecting primers and detection methods, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve problems such as limited sensitivity, and achieve the effects of high sensitivity, good accuracy, and short detection time

Inactive Publication Date: 2014-02-26
RES INST OF TROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been reports in the literature on the establishment of a LAMP method for the Flic gene of R. solanacearum, the LAMP method established on this basis has certain limitations in practical applications because it is only used for water samples and has limited sensitivity.

Method used

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  • Detection primer set, detection kit and detection method for rapidly detecting Ralstonia solanacearum
  • Detection primer set, detection kit and detection method for rapidly detecting Ralstonia solanacearum
  • Detection primer set, detection kit and detection method for rapidly detecting Ralstonia solanacearum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Detection of Ralstonia solanacearum in susceptible eucalyptus tissue

[0042] 1. Plant tissue processing

[0043] Take 5g of vascular bundle tissue and add 10mL of sterile water to grind it to obtain a sample treatment solution for bacterial DNA extraction.

[0044] 2. Bacterial DNA extraction

[0045] Take 1mL sample treatment solution and centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50μLTE to fully suspend and mix, put in 100℃ water bath for 10min, ice bath for 3min, centrifuge at 12000r / min for 5min, take the supernatant for later use, and obtain Template DNA.

[0046] 3. LAMP amplification

[0047] The loop-mediated isothermal amplification (LAMP) reaction solution consists of 25 μL per tube: 2.5 μL 10× Thermopol reaction buffer, 0.4 μL 10 mM dNTPs, 0.5 μL 10 uM upstream external primer F3, 0.5 μL 10 uM downstream external primer B3, 1 μL 40 uM upstream internal primer FIP, 1 μL 40uM downstream inner primer ...

Embodiment 2

[0051] Embodiment 2: Detection of Ralstonia solanacearum in healthy casuarina plant tissue

[0052] 1. Plant tissue processing

[0053] Take 5g of vascular bundle tissue and add 10mL of sterile water, aseptically grind to obtain the sample treatment solution.

[0054] 2. Bacterial DNA extraction

[0055] Add the sample treatment solution to 100ml TTC-LB enrichment solution and incubate at 37°C for 6-8h, take 1ml of the enrichment solution, centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50μL TE to fully suspend and mix, Water bath at 100°C for 10 minutes, ice bath for 3 minutes, centrifuge at 12,000 r / min for 5 minutes, take the supernatant for later use, and obtain the template DNA to be detected.

[0056] 3. LAMP amplification

[0057] The loop-mediated isothermal amplification (LAMP) reaction solution consists of 25 μL per tube: 2.5 μL 10× Thermopol reaction buffer, 0.4 μL 10 mM dNTPs, 0.5 μL 10 uM upstream external primer F3, 0.5 μL...

Embodiment 3

[0061] Embodiment 3: specificity experiment

[0062] Collect 41 strains of R. solanacearum and non-R. solanacearum strains (see Table 1 for test strains). After culturing the strains in nutrient broth (NB) at 30°C for 24 hours, take 1 mL of bacterial liquid, centrifuge at 12,000 r / min for 5 min, and discard the supernatant , collect the cells, add 50 μL TE to fully suspend and mix well, place in 100°C water bath for 10 min, ice bath for 3 min, centrifuge at 12000 r / min for 5 min, take the supernatant for later use, and obtain the template DNA to be detected.

[0063] Loop-mediated isothermal amplification (LAMP) reaction solution, 25 μL per tube consists of: 2.5 μL 10× Thermopol reaction buffer, 0.4 μL 10 mM dNTPs, 0.5 μL 10 uM upstream outer primer F3, 0.5 μL 10 uM downstream outer primer B3, 1 μL 40 uM upstream inner primer FIP , 1 μL 40uM downstream internal primer BIP, 0.6 μL 100mM MgSO 4 , 12.5 μL 2M betaine, 1 μL Bst DNA polymerase, 1 μL template DNA to be detected and ...

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Abstract

The present invention discloses a detection primer set, a detection kit and a detection method for rapidly detecting Ralstonia solanacearum. According to the present invention, based on the Ralstonia solanacearum 16srRNA specific target gene, a set of the specific detection primer set, the detection kit containing the detection primer set, and the detection method through loop-mediated isothermal amplification by using the detection kit are designed and screened so as to determine whether Ralstonia solanacearum exists in a sample requiring detection; the detection kit and the detection method have advantages of high sensitivity, strong specificity, good accuracy and short detection time, wherein the time from the sample treatment to the result reporting only takes 4 h, PCR instrument and an electrophresis apparatus are not required, the operation is simple, and the detection kit and the detection method have higher specificity compared with other PCR technologies; and the detection primer set, the detection kit and the detection method are especially for self-tests in forestry department and enterprises.

Description

Technical field: [0001] The invention belongs to the field of biological detection, and in particular relates to a detection primer set, a detection kit and a detection method for rapidly detecting R. solanacearum. Background technique: [0002] Plant bacterial wilt is a vascular disease caused by Ralstonia solanacearum (Smith) Yabuuchi et al. It can infect a variety of plants such as peanuts, eucalyptus, casuarinas, etc., causing great damage to agricultural and forestry production. Economic losses. Bacterial wilt can be transmitted not only through diseased plants, but also through latently infected plants. Transmission is more threatening with latently infected plants than with diseased plants. The diseased plants are easy to identify from the appearance, and can be prevented and controlled by strengthening plant quarantine. However, the plants in the incubation period of bacterial wilt are difficult to separate from the normal plants in appearance, so the spreading eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 王胜坤康丽华马海宾陆俊锟江业根
Owner RES INST OF TROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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