CN112501256A - CRSPR-cas13a driven RNA rapid detection method based on double-enzyme signal amplification strategy - Google Patents

Abstract

The invention discloses a rapid RNA detection method driven by CRSPR-cas13a and based on a double-enzyme signal amplification strategy, belonging to the technical field of biological detection, and the rapid RNA detection method based on the double-enzyme signal amplification strategy comprises the following steps: collecting a sample; processing a sample; assembling a magnetic separation probe; amplifying and separating a first-level enzymatic signal specifically triggered by a target sequence; amplifying secondary enzymatic signals and giving signals; the nucleic acid detection sequence is controllable, a proper guide sequence can be designed according to actual requirements to control the identified target sequence, the nucleic acid detection sequence has extremely high specificity and sensitivity, a good color development effect is achieved, the detection result can be judged by naked eyes without depending on an analytical instrument under the qualitative condition, the separation speed is high, the requirement on the instrument is simple, and a plurality of groups of samples can be processed simultaneously, so that the labor and the cost are saved.

Description

CRSPR-cas13a driven RNA rapid detection method based on double-enzyme signal amplification strategy

Technical Field

The invention relates to the technical field of biological detection, in particular to a rapid RNA detection method driven by CRSPR-cas13a and based on a double-enzyme signal amplification strategy.

Background

RNA can be used as an effective biomarker for disease diagnosis. However, in the early stage of disease onset or latent stage, the RNA content in body fluid is low, which puts extremely high requirements on the sensitivity of the detection method. The classical rt-PCR detection method is time-consuming and labor-consuming, depends on precise equipment such as a fluorescent quantitative PCR instrument and the like, and has a certain probability of false positive. In recent years, a high-sensitivity detection method based on CRSPR-CAS13a is proposed, but the existing method still depends on equipment such as a fluorescence quantitative PCR instrument, the operation process is complex, the cost is high, and the early stage investigation of diseases is greatly influenced. The lack of highly sensitive, highly specific, and simple to operate RNA detection methods presents a significant challenge to clinical diagnosis of related diseases. Therefore, the development of a rapid, efficient and reliable RNA detection and analysis method has important scientific research and clinical significance.

Traditionally, RNA detection has been mainly by reverse transcription and PCR techniques. These techniques are either cumbersome and expensive in equipment or complicated in sample handling and are not conducive to clinical use and further popularization. The enzymatic colorimetric method is a commonly used analytical detection method in scientific research and clinic, and is widely applied to the fields of immunodiagnosis, pathogenic microorganism screening, environmental monitoring and the like. The method has the advantages of wide detection range, high sensitivity, controllable reaction system and the like due to the superiority of the enzymatic colorimetric method principle. However, the advantage of enzymatic colorimetry in RNA detection is not obvious, for specific reasons: (1) biological fluid components are relatively complex, and free nucleic acid and the like can interfere detection; (2) the clinically available biological fluid is limited and the RNA content is low, and the concentration of the purified nucleic acid sequence is difficult to reach the detection range. In conclusion, the research and development of the novel CRSPR-cas13 a-driven RNA rapid detection technology based on the double-enzyme signal amplification strategy has important clinical significance and application value.

Disclosure of Invention

The invention aims to provide a rapid RNA detection method driven by CRSPR-cas13a and based on a double-enzyme signal amplification strategy, so as to solve the problems that the components of biological body fluid are relatively complex, free nucleic acid and the like can cause interference on detection, clinically available biological body fluid is limited, the RNA content is low, and the concentration of a purified nucleic acid sequence cannot reach the detection range easily.

