CN114717345A - CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus, test strip and application of method - Google Patents
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Abstract
The invention relates to the technical field of biomedicine, in particular to a CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus, a test strip and application thereof, wherein primer pairs aiming at the staphylococcus aureus are respectively shown as SEQ ID No.1 and SEQ ID No. 2. The CRISPR/Cas9 comprises a Cas9 protein and sgRNAs, and the sequence of the sgRNA is shown in SEQ ID NO. 3. The lateral flow chromatography technology comprises a supporting plate, wherein the upper end surface of the supporting plate comprises a sample 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 combined with a capture probe and a quality control line C combined 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 has the advantages of good specificity, high sensitivity, good specificity, good repeatability, simple operation and strong applicability.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus, a test strip and application thereof.
Background
Staphylococcus aureus, gram-positive coccus, is aerobically or facultatively anaerobic and is arranged in the form of a bunch of grapes. Is a common fixed value bacterium of human skin and nasal cavity, is also the staphylococcus which causes clinical infection most commonly, causes local suppurative infection of furuncle, carbuncle, folliculitis, surgical wound and wound, and can cause suppurative infection of deep tissue 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 detecting staphylococcus aureus rapidly, reliably and conveniently.
There are many methods currently developed for detecting staphylococcus aureus, and according to the research of scholars at home and abroad, there are three main detection methods for detecting the infection of staphylococcus aureus: conventional culture methods, immunological methods and molecular biological methods. Most methods do not allow accurate and rapid conclusions to be drawn. For example, the traditional culture method has high accuracy and the lowest detection limit can reach zero. But 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 one of the most mature detection technologies and 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 a plurality of samples with complex components; compared with the traditional culture method, the PCR technology in the molecular biological method has the characteristics of strong specificity, simplicity and high sensitivity, but needs a PCR instrument with a temperature control function, and the wide application of the PCR technology is greatly limited by hybridization after the hydrolysis of an electrophoresis amplification product or a special instrument detection characterization means. Therefore, the invention provides a simple, convenient and sensitive staphylococcus aureus detection method, which has important significance for areas with limited resources.
Disclosure of Invention
The invention aims to provide a CRISPR/Cas 9-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 purpose, the invention provides the following technical scheme: the CRISPR/Cas9 mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus comprises the following steps:
s1: extracting DNA in a staphylococcus aureus sample to be detected, cracking the sample to be detected by using a cracking solution and a proteinase K solution, adding a carboxyl modified magnetic bead and a coating solution, washing the obtained DNA by using a washing solution, and then re-suspending by using a re-suspending solution to obtain the DNA of the sample to be detected;
s2: adopting a primer pair aiming at staphylococcus aureus to perform RPA amplification for a period of time at a constant temperature to obtain a double-stranded product with one end functionalized by FITC;
s3: mixing a proper amount of Cas9 protein, sgRNA and a double-stranded product in a loading buffer solution, incubating for a period of time, adding the compound into a detection zone 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 the detection zone.
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 labeled with FITC.
Preferably, the sequence of the sgRNA is shown in SEQ ID NO. 3.
Preferably, the lysis solution comprises Tris-HCl buffer solution, ethylene diamine 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 the ethylene diamine tetraacetic acid is 1-25mmol/L, the concentration of the sodium chloride is 100-600mmol/L, and the mass-volume ratio of the sodium dodecyl sulfate to the lysis solution is 0.5-1.5g/100 mL;
the final concentration of the proteinase K solution is 20 mg/mL;
the coating liquid comprises polyethylene glycol and sodium chloride, 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 0.2-2.0 mol/L;
the washing solution comprises an aqueous solution with the ethanol content of 50-75 vt%;
the resuspension comprises a Tris-HCl buffer solution and ethylenediaminetetraacetic acid, wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, the concentration of the Tris-HCl buffer solution is 5-20mmol/L, and the concentration of the ethylenediaminetetraacetic acid is 1-25 mmol/L.
