CN113699148A - Ultrasensitive antibody detection method - Google Patents
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- CN113699148A CN113699148A CN202110800264.5A CN202110800264A CN113699148A CN 113699148 A CN113699148 A CN 113699148A CN 202110800264 A CN202110800264 A CN 202110800264A CN 113699148 A CN113699148 A CN 113699148A
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Abstract
The present invention discloses an ultrasensitive antibody detection method, in particular, it relates to an ultrasensitive detection method for SARS-CoV-2 antibody. The invention also provides a crRNA, a probe, a conjugate, a combined product, a test paper, a kit and the like for the method, which can be used for detecting the antibody (such as an anti-SARS-CoV-2 antibody) generated by the immune reaction caused by the antigen in an organism, has very high sensitivity, the detection limit can be as low as 1aM, the operation is simple, any washing step is not needed, the method can be flexibly used for detecting the antibody, and has higher development and application values.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to an ultrasensitive antibody detection method, and especially relates to an ultrasensitive detection method for SARS-CoV-2 antibody.
Background
Serological tests to determine antibody levels in the blood are important tools for diagnosing infectious diseases, such as novel coronavirus disease (COVID-19). Although nasopharyngeal swab Nucleic Acid Test (NAT) is the primary method for identifying severely SARS-CoV-2 infected individuals, sensitive detection of antibodies against SARS-CoV-2 can help identify symptomatic patients with negative NAT results, or diagnose infection in NAT restricted resource restricted areas. In addition, serological antibody testing also provides basic information about disease progression and patient immunity, which is critical for predicting the prevalence of SARS-CoV-2, for therapeutic development, and for evaluating the effectiveness of the COVID-19 vaccine.
To date, a number of immunoassays for anti-SARS-Cov-2 antibodies have been developed, but have their limitations. The most widely used enzyme-linked immunosorbent assays (ELISA) usually require cumbersome washing and incubation steps, which are not suitable for use outside the laboratory (S.K.Elledge, et al, Nat Biotechnol 2021, DOI:10.1038/s 41587-021-00878-8). Several highly automated chemiluminescent immunoassays (CLIA) have been developed and commercialized, but can only be used in a laboratory environment due to the need for highly specialized instrumentation (D.D. Rhoads, et al., J Clin Microbiol 2020,58, DOI: 10.1128/JCM.00760-20). Lateral Flow Assay (LFA) detection is fast and portable, but is generally unreliable due to its qualitative nature and poor sensitivity (C.H.GeurtsvanKessel, et al, Nat Commun 2020,11,3436, DOI:10.1038/s 41467-020-. Furthermore, all of the above assays lack the sensitivity to detect antibodies early in infection and require at least one week to reach detectable antibody concentrations at the onset of symptoms. Ultra-sensitive detection of anti-SARS-CoV-2 antibodies with an Analytical sensitivity that is improved by 1000-fold compared to standard ELISA is possible, but can only be achieved by using a highly specialized and expensive single molecule array (Simoa) platform (M. Norman, et al, Nat Biomed Eng 2020,4, 1180-.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an ultrasensitive antibody detection method, which is realized by the following technical scheme:
a target nucleic acid is designed which consists of fragments S1, S2 and S3 linked in this order, i.e.which can be designated S1-S2-S3.
Specifically, the lengths of S1 and S3 may independently be 5nt or more, e.g., 5-45nt, 5-20nt, 5-10nt, e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 nt.
In some embodiments of the invention, S1 is 7nt in length.
In some embodiments of the invention, S3 is 8nt in length.
In some embodiments of the invention, S1 is GGTCGAG.
In some embodiments of the invention, S3 is CGGCGACG.
Specifically, the length of S2 may be 5-8nt, e.g., 5, 6, 7, 8 nt; in some embodiments of the invention, S2 is 5nt in length.
In some embodiments of the invention, S2 is CTGGA.
In some embodiments of the invention, the target nucleic acid is CGTCGCCGTCCAGGGTCGAG.
In a first aspect, the present invention provides a crRNA comprising: (1) a framework nucleic acid fragment that interacts with the Cas nuclease, and (2) a specific nucleic acid fragment that binds to a target nucleic acid.
In some embodiments of the invention, the crRNA consists of (1) a framework nucleic acid fragment that interacts with the Cas nuclease, and (2) a specific nucleic acid fragment that binds to the target nucleic acid.
Specifically, the length of the specific nucleic acid fragment is more than 18 bt, such as 20-100bt, 20-50bt, such as 18, 20, 22, 24, 25, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 bt; in some embodiments of the invention, the specific nucleic acid fragment is 20bt in length.
Specifically, the specific nucleic acid fragment consists of fragments S1 ', S2' and S3 'linked in sequence, i.e., it can be represented as S1' -S2 '-S3'.
Specifically, the lengths of S1 'and S3' may independently be 5nt or more, e.g., 5-45nt, 5-20nt, 5-10nt, e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 nt.
In some embodiments of the invention, S1' is 7nt in length.
In some embodiments of the invention, S3' is 8nt in length.
In some embodiments of the invention, S1' is CUCGACC.
In some embodiments of the invention, S3' is CGUCGCCG.
Specifically, S2' may be 5-8nt in length, e.g., 5, 6, 7, 8 nt; in some embodiments of the invention, S2' is 5nt in length.
In some embodiments of the invention, S2' is UCCAG.
In some embodiments of the invention, the specific nucleic acid fragment comprises or consists of the nucleotide sequence set forth in SEQ ID NO. 1.
In some embodiments of the invention, the framework nucleic acid fragment comprises or consists of the nucleotide sequence set forth in SEQ ID NO. 2.
In some embodiments of the invention, the crRNA comprises or consists of the nucleotide sequence set forth in SEQ ID NO. 3.
In a second aspect, the present invention provides a single-stranded DNA probe comprising a fragment SaFragment SaIdentical to the fragments S1-S2 (i.e.complementary to the fragments S1 '-S2').
In particular, the fragment SaComprises or consists of the nucleotide sequence shown as SEQ ID NO. 4.
More specifically, the single-stranded DNA probe further comprises a fragment SrOf the fragment SaAnd (4) connecting.
In particular, the fragment SrThe length of (a) may be 10 to 100nt, for example 15 to 50nt, 20 to 40nt, 25 to 35nt, 30 nt.
In particular, the fragment SrComprises the nucleotide sequence shown as SEQ ID NO. 5, orConsisting of it.
In some embodiments, the single-stranded DNA probe comprises or consists of the nucleotide sequence set forth in SEQ ID NO 6.
In a third aspect, the present invention provides a single-stranded DNA probe comprising a fragment SbFragment SbComplementary to the fragment S2-S3 (i.e., corresponding to the fragment S2 '-S3', i.e., replacing U with T in the fragment S2 '-S3' (if the fragment S2 '-S3' contains U)).
In particular, the fragment SbComprises or consists of the nucleotide sequence shown as SEQ ID NO. 7.
More specifically, the single-stranded DNA probe further comprises a PAM sequence, which is complementary to the fragment SbAnd (4) connecting.
Specifically, the PAM sequence is TTTX, where X can be any deoxynucleotide (A, T, C, G); in some embodiments of the invention, the PAM sequence is TTTA.
In particular, the fragment PAM-SbComprises or consists of the nucleotide sequence shown as SEQ ID NO. 8.
More specifically, the single-stranded DNA probe further comprises a fragment Sr', which is identical to the fragment PAM-SbAnd (4) connecting.
