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Primer group and kit for identifying ochrata ochrata and application of primer group and kit

An ochre flower fungus, primer set technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc. specific effect

Pending Publication Date: 2022-03-15
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional detection methods, such as morphological identification, HPLC, PCR, etc., are not suitable for remote areas where the economy is underdeveloped, the knowledge of poisonous mushrooms is short, and the equipment is limited. Therefore, there is an urgent need for an easy-to-operate, A new rapid detection method with high sensitivity, suitable for grassroots and field application

Method used

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  • Primer group and kit for identifying ochrata ochrata and application of primer group and kit
  • Primer group and kit for identifying ochrata ochrata and application of primer group and kit
  • Primer group and kit for identifying ochrata ochrata and application of primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] LAMP primer design and screening

[0062] First, a total of 10 ITS sequences (MZ567199.1, MG846961.1, MG846962.1, MG846960.1, MG846959.1, MG846958.1, MG846957.1, MG846956.1, MG846955. 1 and MG846954.1), after comparing the homologous sequences, the conserved segment within the species of S. ochrenae was selected as the region for primer design.

[0063] In order to ensure the specificity of the primers, the ITS sequences (ITS sequence of Gyromitra esculenta of MZ567191.1, ITS sequence of Gyromitra gigas of MH938663.1, ITS sequence Multiple sequence alignment was performed for Gyromitra ambigua of MT373905.1 and Gyromitrabrunnea of ​​MZ667895.1 with ITS sequence, and specific regions were searched for primer binding sites (taking MG846960.1 as an example, the selected primer design region was 1-171 point).

[0064] Select the related species of the above 4 kinds of chrysanthemum chrysanthemum, as well as S. rufosa, Lactobacillus amberensis, Fissure capella, Amanita con...

Embodiment 2

[0073] Specific detection of primer sets

[0074] To detect the specificity of the primer set obtained by screening in Example 1, a positive control (Certia chinensis), 10 negative controls (mushrooms and common poisonous and edible mushrooms similar in form to Cervus siennae) and A blank control (ddH 2 O), grouping and test results are shown in Table 1. DNA was extracted using DNA secure New Plant Genomic DNA Extraction Kit (TIANGEN, China).

[0075] 10 μL total LAMP reaction system, including: 5 μL LAMP master mix (2×) [purchased from New England Biolabs, USA, containing: potassium chloride 50 mmol / L, ammonium sulfate 10 mmol / L, magnesium sulfate heptahydrate 3 mmol / L, Tween- 200.1%, 100 μmol / L phenol red dye, 0.8 μL NTPs (10 mmol / L), 0.1 μL Bst 2.0 WarmStartTM DNA polymerase (8U / μL)], 3 μL primer mixture [forward outer primer F3 and reverse outer primer B3 The concentration of the inner primer F2, reverse inner primer B2, forward inner primer F1c and reverse inner primer...

Embodiment 3

[0082] Sensitivity detection of the LAMP primer set obtained by screening in embodiment 1

[0083] Detect the DNA purity and concentration by a spectrophotometer, carry out 10-fold serial dilutions from 10 ng / μL to 1 fg / μL of the chrysanthemum genomic DNA, carry out the LAMP reaction (reaction system and reaction procedure are the same as in Example 2), and observe the end of the reaction After the color of the reaction solution, the experimental results see image 3 and Table 2.

[0084] Table 2 Sensitivity detection results of the LAMP primer set of S.

[0085]

[0086]

[0087] Note: "+" is positive, "—" is negative.

[0088] Depend on image 3 As can be seen from Table 2, the reaction solutions of No. 1 and No. 2 tubes are yellow. It can be seen that the detection limit concentration of the primer set provided by the present invention is 1 ng / μL, which is far lower than many traditional detection methods and is sufficient to meet the actual detection requirements....

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Abstract

The invention relates to the technical field of molecular identification, in particular to a primer group and a kit for identifying Gyromitra infusus and application of the primer group and the kit. The primer group provided by the invention can specifically amplify the nucleic acid of the deer antler, and does not have cross reaction with the nucleic acid of the white beveled fungus, the deer antler, the morchella, the bodybell fungus, the brown tray verruca and the beveled beveled fungus which are easy to confuse, and the nucleic acid of other macro fungi such as the boletus amber, the choriomyelon, the amanita coleopleatus and the boletus mucilaginosus, so that the nucleic acid of the deer antler and the nucleic acid of the boletus mucilaginosus are not subjected to cross reaction; the specificity is strong, and the primer group can be used for identifying the ochracea ochracea. Furthermore, according to the identification method provided by the invention, the specific identification of the deer antler can be completed within 90 minutes by utilizing the primer group, the detection result can be directly observed by naked eyes, and the method has the advantages that the operation is simple, the sensitivity is high (the detection limit is 1ng / mu L), the method is suitable for a base layer, and the field detection can be realized.

Description

technical field [0001] The invention relates to the technical field of molecular identification, in particular to a primer set, a kit and an application thereof for identifying S. Background technique [0002] Poisonous mushrooms are large fungi that can cause poisoning reactions and even death in humans and animals after eating. The toxic substances produced by them are called fungal toxins. For different kinds of poisonous mushrooms, they contain a variety of toxins, which can cause different symptoms of poisoning. Once ingested Gyromitra infula can cause hemolytic disease, the main early symptoms are headache, diarrhea and nausea and vomiting, chills, abdominal pain, hepatosplenomegaly, fever and hemoglobinuria in about two days, severe cases Life can be lost due to acute kidney failure. Because these mushrooms contain maline and methyl hydrazine, which can dissolve and destroy red blood cells, causing acute hemolysis. Once the poisoning incident of chrysanthemum chrys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2537/1376Y02A50/30
Inventor 田恩静谢晓梅高崇华郑媛段仁和曹广乘
Owner JILIN AGRICULTURAL UNIV
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