The concentration of the primer pair for RPA amplification is 200-800 nmol/L;
the constant temperature is 35-42 ℃;
the time for RPA amplification is 15-40 min.
The concentration of the Cas9 is 50-500 nmol/L;
the concentration of the sgRNA is 50-500 nmol/L;
the volume of the double-chain product is 1-20 mu L;
the sample loading buffer solution comprises a PB buffer solution, sodium chloride and polyethylene glycol, wherein the pH value of the PB buffer solution is 7.0-8.6, the concentration 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/100 mL;
the incubation time is 1-10 min.
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 sample loading area, a labeling area, a scribing area and a water absorption area, the sample loading area, the labeling 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, the detection line and the quality control line are respectively combined with a capture probe and a secondary antibody, and the sequence of the capture probe is shown as SEQ ID No. 4.
Preferably, the FITC antibody is selected from a monoclonal antibody of FITC, and the concentration is 0.5-3.0 mg/mL; the gold nanoparticles are 15-40 nm; the capture probe is fixed to the scribing region 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 0.5-4.0 mg/mL; the secondary antibody is a rabbit anti-mouse antibody selected from FITC antibody, and the concentration is 0.5-3.0 mg/mL.
Preferably, the support plate comprises a non-absorbent or hydrophobic cardboard, plastic or polystyrene plate; the material of the sample loading area comprises glass fiber cotton or a polyester film; the scribing region 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/Cas 9-mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus comprises the following steps:
a1: coupling gold nanoparticles with an FITC antibody, adjusting the pH value of the 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 adding a BSA solution for sealing to prepare gold nanoparticles coupled with the FITC antibody;
a2: gold nanoparticles coupled with an FITC antibody are applied to a marking area, a capture probe and streptavidin are mixed and then fixed as a detection line T of a marking area, and a secondary antibody is fixed as a quality control line C of the marking area;
a3: and sequentially arranging the sample loading area, the marking area, the scribing area and the water absorption area on the supporting plate along a set direction to obtain the lateral flow chromatography test strip.
The application of the lateral flow chromatography test strip comprises the following steps:
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, a specific functionalized primer pair aiming at staphylococcus aureus is utilized, when staphylococcus aureus exists, a corresponding FITC (FITC-in-situ hybridization) functionalized double-stranded product at one end is obtained through RPA (reverse transcriptase polymerase chain reaction) amplification, a specific site of the double-stranded product is subjected to unwinding under the target recognition action of a CRISPR/Cas9 system, and then a complex generated by combining Cas9/sgRNA and the double-stranded product is respectively hybridized with a gold-labeled antibody and a capture probe to generate a colored band, so that whether the staphylococcus aureus is infected or not 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 obvious advantages, for example, the isothermal amplification condition avoids the use of expensive temperature control instruments, gets rid of the constraint of the instruments, and is more suitable for field detection; the CRISPR/Cas9 system is adopted to specifically recognize double-stranded products, so that primer dimer interference is avoided; the primers similar to PCR are used, the design is simple, and the cost is low; the packaging is simple, and the product can exist in the form of dry powder, so that the product is beneficial to preservation; the operation is simple, and special technical training and the like are not needed;
4. the detection method disclosed by the invention can realize rapid and accurate detection of the staphylococcus aureus without special expensive instruments and only by a common water bath and a corresponding lateral flow chromatography test strip.
Drawings
FIG. 1 is a schematic diagram of screening and detecting infection with Staphylococcus aureus in an actual clinical specimen by lateral chromatography in example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a CRISPR/Cas 9-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, cracking the sample to be detected by using a cracking solution and a proteinase K solution, adding a carboxyl modified magnetic bead and a coating solution, washing the obtained DNA by using a washing solution, and then re-suspending by using a re-suspending solution to obtain the DNA of the sample to be detected;
s2: adopting a primer pair aiming at staphylococcus aureus to perform RPA amplification for a period of time at a constant temperature to obtain a double-stranded product with one end functionalized by FITC;
s3: mixing a proper amount of Cas9 protein, sgRNA and a double-stranded product in a loading buffer solution, incubating for a period of time, adding the compound into a detection zone 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 the detection zone.