In particular, the length of the fragment Sr' may be 10-100nt, such as 15-60nt, 30-50nt, 35-45nt, 42 nt.
In particular, the fragment Sr' comprises or consists of the nucleotide sequence shown in SEQ ID NO. 9.
In some embodiments, the single-stranded DNA probe comprises or consists of the nucleotide sequence set forth in SEQ ID NO. 10.
In a fourth aspect, the present invention provides a conjugate of a probe according to the third or fourth aspect and an antigen moiety of interest.
Specifically, the target antigen can be any substance that can stimulate the body to produce antibodies and specifically bind to the antibodies, such as viruses, bacteria, bacterial exotoxins, and the like.
In some embodiments of the invention, the antigen of interest is a virus.
Specifically, the virus may be a virus of the family adenoviridae, herpesviridae (e.g., EBV), papovaviridae, picornaviridae, poxviridae (e.g., variola virus), hepadnaviridae (e.g., hepatitis B virus), coronaviridae (e.g., HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.), Ponaviridae, filoviridae (e.g., Ebola virus), orthomyxoviridae (e.g., influenza virus), Paramyxoviridae, retroviridae (e.g., HIV), reoviridae, rhabdoviridae (e.g., rabies virus), Flaviviridae.
In some embodiments of the invention, the virus is a coronavirus, specifically HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, and the like.
In some embodiments of the invention, the virus is an orthomyxovirus, such as an influenza virus (e.g., influenza a, influenza b, influenza c, etc.).
In some embodiments of the invention, the virus is a paramyxovirus, such as human parainfluenza virus type 1 (HPIV), HPIV type 2, HPIV type 3, HPIV type 4, sendai virus, mumps virus, measles virus, respiratory syncytial virus, newcastle disease virus, and the like.
In the above, the virus is a flavivirus, such as dengue virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.
In particular, the antigen portion of interest in the conjugate may be a viral protein, for example, the entire protein of a virus or a portion thereof.
In some embodiments of the invention, in the conjugate, the antigen of interest is SARS-CoV-2; specifically, the antigen portion of interest in the conjugate may be one or more of the structural proteins of SARS-CoV-2 (e.g., S protein, M protein, E protein, N protein), or a portion of each structural protein (e.g., the Receptor Binding Domain (RBD) of the S protein).
Specifically, the conjugate further comprises an linker, and the probe is coupled to the target antigen moiety through the linker.
Specifically, the linker may be any suitable linker, such as a linker formed by a label-anti-label complex, wherein the combination of label/anti-label in the label-anti-label complex includes, but is not limited to: biotin or its derivatives/streptavidin, biotin or its derivatives/avidin, biotin or its derivatives/neutravidin, hapten/antibody, antigen/antibody, receptor/ligand, digoxin/digoxigenin, carbohydrate/lectin and polynucleotide/complementary polynucleotide, and the like. In some embodiments of the invention, the linker may be a linker formed by biotin/biotin-bound proteins, including streptavidin, avidin, and neutravidin, as described above, each capable of binding four biotin molecules with a high degree of affinity and specificity.
In some embodiments of the invention, the linker may be-biotin-streptavidin-biotin-.
In some embodiments of the invention, the conjugate can be a probe-biotin-streptavidin-biotin-target antigen moiety (e.g., the RBD of the SARS-CoV-2S protein).
In a fifth aspect, the present invention provides a conjugate of a probe according to the third or fourth aspect of the present invention and a ligand moiety of an antibody of interest.
In particular, the antibody of interest is an antibody against an antigen of interest, the antigen of interest having the definition as described in the fourth aspect of the invention.
Specifically, the target antibody may be any of IgG, IgA, IgM, IgD, IgE, in particular IgG, IgM.
In some embodiments of the invention, the antibody of interest is a SARS-CoV-2 antibody, in particular an anti-SARS-CoV-2 IgG, SARS-CoV-2 IgM.
In some embodiments, the ligand is an antibody, particularly a monoclonal antibody; specifically, when the target antibody is SARS-CoV-2 antibody, the ligand is an antibody against SARS-CoV-2 antibody, for example, when the target antibody is SARS-CoV-2IgG or SARS-CoV-2IgM, the ligand may be an antibody against SARS-CoV-2IgG or an antibody against SARS-CoV-2IgM, respectively.
Specifically, the conjugate further comprises an linker, and the probe is coupled to the ligand moiety via the linker.
In particular, the linker may be any suitable linker having the definition as set forth in the fourth aspect of the invention; in some embodiments of the invention, the linker may be-biotin-streptavidin-biotin-.
In some embodiments of the invention, the conjugate may be a ligand of probe-biotin-streptavidin-biotin-target antibody.
In a sixth aspect, the present invention provides a combination comprising a probe according to the second aspect of the present invention and a probe according to the third aspect of the present invention.
In particular, the combination product further comprises a target antigen material, a ligand material, as described above, and optionally, a substance required for forming a linker.
In particular, the antigenic material of interest may be a viral protein, e.g., the entire protein of a virus or a partial protein thereof; in some embodiments of the invention, the antigenic material of interest is one or more of the structural proteins of SARS-CoV-2 (e.g., S protein, M protein, E protein, N protein), or a portion of a structural protein (e.g., the Receptor Binding Domain (RBD) of the S protein).
Specifically, the ligand raw material is an antibody of an anti-target antigen antibody; for example, when the target antigen is SARS-CoV-2 and the target antibody is anti-SARS-CoV-2 IgG or SARS-CoV-2IgM, the ligand material may be an anti-IgG antibody or an anti-IgM antibody, respectively.
Specifically, the linker may be a linker formed by a label-anti-label complex, and in this case, the substance required for forming the linker may include a label and an anti-label, and the combination of the label/anti-label is as described in the third aspect of the present invention, in particular, biotin and a biotin-binding protein (e.g., streptavidin, avidin, and neutravidin).
In particular, the combination product may further comprise reagents required for DNA polymerization, such as DNA polymerases (e.g.T 4 polymerase), dNTPs, Mg2+Buffer systems, and the like.
In particular, each component of the combination product is separately packaged.
In a seventh aspect, the present invention provides a combination comprising a conjugate according to the fourth aspect of the invention and a conjugate according to the fifth aspect of the invention.
In particular, the combination product further comprises reagents required for DNA polymerization, such as DNA polymerase (e.g., T4 polymerase), dNTP, Mg2+Buffer systems, and the like.
In particular, each component of the combination product is separately packaged.
In an eighth aspect, the present invention provides a combination product comprising a crRNA according to the first aspect of the present invention, a crisp sper protein, and a signaling reporter molecule.
Specifically, the crisp protein is a crisp protein with side cleavage activity, such as Cas12a, Cas12b, Cas13a, Cas13b, Cas14 and Csm6, especially Cas12 a.
In particular, the signaling reporter comprises single stranded DNA.
Specifically, the single-stranded DNA is 5 to 10nt (e.g., 5, 6, 7, 8, 9, 10nt) in length.
Specifically, the single-stranded DNA is composed of T and A.
In some embodiments of the invention, the single-stranded DNA is TTATT.
In one embodiment of the invention, the 5 'end and the 3' end of the signaling reporter are labeled with a fluorescent reporter group and a fluorescent quencher group, respectively.
Specifically, the fluorescent reporter group may be, for example, FAM, Texas Red, ROX, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, LC RED640, LC RED705, or the like.