In order to achieve the purpose, the invention provides the following technical scheme: a CRSPR-cas13a driven RNA rapid detection method based on a double-enzyme signal amplification strategy comprises the following steps:

s1: collecting samples: including animal or human cell culture fluid collection, animal or human blood sample collection, animal or human urine sample collection, animal or human saliva sample collection;

s2: and (3) processing of a sample: comprises the steps of pretreatment of a sample and acquisition of RNA in the sample;

s3: assembling the magnetic separation probe: the method comprises the steps of preparing a buffer solution, activating an amino-modified signal probe and a carboxyl magnetic bead, and combining the amino-modified signal probe and the carboxyl magnetic bead;

s4: target sequence specific triggered primary enzymatic signal amplification and isolation: adding RNA obtained from a sample into 100 mu L of modified signal probe magnetic beads, synchronously adding 10nM purified LluCas 13a and 10nM crRNA, fully and uniformly mixing, standing at room temperature for 10-30min for incubation, placing the incubated sample in a magnetic separation frame, standing, and collecting a solution part;

s5: secondary enzymatic signal amplification and signal presentation: adding 50ul of the first-stage reaction product, namely the recovered liquid part, prepared in the step S4 into a 96-pore plate, adding 50ul of a reaction substrate at the same time, reacting for 10-30min, and observing color change to avoid missing the optimal measurement opportunity;

s6: and (3) detecting a signal of the target nucleic acid sequence.

Preferably, the sample collection in step S1 includes the following steps:

a1: collecting culture solution of animal or human cells: the cell culture solution is DMEM, calf serum (FCS) with the volume fraction of 10% is added, the cells are digested by trypsin, transferred to a cell culture dish and cultured in a saturated water vapor carbon dioxide incubator with the temperature of 37 ℃ and the concentration of 5% CO2 for 24 hours to ensure that the cells are converged for 50% -60%, then the cells are cultured for 24-48 hours by using serum-free DMEM culture solution, and the serum-free DMEM cell culture solution is recovered, stored at the temperature of 4 ℃ in a short term and stored at the temperature of-80 ℃ for a long term;

a2: animal or human body fluid sample collection: blood is drawn from the veins of animals or humans, anticoagulant is added, and the cells in the blood are removed by centrifugation to obtain blood plasma; or collecting blood serum after blood coagulation, storing the obtained blood sample at-80 deg.C or immediately using, collecting urine of animal or human into urine collector to obtain urine sample, avoiding external pollution to urine sample as much as possible, and immediately using or storing at-80 deg.C for use;

a3: cell collection: adherent and histiocyte are obtained by centrifuging for 5 minutes at 1000 rpm after being digested by 0.2 percent trypsin for 1 minute, if not used immediately, the adherent and histiocyte are frozen and stored in minus eighty refrigerator or liquid nitrogen, if not used immediately, the nonadherent cells are directly centrifuged, and if not used immediately, the adherent and histiocyte are frozen and stored in minus eighty refrigerator or liquid nitrogen.

Preferably, the sample processing in step S2 includes the following steps:

b1: pretreatment of a sample: centrifuging the blood sample at 1500 rpm for 30min to remove cells and debris under the premise of adding anticoagulant, obtaining plasma components, and concentrating urine and cell culture solution and then performing lower operation;

b2: RNA extraction: the RNA is obtained from the treated sample by a commercial kit or by a trizol method.

Preferably, the assembling of the magnetic separation probe in the step S3 includes the steps of:

c1: activated carboxyl magnetic beads: weighing 9.76g of MES, 29.22g of NaCl, 0.191g of EDC, 0.115g of NHS, 9.76g of MES and 29.22g of NaCl by an electronic balance, dissolving the MES, the MES and the 29.22g of NaCl in 900mL of ultrapure water, adjusting the pH to 6 by using 3mol/L of sodium hydroxide, fixing the volume to 1L, preparing MES buffer solution, EDC and NHS, respectively dissolving the MES buffer solution, the EDC buffer solution and the NHS in a 10mL centrifuge tube, uniformly mixing carboxyl magnetic beads, placing 50 mu L of the mixed solution in a 1.5mL EP tube, magnetically separating to remove a protective reagent, adding 1mL of the buffer solution into the EP tube, uniformly mixing, adding 100 mu L of LEDC solution into the magnetic bead solution, placing the mixed solution at room temperature for half an hour, adding 100 mu L of LNHS solution into the magnetic beads, placing the magnetic beads at room temperature for one hour for activation, placing;

