CN114717345B - CRISPR/Cas9 mediated isothermal nucleic acid amplification method for staphylococcus aureus detection, test strip and application thereof - Google Patents
CRISPR/Cas9 mediated isothermal nucleic acid amplification method for staphylococcus aureus detection, test strip and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biomedicine, in particular to a CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus, a test strip and application thereof, wherein primer pairs for the staphylococcus aureus are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. The CRISPR/Cas9 comprises Cas9 protein and sgRNA, and the sgRNA sequence is shown as SEQ ID NO. 3. The lateral flow chromatography technology comprises a supporting plate, wherein the upper end face of the supporting plate comprises a loading area, a marking area, a scribing area and a water absorption area which are sequentially arranged, a gold-labeled antibody is borne on the marking area, a detection line T combining with a capture probe and a quality control line C combining with a secondary antibody are arranged in the scribing area, and the sequence of the capture probe is shown as SEQ ID NO.4. The detection method provided by the invention has the advantages of good specificity, high sensitivity, good specificity, good repeatability, simplicity in operation and strong applicability.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus, a test strip and application thereof.
Background
Staphylococcus aureus is abbreviated as staphylococcus aureus, gram-positive coccus, aerobic or facultative anaerobic, and is arranged in a grape cluster shape. Is a common constant value bacterium of human skin and nasal cavity, is also a staphylococcus which is the most common cause of clinical infection, often causes local suppurative infection of furuncles, carbuncles, folliculitis, surgical wounds and wounds, and can cause suppurative infection of deep tissues after entering blood. In addition, the toxins produced can cause food poisoning, manifested as acute gastroenteritis. Therefore, in view of the above, it is important to develop a method for rapidly, reliably and conveniently detecting staphylococcus aureus.
There are many methods developed at present for detecting staphylococcus aureus, and according to the research of students at home and abroad, three types of detection methods mainly detect staphylococcus aureus infection: traditional culture methods, immunological methods and molecular biological methods. Most methods do not draw conclusions accurately and quickly. For example, the conventional culture method has high accuracy and the minimum detection limit can reach zero. However, the steps are complicated, the detection time is long, and the requirement of on-site rapid detection cannot be met; ELISA in the immunological method is used as one of the most mature detection technologies, has the characteristics of high sensitivity and strong specificity, but the sensitivity and the specificity of detection are easily influenced by factors such as antibody adsorption capacity, antibody specificity, matrix background interference and the like, and can not meet the detection requirements of various samples with complex components; compared with the traditional culture method, the PCR technology in the molecular biology method has the characteristics of strong specificity, simplicity and high sensitivity, but the PCR technology which has a temperature control function and is greatly limited by hybridization or special instrument detection characterization means after the hydrolysis of the electrophoresis amplification product is widely applied. Therefore, the invention is a simple, convenient and sensitive staphylococcus aureus examination method, and has important significance for areas with limited resources.
Disclosure of Invention
The invention aims to provide a CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus, a test strip and application thereof, so as to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions: a CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detection of staphylococcus aureus, comprising the steps of:
s1: extracting DNA in a staphylococcus aureus sample to be detected, using a lysate and a proteinase K solution to crack the sample to be detected, adding carboxyl modified magnetic beads and a coating solution, washing the obtained DNA by a washing solution, and then re-suspending by a heavy suspension to obtain the DNA of the detection sample;
s2: adopting a primer pair aiming at staphylococcus aureus, and carrying out RPA amplification for a period of time at a constant temperature to obtain a double-chain product with one end being functionalized by FITC;
s3: mixing a proper amount of Cas9 protein, sgRNA and double-stranded products in a loading buffer solution, incubating for a period of time, adding the complex into a detection area of a lateral flow chromatography test strip for detection, and realizing qualitative or quantitative detection of staphylococcus aureus in a sample to be detected according to a color development result of a detection line.
Preferably, the primer pair comprises a first primer and a second primer, the sequences of the first primer and the second primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the 5' end of the first primer is marked by FITC.