Specifically, the fluorescence quenching group may be, for example, TAMRA, DABCYL, ECLIPSE, BHQ-1, BHQ-2, BHQ-3 or the like.
In some embodiments of the invention, the signaling reporter is FAM-TTATT-BHQ-1.
In another embodiment of the invention, the 5 'end and the 3' end of the signaling reporter are labeled with a first label and a second label, respectively, and the first label and the second label are different.
Specifically, the signaling reporter is further labeled with a signal substance different from the label, and the signal substance is further labeled with an antibody to a second label, whereby the signal substance and the second label are located on the same nucleic acid fragment when the signaling reporter is cleaved by the CRISPR detection system.
In particular, the signal substance may be selected from: fluorophores, colorimetric labels, gold nanoparticles, quantum dots, biotin, and other label molecules that can be used for detection (e.g., alkyne groups for raman diffraction imaging, cyclic olefins for click reactions, priming groups for polymer labeling), can also be selected from polypeptide/protein molecules, LNA/PNA, unnatural amino acids and their analogs (e.g., peptidomimetics), unnatural nucleic acids and their analogs (nucleomimetics), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation/assembly-induced emission molecules, rare earth ion ligand molecules, polyoxometalate, etc.).
In particular, the combination product may also comprise other agents required for crisp sper cleavage, e.g. buffer systems, etc.
In particular, the combination product may also comprise a positive control and/or a negative control.
In particular, each component of the combination product is separately packaged.
In a ninth aspect, the present invention provides a test strip, which comprises a base plate, a sample pad, a conjugate pad, a chromatography matrix and an absorbent pad, wherein the sample pad, the conjugate pad, the chromatography matrix and the absorbent pad are sequentially adhered to the base plate and connected in an overlapping manner.
In particular, the conjugate pad contains a first anti-label signal substance (e.g., a signal substance as described above) for a first label.
Specifically, a quality control (C) region is arranged on one side of the chromatography matrix close to the combination pad, and a detection (T) region is arranged on one side close to the absorption pad.
Specifically, the control region is coated with a second anti-label for the second label.
In particular, the detection zone is coated with an anti-primary anti-label to the primary anti-label.
In some embodiments of the invention, the signaling species is gold nanoparticles.
In some embodiments of the invention, the first label is FAM and the first anti-label is an anti-FAM antibody, e.g., a rabbit anti-FAM polyclonal antibody.
In some embodiments of the invention, the second marker is digoxin and the second anti-marker is an anti-digoxin antibody, e.g., a goat anti-digoxin polyclonal antibody.
In some embodiments of the invention, the anti-first anti-marker is an antibody directed against the first anti-marker, e.g., a goat anti-rabbit antibody.
In particular, the chromatography medium may be a nitrocellulose membrane.
In particular, the sample pad and the conjugate pad may be glass cellulose membranes.
In particular, the base plate may be a PVC base plate.
In a tenth aspect, the invention provides a primer, which comprises a primer 1 and a primer 2, wherein the primer 1 and the primer 2 are shown as SEQ ID NO. 11 and SEQ ID NO. 12 respectively.
In an eleventh aspect, the present invention provides an amplification combination comprising a primer according to the tenth aspect of the invention.
In particular, the amplification combination product may be used to perform any of the following amplification methods: recombinase Polymerase Amplification (RPA), PCR amplification, NASBA isothermal amplification, loop-mediated isothermal amplification, strand displacement amplification, helicase-dependent amplification, nickase amplification reactions, and the like, particularly RPA.
In particular, the amplification combination further comprises reagents required for nucleic acid amplification, e.g., reagents required for RPA, including: recombinase capable of binding single-stranded nucleic acid, single-stranded DNA binding protein (SSB), strand displacement DNA polymerase, dNTP, Mg2+Buffer systems, and the like.
Specifically, each component of the amplification combination product is separately packaged.
In a twelfth aspect, the present invention provides a kit comprising a combination according to the sixth aspect of the invention and a combination according to the eighth aspect of the invention.
Specifically, the kit may further comprise a test strip according to the ninth aspect of the present invention.
In particular, the kit may further comprise an amplification combination according to the eleventh aspect of the invention.
In a thirteenth aspect, the present invention provides a kit comprising a combination according to the seventh aspect of the invention and a combination according to the eighth aspect of the invention.
Specifically, the kit may further comprise a test strip according to the ninth aspect of the present invention.
In particular, the kit may further comprise an amplification combination according to the eleventh aspect of the invention.
In a fourteenth aspect, the present invention provides a use of the crRNA of the first aspect, the probe of the second aspect, the probe of the fourth aspect, the conjugate of the fifth aspect, the combination product of the seventh aspect, the combination product of the eighth aspect, the dipstick of the ninth aspect, the primer of the tenth aspect, the combination product of the eleventh aspect, and the kit of the twelfth aspect, in the preparation of a product for detecting an antibody against a target antigen.
In some embodiments of the invention, the antibody is a SARS-CoV-2 antibody, e.g., anti-SARS-CoV-2 IgG, anti-SARS-CoV-2 IgM.
Specifically, the sample for detecting the target antibody may be blood of the subject or a fraction thereof (e.g., serum), particularly serum, or a product thereof after dilution.
In particular, the subject is an animal, particularly a mammal, e.g. bat, masked palm, primate, particularly a human, carrying or suspected to carry an antigen of interest (such as SARS-CoV-2).
In particular, the test products described above can be used for diagnostic purposes as well as for non-diagnostic purposes.
In a fifteenth aspect, the present invention provides the use of the crRNA of the first aspect, the probe of the second aspect, the probe of the fourth aspect, the conjugate of the fifth aspect, the combination product of the seventh aspect, the combination product of the eighth aspect, the dipstick of the ninth aspect, the primer of the tenth aspect, the combination product of the eleventh aspect, and the kit of the twelfth aspect, in the preparation of a product for diagnosing a disease or disorder caused by a target antigen.
In particular, the above-mentioned diseases or conditions may be selected from: one or more of rhinitis, sinusitis, croup, pharyngitis, tonsillitis, parotitis, laryngitis, tracheitis, asthma, pneumonia, influenza, Zika virus disease, acquired immunodeficiency syndrome (AIDS), etc.
In some embodiments of the invention, the disease is novel coronavirus pneumonia (COVID-19) (the target antigen is SARS-CoV-2).
In a sixteenth aspect, the present invention provides an antibody detection method, comprising the steps of:
(1) mixing a sample to be tested with the combination product of the seventh aspect of the invention, and incubating;
(2) mixing the incubation product of step (1) with the combination product according to the eighth aspect of the invention, and incubating;
(3) and (6) detecting.
In particular, the sample to be tested is the blood of the subject or a fraction thereof (e.g. serum), in particular serum, or a product thereof after dilution.
Specifically, in the incubation system in step (1), the concentration of the DNA polymerase (e.g., T4 polymerase) is 1-6U/50. mu.L of the incubation system, particularly 2U/50. mu.L of the incubation system.
Specifically, in the incubation system in step (1), the concentration of the conjugate described in the fourth aspect and the conjugate described in the fifth aspect is 100fM-10pM per 50. mu.L of the incubation system, and particularly 10pM per 50. mu.L of the incubation system.
Specifically, in the incubation system in step (1), the sample concentration may be 5. mu.L/50. mu.L of the incubation system.
Specifically, the incubation temperature in step (1) may be 37-42 ℃, particularly 37 ℃.
