CN113687066A - Molecular lateral chromatography detection method based on CRISPR system and detection test strip - Google Patents
Molecular lateral chromatography detection method based on CRISPR system and detection test strip Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention relates to the technical field of molecular detection, in particular to a molecular lateral chromatography detection method and a detection test strip based on a CRISPR system. The labeled probe contains a nucleic acid probe and a label which are connected, the Cas protein contains a guide sequence which can target a molecule to be detected, and when the detection sample contains the molecule to be detected, the Cas protein is activated to cut the nucleic acid probe in the labeled probe, so that the label originally fixed on the lateral chromatography test paper or the accessory is released. The invention fixes the labeled probe, releases the labeled substance after being cut and detects the labeled substance by a sandwich method or a capture method test strip, thereby improving the specificity of detection and avoiding the problem of false positive in the prior art.
Description
Technical Field
The invention relates to the technical field of molecular detection, in particular to a molecular lateral chromatography detection method and a detection test strip based on a CRISPR system.
Background
CRISPR molecular detection is a novel molecular detection technology firstly developed by frontier team of Border institute in 2017, and the main principle is that after Cas protein is contacted and combined with a target fragment, the indiscriminate trans-cleavage function of the Cas protein is activated, and the characteristic that all nearby nucleic acid sequences can be cleaved is utilized, and a detection method for judging whether the target fragment exists or not is used for detecting the cleavage condition of a probe sequence (Gootenberg, Jonathan, S, et al. nucleic acid detection with CRISPR-Cas13a/C2C2.[ J ]. Science, 2017.).
In 2019, the Sherlock technique was introduced by the Zhang team combining the CRISPR method with lateral chromatography test paper (Kellner M J, Koob J G, Gootenberg J S, et al. SHErlock: nucleic acid detection with CRISPR nucleotides [ J ]. Nature Protocols,2019,14 (10)). The method introduces a section of probe sequence which is respectively marked with Biotin (Biotin) and Fluorescein (FAM) at two ends into the system. The lateral chromatography test strip can detect sequences containing Biotin and FAM at the same time: avidin (SA) capable of binding Biotin was coated on the proximal band, an anti-FAM antibody was labeled on the colloidal gold, and a secondary antibody capable of binding to the anti-FAM antibody was coated on the distal band. This is a sandwich test strip, and when a sample contains sequences of both Biotin and FAM, a SA-Biotin-probe-FAM-anti-FAM antibody-colloidal gold complex is formed at the proximal strip, thereby developing color.
The Zhang Pioneer group used the sandwich test strip in a competitive manner. When the sample is negative, the CRISPR system is not activated, the probe is complete, and the probe sequence marked with Biotin (Biotin) and Fluorescein (FAM) cannot be cut, so that all the colloidal gold is completely captured by the SA strip at the near end of the test strip by a sandwich method and cannot reach the line at the far end of the test strip.
When the sample is positive, the probe sequences with Biotin (Biotin) and Fluorescein (FAM) respectively marked at the two ends are cut off, an SA-Biotin-probe-FAM-anti-FAM antibody-colloidal gold complex cannot be formed at the near-end strip, and the colloidal gold crosses the near-end strip to reach the far-end strip for color development to show a positive result.
In the method developed by the Zhang Feng team, the sandwich test strip is interpreted by adopting a competition method: the strip at the far end is positive when developing color, and is negative when not developing color, and the strip at the far end is the quality control line in the conventional sandwich test strip.
All detection methods (such as STOPcovid, STOPcovid2 and HOLMESV2) combining the CRISPR system and the test strip after 2019 adopt the same test strip and interpretation mode.
In the chinese patent application CN112708660A, the same probe is fixed on microsphere carriers of various materials, microspheres are added into a liquid phase system of Cas protein reaction, and after the reaction is finished, the microspheres are separated, and then the test paper is still used for interpretation.
The method has a serious defect that the sandwich method test strip is difficult to ensure that all the colloidal gold is captured by the near-end strip, which can be seen from the fact that when the conventional sandwich method test strip is strong positive, the far-end quality control line strip still exists. It can also be seen from the original literature pictures of SHERLOCKv2, STOPcovid2, HOLMESv2, etc., that there is weak coloration at the distal band of the negative test strip in the literature. In this regard, the group of the present inventors also tried using test strips from the same source in the literature, and the distal band did exist when negative, i.e., there was a weak false positive. More seriously, if no probe is added to the system, the distal line appears directly, i.e., a strong false positive appears. Aiming at weak false positive, a feasible improvement method designed by the inventor team of the application is to change the interpretation mode, namely, the far-end strip is specified to be negative than the near-end strip in color weakness, but the system sensitivity is greatly reduced, and the colorimetric interpretation raises the requirement on a user.
In addition, the existing competitive method mode is difficult to develop multi-index joint detection.
Disclosure of Invention
In order to solve the two defects in the prior art, the invention aims to provide a molecular lateral chromatography detection method and a detection test strip based on a CRISPR system, which have high sensitivity and no false positive, and can realize multi-index combined visual detection based on the CRIPSR system.
