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CN-122012761-A - Primer, kit and application for rapidly detecting aeromonas salmonicida by RPA-CRISPRCas12a

CN122012761ACN 122012761 ACN122012761 ACN 122012761ACN-122012761-A

Abstract

The invention discloses a primer, a kit and application for rapidly detecting aeromonas salmonicida by RPA-CRISPRCas12a, and relates to the technical field of aquaculture disease prevention and control. The invention solves the problems of time and labor consumption, easy pollution and need of matched professional detection equipment in the existing detection method, and the RPA amplification primer consists of a forward primer fstA-RPA-F2 and a reverse primer fstA-RPA-R2. The kit contains the RPA amplification primer and the crRNA. The method for rapidly detecting the aeromonas salmonicida is a test paper method and a fluorescence method. The method provided by the invention has the advantages of good inclusion, strong specificity, high sensitivity and the like, and the RPA product obtained by detecting based on the RPA primer and crRNA utilizes a lateral flow chromatography test strip and fluorescence detection to realize the purpose of portable visual rapid detection on site or a culture base, and has high clinical application value in early detection of diseases, epidemiological investigation and comprehensive prevention and control.

Inventors

  • ZHAO RAN
  • WANG JING
  • WANG DI
  • LI SHAOWU
  • SUN ZHIPENG

Assignees

  • 中国水产科学研究院黑龙江水产研究所

Dates

Publication Date
20260512
Application Date
20260306

Claims (7)