Preferably, the sgRNA has a sequence shown in SEQ ID NO. 3.
Preferably, the lysate comprises Tris-HCl buffer solution, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate, wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, the concentration is 5-20mmol/L, the concentration of ethylenediamine tetraacetic acid is 1-25mmol/L, the concentration of sodium chloride is 100-600mmol/L, and the mass-volume ratio of the sodium dodecyl sulfate to the lysate is 0.5-1.5g/100mL;
the final concentration of the proteinase K solution is 20mg/mL;
the coating liquid comprises polyethylene glycol and sodium chloride, wherein the mass average molecular weight of the polyethylene glycol is 400-20000, the mass volume ratio of the polyethylene glycol to the coating liquid is 10-20g/100mL, and the concentration of the sodium chloride in the coating liquid is 0.2-2.0mol/L;
the washing liquid comprises an aqueous solution with the ethanol content of 50-75vt percent;
the heavy suspension comprises Tris-HCl buffer solution and ethylenediamine tetraacetic acid, wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, the concentration is 5-20mmol/L, and the concentration of the ethylenediamine tetraacetic acid is 1-25mmol/L.
The concentration of the primer pair for RPA amplification is 200-800nmol/L;
the constant temperature is 35-42 ℃;
the RPA amplification time is 15-40min.
The concentration of the Cas9 is 50-500nmol/L;
the concentration of the sgRNA is 50-500nmol/L;
the volume of the double-stranded product is 1-20 mu L;
the loading buffer solution comprises PB buffer solution, sodium chloride and polyethylene glycol, wherein the pH value of the PB buffer solution is 7.0-8.6, the concentration of the PB buffer solution is 10-100nmol/L, the concentration of the sodium chloride is 50-500mmol/L, the volume of the buffer solution is 5-15 mu L, the molecular weight of the polyethylene glycol is 200-10000, and the mass-volume ratio of the polyethylene glycol to the buffer solution is 0.5-2.0g/100mL;
the incubation time is 1-10min.
A lateral flow chromatography test strip;
the lateral flow chromatography test strip comprises a support plate, wherein the upper end face of the support plate comprises a loading area, a marking area and a water absorption area, the loading area, the marking area and the water absorption area are sequentially arranged along a set direction, a detection line T and a quality control line C are arranged in the marking area, a capture probe and a secondary antibody are respectively combined with the detection line and the quality control line, and the sequence of the capture probe is shown as SEQ ID NO.4.
Preferably, the FITC antibody is selected from monoclonal antibodies of FITC, and the concentration is 0.5-3.0mg/mL; the gold nano particles are 15-40nm; the capture probe is fixed to the streaking area through the action of streptavidin-biotin, the concentration of the capture probe is 5-100 mu mol/L, and the concentration of the streptavidin is 0.5-4.0mg/mL; the secondary antibody is rabbit anti-mouse antibody selected from FITC antibody, and the concentration is 0.5-3.0mg/mL.
Preferably, the support plate comprises a cardboard, a plastic plate or a polystyrene plate that is non-absorbent or hydrophobic; the material of the loading area comprises glass fiber cotton or polyester film; the scribing area is made of a nitrocellulose membrane or a pure cellulose membrane; the material of the water absorption area comprises filter paper.
The preparation method of the lateral flow chromatography test strip in the CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus comprises the following steps:
a1: coupling gold nanoparticles with FITC antibody, regulating the pH value of a gold nanoparticle solution to 6.0-7.0 by using a potassium carbonate solution with the concentration of 10-100mmol/L, adding the FITC antibody for incubation, and then adding a BSA solution for blocking to obtain FITC antibody-coupled gold nanoparticles;
a2: applying FITC antibody coupled gold nanoparticles to a marking area, mixing a capture probe and streptavidin, fixing the mixture to form a detection line T of a marking area, and fixing a secondary antibody to form a quality control line C of the marking area;
a3: and sequentially arranging the loading area, the marking area, the scribing area and the water absorbing area on the support supporting plate along the set direction to obtain the lateral flow chromatography test strip.