In order to achieve the above purpose, the invention specifically provides the following technical scheme:
a molecular lateral chromatography detection method based on a CRISPR system, the method comprising:
cleavage of a label probe comprising a nucleic acid probe and a label linked together, the nucleic acid probe being cleaved when the nucleic acid probe contacts the activated Cas protein;
release of the label: the labeled probe is fixedly arranged on the lateral chromatography test paper, and when the nucleic acid probe is cut, the label is released from the fixed position;
detection of the marker: the lateral chromatography test paper adopts a sandwich method or a capture method to detect the released marker, thereby realizing the detection of the molecules to be detected.
Further, the nucleic acid probe can be cleaved by the activated Cas protein or the engineered mini-Cas protein.
Further, the nucleic acid probe is any one or more of a double-stranded DNA sequence, a single-stranded RNA sequence and a double-stranded RNA sequence.
Further, the labeled probe is fixedly arranged on the lateral chromatography test paper; preferably, the labeled probe is fixedly arranged on a solid phase carrier; more preferably, the labeled probe is immobilized on a magnetic particle.
Further, the marker comprises not less than two antigenic determinants.
Further, the Cas protein is arranged on the lateral chromatography test paper, the number of the lateral chromatography test paper is not less than two, and the Cas protein on each lateral chromatography test paper comprises different guide sequences.
When a capture method is used, the label is a molecule labeled with a colored dye or a colored microsphere, and the molecule can be captured by the lateral chromatography test paper.
The molecular lateral chromatography detection test strip based on the CRISPR system comprises a lateral chromatography test strip, wherein the lateral chromatography test strip is provided with a Cas protein, the Cas protein is provided with a guide sequence capable of targeting a molecule to be detected, the test strip further comprises a fixed marker probe, and the marker probe comprises a nucleic acid probe and a marker which are connected.
Further, the marker is composed of colored or colorless microspheres or colloidal gold, and the microspheres contain at least one antigenic determinant.
Further, the microspheres comprise antibodies or antigens, and can be detected by a capture method.
Further, the lateral chromatography test paper is sequentially provided with a sample pad, a combination pad, an NC membrane and a water absorption pad;
the Cas protein is disposed on the sample pad;
the labeled probe is fixedly arranged on the sample pad;
a first anti-marker antibody-labeled carrier complex is disposed on the conjugate pad;
the NC membrane is provided with a detection line, and the detection line is coated with a marker capture object.
Furthermore, the sample pad, the combination pad, the NC membrane and the water absorption pad are sequentially adhered to the back plate.
Furthermore, the combination pad, the NC membrane and the water absorption pad are sequentially adhered to the back plate, and the sample pad is independently arranged.
Further, one end of the nucleic acid probe is fixedly combined with the sample pad, and the other end of the nucleic acid probe is coupled with the label.
Further, the nucleic acid probe is provided with an avidin label, the sample pad is chemically coupled with streptavidin, and the labeled probe is fixed on the sample pad through a biotin-avidin system.
Furthermore, one end of the labeled probe is provided with a biotin label, contains a region which can be cut by the Cas protein, and the other end of the labeled probe contains another small molecule label which is combined by an antibody of the small molecule, wherein the antibody of the small molecule contains two or more different small molecule labels; streptavidin is chemically coupled on the sample pad, and the labeled probe is fixed on the sample pad through a biotin-avidin system.
Or, one end of the labeled probe is provided with a biotin label and contains a sequence which can be cut by the Cas protein, and the other end of the labeled probe is coupled with a molecule and contains at least two antigenic determinants; streptavidin is chemically coupled on the sample pad, and the labeled probe is fixed on the sample pad through a biotin-avidin system.
Further, the labeled probe is fixed on the sample pad by a chemical coupling mode.
Further, the labeling probe is coupled with a magnetic particle to obtain a magnetic particle-nucleic acid probe-label compound, and the magnetic particle-labeling probe is arranged on the sample pad.
Further, a magnet is provided on the back of the sample pad.
Furthermore, the magnetic particles are streptavidin magnetic particles, the labeling probes are provided with biotin labels, and the magnetic particles and the labeling probes are connected through a biotin-avidin system.
Furthermore, the labeled probe is obtained by mixing a first labeled probe and a second labeled probe, the first labeled probe is provided with a biotin label at one end, the other end is provided with another small molecule label, and a marker region is complementarily combined with a sequence containing the other small molecule label.
Further, the marker is a macromolecule containing not less than two epitopes, and the first anti-marker antibody and the second anti-marker antibody are antibodies against different epitopes of the marker.
Further, the markers are two different small molecule substances, and the first anti-marker antibody and the second anti-marker antibody are antibodies against the two small molecule substances respectively.
Further, the label is a molecule marked by a colored dye, and the label capture object is an antibody aiming at the molecule.
Further, the label comprises a colored microsphere, the microsphere comprises an antibody, and the label capture substance can be captured by the antibody; colored microspheres include, but are not limited to, colloidal gold, colloidal carbon, red latex microspheres.
