RPA (Recombinase polymerase amplification) primer, probe, kit and detection method for detecting ralstonia solanacearum

A technology of tobacco bacterial wilt bacteria and detection method, which can be applied in the directions of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc. High, false positives, low equipment requirements

Inactive Publication Date: 2018-11-23
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, RPA technology has been widely used in the rapid detection of viruses, bacteria, mycoplasma and animal parasitic nematodes, but there is no report on the detection of tobacco R. solanacearum

Method used

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  • RPA (Recombinase polymerase amplification) primer, probe, kit and detection method for detecting ralstonia solanacearum
  • RPA (Recombinase polymerase amplification) primer, probe, kit and detection method for detecting ralstonia solanacearum
  • RPA (Recombinase polymerase amplification) primer, probe, kit and detection method for detecting ralstonia solanacearum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039]R. solanacearum was used for the screening of RPA primers and probes. Taking the Rip TALI-9 gene of R. solanacearum as the target gene; according to the requirements of the TwistDX operation manual, the primers that can be used in the RPA kit (TwistAmp nfo) were designed and screened, and the primers with high amplification efficiency, sensitivity and specificity were obtained. right. The sequences of the best primers obtained by screening (ie, the upstream and downstream primers of the RPA primers) are shown in SEQ ID No: 1 and SEQ ID No: 2. Then the above primers were modified, and a biotin (Biotin) labeling site was added to the 5' end of the downstream primer of the RPA primer. In addition, a probe with a size of 45bp-54bp was designed according to the amplified fragment of the RPA primer. The 'end was labeled with FAM, the 3' end was modified with C3-Spacer, and the probe was modified with THF at a distance of 34 bp from the 5' end. Finally, according to the requi...

Embodiment 2

[0044] Establishment of RPA Detection Method for Tobacco R. solanacearum

[0045] The detection method for detecting tobacco bacterial wilt bacteria of the present embodiment comprises the following steps:

[0046] (1) Extract the DNA in the sample;

[0047] (2) using the DNA extracted in step (1) as the DNA template to be detected, using the RPA primers and RPA probes described in Example 1 to carry out RPA amplification reaction in an RPA reaction tube;

[0048] The reaction conditions of the RPA amplification reaction in step (2) are: react at 38° C. for 20 minutes, and then terminate the reaction on ice;

[0049] The reaction system of the RPA amplification reaction in step (2) is calculated as 50 μL:

[0050]

[0051] (3) Analyze the RPA amplification product; take the RPA amplification product and mix it with the buffer to prepare a mixed solution (take the above-mentioned 1 μL of the RPA amplification product and add it to 49 μL of PBST buffer and mix, wherein the ...

Embodiment 3

[0055] Specific detection of RPA primers and probes for R. solanacearum

[0056] The detection method for detecting R. solanacearum of the present embodiment is the same as the embodiment 2, and the difference is:

[0057] The samples are respectively R. solanacearum, Flavobacterium, Arthrobacter, Escherichia coli, Pseudomonas, Arthrobacter saprophyte, Bacillus amyloliquefaciens, Paenibacillus, Bacillus subtilis, Serratia marcescens. fungus, rice bacterial leaf spot, Phytophthora nicotianae and tobacco root black rot.

[0058] like figure 2 shown, figure 2 The symbol "1" in the refers to the detection result of tobacco wilt bacteria, figure 2 The symbol "2" in the genus refers to the detection result of Flavobacterium, figure 2 The symbol "3" in the refers to the detection result of Arthrobacter, figure 2 The symbol "4" in the refers to the detection result of Escherichia coli, figure 2 The symbol "5" in the refers to the detection result of Pseudomonas, figure 2...

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Abstract

The invention discloses an RPA (Recombinase polymerase amplification) primer, a probe, a kit and a detection method for detecting ralstonia solanaceraum and belongs to the technical field of biological safety. The RPA primer is designed based on Rip TALI-9 gene of the ralstonia solanaceraum, and a sequence of the RPA primer is shown as SEQ ID No:1 and 2; the PRA probe is designed according to an amplification area of the RPA primer, and a sequence size of the PRA probe is 45 bp to 54 bp; the 5' end is labeled by FAM, and the 3' end is modified by C3-Spacer; furthermore, THF modification is performed at distance to the 34 bp of the 5' end at the middle of the sequence of the RPA probe. The invention further provides the kit containing the RPA primer and the probe. The invention further provides the detection which is constructed based on the RPA primer and the probe. According to the detection method, DNA extracted from a sample is utilized as a template to perform RPA amplification reaction. According to the detection method disclosed by the invention, an RPA technology is applied to molecular detection of the ralstonia solanaceraum for the first time. The detection method has thecharacteristics of strong specificity, high flexibility, good practicability, lower requirement for instruments and equipment; a novel method is provided for diagnosing pseudomonas solanacearum, preventing and treating diseases and reducing pesticide use amount.

Description

technical field [0001] The invention belongs to the technical field of biological safety, and in particular relates to an RPA primer, a probe, a kit and a detection method for detecting R. solanacearum. Background technique [0002] Tobacco bacterial wilt is a bacterial disease caused by Ralstonia solanacearum, which mainly occurs in tropical and subtropical regions with high temperature. It is extremely harmful to continuous cropping tobacco and is a devastating disease in tobacco production. . Tobacco bacterial wilt is a soil-dwelling fungus, overwintering in plant diseased bodies, and colonizing the vascular bundle after invading from the roots of tobacco plants, causing withering, death and systemic necrosis of roots. High temperature (above 30°C) and high humidity (above 90% relative humidity) are the main conditions for the prevalence of bacterial wilt disease in tobacco. Due to the lack of tobacco varieties with high resistance to bacterial wilt and the poor chemical...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2522/101
Inventor 潘月敏鞠玉亮周本国沈鹏飞许大凤张丽娜
Owner ANHUI AGRICULTURAL UNIVERSITY
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