c2: and (3) binding of carboxyl magnetic beads and signal probes: dissolving the activated carboxyl magnetic beads in 1ml PBS buffer solution, adding a signal probe, shaking at room temperature for 24 hours, placing the solution after reaction in a magnetic separation frame, standing for 30 seconds, removing a liquid part, keeping the magnetic beads, changing the potential of the modified magnetic beads, and detecting by using a probe with fluorescence to find that the probe can be combined with the surfaces of the magnetic beads;

c3: activation of streptavidin horseradish peroxidase: weighing 0.292g of EDTA, 29.22g of NaCl and 2.423g of TRIS in a 1L beaker by an electronic balance, adding water to a constant volume of 800ml, uniformly stirring under a magnetic stirrer, transferring the obtained solution to a 1L graduated cylinder to a constant volume of 1L, adjusting the pH value to 7.5 by using 1mol/L of HCl, filtering the obtained solution by using filter paper to obtain a reaction solution, subpackaging for later use, taking 125ul of a streptavidin horseradish peroxidase mother solution, placing the streptavidin mother solution in a 1.5ml EP tube, and adding 375 mu L of the reaction solution to obtain activated streptavidin;

c4: streptavidin-coupled horseradish peroxidase bound to the probe: weighing 0.05MNaCl, 20mM Tris-HCl and 7.51mM EDTA in a beaker by an electronic balance, uniformly stirring under a magnetic stirrer, adding 500 mu L of reaction liquid I and activated streptavidin horse radish peroxidase into magnetic beads for mixing, placing the mixed liquid in a constant temperature shaking table at 60r/s for reacting for 30 minutes at room temperature, placing the mixed liquid in a magnetic separation frame, standing for 30 seconds, removing supernatant, and reserving magnetic beads combined with probes, namely magnetic beads modified by signal probes for later use, wherein the modified magnetic beads have horse radish peroxidase activity and can enable substrates to generate color reaction.

Preferably, the detection signal of the target nucleic acid sequence comprises the following steps:

d1: semi-quantitative analysis: placing the reaction system after termination, namely a 96-well plate, into an enzyme-labeling instrument, measuring the absorption value at 450nm, and comparing the absorption value with a control group and a positive group to obtain a relative value;

d2: quantitative analysis: setting the concentration gradient of the target sequence in the step S3, completing the subsequent operation according to the process, placing the reaction system after termination, namely a 96-well plate into an enzyme-labeling instrument, measuring the absorption value at 450nm, performing linear regression on the measured value, and comparing to obtain the concentration of the target sequence of the sample group;

d3: and (3) qualitative analysis: comparing the reaction systems after termination, observing the difference with the control group, and if the difference is significant, indicating that the target sequence exists.

Compared with the prior art, the invention has the beneficial effects that: the nucleic acid detection sequence is controllable, a proper guide sequence can be designed according to actual requirements to control the recognized target sequence, the method has extremely high specificity based on CAS13a mediated enzymatic reaction, the occurrence of reaction cannot be driven due to mismatching of more than two bases in the target sequence, secondary enzymatic signal amplification adopted by the method has extremely high sensitivity, and has higher detection offline compared with the traditional single enzymatic reaction.

Drawings

FIG. 1 is a schematic diagram of the present invention;

FIG. 2 is a confocal representation of probe loading onto the surface of a magnetic bead;

FIG. 3 specific detection of the dual enzyme signal amplification strategy.