Further, the detection test strip comprises at least two lateral chromatography test strips, and the Cas protein on each lateral chromatography test strip comprises different guide sequences.
Further, the Cas protein comprises a first Cas protein and a second Cas protein, wherein the first Cas protein is a Cas12 protein containing a guide sequence of a targetable novel coronavirus ORF1ab gene, and the second Cas protein is a Cas12 protein containing a guide sequence of a targetable human mucosal protein MUC5 a.
Further, the lateral chromatography test paper is provided with at least two Cas proteins, the lateral chromatography test paper is provided with at least two labeled probes, the number of the Cas proteins is the same as that of the labeled probes, different nucleic acid probes are arranged on the different labeled probes, and different markers are arranged on the different labeled probes.
Further, the lateral chromatography test paper is provided with at least two Cas proteins, the lateral chromatography test paper is provided with at least two labeled probes, different labeled probes are provided with different nucleic acid probes, and different labeled probes are provided with different markers.
Further, the labeling carrier comprises one or more of colloidal gold, latex microspheres, colloidal carbon, magnetic microspheres or fluorescent microspheres.
In the scheme of the capture method, the lateral chromatography test paper is sequentially provided with a sample pad, an NC membrane and a water absorption pad;
the Cas protein is disposed on the sample pad;
the labeled probe is fixedly arranged on the sample pad;
the NC membrane is provided with a detection line, and the detection line is coated with molecules capable of capturing markers.
The detection principle of the invention is as follows: the lateral chromatography test paper is provided with a fixed marker probe and a Cas protein to realize the visual detection of the lateral chromatography of the molecule based on the CRISPR system, the marker probe comprises a nucleic acid probe and a marker which are connected together, the Cas protein comprises a guide sequence which can target the molecule to be detected, when a detection sample contains the molecule to be detected, the guide sequence in the Cas protein identifies the molecule to be detected, the Cas protein is activated, and then the nucleic acid probe in the marker probe is cut, so that the marker originally fixed in the marker probe of the lateral chromatography test paper is released, and the lateral chromatography test paper is also provided with a system which can detect the marker by a sandwich method or a capture method, so that the detection of the marker is realized, and the visual detection of the lateral chromatography of the molecule to be detected is further realized.
Compared with the prior art, the invention has the beneficial effects that:
(1) the specificity is high: according to the invention, the fixed labeled probe is arranged on the lateral chromatography test paper, so that the labeled probe is cut only when a substance to be detected exists in a detection sample, and the labeled substance is released and detected, thereby greatly improving the detection specificity and avoiding the problem of false positive caused by incomplete capture of a near-end strip or no addition of the probe in the prior art.
(2) The operation is convenient: according to the invention, the labeled probe and the Cas protein are arranged on the sample pad of the test strip, so that the steps of adding the Cas protein and the probe after amplification is finished and mixing uniformly by blowing in the prior art can be eliminated. The sample pad of the test strip is of a porous structure, and the test strip presents a distributed uniform reaction.
(3) Accelerating the recognition speed of the target molecule by the Cas protein: on one hand, because the Cas protein and the probe are in a solid phase state, the concentration of the target molecules in the sample is not reduced as the target molecules are added into a liquid system; on the other hand, by adjusting the sample pad formula to reduce the release rate of the Cas protein, the Cas protein can contact more samples or sample amplification products, which is equivalent to increase the apparent concentration of the target molecules, and the requirement of the Cas protein on the number or concentration of the target molecules for recognition can be achieved more quickly.
(4) The invention can carry out single-tube multi-joint detection, and can realize the simultaneous detection of a plurality of indexes by arranging a plurality of lateral chromatography test strips, wherein the Cas protein on each lateral chromatography test strip comprises different guide sequences, and a reaction product in the same tube can be detected by a plurality of lateral chromatography test strips.
(5) The invention can realize single-tube multi-joint detection of a single test strip. The single-tube multi-joint detection is realized by arranging different types of trans-cutting probe sequences and corresponding different types of Cas proteins containing different guide sequences on the test paper. For example, Cas13 of a trans-cleaved single-stranded RNA and Cas12 of a trans-cleaved DNA sequence, and the corresponding different probes, respectively.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
Example 1 lateral chromatography test strip for labeled probe
The lateral chromatography test paper is provided with a sample pad, a combination pad, an NC membrane and a water absorption pad on a bottom plate in sequence; the sample pad is provided with a Cas12 protein and an immobilized label probe. The Cas12 protein comprises a guide sequence capable of targeting a novel coronavirus ORF1ab gene, the labeled probe is a nucleic acid sequence containing two small molecular markers of FAM and digoxin, and biotin is labeled at the fixed end of the probe label for facilitating the fixation of the probe; an anti-FAM antibody-red latex microsphere compound is arranged on the bonding pad; the NC membrane is provided with a detection line and a quality control line, the detection line is coated with an anti-digoxin antibody, and the quality control line is coated with a goat anti-mouse antibody.