Application of lateral flow chromatography test strips:
the lateral flow chromatography test strip is used for detecting a sample of staphylococcus aureus.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, when staphylococcus aureus exists, a corresponding double-stranded product with one end functionalized by FITC is obtained through RPA amplification, a specific site of the double-stranded product is subject to unwinding by means of the targeted recognition effect of a CRISPR/Cas9 system, and then a complex generated by combining Cas9/sgRNA with the double-stranded product is hybridized with a gold-labeled antibody and a capture probe respectively to generate a colored strip, so that whether the double-stranded product is infected by staphylococcus aureus is judged;
2. the detection method has the advantages of good specificity, high sensitivity (10 CFU/mL), good specificity, good repeatability, simple operation and strong applicability;
3. compared with the traditional PCR technology, the recombinase polymerization amplification technology adopted by the invention has remarkable advantages, such as isothermal amplification conditions avoid the use of expensive temperature control instruments, get rid of the constraint of the instruments, and are more suitable for field detection; the CRISPR/Cas9 system is adopted to specifically identify double-stranded products, so that primer dimer interference is avoided; the primer similar to PCR is used, the design is simple, and the cost is low; the packaging is simple, and the powder can exist in a dry powder form, so that the powder is favorable for preservation; the operation is simple, special technical training and the like are not needed;
4. the detection method of the invention does not need a special precious instrument, and can realize the rapid and accurate detection of staphylococcus aureus only by a common water bath kettle and a corresponding lateral flow chromatography test strip.
Drawings
FIG. 1 is a schematic representation of the screening and detection of Staphylococcus aureus infection in an actual clinical sample using a lateral flow chromatography method in example 1 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus, which comprises the following steps:
s1: extracting DNA in a staphylococcus aureus sample to be detected, using a lysate and a proteinase K solution to crack the sample to be detected, adding carboxyl modified magnetic beads and a coating solution, washing the obtained DNA by a washing solution, and then re-suspending by a heavy suspension to obtain the DNA of the detection sample;
s2: adopting a primer pair aiming at staphylococcus aureus, and carrying out RPA amplification for a period of time at a constant temperature to obtain a double-chain product with one end being functionalized by FITC;
s3: mixing a proper amount of Cas9 protein, sgRNA and double-stranded products in a loading buffer solution, incubating for a period of time, adding the complex into a detection area of a lateral flow chromatography test strip for detection, and realizing qualitative or quantitative detection of staphylococcus aureus in a sample to be detected according to a color development result of a detection line.
The primer pair comprises a first primer and a second primer, the specific primers are specific to staphylococcus aureus, the specific and effective amplification of the target staphylococcus aureus can be realized, the sequences of the first primer and the second primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, the 5' end of the first primer is marked by FITC, the first primer is an upstream primer functionalized by FITC, and the specific sequences of the first primer and the second primer are shown in a sequence table in detail; the invention realizes effective amplification of staphylococcus aureus based on the combination of the upstream primer and the downstream primer, and realizes the FITC mark at one end of an amplified product, thereby being a key design for detecting staphylococcus aureus based on lateral flow chromatography of the amplified product.
The sgRNA has a sequence shown in SEQ ID NO.3, and the specific sequence is shown in a sequence table.
A lateral flow chromatography test strip;
the lateral flow chromatography test strip is used for detecting a sample of staphylococcus aureus and comprises a support plate, the upper end face of the support plate comprises a loading area, a marking area, a scribing area and a water absorption area, the loading area, the marking area, the scribing area and the water absorption area are sequentially arranged along a set direction, a detection line T and a quality control line C are arranged in the scribing area, a capture probe and a secondary antibody are respectively combined with the detection line and the quality control line, the sequence of the capture probe is shown as SEQ ID NO.4, and the specific sequence is shown in a sequence table.