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 CRSPR-cas13a driven RNA rapid detection method based on a double-enzyme signal amplification strategy, the nucleic acid detection sequence is controllable, a proper guide sequence can be designed according to the actual requirement to control the identified target sequence, the method has extremely high specificity and extremely high sensitivity, has better color development effect, can judge the detection result by naked eyes without depending on an analytical instrument under the qualitative condition, has high separation speed, has simple requirements on the instrument, can simultaneously process a plurality of groups of samples, thereby saving the labor and the cost, please refer to FIG. 1,

the rapid RNA detection method based on the double-enzyme signal amplification strategy comprises the following steps:

s1: collecting samples: comprises the steps of animal or human cell culture fluid collection, animal or human blood sample collection, animal or human urine sample collection, animal or human saliva sample collection, and comprises the following steps:

a1: collecting culture solution of animal or human cells: the cell culture solution is DMEM, calf serum (FCS) with the volume fraction of 10% is added, the cells are digested by trypsin, transferred to a cell culture dish and cultured in a saturated water vapor carbon dioxide incubator with the temperature of 37 ℃ and the concentration of 5% CO2 for 24 hours to ensure that the cells are converged for 50% -60%, then the cells are cultured for 24-48 hours by using serum-free DMEM culture solution, and the serum-free DMEM cell culture solution is recovered, stored at the temperature of 4 ℃ in a short term and stored at the temperature of-80 ℃ for a long term;

a2: animal or human body fluid sample collection: blood is drawn from the veins of animals or humans, anticoagulant is added, and the cells in the blood are removed by centrifugation to obtain blood plasma; or collecting blood serum after blood coagulation, storing the obtained blood sample at-80 deg.C or immediately using, collecting urine of animal or human into urine collector to obtain urine sample, avoiding external pollution to urine sample as much as possible, and immediately using or storing at-80 deg.C for use;

a3: cell collection: after being digested by 0.2 percent trypsin for 1 minute, adherent cells and histiocytes are obtained by centrifugation for 5 minutes at 1000 rpm, if the adherent cells and the histiocytes are not used immediately, the adherent cells and the histiocytes are frozen and stored in minus eighty refrigerator or liquid nitrogen, if the adherent cells and the histiocytes are not used immediately, the non-adherent cells and the histiocytes are directly centrifuged, and if the non-adherent cells and the histiocytes are not used immediately, the adherent cells and the histiocytes are frozen and stored in minus;

s2: and (3) processing of a sample: the method comprises the steps of pretreatment of a sample and acquisition of RNA in the sample, and comprises the following steps:

b1: pretreatment of a sample: centrifuging the blood sample at 1500 rpm for 30min to remove cells and debris under the premise of adding anticoagulant, obtaining plasma components, and concentrating urine and cell culture solution and then performing lower operation;

b2: RNA extraction: obtaining RNA from the treated sample by a commercial kit or a trizol method;

s3: assembling the magnetic separation probe: the method comprises the following steps of preparing a buffer solution, activating an amino-modified signal probe and a carboxyl magnetic bead, and combining the amino-modified signal probe and the carboxyl magnetic bead, wherein the method comprises the following steps:

c1: activated carboxyl magnetic beads: weighing 9.76g of MES, 29.22g of NaCl, 0.191g of EDC, 0.115g of NHS, 9.76g of MES and 29.22g of NaCl by an electronic balance, dissolving the MES, the MES and the 29.22g of NaCl in 900mL of ultrapure water, adjusting the pH to 6 by using 3mol/L of sodium hydroxide, fixing the volume to 1L, preparing MES buffer solution, EDC and NHS, respectively dissolving the MES buffer solution, the EDC buffer solution and the NHS in a 10mL centrifuge tube, uniformly mixing carboxyl magnetic beads, placing 50 mu L of the mixed solution in a 1.5mL EP tube, magnetically separating to remove a protective reagent, adding 1mL of the buffer solution into the EP tube, uniformly mixing, adding 100 mu L of LEDC solution into the magnetic bead solution, placing the mixed solution at room temperature for half an hour, adding 100 mu L of LNHS solution into the magnetic beads, placing the magnetic beads at room temperature for one hour for activation, placing;