The primer pair comprises a first primer and a second primer, the specific primers for staphylococcus aureus can realize the specific and effective amplification of target staphylococcus aureus, 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 FITC functionalized upstream primer, and the specific sequences of the first primer and the second primer are shown in a sequence table in detail; based on the combination of the upstream primer and the downstream primer, the invention realizes effective amplification of staphylococcus aureus, realizes one end FITC labeling of an amplification product, and is a key design for detecting staphylococcus aureus based on lateral flow chromatography of the amplification product.
The sequence of sgRNA is shown in SEQ ID NO.3, and the specific sequence representation is detailed 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 supporting plate, wherein the upper end face of the supporting plate comprises a sample loading area, a marking area and a water absorption area, the sample 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, the detection line and the quality control line are respectively combined with a capture probe and a secondary antibody, the sequence of the capture probe is shown as SEQ ID NO.4, and the specific sequence is shown in a sequence table in detail.
The FITC antibody is selected from a monoclonal antibody of FITC, and the concentration is 0.5-3.0 mg/mL; the gold nanoparticles are 15-40 nm; the capture probe is fixed on the scribing region 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 0.5-4.0 mg/mL; the secondary antibody is rabbit anti-mouse antibody selected from FITC antibody, and the concentration is 0.5-3.0 mg/mL.
The support plate comprises a non-absorbent or hydrophobic paperboard, plastic plate or polystyrene plate; the material of the sample loading area comprises glass fiber cotton or polyester film; the material of the scribing region comprises 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/Cas 9-mediated isothermal nucleic acid amplification method for detecting staphylococcus aureus comprises the following steps:
a1: coupling gold nanoparticles with an FITC antibody, adjusting the pH value of the 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 adding a BSA solution for sealing to prepare gold nanoparticles coupled with the FITC antibody;
a2: gold nanoparticles coupled with an FITC antibody are applied to a marking area, a capture probe and streptavidin are mixed and then fixed as a detection line T of a marking area, and a secondary antibody is fixed as a quality control line C of the marking area;
a3: and sequentially arranging the sample loading area, the marking area, the scribing area and the water absorption area on the supporting plate along a set direction to obtain the lateral flow chromatography test strip.
In some more specific embodiments, the method for preparing the nucleic acid test strip for detecting staphylococcus aureus comprises the following steps:
1) preparing FITC antibody-conjugated gold nanoparticles: coupling a FITC antibody and gold nanoparticles wrapped by trisodium citrate to prepare FITC antibody-coupled gold nanoparticles;
2) preparation of the labeling region: dripping a proper amount of FITC antibody-coupled gold nanoparticles into each labeling area, drying in an oven at 30-36 ℃, and storing for later use;
3) preparing a scribing area: respectively spraying capture probes combined with streptavidin to a detection line T of a line drawing area by using a film spraying instrument, spraying secondary antibody to a quality control line C of the line drawing area, drying in an oven at 30-36 ℃, and storing for later use;
4) assembling the test strip: and sequentially assembling the sample loading area, the marking area, the scribing area and the water absorption area on the support plate to form the lateral flow chromatography test strip for detecting the staphylococcus aureus infection.
Further, the preparation method of the gold nanoparticles coupled with the FITC antibody in the step 1) specifically comprises the following steps: dropwise adding potassium carbonate solution (10-100mmol/L) with a proper concentration into the gold nanoparticle solution to ensure that the pH value of the reaction system is 6.0-7.0; adding a proper amount of FITC antibody, and incubating at room temperature for 0.5-2 h; adding BSA solution with the mass ratio of 5-15%, and incubating at room temperature for 0.5-2 h; centrifuging (7000 plus 10000g, 7-15 min), removing supernatant, adding BSA solution with the mass volume ratio of 0.5-3% for resuspension, and storing at 4 ℃.