The preparation method of the labeled probe lateral chromatography test strip comprises the following steps:
(1) two sequences were designed: t (Biotin-labeled) TTTTTTTTTCCTTCCTTCTCTC(FAM) and G (digoxigenin-labeled) AGAGAAGGAAGG were synthesized by commercial primer synthesis, and the two were mixed in an equimolar ratio and reacted for 10min before use.
(2) Sample pad preparation: streptavidin coupling is fixed on a sample pad through a chemical method, then the synthesized labeled probe is sprayed on the sample pad, after 30 minutes of reaction, the sample pad is washed three times by PBS (phosphate buffer solution) containing 0.5% BSA and 5% sucrose, unbound labeled probe is washed away, then Cas12 protein containing a guide sequence of targeting novel coronavirus ORF1ab gene is added into a sample pad treatment solution, and the sample pad is dried for 2 hours at 40 ℃ after being treated by the treatment solution.
(3) Preparing a bonding pad: labeling the anti-FAM antibody with red latex microspheres, and spraying a bonding pad: activating red latex microspheres by EDC \ NHS, adding an anti-FAM mouse monoclonal antibody, reacting for 1 hour in an oven at 37 ℃, adding a mixture of BSA and glycine to seal the latex microspheres, spraying the mixture on a bonding pad after centrifugal redissolution, and drying for 2 hours at 40 ℃;
(4) coating with an NC film: respectively preparing 0.5mg/mL digoxin antibody and 0.5mg/mL goat anti-mouse antibody as detection line coating working solution and quality control line coating working solution by using 1xPBS and 0.5% sucrose as solvents, sticking an NC membrane to a PVC (polyvinyl chloride) bottom plate, then scratching the prepared coating working solution on the NC membrane by using a gold-spraying film scratching instrument, and drying for 12 hours at 35 ℃ to obtain a coated NC membrane;
(5) assembling and cutting strips: overlapping the cut absorbent paper, the combining pad and the sample pad in sequence, and adhering the absorbent paper, the combining pad and the sample pad to an NC (numerical control) coated film to obtain a large plate; and cutting the obtained large plate into pieces with the width of 3mm by using a slitter to obtain the lateral chromatography test strip.
The use method of the lateral chromatography test strip of the embodiment comprises the following steps: the sample to be detected is cracked in an extraction-free mode, amplified in PCR or LAMP, RPA and other modes, inserted into the test strip, and reacted for 5 minutes before the result is judged. And judging whether the sample contains the novel coronavirus ORF1ab gene or not through the color development conditions of the detection line and the quality control line.
Example 2 lateral chromatography test strip for labeled probe
The lateral chromatography test paper is provided with a sample pad, a combination pad, an NC membrane and a water absorption pad on a bottom plate in sequence; the sample pad is provided with a Cas12 protein and an immobilized label probe. The Cas12 protein is provided with a guide sequence capable of targeting a human mucosal protein MUC5a, the labeled probe is a nucleic acid sequence for labeling two small molecular markers of DNP and TAMRA, and biotin is labeled at one end of the probe for facilitating the fixation of the probe; the combination pad is provided with an anti-DNP antibody-red latex microsphere compound and an IgY antibody-red latex microsphere compound; the NC membrane is provided with a detection line and a quality control line, the detection line is coated with an anti-TRMRA antibody, and the quality control line is coated with a rabbit anti-chicken IgY antibody.
The preparation method of the labeled probe lateral chromatography test strip comprises the following steps:
(1) the labeled probe was synthesized, and the labeled probe sequence T (Biotin-labeled) TTTTTTTTTCCCCCCCCCCCC (FAM-labeled) was sent to the primer synthesis supplier for synthesis. The FAM antibody is labeled with DNP and TAMRA and then mixed with the labeled probe for use.
(2) Sample pad preparation: streptavidin coupling is fixed on a sample pad through a chemical method, then the synthesized labeled probe is sprayed on the sample pad, after reaction for 30 minutes, the sample pad is washed three times by PBS (phosphate buffer solution) containing 0.5% BSA and 5% sucrose, unbound labeled probe is washed away, Cas12 protein containing a targeted human mucosal protein MUC5a gene guide sequence is added into a sample pad treatment solution, and after treatment by the sample pad treatment solution, the sample pad is dried for 2 hours at 40 ℃.
(3) Preparing a bonding pad: mixing and labeling the anti-DNP antibody and the chicken IgY with red latex microspheres, and spraying a binding pad: activating the red latex microspheres by EDC \ NHS, respectively adding an anti-DNP antibody and chicken IgY, reacting for 1 hour in a 37 ℃ oven, adding a BSA and glycine mixture to seal the latex microspheres, centrifuging, redissolving, and spraying on a bonding pad. Drying for 2 hours at 40 ℃.
(4) Coating with an NC film: adopting 1xPBS and 0.5% sucrose as solvents, respectively preparing 0.5mg/mL anti-TAMRA antibody and 0.5mg/mL rabbit anti-chicken IgY antibody as detection line coating working solution and quality control line coating working solution, pasting an NC membrane on a PVC base plate, then adopting a gold spraying membrane scribing instrument to scribe the prepared coating working solution on the NC membrane, and drying for 12 hours at 35 ℃ to obtain the coating NC membrane.