In particular, the incubation time in step (1) may be 10-30 minutes, for example 20 minutes.
In some embodiments of the invention, step (1) further comprises the step of amplifying the incubation product.
Specifically, the amplification step comprises: the incubation product is mixed with the combination product according to the eleventh aspect of the invention and subjected to amplification incubation.
In particular, the temperature of the amplification incubation may be 37-42 ℃, in particular 37 ℃.
In particular, the time of amplification incubation may be 10-30 minutes, for example 20 minutes.
Specifically, the incubation temperature in step (2) may be 37-42 ℃, particularly 37 ℃.
Specifically, the incubation time in step (2) may be 10-120 minutes; when step (1) does not comprise an amplification step, the incubation time in step (2) may be 50-120 minutes; where step (1) comprises an amplification step, the incubation time in step (2) may be 10-60 minutes.
In some embodiments of the invention, the detecting of step (3) comprises the step of detecting fluorescence from the incubated product of step (2).
In still other embodiments of the present invention, the detecting in step (3) comprises the step of applying the incubation product of step (2) to the strip according to the ninth aspect of the present invention.
In some embodiments of the invention, the above-described antibody detection method is a SARS-CoV-2 antibody detection method.
Specifically, the antibody detection method may be a quantitative detection method or a qualitative detection method.
In particular, the antibody detection methods described above may be used for diagnostic as well as non-diagnostic purposes.
In a seventeenth aspect, the present invention provides an antibody detection method, comprising the steps of:
(1) mixing a sample to be tested with the conjugate and the reagent required by DNA polymerization in the fourth and fifth aspects of the invention, and incubating;
(2) mixing the incubation product of step (2) with crRNA, CRISPER protein, and signaling reporter of the first aspect of the invention, and incubating;
(3) and (6) detecting.
In particular, the sample to be tested is the blood of the subject or a fraction thereof (e.g. serum), in particular serum, or a product thereof after dilution.
Specifically, in step (1), the reagents required for DNA polymerization include, for example, DNA polymerase (e.g., T4 polymerase), dNTP, Mg2+Buffer systems, and the like.
Specifically, in the incubation system in step (1), the concentration of the DNA polymerase (e.g., T4 polymerase) is 1-6U/50. mu.L of the incubation system, particularly 2U/50. mu.L of the incubation system.
Specifically, in the incubation system in step (1), the concentration of the conjugate described in the fourth aspect and the conjugate described in the fifth aspect is 100fM-10pM per 50. mu.L of the incubation system, and particularly 10pM per 50. mu.L of the incubation system.
Specifically, in the incubation system in step (1), the sample concentration may be 5. mu.L/50. mu.L of the incubation system.
Specifically, the incubation temperature in step (1) may be 37-42 ℃, particularly 37 ℃.
In particular, the incubation time in step (1) may be 10-30 minutes, for example 20 minutes.
In some embodiments of the invention, step (1) further comprises the step of amplifying the incubation product.
Specifically, the amplification step comprises: the incubation product is mixed with the combination product according to the eleventh aspect of the invention and subjected to amplification incubation.
In particular, the temperature of the amplification incubation may be 37-42 ℃, in particular 37 ℃.
In particular, the time of amplification incubation may be 10-30 minutes, for example 20 minutes.
Specifically, the incubation temperature in step (2) may be 37-42 ℃, particularly 37 ℃.
Specifically, the incubation time in step (2) may be 10-120 minutes; when step (1) does not comprise an amplification step, the incubation time in step (2) may be 50-120 minutes; where step (1) comprises an amplification step, the incubation time in step (2) may be 10-60 minutes.
In some embodiments of the invention, the detecting of step (3) comprises the step of detecting fluorescence from the incubated product of step (2).
In still other embodiments of the present invention, the detecting in step (3) comprises the step of applying the incubation product of step (2) to the strip according to the ninth aspect of the present invention.
In particular, the method may further comprise the step of preparing the conjugate according to the fourth and fifth aspects of the invention.
In some embodiments of the invention, the step of preparing the conjugate of the fourth aspect may comprise:
the probe according to the second or third aspect of the present invention is biotinylated, then mixed with streptavidin, incubated, then mixed with the antigen source of interest (e.g., SARS-CoV-2 spike protein RBD), and incubated.
In some embodiments of the invention, the step of preparing the conjugate of the fifth aspect may comprise:
the probes according to the third or second aspect of the present invention are biotinylated, then mixed with streptavidin, incubated, then mixed with a ligand for a target antibody (e.g., an anti-human IgG antibody or an anti-human IgM antibody), and incubated.
In some embodiments of the invention, the above-described antibody detection method is a SARS-CoV-2 antibody detection method.
In particular, the antibody detection methods described above may be used for diagnostic as well as non-diagnostic purposes.
In an eighteenth aspect, the present invention provides an antibody detection method, comprising the steps of:
(a) designing a target nucleic acid consisting of fragments S1, S2, and S3 connected in sequence, that is, it can be represented as S1-S2-S3;
(b) designing a crRNA comprising: (1) a framework nucleic acid fragment that interacts with the Cas nuclease, and (2) a specific nucleic acid fragment that binds to a target nucleic acid; wherein the specific nucleic acid fragment consists of the fragments S1 ', S2 ' and S3 ' linked in sequence, i.e. it can be represented as S1 ' -S2 ' -S3 ', S1, S2 and S3 complementary to S1 ', S2 ' and S3 ', respectively;
(c) designing single-stranded DNA probes 1 and 2, wherein the single-stranded DNA probe 1 comprises a fragment SaFragment SaIdentical to the fragment S1-S2 (i.e.complementary to the fragment S1 '-S2'),single-stranded DNA Probe 2 comprising fragment SbFragment SbComplementary to the fragments S2-S3 (i.e.corresponding to fragments S2 '-S3').
Wherein the fragments S1, S2 and S3, the fragments S1 ', S2 ' and S3 ', and the fragment Sa、SbHaving the corresponding definitions of the invention as described above.
Specifically, the target nucleic acid, crRNA, and single-stranded DNA probe are defined as above.
Specifically, the method further includes:
(d) a step of coupling the single-stranded DNA probes 1 and 2 with a target antigen portion and a ligand portion of a target antibody, respectively, or coupling the single-stranded DNA probes 2 and 1 with a target antigen portion and a ligand portion of a target antibody, respectively.
Specifically, the method further includes:
(e) the conjugate is mixed with the sample to be tested and the reagent required for DNA polymerization, and incubated.
Specifically, the reagents required for DNA polymerization include, for example, DNA polymerase (e.g., T4 polymerase), dNTP, Mg2+Buffer systems, and the like.
Specifically, the concentration of DNA polymerase (e.g., T4 polymerase) in the incubation system is 1-6U/50. mu.L of incubation system, particularly 2U/50. mu.L of incubation system.
Specifically, the concentration of the conjugate in the incubation system was 100fM-10 pM/50. mu.L of the incubation system, specifically 10 pM/50. mu.L of the incubation system.
In particular, the incubation temperature may be 37-42 ℃, in particular 37 ℃.
In particular, the incubation time may be 10-30 minutes, for example 20 minutes.
In some embodiments of the invention, step (e) further comprises the step of amplifying the incubated product.
Specifically, the method further includes:
(f) mixing the incubation product (or amplification product) of step (e) with the designed crRNA, CRISPER protein, and signal reporter molecule, and incubating.
In particular, the incubation temperature may be 37-42 ℃, in particular 37 ℃.