FITC antibody is selected from monoclonal antibody of FITC with concentration of 0.5-3.0mg/mL; gold nanoparticles are 15-40nm; the capture probe is fixed to the streaking area through the action of streptavidin-biotin, the concentration of the capture probe is 5-100 mu mol/L, and the concentration of streptavidin is 0.5-4.0mg/mL; the secondary antibody is rabbit anti-mouse antibody selected from FITC antibody, and the concentration is 0.5-3.0mg/mL.
The supporting plate comprises a paper plate, a plastic plate or a polystyrene plate which is not water-absorbing or hydrophobic; the material of the sample loading area comprises glass fiber cotton or polyester film; the scribing area is made of nitrocellulose membrane or pure cellulose membrane; the material of the water absorption area comprises filter paper.
The preparation method of the lateral flow chromatography test strip in the CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus comprises the following steps:
a1: coupling gold nanoparticles with FITC antibody, regulating the pH value of a gold nanoparticle solution to 6.0-7.0 by using a potassium carbonate solution with the concentration of 10-100mmol/L, adding the FITC antibody for incubation, and then adding a BSA solution for blocking to obtain FITC antibody-coupled gold nanoparticles;
a2: applying FITC antibody coupled gold nanoparticles to a marking area, mixing a capture probe and streptavidin, fixing the mixture to form a detection line T of a marking area, and fixing a secondary antibody to form a quality control line C of the marking area;
a3: and sequentially arranging the loading area, the marking area, the scribing area and the water absorbing area on the support supporting plate along the set direction to obtain the lateral flow chromatography test strip.
In some more specific embodiments, the method for preparing a nucleic acid test strip for detecting staphylococcus aureus comprises the following steps:
1) Preparation of FITC antibody-conjugated gold nanoparticles: coupling the FITC antibody with gold nanoparticles coated with trisodium citrate to prepare FITC antibody-coupled gold nanoparticles;
2) Preparation of the marker region: dripping a proper amount of FITC antibody-coupled gold nanoparticles into each marking area, drying in a baking oven at 30-36 ℃ and preserving for later use;
3) Preparation of a scribing area: spraying a capture probe combined with streptavidin to a detection line T of a line drawing area by using a film spraying instrument, spraying a secondary antibody to a quality control line C of the line drawing area, drying in a baking oven at 30-36 ℃ and preserving for later use;
4) Assembling a test strip: the sample loading area, the marking area, the scribing area and the water absorbing area are sequentially assembled on the supporting plate to form the lateral flow chromatography test strip for detecting staphylococcus aureus infection.
Further, the preparation method of the FITC antibody-coupled gold nanoparticle in the step 1) specifically comprises the following steps: dropwise adding a proper amount of potassium carbonate solution (10-100 mmol/L) into the gold nanoparticle solution to enable the pH value of a reaction system to be 6.0-7.0; adding a proper amount of FITC antibody, and incubating for 0.5-2h at room temperature; adding a BSA solution with the mass ratio of 5% -15%, and incubating for 0.5-2h at room temperature; centrifuging (7000-10000 g, 7-15 min), removing supernatant, adding BSA solution with mass-volume ratio of 0.5-3%, resuspending, and preserving at 4deg.C.
Further, the gold nanoparticles have a particle diameter of 15-40nm.
In some embodiments, the detection method specifically includes: extracting DNA of a staphylococcus aureus sample to be detected by using a magnetic bead method, and performing RPA amplification by using a functionalized primer pair specific to the staphylococcus aureus to obtain a double-stranded product with one end being functionalized by FITC; mixing the amplification product with the Cas9/sgRNA to obtain a double-stranded composite product which is recognized and unwound by the Cas 9/sgRNA; the composite product is directly dripped into a loading area of the test strip, flows through the marking area and the scribing area, and is connected onto a detection line T by using the FITC antibody coupled gold nanoparticles, and the color depth on the detection line is directly observed by naked eyes, so that the rapid and accurate detection of staphylococcus aureus is realized.