(5) Assembling and cutting strips: overlapping the cut absorbent paper, the combining pad and the sample pad in sequence, and adhering the absorbent paper, the combining pad and the sample pad to an NC (numerical control) coated film to obtain a large plate; and cutting the obtained large plate into pieces with the width of 3mm by using a slitter to obtain the lateral chromatography test strip.
The use method of the lateral chromatography test strip of the embodiment comprises the following steps: the sample to be detected is cracked in an extraction-free mode, amplified in PCR or LAMP, RPA and other modes, inserted into the test strip, and reacted for 5 minutes before the result is judged. And judging whether the sample contains the human mucosal protein MUC5a sequence or not through the color development conditions of the detection line and the quality control line.
In this embodiment, the FAM antibody is labeled with a small molecule, and two monoclonal antibodies against the FAM antibody can be used to detect the FAM antibody by a sandwich method.
Example 3 lateral chromatography test strip for labeled probe
The lateral chromatography test paper is provided with a sample pad, an NC membrane and a water absorption pad on a bottom plate in sequence; the sample pad is provided with a Cas12 protein and an immobilized label probe. The Cas12 protein is provided with a guide sequence capable of targeting human mucosal protein MUC5a, the labeled probe is an anti-TRMRA antibody labeled by colloidal gold, and biotin is labeled at one end of the probe for facilitating the fixation of the probe; the NC membrane is provided with a detection line and a quality control line, the detection line is coated with TRMRA-BSA whole antigen, and the quality control line is coated with rabbit anti-chicken IgY antibody.
The preparation method of the labeled probe lateral chromatography test strip comprises the following steps:
(1) a labeled probe was synthesized by sending a labeled probe sequence T (biotin label) TTTTTTTTTCCCCCCCCC (-SH) C (-SH) C (-SH) to a primer synthesis supplier. Adding the probe into a PVP stable 20nm colloidal gold solution, then adding an anti-TRMRA antibody for marking, sealing, centrifuging and redissolving for later use.
(2) Sample pad preparation: streptavidin coupling is fixed on a sample pad through a chemical method, then the synthesized labeled probe is sprayed on the sample pad, after reaction for 30 minutes, the sample pad is washed three times by PBS (phosphate buffer solution) containing 0.5% BSA and 5% sucrose, unbound labeled probe is washed away, Cas12 protein containing a targeted human mucosal protein MUC5a gene guide sequence is added into a sample pad treatment solution, and after treatment by the sample pad treatment solution, the sample pad is dried for 2 hours at 40 ℃.
(3) Preparing a bonding pad: the chicken IgY is marked by colloidal gold and then sprayed on a bonding pad, and is dried for 2 hours at 40 ℃.
(4) Coating with an NC film: adopting 1xPBS and 0.5% sucrose as solvents, respectively preparing 0.5mg/mL TAMRA-BSA holoantigen and 0.5mg/mL rabbit anti-chicken IgY antibody as detection line coating working solution and quality control line coating working solution, pasting an NC membrane on a PVC base plate, then adopting a gold spraying membrane scribing instrument to scribe the prepared coating working solution on the NC membrane, and drying for 12 hours at 35 ℃ to obtain the coating NC membrane.
(5) Assembling and cutting strips: overlapping the cut absorbent paper, the combining pad and the sample pad in sequence, and adhering the absorbent paper, the combining pad and the sample pad to an NC (numerical control) coated film to obtain a large plate; and cutting the obtained large plate into pieces with the width of 3mm by using a slitter to obtain the lateral chromatography test strip.
The use method of the lateral chromatography test strip of the embodiment comprises the following steps: the test strip is inserted after the sample to be tested is cracked in an extraction-free mode and amplified in PCR or LAMP, RPA and other modes, the result is judged after 5 minutes of reaction, if the sample contains a human mucosal protein MUC5a sequence, the antibody marked with anti-TAMRA is released and reaches the detection line to be combined with TAMRA-BSA whole antigen for capture. And judging whether the sample contains the human mucosal protein MUC5a sequence or not through the color development conditions of the detection line and the quality control line.
Example 4 lateral chromatography test strip of magnetic particle labeled probe
The lateral chromatography test paper is provided with a sample pad, a combination pad, an NC membrane and a water absorption pad on a bottom plate in sequence; cas12 protein and a labeled probe labeled on magnetic particles are arranged at the sample pad, and a sheet-shaped magnetic stripe is adhered to the back of the bottom plate. The Cas12 protein is provided with a guide sequence capable of targeting a novel coronavirus ORF1ab gene, and the marker probe is a nucleic acid sequence containing two small molecular markers of FAM and digoxin; an anti-FAM antibody-red latex microsphere compound is arranged on the bonding pad; the NC membrane is provided with a detection line and a quality control line, the detection line is coated with an anti-digoxin antibody, and the quality control line is coated with a goat anti-mouse antibody. The function of fixing the marking probe at the position of the sample pad is achieved through the magnetic particles and the magnetic strip.