c2: and (3) binding of carboxyl magnetic beads and signal probes: dissolving the activated carboxyl magnetic beads in 1ml PBS buffer solution, adding a signal probe, shaking at room temperature for 24 hours, placing the solution after reaction in a magnetic separation frame, standing for 30 seconds, removing a liquid part, keeping the magnetic beads, changing the potential of the modified magnetic beads, and detecting by using a probe with fluorescence to find that the probe can be combined with the surfaces of the magnetic beads;

c3: activation of streptavidin horseradish peroxidase: weighing 0.292g of EDTA, 29.22g of NaCl and 2.423g of TRIS in a 1L beaker by an electronic balance, adding water to a constant volume of 800ml, uniformly stirring under a magnetic stirrer, transferring the obtained solution to a 1L graduated cylinder to a constant volume of 1L, adjusting the pH value to 7.5 by using 1mol/L of HCl, filtering the obtained solution by using filter paper to obtain a reaction solution, subpackaging for later use, taking 125ul of a streptavidin horseradish peroxidase mother solution, placing the streptavidin mother solution in a 1.5ml EP tube, and adding 375 mu L of the reaction solution to obtain activated streptavidin;

c4: streptavidin-coupled horseradish peroxidase bound to the probe: weighing 0.05MNaCl, 20mM Tris-HCl and 7.51mM EDTA in a beaker by an electronic balance, uniformly stirring under a magnetic stirrer, adding 500 mu L of reaction liquid I and activated streptavidin horse radish peroxidase into magnetic beads for mixing, placing the mixed liquid in a constant-temperature shaking table for 60r/s reaction at room temperature for 30 minutes, placing the mixed liquid in a magnetic separation frame, standing for 30 seconds, removing supernatant, and reserving magnetic beads combined with probes, namely magnetic beads modified by signal probes for later use, wherein the modified magnetic beads have horse radish peroxidase activity and can enable substrates to generate color reaction;

s4: target sequence specific triggered primary enzymatic signal amplification and isolation: target sequence specific triggered primary enzymatic signal amplification and isolation: adding RNA obtained from a sample into 100 mu L of modified signal probe magnetic beads, synchronously adding 10nM purified LluCas 13a and 10nM crRNA, fully and uniformly mixing, standing at room temperature for 10-30min for incubation, placing the incubated sample in a magnetic separation frame, standing, and collecting a solution part;

s5: secondary enzymatic signal amplification and signal presentation: adding 50ul of the first-stage reaction product, namely the recovered liquid part, prepared in the step S4 into a 96-pore plate, adding 50ul of a reaction substrate at the same time, reacting for 10-30min, and observing color change to avoid missing the optimal measurement opportunity;

s6: the detection signal of the target nucleic acid sequence comprises the following steps:

d1: semi-quantitative analysis: placing the reaction system after termination, namely a 96-well plate, into an enzyme-labeling instrument, measuring the absorption value at 450nm, and comparing the absorption value with a control group and a positive group to obtain a relative value;

d2: quantitative analysis: setting the concentration gradient of the target sequence in the step S3, completing the subsequent operation according to the process, placing the reaction system after termination, namely a 96-well plate into an enzyme-labeling instrument, measuring the absorption value at 450nm, performing linear regression on the measured value, and comparing to obtain the concentration of the target sequence of the sample group;

d3: and (3) qualitative analysis: comparing the reaction systems after termination, observing the difference with the control group, and if the difference is significant, indicating that the target sequence exists.