In some embodiments, the method of detecting staphylococcus aureus specifically comprises: the method comprises the steps of (1) cracking a sample of staphylococcus aureus to be detected by adopting a cell cracking solution and a proteinase K solution, adding carboxyl modified magnetic beads and a coating solution, washing the obtained DNA by using a washing solution, and then re-suspending the DNA by using a heavy suspension to obtain the DNA in the sample of staphylococcus aureus to be detected; adopting a primer pair aiming at staphylococcus aureus, and carrying out RPA amplification for a period of time at a constant temperature to obtain a double-chain product with one end being functionalized by FITC; mixing a proper amount of Cas9 protein, sgRNA and double-stranded products in a loading buffer solution, incubating for a period of time, adding the complex into a detection area of a lateral flow chromatography test strip for detection, and realizing qualitative or quantitative detection of staphylococcus aureus in a sample to be detected according to a color development result of a detection line.
Further, the magnetic beads used in the magnetic bead method are carboxyl modified magnetic nano particles, and the specific scheme is as follows: taking 100-500 mu L of staphylococcus aureus sample, adding 300-1000 mu L of cell lysate and 10-50 mu L of proteinase K solution to completely lyse the sample; adding 10-20 mug carboxyl modified magnetic beads and 200-750 mug coating liquid to adsorb and separate staphylococcus aureus DNA, washing with 500-1500 mug washing liquid, adding 30-90 mug heavy suspension to resuspend, getting the supernatant as the extracted DNA.
Further, the lysate is a solution containing Tris-HCl buffer, ethylenediamine tetraacetic acid (EDTA), sodium chloride (NaCl) and Sodium Dodecyl Sulfate (SDS); wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, and the concentration is 5-20mmol/L; EDTA is 1-25mmol/L, sodium chloride (NaCl) is 100-600mmol/L, and the mass-volume ratio of sodium dodecyl sulfate to lysate is 0.5-1.5g/100mL, i.e., SDS is 0.5-1.5%.
Further, the final concentration of proteinase K solution is 0.25-2.5mg/mL.
Further, the coating liquid mainly comprises polyethylene glycol (PEG) and sodium chloride (NaCl), wherein the average molecular weight of the polyethylene glycol is 400-20000, such as 400, 600, 800, 1000, 2000, 6000, 10000 or 20000, the mass-volume ratio of the polyethylene glycol to the coating liquid is 10-20g/100mL, that is, the mass-volume ratio of the PEG is 10-20%, and the concentration of the sodium chloride in the coating liquid is 0.2-2.0mol/L.
Further, the washing liquid comprises an aqueous solution with an ethanol content of 50-75 vt%.
Further, the main components of the heavy suspension comprise Tris-HCl buffer solution and ethylenediamine tetraacetic acid (EDTA), wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, the concentration is 5-20mmol/L, and the concentration of the ethylenediamine tetraacetic acid (EDTA) is 1.0-25mmol/L.
Further, the concentration of the primer pair for RPA amplification is 200-800nmol/L;
further, the constant temperature is 35-42 ℃;
further, the RPA amplification time is 15-40min.
Further, the concentration of Cas9 is 50-500nmol/L;
further, the sgRNA concentration is 50-500nmol/L;
further, the double-stranded product volume is 1-20. Mu.L;
further, the loading buffer solution comprises PB buffer solution, sodium chloride and polyethylene glycol, wherein the pH value of the PB buffer solution is 7.0-8.6, the concentration of the PB buffer solution is 10-100nmol/L, the concentration of the sodium chloride is 50-500mmol/L, the volume of the buffer solution is 5-15 mu L, the mass average molecular weight of the polyethylene glycol is 200-10000, and the mass volume ratio of the polyethylene glycol to the buffer solution is 0.5-2.0g/100mL;
further, the incubation time is 1-10min.
