WO2021013257A1 - Crispr多靶标检测方法及其试剂盒 - Google Patents

Crispr多靶标检测方法及其试剂盒 Download PDF

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WO2021013257A1
WO2021013257A1 PCT/CN2020/104588 CN2020104588W WO2021013257A1 WO 2021013257 A1 WO2021013257 A1 WO 2021013257A1 CN 2020104588 W CN2020104588 W CN 2020104588W WO 2021013257 A1 WO2021013257 A1 WO 2021013257A1
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nucleic acid
detection system
container
guide rna
cas protein
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王金
李诗渊
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Shanghai Tolo Biotechnology Co Ltd
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Priority to US17/629,545 priority patent/US20220267847A1/en
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Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to a CRISPR multi-target detection method and its kit.
  • Cas12 and Cas13 proteins can be used for nucleic acid detection based on their bypass (or trans) cleavage activity, that is, under the guidance of an artificially designed guide RNA, they bind to nucleic acid fragments of a specific sequence, and then cut single-stranded nucleic acid probes This produces a detectable signal.
  • the difference between Cas12 and Cas13 is that Cas13 protein binds to RNA targets and cleaves RNA probes, while Cas12 binds to DNA targets and cleaves single-stranded DNA probes.
  • RNA reporter probe used in the Cas13 detection process has a greater risk of being degraded by RNase, which results in a higher background signal value of the detection system.
  • the Cas12-based detection method has advantages because it does not require in vitro transcription or RNA reporter probes.
  • the methods and techniques for simultaneously detecting multiple existing targets in one detection system are very important for the expanded application of in vitro diagnostics.
  • a multi-target detection method has been developed based on the Cas13 protein. The key is to take advantage of the different bypass cleavage activities of Cas13 proteins from different species for different RNA reporter probes.
  • Cas12 there is no research report on this characteristic of Cas12; therefore, it is necessary to develop a new method that uses Cas protein for multi-target nucleic acid detection.
  • the purpose of the present invention is to provide a multi-target detection method and kit based on CRISPR-Cas protein.
  • a detection system for detecting target nucleic acid molecules comprising:
  • the guide RNA-reporter nucleic acid composite probes have the structure shown in formula Ia, Ib, Ic or Id,
  • Z1 is the first stem-loop structure region
  • Z2 is no or nucleic acid linking region
  • Z3 is the guide RNA region
  • Z5 is a single-stranded nucleic acid to be cleaved with a detectable label, wherein the detectable label presents different detection states when the single-stranded nucleic acid to be cleaved is cut and uncut, and thus is detected;
  • Z3 and Z5 form a complementary paired double-stranded structural region; and when the target nucleic acid exists in the detection system, Z3 and Z5 do not form the complementary paired structure. Double-stranded structure region;
  • Z4 is none, or a chemical bond or connecting zone used to connect Z3 and Z5;
  • n is a positive integer n ⁇ 1;
  • Cas protein which is a Cas protein with bypass single-stranded nucleic acid cleavage activity.
  • the Cas protein is selected from the following group: Cas12 type, Cas13a type, Cas13b type, Cas14 type, Cas ⁇ , or a combination thereof.
  • the guide RNA-reporter nucleic acid composite probe has a structure as shown in Formula Ia or Ib.
  • the guide RNA-reporter nucleic acid composite probe has a structure shown in Formula Ic or Formula Id.
  • the Cas12 type is selected from the following group: Cas12a, Cas12b, Cas12d, Cas12g, Cas12i, or a combination thereof.
  • the guide RNA-reporter nucleic acid composite probe has a structure of formula IIa:
  • Z1, Z2, Z3, Z4 and Z5 are as described above,
  • the "Z1-Z2-Z3" in formula Ia, formula IIa, formula Ic, formula Ib and "Z3-Z2-Z1" in formula Id are from 5'to 3' direction.
  • the guide RNA-reporter nucleic acid composite probe has a structure of formula IIc:
  • Z1, Z2, Z3, Z4 and Z5 are as described above,
  • the complementary paired double-stranded structural region includes a double-stranded structural region formed by partial or complete complementary pairing of Z3 and Z5.
  • the complementary paired double-stranded structural region is a double-stranded structural region formed by all complementary pairings of Z3 and Z5.
  • the Z1, Z2, and Z3 are used to guide the Cas protein to bind to the target nucleic acid.
  • the Z1 is used to bind or anchor Cas protein.
  • the guide RNA region guides the Cas protein to bind to the target nucleic acid through complementary pairing with the target nucleic acid.
  • Z3, Z4, and Z5 form a second stem-loop structure region, where Z4 is a loop region (including simple structure and complex structure loop region).
  • said Z1 is substantially or entirely composed of RNA.
  • the Z3 is substantially or entirely composed of RNA.
  • the guide RNA-reporter nucleic acid composite probe is single-stranded.
  • the stem-loop structure in Z1 is a crRNA (or CRISPR RNA) stem-loop structure.
  • the length of Z1 is 10-300 nt, preferably 19-100 nt, and more preferably 19-91 nt.
  • the Z2 is a nucleic acid linking region without or with a length of 0-20 nt.
  • the detectable label is a fluorescent group
  • Z5 has a fluorescent group
  • the Z4 and/or Z5 also has a quenching group, if and only if the single strand is to be cleaved
  • the fluorescent signal emitted by the fluorescent group can be detected;
  • the fluorescent groups are different from each other so that they can be distinguished.
  • the detectable label is a fluorescent group
  • Z5 has a quenching group
  • the Z4 and/or Z5 also has a fluorescent group, if and only if the When the single-stranded nucleic acid to be cut is cleaved, the fluorescent signal emitted by the fluorescent group can be detected.
  • the detectable label is a fluorescent group, wherein the fluorescent group is located in any zone of Z5, Z4 and Z3, and the quenching group is located in any zone of Z5, Z4 and Z3 Section, when and only when the single-stranded nucleic acid to be cleaved is cleaved, the fluorescent signal emitted by the fluorescent group can be detected.
  • the fluorescent group and the quenching group are not located at Z3 at the same time.
  • the Z3 contains a nucleic acid sequence that can guide the Cas protein to specifically bind to a target nucleic acid molecule.
  • the length of Z3 is 15-50nt, preferably 16-40nt, more preferably 16-34nt.
  • the Z1, Z2 and Z3 are all RNA nucleic acid sequences.
  • the Z4 is a DNA and/or RNA nucleic acid sequence.
  • the Z5 is a DNA single-stranded nucleic acid sequence, or an RNA single-stranded nucleic acid sequence, or a nucleic acid sequence of both RNA and DNA.
  • the Z5 contains nucleotides based on natural bases, or nucleotides based on natural bases and non-natural bases.
  • the nucleotides include ribonucleic acid, deoxyribonucleic acid, peptide nucleic acid, or a combination thereof.
  • the natural base is selected from the following group: A, T, C, G, U, and I.
  • the length of the Z5 is 3-50 nt, preferably 4-30 nt, more preferably 6-12 nt.
  • the labels carried in the Z5 are a fluorescent group and a quenching group.
  • the fluorescent group and the quenching group are independently located at the 5'end, 3'end and/or middle of the Z5.