Further, the particle size of the gold nanoparticles is 15-40 nm.
In some embodiments, the detection method specifically includes: extracting DNA of a staphylococcus aureus sample to be detected by using a paramagnetic particle method, and performing RPA amplification by using a specific functionalized primer pair aiming at the staphylococcus aureus to obtain a double-stranded product with one end functionalized by FITC; mixing the amplification product with Cas9/sgRNA to obtain a double-stranded composite product which is recognized and uncoiled by Cas 9/sgRNA; and directly dripping the composite product into a sample loading area of the test strip, flowing through a marking area and a scribing area, connecting FITC antibody-coupled gold nanoparticles to a detection line T by using the composite product, and directly observing the color depth on the detection line by naked eyes to realize the rapid and accurate detection of the staphylococcus aureus.
In some embodiments, the method of detecting staphylococcus aureus specifically comprises: cracking a sample of staphylococcus aureus to be detected by adopting cell lysis solution and proteinase K solution, adding carboxyl modified magnetic beads and coating solution, washing the obtained DNA by using washing solution, and then re-suspending by using resuspension solution to obtain the DNA in the sample of staphylococcus aureus to be detected; adopting a primer pair aiming at staphylococcus aureus to perform RPA amplification for a period of time at a constant temperature to obtain a double-stranded product with one end functionalized by FITC; mixing a proper amount of Cas9 protein, sgRNA and a double-stranded product in a loading buffer solution, incubating for a period of time, adding the compound into a detection zone 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 the detection zone.
Further, the magnetic bead used in the magnetic bead method is a magnetic nanoparticle modified by carboxyl, and the specific scheme is as follows: taking 100-; adding 10-20 mu g of carboxyl modified magnetic beads and 750 mu L of 200-1500 mu L of coating liquid for adsorption and separation of the DNA of the staphylococcus aureus, washing by 500-1500 mu L of washing liquid, adding 30-90 mu L of resuspension liquid, and taking supernatant as the extracted DNA.
Further, the lysis solution contains Tris-HCl buffer solution, Ethylene Diamine 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 5-20 mmol/L; the concentration of EDTA is 1-25mmol/L, the concentration of sodium chloride (NaCl) is 100-600mmol/L, and the mass-to-volume ratio of sodium dodecyl sulfate to the lysis solution is 0.5-1.5g/100mL, i.e., the mass-to-volume ratio of SDS is 0.5-1.5%.
Further, the final concentration of proteinase K solution is 0.25-2.5 mg/mL.
Further, the coating solution mainly comprises polyethylene glycol (PEG) and sodium chloride (NaCl), wherein the average molecular weight of the polyethylene glycol is 400-.
Further, the wash solution comprises an aqueous solution having an ethanol content of 50-75 vt%.
Further, the main components of the resuspension comprise Tris-HCl buffer solution and Ethylene Diamine 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 Ethylene Diamine Tetraacetic Acid (EDTA) is 1.0-25 mmol/L.
Further, the concentration of the primer pair for RPA amplification is 200-;
further, the constant temperature is 35-42 ℃;
further, the time for RPA amplification is 15-40 min.
Further, the concentration of Cas9 is 50-500 nmol/L;
further, the concentration of sgRNA is 50-500 nmol/L;
further, the volume of the double-chain product is 1-20 muL;
further, the sample loading buffer solution comprises a PB buffer solution, sodium chloride and polyethylene glycol, wherein the pH value of the PB buffer solution is 7.0-8.6, the concentration 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/100 mL;
further, the incubation time is 1-10 min.