In a nineteenth aspect, the present invention provides a method of diagnosing a disease or condition caused by an antigen of interest.
Specifically, the method may comprise the step of using the method of the sixteenth, seventeenth or eighteenth aspect, or the step of using the crRNA of the first aspect, the probe of the second or the fifth aspect, or the step of using the crRNA of the first aspect, the conjugate of the fourth or the fifth aspect, or the step of using the crRNA of the first aspect, the combination of the seventh aspect, or the combination of the seventh and the eighth aspects.
In particular, the method may further comprise the step of using the amplification combination product of the eleventh aspect.
Specifically, the method may further comprise the step of using the test strip described in the ninth aspect.
In particular, the method may comprise the step of using a kit according to the twelfth or thirteenth aspect.
In particular, the above-mentioned diseases or conditions may be selected from: one or more of rhinitis, sinusitis, croup, pharyngitis, tonsillitis, parotitis, laryngitis, tracheitis, asthma, pneumonia, influenza, Zika virus disease, acquired immunodeficiency syndrome (AIDS), etc.
In some embodiments of the invention, the disease is novel coronavirus pneumonia (COVID-19) (the target antigen is SARS-CoV-2).
The invention provides an ultrasensitive antibody detection method based on CRISPR technology, which can be used for detecting an antibody (such as an anti-SARS-CoV-2 antibody) generated by immune reaction caused by an antigen in an organism, has very high sensitivity, can be detected to the limit of 1aM, is simple to operate, does not need any washing step, can be carried out in a high-throughput manner in a laboratory environment, can also be used as a POC (point of sale) diagnostic tool in an environment with limited resources, and the inventor verifies the clinical effectiveness of the method through a large number of experiments. The invention also provides a crRNA, a probe, a conjugate, a combined product, test paper, a kit and the like for the method, which can be flexibly used for antibody detection and have higher development and application values.
Drawings
FIG. 1 is a schematic workflow diagram of a UCAD assay for the detection of anti-SARS-CoV-2 spike protein RBD mAb (no nucleic acid amplification of pre-designed dsDNA barcodes).
Figure 2 shows the sequence design of the dsDNA template to stimulate the lateral cleavage activity of CRISPR Cas12a, and the strategy to resolve the dsDNA template.
FIG. 3 shows that the close binding of two UCAD probes to the same anti-SARS-CoV-2 antibody via the affinity motif increases the Tm of the double-stranded domain from 10 ℃ to 46 ℃.
Figure 4 shows the kinetic profile of mAb detection using UCAD without RPA. mAb concentrations were 125pM to 8nM and compared to blank (0) (P < 0.01). The threshold value (dashed line) is set to determine the critical time τ to reach the same fluorescence signal. The probe was composed of [ UCAD probe ] ═ 10pM, [ T4 polymerase ] ═ 2 units, [ dNTP ] ═ 40 μ M, [ Cas12a ] ═ 40nM, [ crRNA ] ═ 40nM, and [ FQ-labeled reporter ] ═ 40 nM.
Figure 5 is a schematic flow chart illustrating the operation of UCAD assays.
FIG. 6 shows the optimization of T4 polymerase in UCAD detection of SARS-CoV-2 spike protein RBD human monoclonal antibody (mAb). [ mAb ] ═ 100fM, [ UCAD probe ] ═ 10pM, [ dNTPs ] ═ 40 μ M, [ Cas12a ] ═ 40nM, [ crRNA ] ═ 40nM, [ FQ-labeled reporter ] ═ 40 nM.
FIG. 7 shows the optimization of the concentration of UCAD probes for the detection of SARS-CoV-2 spike protein RBD human monoclonal antibody. [ mAb ] ═ 100fM, [ T4 polymerase ] ═ 2 units, [ dNTPs ] ═ 40 μ M, [ Cas12a ] ═ 40nM, [ crRNA ] ═ 40nM, [ FQ-labeled reporter ] ═ 40 nM.
FIG. 8 shows the kinetic profile of anti-SARS-CoV-2 mAb detected using UCAD at concentrations ranging from 1aM to 1 pM. The threshold value (dashed line) is set to determine the critical time τ to reach the same fluorescence signal.
Fig. 9 shows Δ τ (Δ τ blank- τ sample) as a function of target concentration to establish a calibration curve and compare it to UCAD without RPA amplification. Each error bar represents one standard deviation of triplicate analyses.
FIG. 10 is a schematic workflow diagram showing the analysis of anti-SARS-CoV-2 antibodies in a clinical human blood sample using the UCAD assay.
FIG. 11 shows the kinetic curves for the detection of anti-SARS-CoV-2 IgG and IgM in COVID-19 positive and negative serum samples using UCAD.
FIG. 12 shows the detection of anti-SARS-CoV-2 IgG or IgM in a COVID-19 positive human serum sample diluted in a negative human serum sample using UCAD and a commercially available ELISA kit at a dilution factor of 1 to 100000.
FIG. 13 shows the dilution of anti-SARS-CoV-2 IgG-positive or IgM-positive serum samples with negative human serum and the detection of anti-viral IgG and IgM with UCAD. The probe was composed of [ UCAD probe ] ═ 10pM, [ T4 polymerase ] ═ 2 units, [ dNTP ] ═ 40 μ M, [ Cas12a ] ═ 40nM, [ crRNA ] ═ 40nM, and [ FQ-labeled reporter ] ═ 40 nM.
FIG. 14 shows the evaluation of UCAD for determining anti-SARS-CoV-2 IgG (or IgM) in a human serum sample. A nonparametric t-test of Δ τ was performed on 6 IgG (or IgM) positive serum samples and 6 negative serum samples. The difference in signal between the two groups was statistically significant. P < 0.001.
FIG. 15 shows the kinetic curves for each of the 6 anti-SARS-CoV-2 IgG positive samples and each of the negative human serum samples detected using UCAD. The probe was composed of [ UCAD probe ] ═ 10pM, [ T4 polymerase ] ═ 2 units, [ dNTP ] ═ 40 μ M, [ Cas12a ] ═ 40nM, [ crRNA ] ═ 40nM, and [ FQ-labeled reporter ] ═ 40 nM.
FIG. 16 shows the kinetic curves for each of the 6 anti-SARS-CoV-2 IgM positive samples and each of the negative human serum samples detected using UCAD. The probe was composed of [ UCAD probe ] ═ 10pM, [ T4 polymerase ] ═ 2 units, [ dNTP ] ═ 40 μ M, [ Cas12a ] ═ 40nM, [ crRNA ] ═ 40nM, and [ FQ-labeled reporter ] ═ 40 nM.
FIG. 17 is a schematic representation of UCAD lateral flow readings for the detection of anti-SARS-CoV-2 IgG and IgM.
Figure 18 shows the design of a lateral flow strip that captures all anti-FAM labeled aunps on the C-line in the absence of the target antibody. In the presence of anti-SARS-CoV-2 IgG or IgM, FAM-Dig dual-labeled reporter was cleaved by Cas12a-crRNA, allowing AuNP to escape capture at the C-line and accumulate at the T-line by immobilized secondary antibody.
FIG. 19 shows a UCAD combined with a lateral flow chromatography test strip to visualize anti-SARS-CoV-2 IgG/IgM positive and negative serum samples.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
AuNP gold nanoparticles
UCAD Ultrasensitive CRISPR-based Antibody Detection
dsDNA double stranded DNA
crRNA CRISPR-derived RNA
RBD receptor binding domain, receptor binding domain
mAb monoclonal antibodies
RPA recombinase polymerase amplification
LOD detection limit
nt nucleotides
The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood 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.