While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the embodiments disclosed herein may be used in any combination, provided that there is no structural conflict, and the combinations are not exhaustively described in this specification merely for the sake of brevity and conservation of resources. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims (5)

1. A CRSPR-cas13a driven RNA rapid detection method based on a double-enzyme signal amplification strategy is characterized in that: the rapid RNA detection method based on the double-enzyme signal amplification strategy comprises the following steps:

s1: collecting samples: including animal or human cell culture fluid collection, animal or human blood sample collection, animal or human urine sample collection, animal or human saliva sample collection;

s2: and (3) processing of a sample: comprises the steps of pretreatment of a sample and acquisition of RNA in the sample;

s3: assembling the magnetic separation probe: the method comprises the steps of preparing a buffer solution, activating an amino-modified signal probe and a carboxyl magnetic bead, and combining the amino-modified signal probe and the carboxyl magnetic bead;

s4: target sequence specific triggered primary enzymatic signal amplification and isolation: adding RNA obtained from a sample into 100 mu L of modified signal probe magnetic beads, synchronously adding 10nM purified LluCas 13a and 10nM crRNA, fully and uniformly mixing, standing at room temperature for 10-30min for incubation, placing the incubated sample in a magnetic separation frame, standing, and collecting a solution part;

s5: secondary enzymatic signal amplification and signal presentation: adding 50ul of the first-stage reaction product, namely the recovered liquid part, prepared in the step S4 into a 96-pore plate, adding 50ul of a reaction substrate at the same time, reacting for 10-30min, and observing color change to avoid missing the optimal measurement opportunity;

s6: and (3) detecting a signal of the target nucleic acid sequence.

2. The method for rapidly detecting RNA driven by CRSPR-cas13a and based on a double-enzyme signal amplification strategy according to claim 1, wherein the method comprises the following steps: the sample collection in step S1 includes the following steps:

a1: collecting culture solution of animal or human cells: the cell culture solution is DMEM, calf serum (FCS) with the volume fraction of 10% is added, the cells are digested by trypsin, transferred to a cell culture dish and cultured in a saturated water vapor carbon dioxide incubator with the temperature of 37 ℃ and the concentration of 5% CO2 for 24 hours to ensure that the cells are converged for 50% -60%, then the cells are cultured for 24-48 hours by using serum-free DMEM culture solution, and the serum-free DMEM cell culture solution is recovered, stored at the temperature of 4 ℃ in a short term and stored at the temperature of-80 ℃ for a long term;

a2: animal or human body fluid sample collection: blood is drawn from the veins of animals or humans, anticoagulant is added, and the cells in the blood are removed by centrifugation to obtain blood plasma; or collecting blood serum after blood coagulation, storing the obtained blood sample at-80 deg.C or immediately using, collecting urine of animal or human into urine collector to obtain urine sample, avoiding external pollution to urine sample as much as possible, and immediately using or storing at-80 deg.C for use;

a3: cell collection: adherent and histiocyte are obtained by centrifuging for 5 minutes at 1000 rpm after being digested by 0.2 percent trypsin for 1 minute, if not used immediately, the adherent and histiocyte are frozen and stored in minus eighty refrigerator or liquid nitrogen, if not used immediately, the nonadherent cells are directly centrifuged, and if not used immediately, the adherent and histiocyte are frozen and stored in minus eighty refrigerator or liquid nitrogen.

3. The method for rapidly detecting RNA driven by CRSPR-cas13a and based on a double-enzyme signal amplification strategy according to claim 1, wherein the method comprises the following steps: the sample processing in step S2 includes the steps of:

b1: pretreatment of a sample: centrifuging the blood sample at 1500 rpm for 30min to remove cells and debris under the premise of adding anticoagulant, obtaining plasma components, and concentrating urine and cell culture solution and then performing lower operation;

b2: RNA extraction: the RNA is obtained from the treated sample by a commercial kit or by a trizol method.