In summary, the detection method of the present invention comprises the following steps: preparing gold nanoparticles coupled with FTIC antibody; embedding the line drawing area; thirdly, manufacturing the test strip; extracting the DNA of the staphylococcus aureus sample and amplifying the RPA; and fifthly, visual detection is carried out. According to the invention, a functional primer pair specific to staphylococcus aureus is used, a CRISPR/Cas9 system is used for specifically identifying and de-spinning double-stranded products, the content of the products obtained by amplification is different according to the different content of target staphylococcus aureus in a sample to be detected, and the color depth of a detection line on a test strip is different, so that the relationship between the color depth of the detection line and the corresponding content of staphylococcus aureus is established, and the purpose of sensitively detecting staphylococcus aureus in the sample is further realized.
The technical solutions of the present invention are further explained with reference to the drawings and some preferred embodiments, but the experimental conditions and the setting parameters should not be construed as limitations of the basic technical solutions 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:
preparing gold nanoparticles coupled with FTIC antibody: dropwise adding potassium carbonate solution with proper concentration of 10mmol/L into the gold nanoparticle solution to ensure that the pH value of the reaction system is 6.0; adding a proper amount of FITC antibody, and incubating for 0.5h at room temperature; adding BSA solution with the mass ratio of 5%, and incubating at room temperature for 0.5 h; centrifuging (7000g, 7min), removing supernatant, adding BSA solution with mass volume ratio of 0.5% for resuspension, and storing at 4 ℃.
Embedding the scribed area: and respectively printing the streptavidin binding-capture probe compound and the secondary antibody solution on a detection line T and a quality control line C by using a film spraying instrument, drying in an oven at the temperature of 30 ℃, and storing for later use.
Making the test strip: and respectively assembling the sample loading area, the marking area and the water absorption area on the supporting plate in sequence.
Extraction of DNA and RPA amplification of staphylococcus aureus sample to be detected
The DNA is extracted by a magnetic bead method, and the magnetic nano-particles modified by carboxyl are used as a carrier for extracting the DNA, and the specific implementation mode is as follows: adding 100 mu L of staphylococcus aureus sample, 300 mu L of cell lysate and 10 mu L of proteinase K solution into a clean centrifuge tube, and incubating for 15min at 60 ℃; cooling at room temperature, adding 10 μ g of carboxyl modified magnetic beads and 200 μ L of coating solution, and rotating at room temperature for 3 min; magnetic adsorption, removing supernatant, adding 500 μ L washing solution, and washing twice; magnetic adsorption, removing supernatant, adding 30 mu L of heavy suspension for heavy suspension, magnetic adsorption, and taking supernatant as the extracted DNA.
The specific process of the RPA reaction is as follows: the RPA dry powder reagent was dissolved in 29.5. mu.L of the hybridization buffer, and 0.8. mu.L each of 10. mu.M of the primers for Staphylococcus aureus upstream and downstream and 2. mu.L of the DNA template were added thereto, and the mixture was allowed to react at 40 ℃ for 20min after adding 2.5. mu.L of a 280mM magnesium acetate solution.
Visual detection
In the embodiment, the RPA product in the step (iv) is diluted by a loading buffer solution through a complex formed by incubation with Cas9 protein and sgRNA, and then directly dripped into a loading area of a test strip, reacted for 1min at room temperature, a single-lens reflex camera is used to take pictures, and the intensity of a detection line T is analyzed by ImageJ software.
Example 2
The lateral flow chromatography detection method for detecting staphylococcus aureus in a urine sample provided by the embodiment comprises the following steps:
preparing gold nanoparticles coupled with FTIC antibody: dropwise adding a potassium carbonate solution with a proper concentration of 100mmol/L into the gold nanoparticle solution to ensure that the pH value of the reaction system is 7.0; adding a proper amount of FITC antibody, and incubating for 2h at room temperature; adding BSA solution with the mass ratio of 15%, and incubating at room temperature for 2 h; centrifuging (10000g, 15min), removing supernatant, adding BSA solution with the mass volume ratio of 3% for resuspension, and storing at 4 ℃.