  1. 1. The RPA primer for rapidly detecting the aeromonas salmonicida by the RPA-CRISPRCas12a is characterized in that the RPA amplification primer for rapidly detecting the aeromonas salmonicida by the RPA-CRISPRCas12a consists of a forward primer fstA-RPA-F2 and a reverse primer fstA-RPA-R2, wherein the forward primer fstA-RPA-F2 is 5'-CCAGGAGCCGAGCCAGTAGGCATGATTGGTGA-3' and the reverse primer 5'-CAAAGTGATGGCCAAGCTGGGGCTGGAATACG-3'.
  2. 2. The method is used for rapidly detecting the crRNA of the aeromonas salmonicida by the RPA-CRISPRCas12a, and is characterized in that the crRNA sequence fstA-crRNA2 of the aeromonas salmonicida is 5'-UAAUUUCUACUAAGUGUAGAUUCGGCAAGCGCUAUGUCACCG-3'.
  3. A kit for rapid detection of aeromonas salmonicida by RPA-CRISPRCas12a, comprising the RPA amplification primer of claim 1 and the crRNA of claim 2.
  4. 4. The method for rapidly detecting aeromonas salmonicida is characterized by comprising the following specific steps of: 1. Preparing 50 mu L of RPA reaction total system, 29.5 mu L of RPA reaction reagent PRIMER FREE Rehydration Buffer, 2.4 mu L of primer fstA-RPA-F2 with the concentration of 10 mu mol/L, 2.4 mu L of primer fstA-RPA-R2 with the concentration of 10 mu mol/L, 2.5 mu L of magnesium acetate with the concentration of 280 mmol/L, 1 mu L of DNA template of a sample to be detected and the balance of nucleic-free H 2 O; 2. RPA isothermal amplification, namely reacting 20min under the reaction condition of 39 ℃ to obtain an RPA amplification product; 3. The RPA-CRISPR/Cas12a reaction comprises the steps of configuring a CRISPR reaction system, and reacting at a constant temperature of 37 ℃ for 30min, wherein the CRISPR reaction system comprises 2 mu L of Cas12a Reaction buffer x, 1 mu L of probe with the concentration of 5 mu mol/L, 1 mu L of Cas12a protein with the concentration of 1 mu mol/L, 1 mu L of crRNA with the concentration of 1 mu mol/L as described in claim 2, 4 mu L of RPA amplification product and 11 mu L of nucleic-free H 2 O; 4. Judging the result through a fluorescence curve or the color change of a product in the tube; Wherein the probe sequence and the fluorescent group are the fluorescent ssDNA reporter sequence of 5'-FAM-TTTAATTT-BHQ1-3'.
  5. 5. The method according to claim 4, wherein the preparation method of the RPA reaction system of the first step comprises adding all components except magnesium acetate and DNA template into a reaction tube containing RPA reaction reagent dry powder, thoroughly vortex mixing, centrifuging, opening the reaction tube, adding 2.5. Mu.L of magnesium acetate to the tube cap of the reaction tube, then adding template DNA into the reaction tube, thoroughly mixing and centrifuging.
  6. 6. The method for rapidly detecting aeromonas salmonicida is characterized by comprising the following specific steps of: 1. Preparing 50 mu L of RPA reaction total system, 29.5 mu L of RPA reaction reagent PRIMER FREE Rehydration Buffer, 2.4 mu L of primer fstA-RPA-F2 with the concentration of 10 mu mol/L, 2.4 mu L of primer fstA-RPA-R2 with the concentration of 10 mu mol/L, 2.5 mu L of magnesium acetate with the concentration of 280 mmol/L, 1 mu L of DNA template of a sample to be detected and the balance of nucleic-free H 2 O; 2. RPA isothermal amplification, namely reacting 20min under the reaction condition of 39 ℃ to obtain an RPA amplification product; 3. CRISPR/Cas12a reaction, namely, configuring a CRISPR reaction system, and reacting at a constant temperature of 37 ℃ for 30min, wherein the CRISPR reaction system comprises 2 mu L of Cas12a Reaction buffer x, 1 mu L of probe with concentration of 1 mu mol/L, 1 mu L of Cas12a protein with concentration of 1 mu mol/L, 1 mu L of crRNA with concentration of 1 mu mol/L as set forth in claim 2, 4 mu L of RPA amplification product and 11 mu L of nucleic-free H 2 O; 4. adding 10 mu L of CRISPR/Cas12a reaction product into a 1.5mL centrifuge tube containing 40 mu L of ddH 2 O, mixing, putting into a test strip, and waiting for 5-10 min to obtain a test paper detection result; wherein the probe sequence and the fluorescent group are the test paper type reporter sequence of 5'-FAM-TTTAATTT-Biotin-3'.
  7. 7. The method according to claim 6, wherein the preparation method of the RPA reaction system of the first step comprises adding all components except magnesium acetate and DNA template into a reaction tube containing RPA reaction reagent dry powder, thoroughly vortex mixing, centrifuging, opening the reaction tube, adding 2.5. Mu.L of magnesium acetate to the tube cap of the reaction tube, then adding template DNA into the reaction tube, thoroughly mixing and centrifuging.