  • each guide RNA-reporter nucleic acid composite probe has different fluorescent groups and different or the same quenching groups between each pair.
  • the detection system also contains m target nucleic acid molecules to be detected, where m is a positive integer and m ⁇ n.
  • the target nucleic acid molecule includes a target nucleic acid molecule derived from the group consisting of plants, animals, insects, microorganisms, viruses, or combinations thereof.
  • the target nucleic acid is artificially synthesized or naturally occurring nucleic acid.
  • the target nucleic acid includes wild-type or mutant nucleic acid.
  • the target nucleic acid molecule is target DNA or RNA.
  • the target DNA includes DNA without reverse transcription or DNA obtained by reverse transcription or amplification of RNA (for example, cDNA, etc.).
  • the target RNA includes RNA that has not been transcribed or is transcribed from DNA.
  • the detection includes: qualitative detection or quantitative detection.
  • the detection system further contains (c) buffer.
  • the detection system further contains the target nucleic acid molecule to be detected.
  • the detection system further contains reagents for nucleic acid amplification reactions.
  • the detection system further contains:
  • the concentration of the target nucleic acid molecule to be detected in the system to be detected is 1 ⁇ 10 -9 nM to 1 ⁇ 10 3 nM; preferably 1 ⁇ 10 -8 nM to 1 ⁇ 10 2 nM.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1 ⁇ 10 15 copies/ml, preferably 1 to 10 10 copies/ml, more preferably 1 To 10 5 copies/ml.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1000 copies/ml, preferably 1 to 100 copies/ml, more preferably 1 to 10 copies/ml. Ml.
  • the molar ratio of each guide RNA-reporter nucleic acid composite probe to the corresponding target nucleic acid molecule is 1:1 to 10 14 :1, preferably 10:1 to 10 5 :1, more preferably 20:1 to 10 3 :1.
  • the Cas protein is selected from the group consisting of Cas12a, Cas12b, Cas12d, Cas12g, Cas12i, Cas13a, Cas13b, Cas14 and Cas ⁇ .
  • the Cas12a protein is selected from the group consisting of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a and Lb4Cas12a.
  • the Cas12a protein is LbCas12a or FnCas12a.
  • the Cas12b protein is selected from the group consisting of AaCas12b, AacCas12b, AapCas12b, AbCas12b, AkCas12b, AmCas12b, BhCas12b, BsCas12b, EbCas12b and LsCas12b.
  • the Cas12g protein is Cas12g1.
  • the Cas12i protein is Cas12i1 or Cas12i2.
  • the Cas13a protein is selected from the group consisting of LshCas13a, LwaCas13a, LbaCas13a, LseCas13a, LbmCas13a, LbnCas13a, CamCas13a, CgaCas13a, Cga2Cas13a, PprCas13a, LweCas13a, Lwa2Cas13a, Rbf13Casa13, CasabuCas13a, RbfCas13a, RbfCas13a, LbnCas13a, Cga2Cas13a, PprCas13a, LweCas13a, Lwa2Cas13a, Rbf13Casa13, CasbuCas13a, Rbf13a, LbnCas13a.
  • the Cas13b protein is selected from the group consisting of BzoCas13b, PinCas13b, PbuCas13b, AspCas13b, PsmCas13b, RanCas13b, PauCas13b, PsaCas13b, Pin2Cas13b, CcaCas13b, PguCas13b, PspCas13b, PigCas13b and Pin.
  • the Cas14 protein is selected from the group consisting of Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h and Cas14u.
  • the Cas ⁇ protein is selected from the group consisting of Cas ⁇ -1, Cas ⁇ -2 and Cas ⁇ -3.
  • n is a positive integer between 2 and 200; preferably, n is a positive integer between 2 and 100; more preferably, n is a positive integer between 2 and 20; more preferably Ground, n is a positive integer between 2-10.
  • kits for detecting a target nucleic acid molecule comprising:
  • Z1 is the first stem-loop structure region
  • Z2 is no or nucleic acid linking region
  • Z3 is the guide RNA region
  • Z5 is a single-stranded nucleic acid to be cleaved with a detectable label, wherein the detectable label presents different detection states when the single-stranded nucleic acid to be cleaved is cut and uncut, and thus is detected;
  • Z3 and Z5 form a complementary paired double-stranded structural region; and when the target nucleic acid exists in the detection system, Z3 and Z5 do not form the complementary paired structure. Double-stranded structure region;
  • Z4 is none, or a chemical bond or connecting zone used to connect Z3 and Z5;
  • n is a positive integer n ⁇ 1;
  • the second container and the Cas protein in the second container, the Cas protein is a Cas protein with bypass single-stranded nucleic acid cleavage activity
  • the target nucleic acid molecule is target DNA and/or target RNA.
  • first container, the second container and the third container may be the same container or different containers.
  • the kit further includes:
  • the detection is used to simultaneously detect two or more different target nucleic acid molecules.
  • the detection system further contains reagents for nucleic acid amplification reactions.
  • the fourth container, the fifth container and the sixth container may be the same container or different containers.
  • two, more or all of the first container to the sixth container may be the same container or different containers.
  • a method for detecting target nucleic acid molecules in a sample which includes the following steps:
  • the guide RNA-reporter nucleic acid composite probe is cleaved by the Cas protein, which means that there is a corresponding target nucleic acid molecule in the sample; and the guide RNA-reporter nucleic acid composite probe is not cleaved by the Cas protein, it means that There is no corresponding target nucleic acid molecule in the sample.
  • the sample to be tested includes a sample that has not been amplified and a sample that has undergone amplification (or nucleic acid amplification).
  • the sample to be tested is a sample obtained through amplification.
  • the nucleic acid amplification method is selected from the following group: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification Increase, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
  • the PCR includes high temperature PCR, normal temperature PCR, or low temperature PCR.
  • the detection in step (ii) includes fluorescence detection.
  • the fluorescence detection method uses a microplate reader or a fluorescence spectrophotometer or a fluorescence quantitative PCR instrument for detection.
  • the method is an in vitro detection method.
  • the sample is an in vitro or ex vivo sample.
  • the method is non-diagnostic and non-therapeutic.
  • the method is diagnostic.
  • Figure 1 shows a schematic diagram of the guide RNA-reporter nucleic acid composite probe (Formula Ia).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • FIG. 2 shows the results of CRISPR multi-target detection.
  • Figure 2A shows the colors of 8 different samples
  • Figure 2B is the Green channel: FAM fluorescence is detected, and the detected target sequence is DNMT1-3 sites
  • Figure 2C is the Orange channel: ROX fluorescence is detected, and the detected target sequence is detected. Is the sry site).
  • FIG 3 shows a schematic diagram of another guide RNA-reporter nucleic acid composite probe (formula Ib).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • FIG 4 shows a schematic diagram of another guide RNA-reporter nucleic acid composite probe (formula Ic).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • FIG. 5 shows a schematic diagram of another guide RNA-reporter nucleic acid composite probe (formula Id).
  • F represents a fluorescent group (or other detectable label)
  • Q is a quenching group (or other quenching functional group for quenching the F signal).
