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Loop-mediated isothermal amplification primer group for detecting citrus yellow shoots and kit

A technology of loop-mediated isothermal and amplified primers, which is applied in the field of detection of citrus Huanglongbing pathogens, can solve the problems of high technical requirements, complex processing, and difficulty in distinguishing other diseases, and achieve the effect of expanding the scope of application

Inactive Publication Date: 2017-06-20
张薇
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. Field diagnosis - simple, but easy to confuse, not easy to distinguish from other diseases;
[0005] 2. Electron microscope detection—requires expensive instruments and laboratories, and is easy to miss;
[0006] 3. Immunological detection - requires laboratories and instruments, and the cycle is long;
[0007] 4. Molecular detection—accurate and fast, but the pretreatment is complicated, requires expensive equipment, and requires high technical personnel
[0008] Currently there is no method that can quickly and accurately detect Huanglongbing

Method used

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  • Loop-mediated isothermal amplification primer group for detecting citrus yellow shoots and kit
  • Loop-mediated isothermal amplification primer group for detecting citrus yellow shoots and kit
  • Loop-mediated isothermal amplification primer group for detecting citrus yellow shoots and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0043] A ring-mediated isothermal amplification primer set for detecting Huanglongbing, including primer P1, primer P2, primer P3, primer P4, primer P5 and primer P6, the sequence of the primer P1 is shown in SEQ ID NO: 1 , the sequence of the primer P2 is shown in SEQ ID NO: 2, the sequence of the primer P3 is shown in SEQ ID NO: 3, the sequence of the primer P4 is shown in SEQ ID NO: 4, the sequence of the primer P5 is shown in The sequence is shown in SEQ ID NO:5, and the sequence of the primer P6 is shown in SEQ ID NO:6.

Embodiment 2

[0045] A kit containing the loop-mediated isothermal amplification primer set described in Example 1, the kit also includes a reaction solution and a control group, the reaction solution includes DNA polymerase, buffer, dNTPS, MgSO4 and glycine trimethyl Inner salt of amine; the control includes a positive control and a negative control, the positive control is an Escherichia coli plasmid DNA fragment containing the gene of interest, the sequence of the gene of interest is shown in SEQ ID NO: 7, the The negative control is the reaction solution or double distilled water.

[0046] Generally, the concentration of the primer reagent needs to be configured before use. For convenience, the concentration of the primers P1 and P2 is 40nM, the concentration of the primers P3 and P4 is 5nM, and the concentration of the primers P5 and P6 is 20nM. The volume of the loop-mediated isothermal amplification primer set is 6ul, the volume of the reaction solution is 15ul, and the volume of eac...

Embodiment 3

[0050] figure 1 For using the kit described in embodiment 2 to detect the method flow diagram of whether citrus is infected by Huanglongbing pathogen, described method comprises the following steps:

[0051] (1) Sample processing: Take citrus leaves suspected to be infected with Huanglongbing, freeze and crush them with liquid nitrogen or mechanically crush them, add 1ml of cell lysate to extract the precipitated nucleic acid, and dilute it as a sample;

[0052] (2) Primer treatment: Primer P1, primer P2, primer P3, primer P4, primer P5, and primer P6 were mixed in equal volumes to obtain primer reagents. Before mixing, the concentration of primer P1 and primer P2 was 40 nM, and primer P3 And the concentration of primer P4 is 5nM, the concentration of primer P5 and primer P6 is 20nM;

[0053] (3) Loop-mediated isothermal amplification reaction: After mixing the sample, primer reagent and reaction solution evenly, it was used as the detection group; after the positive control ...

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Abstract

The invention provides a loop-mediated isothermal amplification primer group capable of quickly and accurately detecting citrus yellow shoots and a kit. The loop-mediated isothermal amplification primer group comprises primers P1, P2, P3, P4, P5 and P6, wherein the sequence of the primer P1 is shown as SEQ ID NO: 1, the sequence of the primer P2 is shown as SEQ ID NO: 2, the sequence of the primer P3 is shown as SEQ ID NO: 3, the sequence of the primer P4 is shown as SEQ ID NO: 4, the sequence of the primer P5 is shown as SEQ ID NO: 5, and the sequence of the primer P6 is shown as SEQ ID NO: 6. The kit comprises the loop-mediated isothermal amplification primer group and a reaction solution, and can quickly and accurately detect whether citruses are infected with citrus yellow shoot pathogens.

Description

technical field [0001] The invention belongs to the technical field of citrus Huanglongbing pathogen detection, and in particular relates to a loop-mediated isothermal amplification primer set and a kit for detecting Huanglongbing. Background technique [0002] Citrus Huanglongbing is a devastating disease of citrus production in the world. The pathogen of citrus greening disease is a Gram-negative bacillus, which can infect a variety of Rutaceae plants including Citrus, Hovenia, Kumquat and Murata and produce devastating consequences, which is extremely harmful. There is no effective cure for the plants infected by Huanglongbing bacteria, so they can only be cut down to prevent the epidemic from spreading, causing huge losses to farmers. [0003] The current diagnostic methods for Huanglongbing and their advantages and disadvantages are as follows: [0004] 1. Field diagnosis - simple, but easy to confuse, not easy to distinguish from other diseases; [0005] 2. Electron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12Q1/04
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 张薇王耕王振超陈安东
Owner 张薇
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