In summary, the implementation steps of the detection method of the invention are as follows: (1) preparing FTIC antibody coupled gold nano particles; (2) embedding a line drawing area; (3) manufacturing a test strip; (4) extracting staphylococcus aureus sample DNA and amplifying RPA; (5) and (5) visual detection. According to the invention, a functional primer pair specific to staphylococcus aureus is used, a CRISPR/Cas9 system is utilized to specifically identify and unwind double-stranded products, the content of amplified products is different according to the difference of the content of target staphylococcus aureus in a sample to be detected, and the color depth of a detection line on a test strip is also different, so that the relation between the color depth of the detection line and the content of the corresponding staphylococcus aureus is established, and the aim of sensitively detecting the staphylococcus aureus in the sample is further realized.
The following description of the present invention is further provided with reference to the accompanying drawings and several preferred embodiments, but the experimental conditions and setting parameters should not be construed as limiting the basic technical scheme of the present invention. And the scope of the present invention is not limited to the following examples.
Example 1
The lateral flow chromatography detection method for detecting staphylococcus aureus in a blood sample provided by the embodiment comprises the following steps:
(1) preparation of FTIC antibody-conjugated gold nanoparticles: dropwise adding 10mmol/L potassium carbonate solution with proper concentration into the gold nanoparticle solution to enable the pH value of a reaction system to be 6.0; adding a proper amount of FITC antibody, and incubating for 0.5h at room temperature; adding a BSA solution with the mass ratio of 5%, and incubating for 0.5h at room temperature; centrifugation (70000 g,7 min), removal of supernatant, resuspension with 0.5% by mass/volume BSA solution and storage at 4 ℃.
(2) Embedding a scribing region: printing the streptavidin combined-capture probe compound and the secondary antibody solution to a detection line T and a quality control line C respectively by using a film spraying instrument, drying in a baking oven at 30 ℃ and storing for later use.
(3) And (3) manufacturing a test strip: and respectively assembling the loading area, the marking area, the scribing area and the water absorbing area on the supporting plate in sequence.
(4) Extraction and RPA amplification of staphylococcus aureus sample DNA to be detected
The DNA is extracted by a magnetic bead method, and carboxyl modified magnetic nano particles are used as a carrier for DNA extraction, and the specific implementation mode is as follows: 100. Mu.L of staphylococcus aureus sample, 300. Mu.L of cell lysate and 10. Mu.L of proteinase K solution are added into a clean centrifuge tube, and incubated at 60 ℃ for 15min; cooling at room temperature, adding 10 μg carboxyl modified magnetic beads and 200 μl coating solution, and rotating at room temperature for 3min; magnetically adsorbing, removing supernatant, and adding 500 mu L of washing liquid to wash twice; magnetically adsorbing, removing supernatant, adding 30 mu L of heavy suspension for resuspension, magnetically adsorbing, and taking the supernatant as the extracted DNA.
The RPA reaction comprises the following specific processes: the RPA dry powder reagent was dissolved in 29.5. Mu.L of hybridization buffer, 0.8. Mu.L of each of 10. Mu.M of upstream and downstream primers of Staphylococcus aureus was added, 2. Mu.L of DNA template was added, water was added to 47.5. Mu.L, and the mixture was immediately reacted at 40℃for 20 minutes after adding 2.5. Mu.L of 280mM magnesium acetate solution.
(5) Visual inspection
In the embodiment, the RPA product in step (4) is directly dripped into the loading area of the test strip after being diluted by the loading buffer solution through the complex formed after incubation with the Cas9 protein and the sgRNA, the reaction is carried out for 1min at room temperature, a picture is taken by using a single-phase inverter, and the intensity of the detection line T is analyzed by using ImageJ software.
Example 2
The lateral flow chromatography detection method for detecting staphylococcus aureus in urine samples provided by the embodiment comprises the following steps:
(1) preparation of FTIC antibody-conjugated gold nanoparticles: dropwise adding 100mmol/L potassium carbonate solution with proper concentration into the gold nanoparticle solution to enable the pH value of a reaction system to be 7.0; adding a proper amount of FITC antibody, and incubating for 2 hours at room temperature; adding a BSA solution with the mass ratio of 15%, and incubating for 2 hours at room temperature; centrifugation (10000 g,15 min), removal of supernatant, resuspension with 3% by weight of BSA solution and storage at 4 ℃.