  • Figure 6 shows the structure of the guide RNA-reporter nucleic acid composite probe in Example 2 or Example 3.
  • Figure 7 shows the results of the Cas12b multi-target detection test.
  • Figure 8 shows the results of the Cas14a1 multi-target detection test.
  • the inventors developed a method based on CRISPR technology that can simultaneously detect multiple target nucleic acid molecules in the same detection system for the first time.
  • the present inventors developed a guide RNA-reporter nucleic acid composite probe in which the first stem-loop structure region of RNA, the guide RNA region, the connecting region, and the fluorescent group and quenching group are connected in series.
  • CRISPR refers to clustered regularly spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats), which are part of the immune system of many prokaryotes.
  • Cas protein refers to CRISPR-associated protein, which is a related protein in the CRISPR system.
  • Cas12a (formerly known as “Cpf1”) refers to a crRNA-dependent endonuclease, which is a V-A type enzyme in the CRISPR system classification.
  • Cas12b and “C2c1” are used interchangeably and refer to the sgRNA-dependent endonuclease, which is a V-B type enzyme in the CRISPR system classification.
  • Cas12c and “C2c3” are used interchangeably and refer to tracrRNA:crRNA (or sgRNA)-dependent endonuclease, which is a V-C type enzyme in the CRISPR system classification.
  • Cas12d and “CasY” are used interchangeably and refer to scoutRNA:crRNA-dependent endonuclease, which is a V-D type enzyme in the CRISPR system classification.
  • Cas12g refers to a tracrRNA:crRNA (or sgRNA) dependent RNase, which is a V-G type enzyme in the CRISPR system classification.
  • Cas12i refers to a crRNA-dependent endonuclease, which is a type V-I enzyme in the CRISPR system classification.
  • Cas13a and C2c2 are used interchangeably and refer to crRNA-dependent endonucleases, which are type VI-A enzymes in the CRISPR system classification.
  • Cas13b refers to a crRNA-dependent endonuclease, which is a VI-B type enzyme in the CRISPR system classification.
  • Cas14 refers to a tracrRNA:crRNA (or sgRNA) dependent endonuclease, which is a V-F type enzyme in the CRISPR system classification.
  • Cas ⁇ and Cas12j are used interchangeably and refer to crRNA-dependent endonucleases, which belong to the V-type enzyme in the CRISPR system classification.
  • PCR refers to "polymerase chain reaction", a method used to amplify large amounts of DNA fragments of interest.
  • guide RNA-reporter nucleic acid composite probe of the present invention As used herein, the terms "guide RNA-reporter nucleic acid composite probe of the present invention”, “composite probe of the present invention”, and “probe of the present invention” are used interchangeably and refer to the target nucleic acid described in the present invention.
  • Molecular probes include guide RNA-reporter nucleic acid composite probes with Ia or Ib. It should be understood that the term also includes different forms of pairing, partial pairing or no pairing formed between Z3 and Z5 in the composite probe. For example, formula IIa is the state where Z3 and Z5 in the composite probe of formula Ia of the present invention form a pair.
  • a guide RNA-reporter nucleic acid composite probe with a novel structure is provided.
  • a representative composite probe has the structure shown in formula Ia,
  • Z1, Z2, Z3, Z4, and Z5 are as described above.
  • Z1, Z2, Z3, Z4, and Z5 are as described above.
  • the composite probe of formula Ic or formula Id is used in conjunction with the Cas protein of Cas13b type.
  • the labels carried in the Z5 are a fluorescent group and a quenching group, and the fluorescent group and the quenching group are each independently located at the 5'end of the nucleic acid probe , 3'end and/or middle.
  • RNA-reporter nucleic acid composite probe the structure of a representative guide RNA-reporter nucleic acid composite probe is shown in Figure 1.
  • a DNA sequence is added to the 3'end of the guide RNA, a quenching group (Q) is added in the middle, and a fluorescent group (F) is added to the 3'end.
  • Q quenching group
  • F fluorescent group
  • the sequence of the composite probe not only functions as a guide RNA, but also functions as a fluorescent probe.
  • the terminal sequence (Z5) of the DNA is complementary to a part of the sequence base (Z3) of the guide RNA to form a hairpin structure.
  • the composite probe of the present invention binds to the Cas12a protein and binds to the target sequence.
  • the hairpin structure (that is, the pairing structure of Z3 and Z5 will untie) will open, and the side of Cas12a will be activated Pathway single-stranded DNA cleavage activity, thereby cleaving the DNA part of the composite probe (such as Z5), causing the fluorescent group to be separated from the quenching group, thereby causing the quenching group to lose its quenching function and emit fluorescence.
  • the Cas12 bypass cleavage activity activated by other targets will not affect the unbound target.
  • the composite probes are chopped and will not interfere with each other.
  • reaction system for detecting one or more (especially simultaneous detection of multiple) target nucleic acid molecules, the reaction system comprising:
  • n kinds of the guide RNA-reporter nucleic acid composite probes of the present invention preferably, n is 2-500 or 2-200;
  • Cas protein which is a Cas protein with bypass single-stranded nucleic acid cleavage activity.
  • the detection system provided by the present invention can detect m target nucleic acid molecules to be detected, where m is a positive integer and m ⁇ n.
  • the detection includes: qualitative detection or quantitative detection.
  • the detection system further contains:
  • the concentration of the target nucleic acid molecule to be detected in the system to be detected is 1 ⁇ 10 -9 nM to 1 ⁇ 10 3 nM; preferably 1 ⁇ 10 -8 nM to 1 ⁇ 10 2 nM.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1 ⁇ 10 15 copies/ml, preferably 1 to 10 10 copies/ml, more preferably 1 To 10 5 copies/ml.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1 to 1000 copies/ml, preferably 1 to 100 copies/ml, more preferably 1 to 10 copies/ml. Ml.
  • the molar ratio of each guide RNA-reporter nucleic acid composite probe to the corresponding target nucleic acid molecule is 1:1 to 10 14 :1, preferably 10:1 to 10 5 :1, more preferably 20:1 to 10 3 :1.
  • a kit for detecting one or more target nucleic acid molecules (especially detecting multiple simultaneously) is provided, and the kit includes:
  • n a positive integer of 2-500 or 2-200
  • the second container and the Cas protein in the second container, the Cas protein is a Cas protein with bypass single-stranded nucleic acid cleavage activity
  • the detection system further contains reagents for nucleic acid amplification reactions. That is, in the detection system of the present invention, the target nucleic acid molecule can be amplified, and the amplified target nucleic acid molecule can be detected, which has the function of signal amplification.
  • the fourth container, the fifth container, and the sixth container may be the same container or different containers.
  • two, more or all of the first container to the sixth container may be the same container or different containers.
  • a method for simultaneously detecting multiple target nucleic acid molecules in a sample which includes the following steps:
  • the guide RNA-reporter nucleic acid composite probe is cleaved by the Cas protein, which means that there is a corresponding target nucleic acid molecule in the sample; and the guide RNA-reporter nucleic acid composite probe is not cleaved by the Cas protein, it means that There is no corresponding target nucleic acid molecule in the sample.