Some of the reagents and materials used in the experiments of the examples are as follows:
magnesium chloride hexahydrate (MgCl)2·6H2O), sodium chloride (NaCl), TWEEN 20 and biotin were purchased from tancool (shanghai, china).
Cas12a (Cpf1) V3 was purchased from Integrated DNA Technologies (Coralville, IA). What is needed isBuffer, deoxynucleotide (dNTP) solution mixture, T4 polymerase, streptavidin, biotinylated anti-human IgG antibody, and biotinylated anti-human IgM antibody were purchased from Sangon Biotech (Shanghai, China). All DNA sequences were purified using high performance liquid chromatography. Anti-digoxin rabbit polyclonal antibodies were purchased from ABclonal Biotech (wuhan, china). Gold nanoparticle (AuNP) (20nm) solutions were purchased from sierra biotechnology limited (sienna, china). Biotinylated SARS-Cov-2 Spike RBD-His recombinant protein was purchased from Sino Biological (Beijing, China). SARS-CoV-2 Spike RBD monoclonal antibody (clone name: OTIH 401) was purchased from Origene (Beijing, China). Twist DxTMRPA base kit was purchased from twist dx (Cambridge, u.k). SARS-CoV-2IgG and IgM ELISA kits, and all anti-SARS-CoV-2 IgG and IgM positive and negative human serum samples were purchased from BGI genetics (Shenzhen, China).
Nitrocellulose membranes were purchased from Sigma Aldrich (cheng du, china).
Example 1
UCAD theory of operation:
the detection of immunoglobulin g (igg) and immunoglobulin m (igm) against SARS-CoV-2 is translated into the formation of a rationally designed DNA barcode that can be exponentially amplified and specifically detected using CRISPR-Cas12a (workflow shown in figure 1).
A double stranded dna (dsdna) template that can be detected by the crRNA-Cas12a complex was first designed (as shown in figure 2). The dsDNA template is then split into two single-stranded dna (ssdna) probes, leaving only 5-8nt complementary domains (blue domains). The Tm of the short complementary domains is estimated by DNA Analyzer software to be only-10 ℃ so they cannot form stable duplexes at 37 ℃. The two ssDNA probes were then conjugated to SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and anti-human IgG (or IgM) antibodies, respectively. RBD was chosen because of its high specificity, and ability to distinguish SARS-CoV-2 from other SARS-like coronaviruses. The two ssDNA probes can then bind to the same anti-SARS-CoV-2 antibody and bring the two complementary domains into close proximity. A stable duplex can then be formed, with an estimated Tm of 46 ℃ (as shown in figure 3). The CRISPR-detectable dsDNA template can then be regenerated and used as a barcode for detection of anti-SARS-CoV-2 antibodies, driven by T4 polymerase.
Direct quantification of dsDNA barcodes by target-induced lateral cleavage activity of Cas12a resulted in linear detection of SARS-CoV-2 spike protein RBD human monoclonal antibody (mAb).
The experimental procedure was as follows:
preparation of UCAD probe:
mu.L of 2.5. mu.M biotinylated ssDNA probe 1 (as shown in Table 1) was mixed with an equal volume of 2.5. mu.M streptavidin and incubated at 37 ℃ for 30 minutes. After cooling to room temperature, 50. mu.L of 1.25. mu.M biotinylated SARS-CoV-2 spike protein RBD was mixed with the reaction solution and incubated at 25 ℃ for an additional 30 minutes to give ssDNA probe 1-RBD conjugate. ssDNA probe 2 (shown in table 1) was conjugated with anti-human IgG antibody or anti-human IgM antibody using the same protocol to obtain ssDNA probe 2-anti-human IgG antibody/anti-human IgM antibody conjugate. After conjugation, the prepared conjugates were diluted to 250nM with Tris-biotin buffer (20mM Tris-HCl, 0.01% BSA, 1mM biotin) and stored at 4 ℃ until use.
Preparation of antibody-modified AuNPs
Mu.l of 9.5. mu.g/mL rabbit anti-FAM polyclonal antibody was added to 900. mu.l of 7X 1011in/mL AuNP solution and incubated for 90 minutes at room temperature. Then 500. mu.L of 5% BSA was added to the reaction solution and incubated at room temperature for another 1 hour. The reaction solution was centrifuged at 17000g for 1 hour at 4 ℃ and AuNP precipitate was collected. The pellet was washed twice with PBS buffer and then resuspended in 20mM Na3PO45% BSA, 0.25 % TWEEN 20 and 10% sucrose in 1mL buffer. Antibody-modified aunps were stored at 4 ℃ until use.
Construction of Lateral Flow test Strip (Lateral Flow Strip)
The lateral flow chromatography test strip consists of a sample pad, a combination pad, a nitrocellulose membrane and an adsorption pad. The sample pad was saturated with a buffer containing 0.25% Triton X-100, 0.05M Tris-HCl and 0.15M sodium chloride. An optimized volume of AuNP-anti FAM conjugate was loaded onto the binding pad. Goat anti-digoxin polyclonal antibody was dispensed onto nitrocellulose membrane at the control (C) line and goat anti-rabbit IgG was dispensed at the test (T) line using XYZ platform dispenser HM3030 (shanghai gold-labelled biotechnology limited). All membranes and mats were dried at 37 ℃ for 2 hours, then assembled and cut.
Protein detection using UCAD
(1) For a typical assay, 50. mu.L of the reaction mixture contained 5. mu.L of samples at various concentrations, 10pM ssDNA probe 1-RBD conjugate, 10pM ssDNA probe 2-anti-human IgG antibody/anti-human IgM antibody conjugate, 40. mu.M dNTP, and 2 units of T4 polymerase (at 1 XNEBuffer)TM2 buffer solution). The solution was incubated at 37 ℃ for 20 minutes. (the sequence formed by the ssDNA probe 1 after primer extension is shown as SEQ ID NO:13, which is a target sequence recognizable by cas12a, and the sequence formed by the ssDNA probe 2 after primer extension is shown as SEQ ID NO:14, which is complementary to the sequence shown as SEQ ID NO: 13).
(2) Mixing 10. mu.L of the solution with TwistDxTMThe reagents provided in the RPA base kit were mixed and incubated at 37 ℃ for 20 minutes.
(3) For fluorescence readings, the incubation product of step (1) or the RPA product of step (2) was mixed with 40nM Cas12a, 40nM crRNA (as shown in Table 1), and 40nM FAM-BHQ-1 labeled ssDNA reporter (as shown in Table 1) in a 96-well microplate at 100. mu.L 1 XNEBuffer TM2, mixing. Fluorescence was measured immediately and every 1 minute at 37 ℃ for 2 hours using a staining 5 cell imaging multimodal reader (Biotek) with excitation/emission at 495/520 nm.
(3') for lateral flow readings, incubation products of step (1) or RPA amplification products of step (2) were mixed with 40nM Cas12a, 40nM crRNA (as shown in Table 1) and 40nM FAM-Dig ssDNA reporter (as shown in Table 1) at 20. mu.L of 1 XNEBuffer TM2 for 30 minutes at 37 ℃ and then loaded onto the sample pad of the lateral flow strip with 30 μ L of 4 × SSC buffer. After development, the results on the lateral flow chromatography test strip can be read or used directly with the naked eyeA digital camera.