The preparation method of the labeled probe lateral chromatography test strip comprises the following steps:
(1) label probes were synthesized, two sequences were synthesized: t (Biotin marker) TTTTTTTTTCCTTCCTTCTCTC (FAM marker) and G (digoxin marker) AGAGAAGGAAGG, which were mixed in an equimolar ratio and reacted for 10min before use.
(2) Sample pad preparation: streptavidin is coupled to the magnetic particles through a commercialized method, the synthesized labeled probe is reacted with magnetic beads after redissolution, the probe is bonded to the magnetic beads through the avidin, and then the magnetic beads are washed three times to remove the unbound labeled probe. The sample pad was treated with a treatment solution containing two Cas proteins, and magnetic beads were sprayed on the sample pad.
(3) Preparing a bonding pad: labeling FAM antibody with red latex microspheres, spraying a binding pad: activating the red latex microspheres by EDC \ NHS, adding an anti-FAM mouse monoclonal antibody, reacting for 1 hour in an oven at 37 ℃, adding a mixture of BSA and glycine to seal the latex microspheres, centrifuging, redissolving, and spraying on a bonding pad. Drying for 2h at 40 ℃;
(4) coating with an NC film: respectively preparing 0.5mg/mL digoxin antibody and 0.5mg/mL goat anti-mouse antibody as detection line coating working solution and quality control line coating working solution by using 1xPBS and 0.5% sucrose as solvents, sticking an NC membrane to a PVC (polyvinyl chloride) bottom plate, then coating the prepared coating working solution on the NC membrane by using a gold spraying membrane-scribing instrument, and drying for 12 hours at 35 ℃ to obtain a coated NC membrane;
(5) assembling and cutting strips: overlapping the cut absorbent paper, the combination pad and the sample pad in sequence, adhering the absorbent paper, the combination pad and the sample pad to the coated NC film, and adhering a flaky magnetic stripe to the sample pad on the back of the bottom plate to obtain a large plate; and cutting the obtained large plate into pieces with the width of 3mm by using a slitter to obtain the lateral chromatography test strip.
The use method of the lateral chromatography test strip of the embodiment comprises the following steps: the sample to be detected is cracked in an extraction-free mode, amplified in PCR or LAMP, RPA and other modes, inserted into the test strip, and reacted for 10 minutes before the result is judged. And judging whether the sample contains the novel coronavirus ORF1ab gene or not through the color development conditions of the detection line and the quality control line.
Example 5 molecular lateral chromatography multi-index joint detection test paper based on CRISPR system
The test strip comprises two lateral chromatography test strips, and specifically, the two test strips in the embodiment 1 and the embodiment 2 can be placed side by side. The test paper of the embodiment 1 is used for detecting and judging whether a sample contains a novel coronavirus ORF1ab gene or not, the test paper 2 is used for internal quality control, a human mucosal protein MUC5a sequence is determined, and the sequence is effectively amplified.
The use method of the lateral chromatography test strip of the embodiment comprises the following steps: the human nasal cavity collection sample to be detected is cracked in an extraction-free mode, and then the novel coronavirus ORF1ab gene and the human mucosal protein MUC5a gene are amplified simultaneously in PCR (polymerase chain reaction) or LAMP (loop-mediated isothermal amplification), RPA (reverse transcription) and other modes. Then the test strips are inserted in parallel, and the result is judged after the reaction is carried out for 10 minutes. The test strip of example 2 is positive, and the test strip is used as internal quality control to determine that the human mucosal components are collected in the sample, and the test strip of example 1 is used for judging whether the sample contains the novel coronavirus ORF1ab gene.
Example 6 molecular lateral chromatography test strip based on CRISPR system
In this example, primers (shown in SEQ ID NO: 1-2) recommended by the novel coronavirus pneumonia prevention and control program (eighth edition) were used to amplify the novel coronavirus ORF1ab gene, and the labeling was performed using a pseudovirus method, followed by detection using the test strip.
(1) Preparing a probe: the mixture was reacted for 10min by mixing 200nM of T (biotin-labeled) TTTTTTTTTCCTTCCTTCTCTC (FAM-labeled) and 200nM of G (digoxin-labeled) AGAGAAGGAAGG.
(2) Preparation of Cas protein containing guide sequence: the leader GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGC AAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACCACUUAAAAACACAGUCU GU (SEQ ID NO: 3) was mixed at a concentration of 10uM with AapCas12b protein at a concentration of 10uM, and reacted at 30 ℃ for 30 minutes for further use. AapCas12b protein was purchased from Touhong harbor Biotech Co.
(3) Sample pad preparation:
processing a sample pad: the glass fiber pad was washed and treated in a fume hood with 2% 3-mercaptopropyl) trimethoxysilane and 2mM N-gamma-maleimidobutyryl-oxysuccinimide) in sequence. And after cleaning, soaking the cleaned product in 100ng/ml streptavidin solution, and after cleaning, drying the cleaned product in an oven at 37 ℃ for 2h for later use.
