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CN-122012777-A - Primer group for rapidly detecting amanita pseudooomycetes, application of primer group and kit containing primer group

CN122012777ACN 122012777 ACN122012777 ACN 122012777ACN-122012777-A

Abstract

The invention relates to a primer group for rapidly detecting amanita pseudooomycetes, application of the primer group and a kit containing the primer group, which belong to the technical field of nucleic acid detection, and the nucleotide sequence of the primer group is shown as SEQ ID No. 1-5; the invention also discloses application of the primer group, a kit containing the primer group and an LAMP amplification method for carrying out LAMP amplification on amanita pseudoopathiae genes by using the primer group, wherein the method adopts LAMP amplification to detect the amanita pseudoopae, can detect only 10-20mg of sample, can finish sample DNA extraction and detection result judgment in 1 hour, and has high detection sensitivity and stable and reliable result.

Inventors

  • GUO QIXIN
  • ZHANG LEI
  • SHU PING
  • CHEN PENGYUN
  • ZHANG YAN
  • XU XING
  • YANG WEIHUA
  • MAO YONGYANG

Assignees

  • 大理白族自治州检验检测院

Dates

Publication Date
20260512
Application Date
20260205

Claims (9)

  1. 1. The Primer group for rapidly detecting amanita pseudooomycetes is characterized by comprising primers A-NEO Primer F3, A-NEO Primer B3, A-NEO PRIMER FIP, A-NEO Primer BIP and A-NEO Primer LF, wherein the nucleotide sequence of the Primer group is as follows; the nucleotide sequence of the A-NEO F3 is shown as SEQ ID No. 1; The nucleotide sequence of the A-NEO B3 is shown as SEQ ID No. 2; the nucleotide sequence of the A-NEO FIP is shown in SEQ ID No. 3; the nucleotide sequence of NEO BIP is shown as SEQ ID No. 4; the nucleotide sequence of the A-NEO LF is shown as SEQ ID No. 5.
  2. 2. The use of the primer set according to claim 1 for detecting amanita pseudooomycetes.
  3. 3. A kit for detecting amanita pseudoovale, comprising the primer according to claim 1.
  4. 4. A method for rapid detection of amanita qualis, characterized in that the primer set according to claim 1 is used.
  5. 5. The method according to claim 4, wherein the Amanita pseudoganii is detected after amplification by LAMP using the primer set according to claim 1.
  6. 6. The method according to claim 4, comprising the steps of: (1) Rapidly extracting amanita pseudoootheca DNA by a filter paper method; (2) Amplification using sample DNA as a template for LAMP amplification, wherein the nucleic acid molecule primer set is as defined in claim 1: (3) Observing color change of the LAMP amplification product obtained in the step (2), and after the amplification reaction is finished, changing the color of the reaction solution from violet to sky blue, thereby identifying that the sample is amanita pseudooomycetes or contains amanita pseudooomycetes.
  7. 7. The method according to claim 6, wherein in step (1), the following are specifically included: s1, selecting a fungus cover part of a sample, and grinding the fungus cover part into powder in liquid nitrogen; S2, taking a ground sample, putting the ground sample into a 1.5mL centrifuge tube, adding a cracking solution, and swirling the cracking solution to uniformly mix the cracking solution to obtain a crude extract; s3, immersing the nucleic acid binding region of the filter paper strip into the crude extract for 3-5S each time, wherein the total time is 3 times; s4, immersing the filter paper strip into the eluent to wash out impurities.
  8. 8. The method of claim 7, wherein the step of determining the position of the probe is performed, The lysate consists of mixed solution of 0.06% SDS, 0.125 mol/L NaCl, 0.01mol/L Tris-HCl buffer solution and 1mmol/L EDTA, wherein the pH=8.0; The eluent consists of a mixed solution of 0.01mol/L Tris-HCl buffer solution and 1.5% Tween-20, wherein the pH=7.8-8.2; The filter paper strip used has a pore size of 1-3 μm and a specification of (40-50) mm× mm, and consists of a hydrophobic region with one end immersed in melted solid paraffin to form (40-50) mm× mm and a DNA junction region with one end (2-4) mm× mm.
  9. 9. The method of claim 6, wherein the LAMP amplification reaction system of step (2) is, 320U/mL of 1×Isothermal Amplificationg buffer,MgSO 4 4~8mmol,dNTP mix 1.4μM,F3/B3 0.16μM,FIP/BIP 1.20μM,LF 0.32μM,Bst DNA polymerase containing 2 mmolMg 2+ , 120 mu M of HNB, template extracted by a DNA filter paper strip method and the balance of ddH 2 O.