  • the sample to be tested includes unamplified samples and amplified (or nucleic acid amplified) samples, and may also include untranscribed samples and transcribed samples.
  • the nucleic acid amplification method is selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription mediator Guided amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
  • the PCR includes high temperature PCR, normal temperature PCR, and/or low temperature PCR.
  • the detection in step (ii) includes fluorescence detection.
  • the fluorescence detection method uses a microplate reader or a fluorescence spectrophotometer for detection.
  • the method is an in vitro detection method.
  • the sample is an in vitro or ex vivo sample.
  • the method is non-diagnostic and non-therapeutic.
  • the multi-target detection method of the present invention realizes the detection of multiple target nucleic acid molecules at the same time with extremely high sensitivity in the same detection system, and can detect nucleic acid molecules (such as DNA) with a concentration of 10 -17 M.
  • Multi-purpose It can detect different nucleic acid samples, including DNA samples and RNA samples.
  • the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 1.
  • two kinds of probes are designed and synthesized, the sequence is as follows:
  • the target sequence needs to be amplified.
  • PCR or any other amplification method can be used.
  • isothermal LAMP amplification is used.
  • LAMP amplification reaction After heating male saliva at 95°C for 10 minutes, it serves as a template. The total volume of each reaction system is 20 ⁇ L, and two types of primers are added to respectively amplify the gene DNMT1-3 on the autosomes and the gene sry unique to the male Y chromosome (see primer table 2 for sequence). The specific primer amounts are 1.6 ⁇ M FIP and BIP, 0.2 ⁇ M F3 and B3, 0.4 ⁇ M LoopF and LoopB.
  • the kit used for the LAMP reaction is LAMP Kit (NEB).
  • the LAMP reaction program is 65°C for 40 minutes. The above products are called DNM and sry.
  • IS6110-1 fragment was amplified using the Mycobacterium tuberculosis genome as a template, and the amplified product was called IS-1.
  • Probes add 0.5 ⁇ L each (3 probes of 10 ⁇ M)
  • Figure 2B is the Green channel, which detects FAM fluorescence against the DNM target.
  • the samples that can detect significant rising curves are samples 2, 5, 6 and 8, respectively;
  • Figure 2C is the Orange channel, which is The ROX fluorescence of the sry target was detected, and samples 4, 6, 7 and 8 could be detected with a significant rising curve.
  • the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • Figure 6 the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • two kinds of probes are designed and synthesized, the sequence is as follows:
  • the transcription template was prepared by annealing T7-crRNA-F with the synthetic oligonucleotide Cas12b_tracrRNA (Table 3). Specifically, the paired oligonucleotides (4 ⁇ M) were annealed in 1 ⁇ PCR buffer (Transgen Biotech) with a total volume of 50 ⁇ L, and then an annealing procedure was performed: initial denaturation at 95°C for 5 minutes, and then cooling from 95°C to At 20°C, use a thermal cycler to reduce 1°C per minute. T7 high-yield transcription kit was used to synthesize tracrRNA, and the reaction was carried out at 37°C overnight (about 16h). Use DNase I to process template DNA, then use RNA purification and concentration kit to purify RNA, and quantify it with NanoDrop 2000C, and store it in a refrigerator at -80°C.
  • Cas12b_tracrRNA SEQ ID NO: 23
  • the target sequence needs to be amplified.
  • PCR or any other amplification method can be used.
  • the PCR amplification method is used.
  • PCR reaction separately extract Salmonella genomic DNA and E. coli O157 genomic DNA as templates for PCR amplification.
  • the total volume of each reaction system is 20 ⁇ L, and two types of primers are added to respectively amplify the specific fragments in Salmonella (product named SE) and E. coli O157 (product named O157) (see primer table 4 for sequence) .
  • the PCR reaction program is 95°C for 2 minutes, and then 35 cycles of 98°C for 10s, 60°C for 15s, and 72°C for 10s. After the PCR is completed, the product is directly used in the Cas12b reaction.
  • reaction samples There are two reaction samples, the difference is that the added template is different, they are the PCR amplification products of SE and O157 respectively, and the reaction system with sterile water is used as a negative control.
  • the fluorescence quantitative PCR instrument used in this reaction is ABI StepOne Plus, and the detected signal is FAM fluorescence.
  • the result is shown in Figure 7.
  • the FAM fluorescence signal of the reaction group added with the SE template is very low, while the FAM fluorescence signal of the reaction group added with the O157 template increases rapidly , And the signal intensity of the SE group formed a significant difference.
  • results of ROX fluorescence detection using a microplate reader show that the O157 template cannot activate the cleavage of the SE reporter nucleic acid probe, while the SE template can activate the cleavage of the SE reporter nucleic acid probe.
  • the above results show that once the corresponding target exists in the detection system, the corresponding fluorescent signal can be detected, which is consistent with the expected result; on the contrary, the existence of a certain target can be judged according to the type of the detected fluorescent signal.
  • the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • Figure 6 the structure of the guide RNA-reporter nucleic acid composite probe is shown in Figure 6, which consists of tracrRNA and crRNA reporter nucleic acid probes.
  • two kinds of probes are designed and synthesized, the sequence is as follows:
  • tracrRNA with Cas14a1 was synthesized and cloned into the pUC57 vector. Then use T7-crRNA-F and Cas14a-tracr-R primer pair (Table 6) for amplification, and the amplified product is purified and used to transcribe tracrRNA.
  • the tracrRNA was synthesized using 200ng transcription template and T7 high-yield transcription kit, and the reaction was carried out overnight (about 16h) at 37°C.
  • Use DNase I to process template DNA, then use RNA purification and concentration kit to purify RNA, and quantify it with NanoDrop 2000C, and store it in a refrigerator at -80°C.
  • Synthetic Cas14a1 tracrRNA sequence with T7 promoter (SEQ ID NO: 32):
  • Tris buffer solution 50mM Tris-HCl[pH 8.3], 75mM KCl, 3mM MgCl 2
  • Tris buffer solution 50mM Tris-HCl[pH 8.3], 75mM KCl, 3mM MgCl 2
  • an annealing reaction is performed on a PCR machine. Initial denaturation at 85°C for 5 minutes, then cooling from 85°C to 25°C, using a thermal cycler to reduce 3°C every minute.
  • the complex probe after annealing can be used in the cleavage reaction of Cas14 to detect the target nucleic acid.
  • the target sequence needs to be amplified.
  • PCR or any other amplification method can be used.
  • the PCR amplification method is used.
  • PCR reaction separately extract Salmonella genomic DNA and E. coli O157 genomic DNA as templates for PCR amplification.
  • the total volume of each reaction system is 20 ⁇ L, and two types of primers are added for amplification. In each pair of primers, the 5'end of one of them is phosphorylated.
  • the above-mentioned primer pairs were used to amplify the specific fragment in Salmonella (product named SE-ps) and the specific fragment in E. coli O157 (product named O157-ps) (see primer table 6 for the sequence).
  • SE-ps specific fragment in Salmonella
  • E. coli O157-ps product named O157-ps
  • the PCR reaction program is 95°C for 2 minutes, and then 35 cycles of 98°C for 10s, 60°C for 15s, and 72°C for 10s. After the PCR is completed, the product is directly used in the Cas14 reaction.