② mixing the prepared probe and Cas protein containing guide sequence, and adding 10 XNEB Buffer 3.1, 0.5% BSA and 2% sucrose, 100mM taurocholic acid, 15mM MgSO4And 50u/ul of RNase inhibitor and sample pad treating solution, with which the pad washed in the step (1) was sprayed. The sample pad was dried at 37 degrees celsius for 2 hours after treatment with the treatment solution.
(4) Preparing a bonding pad: activating red latex microspheres by EDC \ NHS, adding an anti-FAM mouse monoclonal antibody, reacting for 1 hour in an oven at 37 ℃, adding a mixture of BSA and glycine to seal the latex microspheres, spraying the mixture on a bonding pad after centrifugal redissolution, and drying for 2 hours at 40 ℃;
(5) coating with an NC film: respectively preparing 0.5mg/mL digoxin antibody and 0.5mg/mL goat anti-mouse antibody as detection line coating working solution and quality control line coating working solution by using 1xPBS and 0.5% sucrose as solvents, sticking an NC membrane to a PVC (polyvinyl chloride) bottom plate, then scratching the prepared coating working solution on the NC membrane by using a gold-spraying film scratching instrument, and drying for 12 hours at 35 ℃ to obtain a coated NC membrane;
(6) assembling and cutting strips: overlapping the cut absorbent paper, the combining pad and the sample pad in sequence, and adhering the absorbent paper, the combining pad and the sample pad to an NC (numerical control) coated film to obtain a large plate; and cutting the obtained large plate into pieces with the width of 3mm by using a slitter to obtain the lateral chromatography test strip.
(7) The negative nasal swab sample and the sample added with the novel coronavirus ORF1ab pseudovirus are subjected to isothermal amplification by using an RAA test strip respectively. The novel coronavirus ORF1ab pseudovirus was purchased from Shanghai Fubai Australian organism, reaction system 50 ul.
(8) Reaction of the test strip: after the amplification is completed, the sample pad end of the test strip is inserted into the reaction system for reaction for 20 minutes. The amplification product reached the sample pad by capillary electrophoresis, but did not reach the conjugate pad and NC membrane.
(9) After the reaction is finished, 50ul PBS is added into the tube, the test strip is colored, and the result is judged according to the color development condition.
The test strip can detect 500 copies/mL by detecting 10 nasal swabs added with different concentrations and without adding the novel coronavirus ORF1ab pseudovirus.
The experimental results show that the product has the advantages of strong specificity, high sensitivity, simple and convenient operation and high detection speed, can realize multi-index combined detection, and can better realize molecular lateral chromatography visual detection based on a CRISPR system.
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.
Sequence listing
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Claims (29)
1. A molecular lateral chromatography detection method based on a CRISPR system comprises the steps of labeled probe cutting, label releasing and lateral chromatography test paper detection, and is characterized in that the method comprises the following steps:
and (3) cutting the labeled probe: the label probe comprises a nucleic acid probe and a label which are connected, and when the nucleic acid probe contacts the Cas protein activated by a molecule to be detected, the nucleic acid probe is cut;
release of the label: the label probe is fixedly arranged, the label comprises not less than two antigenic determinants, and when the nucleic acid probe is cut, the label is released from the fixed position;
detection of the marker: the lateral chromatography test paper adopts a sandwich method or a capture method to detect the released marker, thereby realizing the detection of the molecules to be detected.
2. The molecular lateral chromatography detection method based on CRISPR system of claim 1, wherein the nucleic acid probe can be cleaved by activated Cas protein or engineered micro Cas protein.
3. The molecular lateral chromatography detection method based on CRISPR system of claim 1, wherein the nucleic acid probe is any one or more of double-stranded DNA sequence, single-stranded RNA sequence and double-stranded RNA sequence.
4. The molecular lateral chromatography detection method based on CRISPR system of claim 1, wherein said labeled probe is immobilized on said lateral chromatography test paper.
5. The molecular lateral chromatography detection method based on CRISPR system of claim 1, wherein said labeled probe is immobilized on a solid phase carrier.
6. The molecular lateral chromatography detection method based on CRISPR system of claim 5, wherein said labeled probe is immobilized on magnetic particle.
7. A molecular lateral chromatography detection test strip based on a CRISPR system comprises a lateral chromatography test strip and a Cas protein, and is characterized in that the test strip further comprises an immobilized marker probe, the marker probe comprises a nucleic acid probe and a marker which are connected, and the marker comprises not less than two antigenic determinants.
8. The CRISPR system-based molecular lateral chromatography detection strip of claim 7, wherein the label is comprised of a colored or colorless microsphere or colloidal gold, said microsphere comprising not less than one epitope thereon.
9. The CRISPR system-based molecular lateral chromatography detection strip of claim 8, wherein the microspheres comprise antibodies or antigens, which can be detected by capture.
10. The CRISPR system-based molecular lateral chromatography detection strip of claim 7, wherein the lateral chromatography strip is provided with a backing plate, a sample pad, a binding pad, an NC membrane and a water absorbent pad;
the Cas protein is disposed on the sample pad;
the labeled probe is fixedly arranged on the sample pad;
a first anti-marker antibody-labeled carrier complex is disposed on the conjugate pad;
and the NC membrane is provided with a detection line, and the detection line is coated with a second anti-marker antibody.