Description

Primer group for rapidly detecting amanita pseudooomycetes, application of primer group and kit containing primer group Technical Field The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer group for rapidly detecting amanita pseudooomycetes, application of the primer group and a kit comprising the primer group. Background The amanita is similar to the amanita and the amanita deliciosa in morphology, has the same growing season and growing environment, is mixedly grown together and is easy to be eaten by people, and the amanita deliciosa contains internal transcribed spacer ITS genes to express protein amanita peptide toxoid to have severe toxicity, so that acute renal failure type poisoning is caused, renal failure of patients is caused, liver is accumulated, the death rate is extremely high, and the appearance of the amanita deliciosa is as shown in figure 1. In the aspect of the rapid detection method, the molecular biological detection technology is generally developed based on the extracted DNA, the complexity of the nucleic acid extraction method limits the use of a plurality of nucleic acid amplification technologies outside a laboratory environment, the rapid nucleic acid extraction method based on fiber filter paper adsorption effectively avoids ethanol precipitation, phenol-chloroform extraction, centrifugation and column chromatography treatment in the traditional method, reduces sample loss and pollution, improves sample treatment efficiency, compared with the rapid nucleic acid extraction method developed based on magnetic materials and silica matrix materials, the fiber filter paper adsorption method can obtain satisfactory DNA within 1min, has the advantages of simple and convenient operation, low price, rapid extraction and the like, and the cellulose filter paper carrying nucleic acid does not need to elute the method for extracting genome DNA of biological samples, so that the technology does not need to prepare corresponding lysate and eluent in the extraction of genome DNA of mushrooms in the biological samples. At present, aiming at the defects of more methods developed by mushrooms based on molecular biology technology, PCR amplification adopted mostly, long detection time, high dependence degree of detection instruments and the like, the method for rapidly identifying amanita pseudooomycetes is provided, and has positive significance. Disclosure of Invention Aiming at the problems in the background technology, the invention provides a primer group for rapidly detecting amanita pseudoovalis, and the primer group can effectively detect the amanita pseudoovalis. The primer group is used for detection by adopting a loop-mediated isothermal amplification (LAMP) technology, and the amanita pseudooomycetes sample or the amanita pseudooomycetes doped in the edible fungi can be identified within 1h, so that the method is simple, convenient and quick. Based on this, the first object of the present invention is to improve the rapid detection of amanita pseudoovalis, the Primer set consisting of primers A-NEO Primer F3, A-NEO Primer B3, A-NEO PRIMER FIP, A-NEO Primer BIP and A-NEO Primer LF, the nucleotide sequence of the Primer set being, The nucleotide sequence of the A-NEO F3 is shown as SEQ ID No. 1; The nucleotide sequence of the A-NEO B3 is shown as SEQ ID No. 2; the nucleotide sequence of the A-NEO FIP is shown in SEQ ID No. 3; The nucleotide sequence of the A-NEO BIP is shown as SEQ ID No. 4. The nucleotide sequence of the A-NEO LF is shown as SEQ ID No. 5. The second object of the invention is to provide the application of the primer group in detecting amanita pseudooomycetes. The third object of the present invention is to provide a kit for detecting amanita pseudooomycetes comprising the above primer set. The fourth object of the present invention is to provide a method for rapidly detecting amanita pseudoegg cover, which uses the above-mentioned primer set. Further, the Amanita pseudoganii is amplified by LAMP method by using the primer set and then detected. Further, the method comprises the following steps: (1) Rapidly extracting amanita pseudoootheca DNA by a filter paper method; (2) Amplification using sample DNA as a template for LAMP amplification, wherein the nucleic acid molecule primer set is as defined in claim 1: (3) And (3) observing color change of the LAMP amplification product in the step (2), and after the amplification reaction is finished, changing the color of a reaction solution from violet to sky blue, thereby identifying that the sample is amanita pseudooomycetes or contains amanita pseudooomycetes. Further, in the step (1), the following are specifically included: s1, selecting a fungus cover part of a sample, and grinding the fungus cover part into powder in liquid nitrogen; S2, taking a ground sample, putting the ground sample into a 1.5mL centrifuge tube, adding a cracking solution, and swirling the crack