(2) Embedding a scribing region: and printing the streptavidin combined-capture probe complex and the secondary antibody solution on a detection line T and a quality control line C respectively by using a film spraying instrument, drying in a 36 ℃ oven, and storing for later use.
(3) And (3) manufacturing a test strip: and respectively assembling the loading area, the marking area, the scribing area and the water absorbing area on the supporting plate in sequence.
(4) Extraction and RPA amplification of staphylococcus aureus sample DNA to be detected
The DNA is extracted by a magnetic bead method, and carboxyl modified magnetic nano particles are used as a carrier for DNA extraction, and the specific implementation mode is as follows: adding 500 mu L of staphylococcus aureus sample, 1000 mu L of cell lysate and 10 mu L of proteinase K solution into a clean centrifuge tube, and incubating at 60 ℃ for 15min; cooling at room temperature, adding 10 μg carboxyl modified magnetic beads and 750 μl coating solution, and rotating at room temperature for 3min; magnetically adsorbing, removing supernatant, and adding 1500 mu L of washing liquid to wash twice; magnetically adsorbing, removing supernatant, adding 90 mu L of heavy suspension for resuspension, magnetically adsorbing, and taking the supernatant as the extracted DNA.
The RPA reaction comprises the following specific processes: the RPA dry powder reagent was dissolved in 29.5. Mu.L of hybridization buffer, 0.8. Mu.L of each of 10. Mu.M of upstream and downstream primers of Staphylococcus aureus was added, 2. Mu.L of DNA template was added, water was added to 47.5. Mu.L, and the mixture was immediately reacted at 40℃for 20 minutes after adding 2.5. Mu.L of 280mM magnesium acetate solution.
(5) Visual inspection
In the embodiment, the RPA product in step (4) is directly dripped into the loading area of the test strip after being diluted by the loading buffer solution through the complex formed after incubation with the Cas9 protein and the sgRNA, the reaction is carried out for 10min at room temperature, a single-phase inverter is used for shooting pictures, and the intensity of the detection line T is analyzed by using ImageJ software.
In summary, by the above technical scheme, when staphylococcus aureus exists, a corresponding double-stranded amplification product with one end being subjected to FITC functionalization is obtained through RPA amplification, the double-stranded amplification product is identified and unwound by using a CRISPR/Cas9 system, a complex is obtained, and then FITC-labeled gold nanoparticles are connected to a detection line by using the complex, so that a colored strip is generated. According to different staphylococcus aureus contents, the amount of the generated compound is different, so that the colors of different detection lines are different in shade, the relation between the colors of different detection lines and the staphylococcus aureus content is established, and the purposes of sensitively screening and detecting the staphylococcus aureus are realized. The method has the advantages of good specificity, high sensitivity, good repeatability, simple operation and strong adaptability, and can detect staphylococcus aureus with the concentration of 10.CFU/mL. The method can realize rapid and accurate detection of staphylococcus aureus by only using a common water bath kettle and a corresponding lateral chromatography test strip without a special precious instrument, and is suitable for detecting hardware resource deficiency areas to carry out related work.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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Claims (3)
1. A CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detection of staphylococcus aureus for non-disease diagnosis purposes, characterized by: the method comprises the following steps:
s1: extracting DNA in a staphylococcus aureus sample to be detected, using a lysate and a proteinase K solution to crack the sample to be detected, adding carboxyl modified magnetic beads and a coating solution, washing the obtained DNA by a washing solution, and then re-suspending by a heavy suspension to obtain the DNA of the detection sample;
s2: adopting a primer pair aiming at staphylococcus aureus, and carrying out RPA amplification for a period of time at a constant temperature to obtain a double-chain product with one end being functionalized by FITC;