4. The method for rapidly detecting RNA driven by CRSPR-cas13a and based on a double-enzyme signal amplification strategy according to claim 1, wherein the method comprises the following steps: the assembling of the magnetic separation probe in the step S3 includes the steps of:

c1: activated carboxyl magnetic beads: weighing 9.76g of MES, 29.22g of NaCl, 0.191g of EDC, 0.115g of NHS, 9.76g of MES and 29.22g of NaCl by an electronic balance, dissolving the MES, the MES and the 29.22g of NaCl in 900mL of ultrapure water, adjusting the pH to 6 by using 3mol/L of sodium hydroxide, fixing the volume to 1L, preparing MES buffer solution, EDC and NHS, respectively dissolving the MES buffer solution, the EDC buffer solution and the NHS in a 10mL centrifuge tube, uniformly mixing carboxyl magnetic beads, placing 50 mu L of the mixed solution in a 1.5mL EP tube, magnetically separating to remove a protective reagent, adding 1mL of the buffer solution into the EP tube, uniformly mixing, adding 100 mu L of LEDC solution into the magnetic bead solution, placing the mixed solution at room temperature for half an hour, adding 100 mu L of LNHS solution into the magnetic beads, placing the magnetic beads at room temperature for one hour for activation, placing;

c2: and (3) binding of carboxyl magnetic beads and signal probes: dissolving the activated carboxyl magnetic beads in 1ml PBS buffer solution, adding a signal probe, shaking at room temperature for 24 hours, placing the solution after reaction in a magnetic separation frame, standing for 30 seconds, removing a liquid part, keeping the magnetic beads, changing the potential of the modified magnetic beads, and detecting by using a probe with fluorescence to find that the probe can be combined with the surfaces of the magnetic beads;

c3: activation of streptavidin horseradish peroxidase: weighing 0.292g of EDTA, 29.22g of NaCl and 2.423g of TRIS in a 1L beaker by an electronic balance, adding water to a constant volume of 800ml, uniformly stirring under a magnetic stirrer, transferring the obtained solution to a 1L graduated cylinder to a constant volume of 1L, adjusting the pH value to 7.5 by using 1mol/L of HCl, filtering the obtained solution by using filter paper to obtain a reaction solution, subpackaging for later use, taking 125ul of a streptavidin horseradish peroxidase mother solution, placing the streptavidin mother solution in a 1.5ml EP tube, and adding 375 mu L of the reaction solution to obtain activated streptavidin;

c4: streptavidin-coupled horseradish peroxidase bound to the probe: weighing 0.05MNaCl, 20mM Tris-HCl and 7.51mM EDTA in a beaker by an electronic balance, uniformly stirring under a magnetic stirrer, adding 500 mu L of reaction liquid I and activated streptavidin horse radish peroxidase into magnetic beads for mixing, placing the mixed liquid in a constant temperature shaking table at 60r/s for reacting for 30 minutes at room temperature, placing the mixed liquid in a magnetic separation frame, standing for 30 seconds, removing supernatant, and reserving magnetic beads combined with probes, namely magnetic beads modified by signal probes for later use, wherein the modified magnetic beads have horse radish peroxidase activity and can enable substrates to generate color reaction.

5. The method for rapidly detecting RNA driven by CRSPR-cas13a and based on a double-enzyme signal amplification strategy according to claim 1, wherein the method comprises the following steps: the step S6 of detecting the signal of the target nucleic acid sequence includes the following steps:

d1: semi-quantitative analysis: placing the reaction system after termination, namely a 96-well plate, into an enzyme-labeling instrument, measuring the absorption value at 450nm, and comparing the absorption value with a control group and a positive group to obtain a relative value;

d2: quantitative analysis: setting the concentration gradient of the target sequence in the step S3, completing the subsequent operation according to the process, placing the reaction system after termination, namely a 96-well plate into an enzyme-labeling instrument, measuring the absorption value at 450nm, performing linear regression on the measured value, and comparing to obtain the concentration of the target sequence of the sample group;

d3: and (3) qualitative analysis: comparing the reaction systems after termination, observing the difference with the control group, and if the difference is significant, indicating that the target sequence exists.

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