  • reaction buffer of 10*Cas14 is: 250mM NaCl, 200mM HEPES, pH 7.5, 10mM DTT, 50% glycerol, and 50mM MgCl 2 .
  • reaction samples There are 2 reaction samples, the difference is that the added template is different, they are the PCR amplification products of SE-ps and O157-ps, and the reaction system with sterile water is used as a negative control.
  • the fluorescence quantitative PCR instrument used in this reaction is ABI StepOne Plus, and the detected signal is FAM fluorescence.
  • the results are shown in Figure 8. After subtracting the background signal (that is, adding sterile water as a template), the FAM fluorescence signal of the reaction group added with the SE-ps template is lower, while the FAM fluorescence signal of the reaction group added with the O157-ps template The signal grows very fast, which is significantly different from the signal strength of the SE-ps group. Based on the above results, it can be inferred that the SE-ps template cannot activate the cleavage of the O157-ps reporter nucleic acid probe, while the O157-ps template can activate the cleavage of the O157-ps reporter nucleic acid probe.
  • results of ROX fluorescence detection using a microplate reader show that the O157-ps template cannot activate the cleavage of the SE-ps reporter nucleic acid probe, while the SE-ps template can activate the cleavage of the SE-ps reporter nucleic acid probe.
  • the above results show that once the corresponding target exists in the detection system, the corresponding fluorescent signal can be detected, which is consistent with the expected result; on the contrary, the existence of a certain target can be judged according to the type of the detected fluorescent signal.

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Abstract

本发明属于核酸检测领域，提供了一种利用CRISPR方法快速检测多靶标的方法和试剂盒。本发明提供了一种用于检测靶标核酸分子的检测体系，包含：(a)n种具有特定结构的向导RNA-报告核酸复合探针；和(b)Cas蛋白，所述Cas蛋白是具有旁路单链核酸切割活性的Cas蛋白。

Description

CRISPR多靶标检测方法及其试剂盒 技术领域
本发明涉及生物技术领域，具体地说，本发明涉及一种CRISPR多靶标检测方法及其试剂盒。
背景技术
利用Cas13或Cas12蛋白的CRISPR诊断方法被誉为下一代检测技术(分别称为SHERLOCK和HOLMES)，因其同时兼具快速、灵敏、特异、简便和廉价的特点。Cas12和Cas13蛋白能用于核酸检测是基于它们旁路(或反式)切割的活力，即在人工设计的向导RNA的引导下，结合特定序列的核酸片段，随之切割单链核酸探针，从而产生可被检测的信号。Cas12和Cas13的差异在于，Cas13蛋白结合RNA标靶并切割RNA探针，而Cas12则结合DNA靶标并切割单链DNA探针。
从原理上来说，若使用Cas13来检测常规的DNA靶标，对靶标DNA进行扩增，并在此过程中引入T7等启动子序列；然后还需要进行体外转录以生成模板RNA，以便Cas13进行识别和结合。此外，在Cas13的检测过程中所使用的RNA报告探针存在较大风险被RNA酶降解，从而导致检测体系的背景信号值较高。相比之下，基于Cas12的检测方法由于无需体外转录也不需要RNA报告探针，因而更具优势。
在一个检测体系中同时检测多个存在靶标的方法和技术对于体外诊断的拓展应用非常重要。目前，基于Cas13蛋白已开发出一种多靶标检测的方法，其关键是利用不同物种来源的Cas13蛋白针对不同RNA报告探针的旁路切割活性不同的特点。然而，Cas12没有这方面特性的研究报导；因此，有必要开发一种全新的方法，利用Cas蛋白进行多靶标核酸的检测。
发明内容
本发明的目的就是提供一种基于CRISPR-Cas蛋白的多靶标检测方法和试剂盒。
在本发明的第一方面，提供了一种用于检测靶标核酸分子的检测体系，所述检测体系包含：
(a)n种向导RNA-报告核酸复合探针，所述的向导RNA-报告核酸复合探针具有如式Ia、Ib、Ic或Id所示结构，
Z1-Z2-Z3-Z4-Z5  (式Ia)
Figure PCTCN2020104588-appb-000001
Z5-Z4-Z3-Z2-Z1  (式Ic)
Figure PCTCN2020104588-appb-000002
其中，
Z1为第一茎环结构区；
Z2为无或核酸连接区；
Z3为向导RNA区；
Z5为带可检测标记的单链待切割核酸，其中，所述的可检测标记在所述单链待切割核酸被切割和未被切割的情况下，呈现不同的检测状态从而被检测出；
其中，当靶标核酸不存在于所述检测体系时，Z3与Z5形成一互补配对的双链结构区；而当靶标核酸存在于所述检测体系时，Z3与Z5不形成所述的互补配对的双链结构区；
Z4为无、或用于连接Z3和Z5的化学键或连接区；
Figure PCTCN2020104588-appb-000003
为碱基互补配对的氢键；
并且，n为n≥1的正整数；和
(b)Cas蛋白，所述Cas蛋白是具有旁路单链核酸切割活性的Cas蛋白。