11. The molecular lateral chromatography detection test strip based on a CRISPR system of claim 10, wherein said NC membrane is provided with a detection line coated with a label capture.
12. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein the sample pad, the conjugate pad, the NC membrane and the absorbent pad are sequentially attached to the backing plate.
13. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein the conjugate pad, NC membrane and absorbent pad are sequentially attached to the backing plate, and the sample pad is separately provided.
14. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein one end of the nucleic acid probe is fixedly attached to the sample pad and the other end of the nucleic acid probe is coupled to the label.
15. The CRISPR system-based molecular lateral chromatography detection test strip of claim 10, wherein the nucleic acid probe is provided with an avidin label, the sample pad is chemically coupled with streptavidin, and the labeled probe is immobilized on the sample pad by a biotin-avidin system.
16. The CRISPR system-based molecular lateral chromatography detection test strip of claim 10, wherein the labeled probe has a biotin label at one end, comprises a region cleavable by Cas protein, has another small molecule label at the other end, and is bound by an antibody of the small molecule, wherein the antibody of the small molecule comprises two or more different small molecule labels; streptavidin is chemically coupled on the sample pad, and the labeled probe is fixed on the sample pad through a biotin-avidin system.
17. The CRISPR system-based molecular lateral chromatography detection test strip of claim 10, wherein the labeled probe has a biotin label at one end and comprises a sequence cleavable by a Cas protein, and the coupling molecule at the other end comprises at least two epitopes; streptavidin is chemically coupled on the sample pad, and the labeled probe is fixed on the sample pad through a biotin-avidin system.
18. The CRISPR system-based molecular lateral chromatography detection test strip of claim 10, wherein the label probe is coupled to a magnetic particle to obtain a magnetic particle-nucleic acid probe-label complex, wherein the magnetic particle-label probe is disposed on the sample pad, and a magnet is disposed on the back of the sample pad.
19. The molecular lateral chromatography detection test strip based on the CRISPR system of claim 10, wherein the magnetic particles are streptavidin magnetic particles, the labeled probe is provided with a biotin label, and the magnetic particles and the labeled probe are connected through a biotin-avidin system.
20. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein the labeled probe is obtained by mixing a first labeled probe with a biotin label at one end and another small molecule label at the other end, and the region of the label is complementarily bound to a sequence comprising the another small molecule label.
21. The CRISPR system-based molecular lateral chromatography detection test strip of claim 10, wherein the marker is a macromolecule comprising not less than two epitopes and the first and second anti-marker antibodies are antibodies against different epitopes of the marker.
22. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein the marker is two different small molecule substances, and the first anti-marker antibody and the second anti-marker antibody are antibodies against the two small molecule substances, respectively.
23. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein the label is a colored dye-labeled molecule and the label capture is an antibody against the molecule.
24. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein the label comprises a colored microsphere comprising an antibody, and the label capture is captured by the antibody; colored microspheres include, but are not limited to, colloidal gold, colloidal carbon, red latex microspheres.
25. The CRISPR system-based molecular lateral chromatography detection strip of claim 10, wherein the detection strip comprises not less than two lateral chromatography strips, and the Cas protein on each lateral chromatography strip comprises a different guide sequence.
26. The CRISPR system-based molecular lateral chromatography detection test strip of claim 25, wherein the Cas protein comprises a first Cas protein and a second Cas protein, the first Cas protein is a Cas12 protein comprising a targetable novel coronavirus ORF1ab gene guide sequence, and the second Cas protein is a Cas12 protein comprising a targetable human mucosal protein MUC5a guide sequence.
27. The molecular lateral chromatography detection test strip based on the CRISPR system of claim 10, wherein the lateral chromatography test strip is provided with not less than two Cas proteins, the lateral chromatography test strip is provided with not less than two labeled probes, the number of the Cas proteins is the same as that of the labeled probes, different nucleic acid probes are provided on the different labeled probes, and different markers are provided on the different labeled probes.
28. The molecular lateral chromatography detection test strip based on the CRISPR system of claim 10, wherein the lateral chromatography test strip is provided with at least two Cas proteins, at least two labeled probes are provided on the lateral chromatography test strip, different nucleic acid probes are provided on the different labeled probes, and different markers are provided on the different labeled probes.
29. The CRISPR system-based molecular lateral chromatography detection test strip of claim 10, wherein the labeling vector comprises one or more of colloidal gold, latex microspheres, nanocarbon, magnetic microspheres, or fluorescent microspheres.
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| CN115541875A (en) * | 2022-09-27 | 2022-12-30 | 江苏迅睿生物技术有限公司 | N gene detection primer and probe group, chromatography test strip, preparation method, detection kit and detection method |
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| CN115389760A (en) * | 2022-10-27 | 2022-11-25 | 艾康生物技术(杭州)有限公司 | Detection reagent for immunoassay test strip |
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