Embedding the scribing area: and respectively printing the streptavidin binding-capture probe compound and the secondary antibody solution on a detection line T and a quality control line C by using a film spraying instrument, drying in an oven at 36 ℃ and storing for later use.
Making the test strip: and respectively assembling the sample loading area, the marking area and the water absorption area on the supporting plate in sequence.
Extraction of DNA and RPA amplification of staphylococcus aureus sample to be detected
The DNA is extracted by a magnetic bead method, and the magnetic nano-particles modified by carboxyl are used as a carrier for extracting the DNA, and the specific implementation mode is as follows: adding 500 mu L of a staphylococcus aureus sample, 1000 mu L of cell lysate and 10 mu L of proteinase K solution into a clean centrifugal tube, and incubating for 15min at 60 ℃; cooling at room temperature, adding 10 μ g of carboxyl modified magnetic beads and 750 μ L of coating solution, and rotating at room temperature for 3 min; magnetic adsorption, removing supernatant, adding 1500 microliter of washing solution and washing twice; magnetic adsorption, removing supernatant, adding 90 μ L heavy suspension for resuspension, magnetic adsorption, and collecting supernatant to obtain the extracted DNA.
The specific process of the RPA reaction is as follows: the RPA dry powder reagent was dissolved in 29.5. mu.L of the hybridization buffer, and 0.8. mu.L each of 10. mu.M of the primers for Staphylococcus aureus upstream and downstream and 2. mu.L of the DNA template were added thereto, and the mixture was allowed to react at 40 ℃ for 20min after adding 2.5. mu.L of a 280mM magnesium acetate solution.
Visual detection
In the embodiment, the RPA product in the step (iv) is diluted by a loading buffer solution through a complex formed by incubation with Cas9 protein and sgRNA, and then directly dripped into a loading area of a test strip, reacted for 10min at room temperature, a single-lens reflex camera is used for taking pictures, and the intensity of a detection line T is analyzed by ImageJ software.
In conclusion, according to the technical scheme, when staphylococcus aureus exists, the corresponding double-stranded amplification product with one end functionalized by FITC is obtained through RPA amplification, a CRISPR/Cas9 system is used for recognizing and de-spinning the double-stranded amplification product to obtain a complex of the double-stranded amplification product, and then the complex is used for connecting FITC-labeled gold nanoparticles to a detection line, so that a colored band is generated. According to different amounts of the generated compounds caused by different contents of the staphylococcus aureus, the colors of different detection lines are different in depth, the relationship between the color depth of the different detection lines and the content of the staphylococcus aureus is established, and the purpose of sensitively screening and detecting the staphylococcus aureus is achieved. The method has the advantages of good specificity, high sensitivity, good repeatability, simple operation and strong adaptability, and can detect the staphylococcus aureus with the concentration of 10. CFU/mL. The method can realize the rapid and accurate detection of the staphylococcus aureus without special expensive instruments and only by a common water bath and a corresponding lateral chromatography test strip, and is suitable for carrying out related work in areas with deficient detection hardware resources.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments 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 (9)
- A CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for staphylococcus aureus detection, comprising: the method comprises the following steps:s1: extracting DNA in a staphylococcus aureus sample to be detected, cracking the sample to be detected by using a cracking solution and a proteinase K solution, adding a carboxyl modified magnetic bead and a coating solution, washing the obtained DNA by using a washing solution, and then re-suspending by using a re-suspending solution to obtain the DNA of the sample to be detected;s2: adopting a primer pair aiming at staphylococcus aureus to perform RPA amplification for a period of time at a constant temperature to obtain a double-stranded product with one end functionalized by FITC;s3: mixing a proper amount of Cas9 protein, sgRNA and a double-stranded product in a loading buffer solution, incubating for a period of time, adding the compound into a detection zone 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 the detection zone.