在另一优选例中，所述的Cas蛋白选自下组：Cas12型、Cas13a型、Cas13b型、Cas14型、CasΦ、或其组合。
在另一优选例中，当Cas蛋白为Cas12型和/或Cas13a型和/或Cas14型时，所述的向导RNA-报告核酸复合探针具有如式Ia或Ib所示结构。
在另一优选例中，当Cas蛋白为Cas13b型时，所述的向导RNA-报告核酸复合探针具有如式Ic或式Id所示结构。
在另一优选例中，所述的Cas12型选自下组：Cas12a、Cas12b、Cas12d、Cas12g、Cas12i、或其组合。
在另一优选例中，当靶标核酸不存在于所述检测体系时，所述的向导RNA-报告核酸复合探针为式IIa结构：
Figure PCTCN2020104588-appb-000004
式中，
Z1、Z2、Z3、Z4和Z5如上所述，
Figure PCTCN2020104588-appb-000005
为碱基互补配对的氢键。
在另一优选例中，在式Ia、式IIa、式Ic的结构式、式Ib中的“Z1-Z2-Z3”和式Id中的“Z3-Z2-Z1”，为从5'至3'方向。
在另一优选例中，当靶标核酸不存在于所述检测体系时，所述的向导RNA-报告核酸复合探针为式IIc结构：
Figure PCTCN2020104588-appb-000006
式中，
Z1、Z2、Z3、Z4和Z5如上所述，
Figure PCTCN2020104588-appb-000007
为碱基互补配对的氢键。
在另一优选例中，所述的互补配对的双链结构区包括Z3和Z5通过部分或全部互补配对所形成的双链结构区。
在另一优选例中，所述的互补配对的双链结构区为Z3和Z5通过全部互补配对所形成的双链结构区。
在另一优选例中，所述的Z1、Z2和Z3用于引导Cas蛋白结合于靶标核酸。
在另一优选例中，所述的Z1用于结合或锚定Cas蛋白。
在另一优选例中，所述向导RNA区通过与靶标核酸的互补配对，引导Cas蛋白结合于所述的靶标核酸。
在另一优选例中，Z3、Z4和Z5形成第二茎环结构区，其中，Z4为环区域(包括简单结构和复杂结构的环区域)。
在另一优选例中，所述的Z1基本上或全部由RNA构成。
在另一优选例中，所述的Z3基本上或全部由RNA构成。
在另一优选例中，所述向导RNA-报告核酸复合探针为单链。
在另一优选例中，所述Z1中的茎环结构为crRNA(或CRISPR RNA)茎环结构。
在另一优选例中，所述Z1的长度为10-300nt，较佳地为19-100nt，更佳地为19-91nt。
在另一优选例中，所述Z2是无或长度为0-20nt的核酸连接区。
在另一优选例中，所述体系中：
(i)n≥2，并且n为正整数；
(ii)所述的可检测标记为荧光基团，并且Z5带有荧光基团，而所述Z4和/或Z5中还带有淬灭基团，当且仅当所述的单链待切割核酸被切割时，可检测到荧光基团所发出的荧光信号；和/或
(iii)在所述的n种向导RNA-报告核酸复合探针中，所述的荧光基团两两不同，从而可被区分。
在另一优选例中，所述的可检测标记为荧光基团，并且Z5带有淬灭基团，而所述Z4和/或Z5中还带有荧光基团，当且仅当所述的单链待切割核酸被切割时，可检测到荧光基团所发出的荧光信号。
在另一优选例中，所述的可检测标记为荧光基团，其中，荧光基团位于Z5、Z4和Z3中任一区段，而淬灭基团位于Z5、Z4和Z3中任一区段，当且仅当所述的单链待切割核酸被切割时，可检测到荧光基团所发出的荧光信号。
在另一优选例中，所述荧光基团和淬灭基团不同时位于Z3。
在另一优选例中，所述Z3中包含可以引导所述Cas蛋白特异性结合靶标核酸分子的核酸序列。
在另一优选例中，所述Z3的长度为15-50nt，较佳地16-40nt，更佳地16-34nt。
在另一优选例中，所述Z1、Z2和Z3均是RNA核酸序列。
在另一优选例中，所述Z4为DNA和/或RNA核酸序列。
在另一优选例中，所述Z5为DNA单链核酸序列，或者RNA单链核酸序列，或同时具有RNA和DNA的核酸序列。
在另一优选例中，所述Z5含有基于天然碱基的核苷酸，或者含有基于天然碱 基和非天然碱基的核苷酸。
在另一优选例中，所述的核苷酸包括核糖核酸、脱氧核糖核酸、肽核酸、或其组合。
在另一优选例中，所述的天然碱基选自下组：A、T、C、G、U、I。
在另一优选例中，所述Z5的长度为3-50nt，较佳地4-30nt，更佳地6-12nt。
在另一优选例中，所述Z5中所带的标记为荧光基团和淬灭基团。
在另一优选例中，且所述的荧光基团和淬灭基团各自独立地位于所述Z5的5'端、3'端和/或中部。
在另一优选例中，在所述反应体系中，各向导RNA-报告核酸复合探针两两之间均带有不同的荧光基团和不同或相同的淬灭基团。
在另一优选例中，所述检测体系中还含有m种待检测的靶标核酸分子，其中，m为正整数，并且m≤n。
在另一优选例中，所述靶标核酸分子包括来源于选自下组的靶标核酸分子：植物、动物、昆虫、微生物、病毒、或其组合。
在另一优选例中，所述的靶标核酸是人工合成或天然存在的核酸。
在另一优选例中，所述的靶标核酸包括野生型或突变型的核酸。
在另一优选例中，所述的靶标核酸分子为靶标DNA或RNA。
在另一优选例中，所述的靶标DNA包括未经逆转录的DNA或由RNA逆转录或扩增而获得的DNA(例如，cDNA等)。
在另一优选例中，所述的靶标RNA包括未经转录或由DNA转录而获得的RNA。
在另一优选例中，所述的检测包括：定性检测或定量检测。
在另一优选例中，所述的检测体系还含有(c)缓冲液。
在另一优选例中，所述的检测体系还含有待检测的靶标核酸分子。
在另一优选例中，所述的检测体系还含有用于核酸扩增反应的试剂。
在另一优选例中，所述的检测体系还含有：
(d1)用于扩增靶标DNA的聚合酶；
(d2)任选的用于反转录的反转录酶；
(d3)任选的用于转录的转录酶；
(d4)用于扩增反应和/或反转录反应的dNTPs；
(d5)用于转录反应的NTPs。
在另一优选例中，所述的待检测的靶标核酸分子在所述待检测体系中的浓度为1×10 -9nM至1×10 3nM；较佳地1×10 -8nM至1×10 2nM。
在另一优选例中，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1至1×10 15拷贝/毫升，较佳地1至10 10拷贝/毫升，更佳地1至10 5拷贝/毫升。
在另一优选例中，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1至1000拷贝/毫升，较佳地1至100拷贝/毫升，更佳地1至10拷贝/毫升。
在另一优选例中，所述的检测体系中，每个向导RNA-报告核酸复合探针与对应的靶标核酸分子的摩尔比为1：1至10 14：1，较佳地10：1至10 5：1，更佳地20：1至10 3：1。
在另一优选例中，所述Cas蛋白选自下组：Cas12a、Cas12b、Cas12d、Cas12g、Cas12i、Cas13a、Cas13b、Cas14和CasΦ。
在另一优选例中，所述的Cas12a蛋白选自下组：FnCas12a、AsCas12a、LbCas12a、Lb5Cas12a、HkCas12a、OsCas12a、TsCas12a、BbCas12a、BoCas12a和Lb4Cas12a。
在另一优选例中，所述Cas12a蛋白为LbCas12a或FnCas12a。
在另一优选例中，所述Cas12b蛋白选自下组：AaCas12b、AacCas12b、AapCas12b、AbCas12b、AkCas12b、AmCas12b、BhCas12b、BsCas12b、EbCas12b和LsCas12b。