s3: mixing a proper amount of Cas9 protein, sgRNA and double-stranded products in a loading buffer solution, incubating for a period of time, adding the complex into a detection area of a lateral flow chromatography test strip for detection, and realizing qualitative or quantitative detection of staphylococcus aureus in a sample to be detected according to a color development result of a detection line;
the primer pair comprises a first primer and a second primer, the sequences of the first primer and the second primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the 5' end of the first primer is marked by FITC;
the sequence of the sgRNA is shown as SEQ ID NO. 3;
the lysate comprises Tris-HCl buffer solution, ethylenediamine tetraacetic acid, sodium chloride and sodium dodecyl sulfate, wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, the concentration is 5-20mmol/L, the concentration of ethylenediamine tetraacetic acid is 1-25mmol/L, the concentration of sodium chloride is 100-600mmol/L, and the mass volume ratio of the sodium dodecyl sulfate to the lysate is 0.5-1.5g/100mL;
the final concentration of the proteinase K solution is 20mg/mL;
the coating liquid comprises polyethylene glycol and sodium chloride, wherein the mass average molecular weight of the polyethylene glycol is 400-20000, the mass volume ratio of the polyethylene glycol to the coating liquid is 10-20g/100mL, and the concentration of the sodium chloride in the coating liquid is 0.2-2.0mol/L;
the washing liquid comprises an aqueous solution with the ethanol content of 50-75vt percent;
the heavy suspension comprises Tris-HCl buffer solution and ethylenediamine tetraacetic acid, wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, the concentration is 5-20mmol/L, and the concentration of the ethylenediamine tetraacetic acid is 1-25mmol/L;
the concentration of the primer pair for RPA amplification is 200-800nmol/L;
the constant temperature is 35-42 ℃;
the RPA amplification time is 15-40min;
the concentration of the Cas9 is 50-500nmol/L;
the concentration of the sgRNA is 50-500nmol/L;
the volume of the double-stranded product is 1-20 mu L;
the loading buffer solution comprises PB buffer solution, sodium chloride and polyethylene glycol, wherein the pH value of the PB buffer solution is 7.0-8.6, the concentration of the PB buffer solution is 10-100nmol/L, the concentration of the sodium chloride is 50-500mmol/L, the volume of the buffer solution is 5-15 mu L, the molecular weight of the polyethylene glycol is 200-10000, and the mass-volume ratio of the polyethylene glycol to the buffer solution is 0.5-2.0g/100mL;
the incubation time is 1-10min;
the lateral flow chromatography test strip comprises a support plate, wherein the upper end surface of the support plate comprises a loading area, a marking area, a scribing area and a water absorption area, wherein the loading area, the marking area, the scribing area and the water absorption area are sequentially arranged along a set direction, a detection line T and a quality control line C are arranged in the scribing area, a capture probe and a secondary antibody are respectively combined with the detection line and the quality control line, and the sequence of the capture probe is shown as SEQ ID NO.4;
the FITC antibody is selected from monoclonal antibodies of FITC, and the concentration is 0.5-3.0mg/mL; the gold nano particles are 15-40nm; the capture probe is fixed to the streaking area through the action of streptavidin-biotin, the concentration of the capture probe is 5-100 mu mol/L, and the concentration of the streptavidin is 0.5-4.0mg/mL; the secondary antibody is rabbit anti-mouse antibody selected from FITC antibody, and the concentration is 0.5-3.0mg/mL.
2. The CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for detection of staphylococcus aureus for non-disease diagnostic purposes according to claim 1, characterized in that: the support plate comprises a paper plate, a plastic plate or a polystyrene plate which is not water-absorbent or hydrophobic; the material of the loading area comprises glass fiber cotton or polyester film; the scribing area is made of a nitrocellulose membrane or a pure cellulose membrane; the material of the water absorption area comprises filter paper.
3. The CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for detection of staphylococcus aureus for non-disease diagnostic purposes according to claim 1, characterized in that: the lateral flow chromatography test strip is used for detecting a sample of staphylococcus aureus.
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