Detection of antibodies Using a commercially available ELISA kit (comparative experiment)
ELISA was performed for anti-SARS-CoV-2 IgG and IgM according to the instructions of the kit manual. For anti-SARS-CoV-2 IgG assay, 10. mu.L of human serum was mixed with 100. mu.L of diluent and incubated for 30 minutes at 37 ℃ in ELISA microplate wells. The plate was then washed 5 times with 350. mu.L of wash buffer, and then 100. mu.L of IgG enzyme working solution was added. Thereafter, the plates were incubated at 37 ℃ for 30 minutes and washed again with 5X 350. mu.L of wash buffer. mu.L of substrate A and 50. mu.L of substrate B were mixed in a microplate well and incubated at 37 ℃ for 10 minutes protected from light, then 50. mu.L of quench solution was added. The OD of each sample was measured immediately after quenching (450-620 nm). To detect anti-SARS-CoV-2 IgM antibodies, 10. mu.L of human serum was mixed with 100. mu.L of diluent and incubated in a microplate well for 60 minutes at 37 ℃. After 5X 350. mu.L of wash, 100. mu.L of IgM enzyme working solution was added to the plate and incubated at 37 ℃ for another 30 minutes. The IgM ELISA plates were incubated with 50. mu.L of substrate A and 50. mu.L of substrate B at 37 ℃ for 10 minutes protected from light, washed 5 times with 350. mu.L of washing buffer. OD values (450-620nm) were measured immediately after addition of 50. mu.L of the quenching solution.
TABLE 1 nucleic acid sequences and modifications
The nucleic acid sequences in Table 1 were those designed by the inventors and then synthesized by the synthetic company.
The experimental results are as follows:
the kinetic profile of mAb detection using UCAD without Recombinase Polymerase Amplification (RPA) is shown in fig. 4, where mAb concentration is 125pM to 8nM and compared to blank (0) (P < 0.01). The threshold value (dashed line) is set to determine the critical time τ to reach the same fluorescence signal. As shown in fig. 4, the LOD of the UCAD detection mAb was 125 pM.
Example 2
To further reduce the LOD of UCAD, the dsDNA barcodes were pre-amplified using RPA and the workflow is shown in FIG. 5.
The concentration of T4 polymerase in UCAD detection SARS-CoV-2 spike protein RBD human monoclonal antibody (mAb) was optimized, and the results are shown in FIG. 6. The fluorescent signals generated in each group (mAb and blank) using different units of T4 polymerase were compared, and 2 units were selected for subsequent experiments due to the greatest signal difference between mAb and blank samples.
The concentration of UCAD probe for detecting SARS-CoV-2 spike protein RBD human monoclonal antibody was optimized, and the results are shown in FIG. 7. The 10pM to 100fM UCAD probes produce fluorescent signals for the mAb that are distinguished from the blank, while the 100pM UCAD probes produce significant non-specific binding. Based on this optimization, a 10pM UCAD probe was selected for subsequent experiments to ensure a greater dynamic range of detection.
By optimizing the concentrations of ssDNA probe and T4 polymerase, the inventors successfully achieved detection of mabs in PBS buffer, where LOD can be as low as 1aM (as shown in fig. 8 and 9).
Example 3
After achieving an ultra-sensitive detection of anti-RBD mAb in the buffer system (as described in examples 1 and 2), the inventors next verified the effectiveness of UCAD against authentic COVID-positive human serum samples, the workflow is shown in fig. 10, the experimental procedure is described in example 1, and the results are shown in fig. 11.
Notably, the UCAD assay successfully detected anti-SARS-CoV-2 IgG (fig. 11A) and IgM (fig. 11B) in commercial COVID positive (or negative) human serum samples, the positive samples had high fluorescence signals, while the negative samples had low background signals.
Notably, the results in fig. 11A and 11B show a significant reduction in background signal compared to the kinetic curve in the buffer system (fig. 8), indicating that high concentrations of human serum albumin help prevent non-specific binding between RBD and anti-human IgG (or IgM) in the absence of the antibody of interest.
Example 4
To further assess the sensitivity of UCAD in clinical serum samples, COVID-positive serum samples were diluted with negative human serum samples at a dilution factor of 1-100000. The experimental procedure is as described in example 1, and the results are shown in FIGS. 12-16.
The results show that UCAD is able to detect anti-SARS-CoV-2 IgG (as shown in fig. 12A and 13A) and IgM (as shown in fig. 12B and 13B) in all diluted samples. By comparison, the standard ELISA kit only worked on 10-fold dilutions of serum samples (as shown in fig. 12A and 12B), indicating that the UCAD assay was at least 10000-fold higher in clinical human serum samples than the standard ELISA.
The inventors have successfully identified 6 IgG positive (as shown in figures 14A and 15) and 6 IgM positive (as shown in figures 14B and 16) human and negative serum samples, further confirming the clinical efficacy of UCAD.
Example 5 the ultimate goal of the inventors was to evaluate the potential of deploying UCAD as a COVID-19 serological test in a point-of-care (POC) setting, and a schematic of UCAD lateral flow readings for the detection of anti-SARS-CoV-2 IgG and IgM is shown in fig. 17. The inventors converted the fluorescent ssDNA reporter labeled with a fluorescence quencher for Cas12a into a ssDNA reporter labeled with FAM at one end and digoxin (Dig) at the other end (as shown in table 1 above), and then constructed lateral flow test strips with anti-Dig immobilized on the control line (C-line) and anti-rabbit IgG immobilized on the test line (T-line). In the absence of the target anti-SARS-CoV-2 antibody, the FAM-Dig labeled ssDNA reporter remained intact, which helped to capture all rabbit anti-FAM labeled gold nanoparticles (AuNPs) at the C-line. In the presence of anti-SARS-CoV-2 IgG or IgM, the ssDNA reporter is cleaved by the target-induced side activity of Cas12 a. As a result, AuNP was captured at the T-line. As shown in fig. 18.
Referring to the experimental procedure described in example 1, samples were tested by a flow-through test strip and the results are shown in FIG. 19. By visualizing the results in FIG. 19, the inventors were able to clearly identify 6 anti-SARS-CoV-2 IgG-positive and 6 IgM-positive human serum samples from uninfected human serum samples.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experience, and preferences of those skilled in the art.
The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.
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Claims (39)
1. A crRNA, comprising: (1) a framework nucleic acid fragment that interacts with the Cas nuclease, and (2) a specific nucleic acid fragment that binds to a target nucleic acid.
2. The crRNA of claim 1, wherein the specific nucleic acid fragment comprises or consists of the nucleotide sequence set forth in SEQ ID NO. 1.
3. The crRNA of claim 1 or 2, wherein the framework nucleic acid segment comprises or consists of the nucleotide sequence set forth in SEQ ID No. 2.
4. The crRNA of claim 1, wherein the crRNA comprises or consists of the nucleotide sequence set forth in SEQ ID NO 3.
5. A nucleic acid consisting of fragments S1, S2 and S3 linked in sequence, wherein S1 and S3 are independently 5nt or more in length, and S2 is 5 to 8nt in length;
preferably, S1 is CGTCGCCG, S3 is GGTCGAG, and S2 is TCCAG.
6. A single-stranded DNA probe comprising a fragment SaThe fragment SaIdentical to fragment S1-S2 of the nucleic acid of claim 5.