- 2. The CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for staphylococcus aureus detection according to claim 1, wherein: 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 labeled by FITC.
- 3. The CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for staphylococcus aureus detection according to claim 1, wherein: the sequence of the sgRNA is shown in SEQ ID NO. 3.
- 4. The CRISPR/Cas 9-mediated isothermal nucleic acid amplification method for staphylococcus aureus detection according to claim 1, wherein: the lysis solution comprises Tris-HCl buffer solution, ethylene diamine 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 the ethylene diamine tetraacetic acid is 1-25mmol/L, the concentration of the sodium chloride is 100-600mmol/L, and the mass-volume ratio of the sodium dodecyl sulfate to the lysis solution is 0.5-1.5g/100 mL;the final concentration of the proteinase K solution is 20 mg/mL;the coating liquid comprises polyethylene glycol and sodium chloride, 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 0.2-2.0 mol/L;the washing solution comprises an aqueous solution with the ethanol content of 50-75 vt%;the resuspension comprises a Tris-HCl buffer solution and ethylenediaminetetraacetic acid, wherein the pH value of the Tris-HCl buffer solution is 7.2-8.8, the concentration of the Tris-HCl buffer solution is 5-20mmol/L, and the concentration of the ethylenediaminetetraacetic acid is 1-25 mmol/L.The concentration of the primer pair for RPA amplification is 200-800 nmol/L;the constant temperature is 35-42 ℃;the time for RPA amplification is 15-40 min.The concentration of the Cas9 is 50-500 nmol/L;the concentration of the sgRNA is 50-500 nmol/L;the volume of the double-chain product is 1-20 mu L;the sample loading buffer solution comprises a PB buffer solution, sodium chloride and polyethylene glycol, wherein the pH value of the PB buffer solution is 7.0-8.6, the concentration 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/100 mL;the incubation time is 1-10 min.
- 5. A lateral flow chromatography test strip as claimed in claim 1, wherein: the lateral flow chromatography test strip comprises a support plate, wherein the upper end face of the support plate comprises a sample loading area, a labeling area, a scribing area and a water absorption area, the sample loading area, the labeling 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, the detection line and the quality control line are respectively combined with a capture probe and a secondary antibody, and the sequence of the capture probe is shown as SEQ ID No. 4.
- 6. The lateral flow chromatography test strip of claim 5, wherein: the FITC antibody is selected from a monoclonal antibody of FITC, and the concentration is 0.5-3.0 mg/mL; the gold nanoparticles are 15-40 nm; the capture probe is fixed to the scribing region 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 0.5-4.0 mg/mL; the secondary antibody is a rabbit anti-mouse antibody selected from FITC antibody, and the concentration is 0.5-3.0 mg/mL.
- 7. The lateral flow chromatography test strip of claim 5, wherein: the support plate comprises a non-absorbent or hydrophobic paperboard, plastic plate or polystyrene plate; the material of the sample loading area comprises glass fiber cotton or a polyester film; the scribing region is made of nitrocellulose membrane or pure cellulose membrane; the material of the water absorption area comprises filter paper.
- 8. The method for preparing the lateral flow chromatography test strip of claim 5, wherein: the method comprises the following steps:a1: coupling gold nanoparticles with an FITC antibody, adjusting the pH value of the 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 adding a BSA solution for sealing to prepare gold nanoparticles coupled with the FITC antibody;a2: gold nanoparticles coupled with an FITC antibody are applied to a marking area, a capture probe and streptavidin are mixed and then fixed as a detection line T of a marking area, and a secondary antibody is fixed as a quality control line C of the marking area;a3: and sequentially arranging the sample loading area, the marking area, the scribing area and the water absorption area on the supporting plate along a set direction to obtain the lateral flow chromatography test strip.
- 9. A lateral flow chromatography test strip according to any one of claims 5 to 8, wherein: the lateral flow chromatography test strip is used for detecting a sample of staphylococcus aureus.
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