在另一优选例中，所述Cas12g蛋白为Cas12g1。
在另一优选例中，所述Cas12i蛋白为Cas12i1或Cas12i2。
在另一优选例中，所述Cas13a蛋白选自下组：LshCas13a、LwaCas13a、LbaCas13a、LseCas13a、LbmCas13a、LbnCas13a、CamCas13a、CgaCas13a、Cga2Cas13a、PprCas13a、LweCas13a、Lwa2Cas13a、LbfCas13a、RcsCas13a、RcrCas13a、RcdCas13a和LbuCas13a。
在另一优选例中，所述Cas13b蛋白选自下组：BzoCas13b、PinCas13b、PbuCas13b、AspCas13b、PsmCas13b、RanCas13b、PauCas13b、PsaCas13b、Pin2Cas13b、CcaCas13b、PguCas13b、PspCas13b、PigCas13b和Pin3Cas13b。
在另一优选例中，所述Cas14蛋白选自下组：Cas14a、Cas14b、Cas14c、Cas14d、Cas14e、Cas14f、Cas14g、Cas14h和Cas14u。
在另一优选例中，所述CasΦ蛋白选自下组：CasΦ-1、CasΦ-2和CasΦ-3。
在另一优选例中，n为2-200之间的正整数；较佳地，n为2-100之间的正整数；更佳地，n为2-20之间的正整数；更佳地，n为2-10之间的正整数。
在本发明的第二方面，提供了一种用于检测靶标核酸分子的试剂盒，所述试剂盒包括：
i)第一容器以及位于第一容器内的n种具有如式Ia、Ib、Ic或Id所示结构的向导RNA-报告核酸复合探针，
Z1-Z2-Z3-Z4-Z5  (式Ia)
Figure PCTCN2020104588-appb-000008
Z5-Z4-Z3-Z2-Z1  (式Ic)
Figure PCTCN2020104588-appb-000009
其中，
Z1为第一茎环结构区；
Z2为无或核酸连接区；
Z3为向导RNA区；
Z5为带可检测标记的单链待切割核酸，其中，所述的可检测标记在所述单链待切割核酸被切割和未被切割的情况下，呈现不同的检测状态从而被检测出；
其中，当靶标核酸不存在于所述检测体系时，Z3与Z5形成一互补配对的双链结构区；而当靶标核酸存在于所述检测体系时，Z3与Z5不形成所述的互补配对的双链结构区；
Z4为无、或用于连接Z3和Z5的化学键或连接区；
Figure PCTCN2020104588-appb-000010
为碱基互补配对的氢键；
并且，n为n≥1的正整数；
ii)第二容器以及位于第二容器内的Cas蛋白，所述Cas蛋白是具有旁路单链核酸切割活性的Cas蛋白；
iii)任选的第三容器以及位于第三容器中的缓冲液。
在另一优选例中，所述的靶标核酸分子为靶标DNA和/或靶标RNA。
在另一优选例中，所述第一容器、第二容器和第三容器可以是相同容器或不同容器。
在另一优选例中，所述试剂盒还包括：
iv)第四容器以及位于第四容器内的用于扩增靶标DNA的聚合酶；
v)任选的第五容器以及位于第五容器内的用于反转录的反转录酶和/或用于转录的转录酶；
vii)第六容器以及位于第六容器内的用于扩增反应和/或反转录反应的dNTPs和/或用于转录反应的NTPs。
在另一优选例中，所述检测用于同时检测两种或多种不同的靶标核酸分子。
在另一优选例中，所述的检测体系还含有用于核酸扩增反应的试剂。
在另一优选例中，所述第四容器、第五容器和第六容器可以是相同容器或不同容器。
在另一优选例中，所述第一容器至第六容器中的二个、多个或全部可以是相同容器或不同容器。
在本发明的第三方面，提供了检测样本中的靶标核酸分子的方法，包括以下步骤：
(i)提供如本发明第一方面所述的用于同时检测多种靶标核酸分子的检测体系，并且所述的检测体系还含有待检测的样本；和
(ii)检测所述检测体系中的向导RNA-报告核酸复合探针是否被Cas蛋白进行切割，所述的切割为针对单链核酸的旁路(或反式)切割；
其中，所述向导RNA-报告核酸复合探针被Cas蛋白切割，则表示所述样本中 存在对应的靶标核酸分子；而所述向导RNA-报告核酸复合探针不被Cas蛋白切割，则表示所述样本中不存在对应的靶标核酸分子。
在另一优选例中，所述的待检测的样本包括未经扩增的样本以及经过扩增(或核酸扩增)的样本。
在另一优选例中，所述的待检测的样本是经过扩增而获得的样本。
在另一优选例中，所述核酸扩增的方法选自下组：PCR扩增、LAMP扩增、RPA扩增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增、滚环扩增、HDA、SPIA、NEAR、TMA和SMAP2。
在另一优选例中，所述的PCR包括高温PCR、常温PCR、或低温PCR。
在另一优选例中，在步骤(ii)中的检测包括荧光检测法。
在另一优选例中，所述荧光检测法采用酶标仪或者荧光分光光度计或者荧光定量PCR仪进行检测。
在另一优选例中，所述的方法是体外检测方法。
在另一优选例中，所述的样本是体外的或离体的样本。
在另一优选例中，所述的方法是非诊断性的和非治疗性的。
在另一优选例中，所述的方法是诊断性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了向导RNA-报告核酸复合探针示意图(式Ia)。图中，F表示荧光基团(或其他可检测标记)，Q为淬灭基团(或其他用于淬灭F信号的淬灭官能团)。
图2显示了CRISPR多靶标检测结果。
其中，图2A显示了代表了8个不同样品的颜色；图2B是Green通道：检测FAM荧光，检测的靶标序列为DNMT1-3位点；图2C是Orange通道：检测ROX荧光，检测的靶标序列为sry位点)。
图3显示了另一种向导RNA-报告核酸复合探针示意图(式Ib)。图中，F表示荧光基团(或其他可检测标记)，Q为淬灭基团(或其他用于淬灭F信号的淬灭官能 团)。
图4显示了另一种向导RNA-报告核酸复合探针示意图(式Ic)。图中，F表示荧光基团(或其他可检测标记)，Q为淬灭基团(或其他用于淬灭F信号的淬灭官能团)。
图5显示了另一种向导RNA-报告核酸复合探针示意图(式Id)。图中，F表示荧光基团(或其他可检测标记)，Q为淬灭基团(或其他用于淬灭F信号的淬灭官能团)。
图6显示了实施例2或实施例3中的向导RNA-报告核酸复合探针的结构。
图7显示了Cas12b多靶标检测测试的结果。
图8显示了Cas14a1多靶标检测测试的结果。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发出一种基于CRISPR技术可以在同一检测体系中同时检测多个靶标核酸分子的方法。具体地，本发明人开发了一种向导RNA-报告核酸复合探针，所述探针中串联有RNA第一茎环结构区、向导RNA区、连接区以及带有荧光基团和淬灭基团的单链待切割核酸；在同一检测体系中，可含有多种不同的所述向导RNA-报告核酸复合探针，所针对的靶标核酸分子不同的各探针中，所带的荧光基团和淬灭基团也不相同。因此，可以根据检测出的不同的荧光信号，来区分被检测出的不同的靶标核酸分子。使用这种基于CRISPR技术的多靶标检测方法，可以同时、快速、准确地检测出同一样本体系中所含有的不同靶标核酸分子。在此基础上完成了本发明。
术语
术语“CRISPR”是指成簇的、规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats)，该序列是许多原核生物的免疫系统的部分。
