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Lamp rapid detection method and kit for chytrid fungus

A detection method and kit technology are applied in the application field of agricultural science and biotechnology to achieve the effects of simple operation, low cost and rapid response.

Active Publication Date: 2016-08-24
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, LAMP technology has not yet been applied to the detection of chytrid

Method used

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  • Lamp rapid detection method and kit for chytrid fungus
  • Lamp rapid detection method and kit for chytrid fungus
  • Lamp rapid detection method and kit for chytrid fungus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: LAMP method detection of chytrid

[0038] The positive sample among the samples to be tested in this embodiment, chytrid, was provided by American expert Joyce, and the negative sample was pure water.

[0039] The specific detection method steps are:

[0040] (1) According to the steps in the instructions of the TIANGEN tissue DNA extraction kit, extract the total DNA nucleic acid of the positive sample and the negative sample as the reaction template;

[0041] (2) Configure the LAMP reaction solution: taking the total reaction volume of 25 μL as an example, the amplification system consists of 5 μL solution 1, 5 μL solution 2 and 15 μL solution 3;

[0042] Wherein solution 1 is a 2× reaction buffer, and the 2× reaction buffer is composed of 900 μL 2×LAMP reaction buffer and 100 μL of a dNTP mixture with a concentration of 25 mM; wherein the composition of the 2×LAMP reaction buffer is: 1 M pH8.8 Tris -HCl 40mL, KCl 1.49g, MgSO 4 1.93g, (NH4) 2 SO 4 ...

Embodiment 2

[0049] Example 2: The specificity experiment of the kit used in the LAMP rapid detection method of chytrid:

[0050] The primers and operation steps in this example are exactly the same as those in Example 1.

[0051] Using the primers and operating steps in Example 1, 7 samples were used to detect the chytrid genome, the mucormyces genome, the saprolegniasis genome, the penicillium genome, the yeast genome, the iridovirus genome, and the herpesvirus genome. The negative sample was pure water.

[0052] The specific detection method is:

[0053] (1) According to the steps in the instructions of the TIANGEN tissue DNA extraction kit, extract the total DNA of the chytrid genome, the mucormyces genome, the saprolegniasis genome, the penicillium genome, the yeast genome, the iridescent virus genome, the herpes virus gene and pure water as Reaction template;

[0054] (2) Configure the LAMP reaction solution: taking the total reaction volume of 25 μL as an example, the amplificat...

Embodiment 3

[0062] Example 3: Sensitivity experiment of the kit used in the LAMP rapid detection method of chytrid:

[0063] The primers and operating steps in this example are exactly the same as in Example 1. The positive sample in the sample to be tested is chytrid provided by American expert Joyce, and the negative sample is pure water.

[0064] It is the result of nucleic acid electrophoresis detection after amplifying genes of different concentration gradients with LAMP primers of chytrid virus. The amplification condition is 62°C and the amplification time is 30 minutes. 1.028×10 7 -1.028×10 1 All copies of the template produced specific products, and no bands were produced in the control group. It can be seen from this figure that the universal LAMP primer designed in this study can detect at least 1.028×10 after amplification for 30 minutes at 62°C 1 A copy of the template (as attached image 3 and Figure 4 ).

[0065] The specific detection steps are as follows:

[0066...

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Abstract

The invention discloses a chytrid LAMP rapid detection method and a kit, and belongs to the field of application of agriculture science biotechnology. The rapid detection method comprises the following steps: (1) extracting total DNA nucleic acid of a to-be detected sample as a reaction template; (2) preparing an LAMP reaction solution according to the solution 1: solution 2: solution 3 volume ratio of 1:1:3; (3) adding the sample total DNA extracted in the step (1) into the LAMP reaction solution prepared in the step (2) and mixing; (4) reacting at water bath with a constant temperature of 62 DEG C for 30 min; and (5) obtaining the diagnosis result, namely employing electrophoresis detection or fluorescence dye technology to perform identification on the product of amplification reaction in the step (4). The method has the advantages of simple operation, low cost, rapid reaction, high specificity and high sensitivity, and is short in consumed time, has the sensitivity higher than that of common PCR by 100 times, and has the characteristics of relatively high accuracy on early-stage diagnosis on chytrid.

Description

technical field [0001] The invention belongs to the application field of agricultural science and biotechnology, and in particular relates to a rapid detection method of LAMP for chytrid fungus and a practical detection kit matched with the method. Background technique [0002] Chytridiomycetes cause amphibian chytridiomycosis. The full name of LAMP technology is Loop-mediated Isothermal Amplification (Loop-mediated Isothermal Amplification). A new type of gene amplification technology invented by Japanese scholar Notomi et al. However, LAMP technology has not been applied to the detection of chytrid at present. Contents of the invention [0003] The purpose of the present invention is to disclose a LAMP rapid detection method for chytrid. [0004] The second object of the present invention is to disclose a kit for the rapid detection method of Chytrium LAMP. [0005] The purpose of the present invention is achieved through the following technical solutions: [0006] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 王晓龙张子群
Owner NORTHEAST FORESTRY UNIVERSITY
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