7. The single-stranded DNA probe of claim 6, further comprising a fragment SrThe fragment SrAnd fragment SaConnecting;
preferably, the fragment SrThe length of (A) is 10-100 nt;
more preferably, the fragment SrComprises or consists of the nucleotide sequence shown as SEQ ID NO. 5.
8. The single-stranded DNA probe of claim 6, wherein the single-stranded DNA probe comprises or consists of the nucleotide sequence set forth in SEQ ID NO. 6.
9. A single-stranded DNA probe comprising a fragment SbThe fragment SbComplementary to fragment S2-S3 of the nucleic acid of claim 5.
10. The single-stranded DNA probe of claim 9, wherein the fragment SbComprises or consists of the nucleotide sequence shown as SEQ ID NO. 7.
11. The single-stranded DNA probe of claim 10, further comprising a PAM sequence, which is complementary to fragment SbConnecting;
preferably, the PAM sequence is TTTX, where X can be any deoxynucleotide;
more preferably, the PAM sequence is TTTA.
12. The single-stranded DNA probe of claim 11, further comprising a fragment Sr ', the fragment Sr' being identical to a fragment PAM-SbConnecting;
preferably, the fragment Sr' comprises or consists of the nucleotide sequence shown in SEQ ID NO. 9.
13. The single-stranded DNA probe of claim 10, wherein the single-stranded DNA probe comprises or consists of the nucleotide sequence set forth in SEQ ID No. 10.
14. A conjugate of a single-stranded DNA probe as claimed in any one of claims 6 to 8, 9 to 13 and an antigenic moiety of interest.
15. The conjugate of claim 14, wherein the antigenic moiety of interest is a viral protein;
preferably, the antigen portion of interest is one or more of the structural proteins of SARS-CoV-2 (e.g., S protein, M protein, E protein, N protein), or a portion of a structural protein (e.g., the Receptor Binding Domain (RBD) of S protein).
16. A conjugate of a single-stranded DNA probe of any one of claims 6-8, 9-13 and a ligand portion of a target antibody.
17. The conjugate according to claim 16, wherein the ligand is an antibody, in particular a monoclonal antibody;
preferably, the target antibody is a SARS-CoV-2 antibody, in particular an anti-SARS-CoV-2 IgG, a SARS-CoV-2IgM, and the ligand is an antibody against SARS-CoV-2 antibody, in particular an antibody against SARS-CoV-2IgG or an antibody against SARS-CoV-2 IgM.
18. A combination comprising a single stranded DNA probe according to any one of claims 6 to 8, 9 to 13.
19. A combination product according to claim 18, further comprising a target antigen source, a ligand source, and optionally a linker forming substance.
20. A combination comprising a conjugate according to any one of claims 14-15, 16-17.
21. A combination product according to claim 20, further comprising reagents required for DNA polymerisation, such as a DNA polymerase (e.g. T4 polymerase), dntps, Mg2+And a buffer system.
22. A combination comprising a crRNA, a CRISPER protein, and a signaling reporter as claimed in any one of claims 1 to 4.
23. A combination product according to claim 22 wherein the crisp sper protein is a crisp sper protein having lateral cleavage activity, for example Cas12a, Cas12b, Cas13a, Cas13b, Cas14, Csm6, in particular Cas12 a.
24. A combination product according to claim 22, wherein the signalling reporter molecule comprises single stranded DNA;
preferably, the single-stranded DNA is 5 to 10nt in length;
preferably, the single-stranded DNA consists of T and a;
more preferably, the single-stranded DNA is TTATT.
25. The combination of claim 24, wherein the 5 'end and the 3' end of the signaling reporter are labeled with a fluorescent reporter and a fluorescent quencher, respectively.
26. A combination product according to claim 24, wherein the signal reporter is labelled at its 5 'end and at its 3' end with a first label and a second label, respectively, and wherein the first label and the second label are different.
27. A primer comprises a primer 1 and a primer 2, wherein the primer 1 and the primer 2 are respectively shown as SEQ ID NO. 11 and SEQ ID NO. 12.
28. An amplification combination comprising the primers of claim 27.
29. An amplification combination according to claim 28, further comprising reagents required for nucleic acid amplification, e.g. reagents required for RPA, comprising: recombinase capable of binding single-stranded nucleic acid, single-stranded DNA binding protein, strand displacement DNA polymerase, dNTP, Mg2+And a buffer system.
30. Use of a crRNA according to any one of claims 1 to 4, a single-stranded DNA probe according to any one of claims 6 to 8, 9 to 13, a conjugate according to any one of claims 14 to 15, 16 to 17, a combination according to any one of claims 20 to 21, 22 to 26, a primer according to claim 27 or an amplification combination according to claim 28 or 29 for the preparation of a product for the detection of antibodies against a target antigen.
31. Use according to claim 30, wherein the antibody is a SARS-CoV-2 antibody, such as anti-SARS-CoV-2 IgG, anti-SARS-CoV-2 IgM.
32. Use of a crRNA according to any one of claims 1 to 4, a single-stranded DNA probe according to any one of claims 6 to 8, 9 to 13, a conjugate according to any one of claims 14 to 15, 16 to 17, a combination according to any one of claims 20 to 21, 22 to 26, a primer according to claim 27 or an amplification combination according to claim 28 or 29 for the preparation of a product for the diagnosis of a disease or condition caused by an antigen of interest.
33. The use of claim 32, wherein the disease is COVID-19.
34. An antibody detection method comprising the steps of:
(1) mixing a sample to be tested with a conjugate of a single-stranded DNA probe 1 and a target antigen part, a conjugate of a single-stranded DNA probe 2 and a ligand part of the antibody and a reagent required by DNA polymerization, and incubating;
(2) mixing the incubation product of step (2) with the crRNA, CRISPER protein, and signaling reporter of any one of claims 1-4, and incubating;
(3) detecting;
wherein the single-stranded DNA probe 1 comprises or consists of a nucleotide sequence shown as SEQ ID NO. 6, and the single-stranded DNA probe 2 comprises or consists of a nucleotide sequence shown as SEQ ID NO. 10; or,
wherein the single-stranded DNA probe 1 comprises or consists of a nucleotide sequence shown by SEQ ID NO. 10, and the single-stranded DNA probe 2 comprises or consists of a nucleotide sequence shown by SEQ ID NO. 6.
35. The method of claim 34, wherein the reagents required for DNA polymerization include a DNA polymerase (e.g., T4 polymerase);
the concentration of the DNA polymerase (e.g., T4 polymerase) in the incubation system in step (1) is 1-6U/50. mu.L of the incubation system, particularly 2U/50. mu.L of the incubation system.
36. The method according to claim 34, wherein the concentration of the conjugate in the incubation system of step (1) is between 100fM and 10pM per 50 μ L of incubation system, in particular 10pM per 50 μ L of incubation system.
37. The method according to claim 34, wherein the incubation temperature is 37-42 ℃, in particular 37 ℃.
38. The method of claim 34, wherein step (1) further comprises the step of amplifying the incubated product.
39. The method of claim 34, wherein the antibody detection method is a SARS-CoV-2 antibody detection method.
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| CN116479175B (en) * | 2023-03-13 | 2024-10-18 | 广州国家实验室 | Detection system, kit and method for detecting human parainfluenza virus nucleic acid |
| CN117904262A (en) * | 2024-01-17 | 2024-04-19 | 华南师范大学 | Nucleic acid detection method based on CRISPR/Cas12 cis-cleavage, test strip, kit and application |
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