Description

Primer, kit and application for rapidly detecting aeromonas salmonicida by RPA-CRISPRCas12a Technical Field The invention relates to the technical field of aquaculture disease prevention and control, in particular to a primer, a kit and application for rapidly detecting aeromonas salmonicida by RPA-CRISPRCas12 a. Background Aeromonas salmonicida (Aeromonas salmonicida) belongs to the genus Aeromonas and is one of main pathogenic bacteria for inducing furuncle and sore diseases of cold water fish. The strain not only infects salmon and trout, but also infects various warm water fishes, causes typical furuncle symptoms, is characterized by clinical symptoms such as furuncle, intestinal swelling, inflammation and the like on the body surface, causes high mortality rate, and brings serious economic loss to the aquaculture industry. Aiming at aeromonas salmonicida, bacterial pathogen detection is mostly adopted at present, however, the method is time-consuming, laborious and easy to pollute, and needs to rely on professional equipment, thus severely restricting the benign development of the aquaculture industry. Disclosure of Invention The invention provides a primer, a kit and application for rapidly detecting aeromonas salmonicida by using RPA-CRISPRCas12a, which are used for solving the problems of time and labor consumption, easiness in pollution and need of matching with professional detection equipment existing in the existing detection method. The RPA amplification primer for rapidly detecting aeromonas salmonicida by using the RPA-CRISPRCas12a consists of a forward primer fstA-RPA-F2 and a reverse primer fstA-RPA-R2, wherein the forward primer fstA-RPA-F2 is 5'-CCAGGAGCCGAGCCAGTAGGCATGATTGGTGA-3' and the reverse primer 5'-CAAAGTGATGGCCAAGCTGGGGCTGGAATACG-3'. The invention is used for rapidly detecting the Salmonella salmonicida aeromonas crRNA sequence fstA-crRNA2 of 5'-UAAUUUCUACUAAGUGUAGAUUCGGCAAGCGCUAUGUCACCG-3' by RPA-CRISPRCas12a The kit for rapidly detecting aeromonas salmonicida by using the RPA-CRISPRCas12a contains the RPA amplification primer and crRNA. The method for rapidly detecting aeromonas salmonicida comprises the following specific steps of: 1. Preparing 50 mu L of RPA reaction total system, namely 29.5 mu L of RPA reaction reagent PRIMER FREE Rehydration Buffer, 2.4 mu L of primer fstA-RPA-F2 with the concentration of 10 mu mol/L, 2.4 mu L of primer fstA-RPA-R2 with the concentration of 10 mu mol/L, 2.5 mu L of magnesium acetate with the concentration of 280 mmol/L (mM), 1 mu L of DNA template of a sample to be detected and the balance of nucleic-free H 2 O; 2. RPA isothermal amplification, namely reacting 20min under the reaction condition of 39 ℃ to obtain an RPA amplification product; 3. The RPA-CRISPR/Cas12a reaction comprises the steps of configuring a CRISPR reaction system, and reacting at a constant temperature of 37 ℃ for 30min, wherein the CRISPR reaction system comprises 2 mu L of Cas12a Reaction buffer x, 1 mu L of probe with the concentration of 5 mu mol/L, 1 mu L of Cas12a protein with the concentration of 1 mu mol/L (mu M), 1 mu L of crRNA (fstA-crRNA 2) with the concentration of 1 mu mol/L, 4 mu L of RPA amplification product and 11 mu L of nucleic-free H 2 O; 4. Judging the result through a fluorescence curve or the color change of a product in the tube; Wherein the probe sequence and the fluorescent group are the fluorescent ssDNA reporter sequence of 5'-FAM-TTTAATTT-BHQ1-3'. Further, the preparation method of the RPA reaction total system comprises the steps of adding all components except magnesium acetate and DNA templates into a reaction tube filled with RPA reaction reagent dry powder, fully vortex mixing, centrifuging, opening the reaction tube, adding 2.5 mu L of magnesium acetate on a tube cover of the reaction tube, then adding template DNA into the reaction tube, fully mixing and centrifuging. The method for rapidly detecting aeromonas salmonicida comprises the following specific steps of: 1. Preparing 50 mu L of RPA reaction total system, 29.5 mu L of RPA reaction reagent PRIMER FREE Rehydration Buffer, 2.4 mu L of primer fstA-RPA-F2 with the concentration of 10 mu mol/L, 2.4 mu L of primer fstA-RPA-R2 with the concentration of 10 mu mol/L, 2.5 mu L of magnesium acetate with the concentration of 280 mmol/L, 1 mu L of DNA template of a sample to be detected and the balance of nucleic-free H 2 O; 2. RPA isothermal amplification, namely reacting 20 min under the reaction condition of 39 ℃ to obtain an RPA amplification product; 3. CRISPR/Cas12a reaction, namely, configuring a CRISPR reaction system, and reacting at a constant temperature of 37 ℃ for 30min, wherein the CRISPR reaction system comprises 2 mu L of Cas12a Reaction buffer x, 1 mu L of probe with concentration of 1 mu mol/L, 1 mu L of Cas12a protein with concentration of 1 mu mol/L, 1 mu L of crRNA (fstA-crRNA 2) with concentration of 1 mu mol/L, 4 mu L of RPA amplification product and 11 mu L of Nuc