术语“Cas蛋白”是指CRISPR-associated蛋白，它是CRISPR系统中的相关蛋白。
术语“Cas12a”(旧称“Cpf1”)是指crRNA依赖的内切酶，它是CRISPR系统分类中V-A型的酶。
术语“Cas12b”，“C2c1”可互换使用，是指sgRNA依赖的内切酶，它是CRISPR系统分类中V-B型的酶。
术语“Cas12c”，“C2c3”可互换使用，是指tracrRNA:crRNA(或sgRNA)依赖的内切酶，它是CRISPR系统分类中V-C型的酶。
术语“Cas12d”，“CasY”可互换使用，是指scoutRNA:crRNA依赖的内切酶，它是CRISPR系统分类中V-D型的酶。
术语“Cas12g”，是指tracrRNA:crRNA(或sgRNA)依赖的RNA酶，它是CRISPR系统分类中V-G型的酶。
术语“Cas12i”，是指crRNA依赖的核酸内切酶，它是CRISPR系统分类中V-I型的酶。
术语“Cas13a”，“C2c2”可互换使用，是指crRNA依赖的内切酶，它是CRISPR系统分类中VI-A型的酶。
术语“Cas13b”，是指crRNA依赖的内切酶，它是CRISPR系统分类中VI-B型的酶。
术语“Cas14”，是指tracrRNA:crRNA(或sgRNA)依赖的内切酶，它是CRISPR系统分类中V-F型的酶。
术语“CasΦ”、“Cas12j”可互换使用，是指crRNA依赖的核酸内切酶，它属于CRISPR系统分类中V型的酶。
术语“PCR”是指“聚合酶链式反应”，用于大量扩增目的DNA片段的一种方法。
向导RNA-报告核酸复合探针
如本文所用，术语“本发明的向导RNA-报告核酸复合探针”、“本发明的复合探针”、“本发明探针”可互换使用，指本发明所述的可用于检测靶标核酸分子的探针，包括具有Ia或Ib所示的向导RNA-报告核酸复合探针。应理解，该术语还包括所述复合探针中Z3和Z5之间形成配对、部分配对或不形成配对的各自不同形式。例如，式IIa为本发明式Ia复合探针中Z3和Z5形成配对的状态。
在本发明中，提供了一种结构新颖的向导RNA-报告核酸复合探针。一种代表性的复合探针具有如式Ia所示结构，
Z1-Z2-Z3-Z4-Z5  (式Ia)
式中，Z1、Z2、Z3、Z4和Z5如上所述。
另一种与式Ia类似的复合探针为式Ib结构，其中，Z1-Z2-Z3和Z5分别为两个独立的分子：
Figure PCTCN2020104588-appb-000011
式中，Z1、Z2、Z3、Z5和
Figure PCTCN2020104588-appb-000012
如上所述。
另一种与式Ia类似的复合探针为式Ic结构，
Z5-Z4-Z3-Z2-Z1  (式Ic)
式中，Z1、Z2、Z3、Z4和Z5如上所述。
另一种与式Ia类似的复合探针为式Id结构，其中，Z1-Z2-Z3和Z5分别为两个独立的分子：
Figure PCTCN2020104588-appb-000013
式中，Z1、Z2、Z3、Z5和
Figure PCTCN2020104588-appb-000014
如上所述。
在本发明中，所述的式Ic或式Id结构的复合探针与Cas13b型的Cas蛋白配合使用。
在另一优选例中，所述Z5中所带的标记为荧光基团和淬灭基团，且所述的荧光基团和淬灭基团各自独立地位于所述核酸探针的5'端、3'端和/或中部。
以Cas12型为例，一种代表性的向导RNA-报告核酸复合探针的结构如图1所示。在该复合探针中，在向导RNA的3'端加入了一段DNA的序列，并且在中间加入了淬灭基团(Q)、3'末端加入了荧光基团(F)。所述复合探针的序列既起到了向导RNA的作用，也起到了荧光探针的作用。
为了便于理解，以Cas12型为例，提供以下原理供参考。然而，应理解，本发明的保护范围不受该原理的限制。在本发明中，在没有靶标DNA的情况下，DNA的末端序列(Z5)与向导RNA的一部分序列碱基(Z3)互补配对，形成一个发夹结构。当存在特异的靶标序列时，本发明的复合探针与Cas12a蛋白结合并结合到靶标序 列上，此时发夹结构(即Z3和Z5的配对结构会解开)会打开，同时激活Cas12a的旁路单链DNA切割活力，从而切割该复合探针的DNA部分(例如Z5)，导致荧光基团与淬灭基团分开，进而使得淬灭基团失去淬灭功能而发出荧光。
在本发明中，即使在同一反应体系或检测体系中加入多种荧光探针时，由于初始状态没有游离的单链DNA，因此被其他靶标激活的Cas12旁路切割活力并不会将未结合靶标的复合探针切碎，也不会相互干扰。
本发明反应体系
在本发明中，提供了一种用于检测一种或多种(尤其是同时检测多种)靶标核酸分子的反应体系，所述反应体系包括：
(a)n种本发明的所述的向导RNA-报告核酸复合探针(优选地，n为2-500或2-200)；和
(b)Cas蛋白，所述Cas蛋白是具有旁路单链核酸切割活性的Cas蛋白。
本发明所提供的检测体系，可检测m种待检测的靶标核酸分子，其中，m为正整数，并且m≤n。
在本发明中，所述的检测包括：定性检测或定量检测。
在本发明的另一个实施方式中，所述的检测体系还含有：
(d1)用于扩增靶标DNA的聚合酶；
(d2)任选的用于反转录的反转录酶；
(d3)任选的用于转录的转录酶；
(d4)用于扩增反应和/或反转录反应的dNTPs；
(d5)用于转录反应的NTPs。
优选地，在本发明所提供的检测体系中，所述的待检测的靶标核酸分子在所述待检测体系中的浓度为1×10 -9nM至1×10 3nM；较佳地1×10 -8nM至1×10 2nM。
在另一优选例中，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1至1×10 15拷贝/毫升，较佳地1至10 10拷贝/毫升，更佳地1至10 5拷贝/毫升。
在另一优选例中，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1至1000拷贝/毫升，较佳地1至100拷贝/毫升，更佳地1至10拷贝/毫升。
在另一优选例中，所述的检测体系中，每个向导RNA-报告核酸复合探针与对应的靶标核酸分子的摩尔比为1：1至10 14：1，较佳地10：1至10 5：1，更佳地20：1至10 3：1。
本发明试剂盒
在本发明中，提供了用于检测一种或多种(尤其是同时检测多种)靶标核酸分子的试剂盒，所述的试剂盒包括：
i)第一容器以及位于第一容器内的n种如本文所述的向导RNA-报告核酸复合探针(优选地，n为2-500或2-200的正整数)；
ii)第二容器以及位于第二容器内的Cas蛋白，所述Cas蛋白是具有旁路单链核酸切割活性的Cas蛋白；
iii)任选的第三容器以及位于第三容器中的缓冲液。
在一个优选的实施方式中，所述的检测体系还含有用于核酸扩增反应的试剂。即本发明的检测体系中，可对靶标核酸分子进行扩增，并对扩增的靶标核酸分子其进行检测，其具有信号放大的作用。
在本发明的一个实施方式中，所述第四容器、第五容器和第六容器可以是相同容器或不同容器。优选地，所述第一容器至第六容器中的二个、多个或全部可以是相同容器或不同容器。
多重检测
在本发明中，提供了一种同时检测样本中的多种靶标核酸分子的方法，包括以下步骤：
(i)提供本发明第一方面所述的用于检测靶标核酸分子的检测体系，并且所述的检测体系还含有待检测的样本；和
(ii)检测所述检测体系中的向导RNA-报告核酸复合探针是否被Cas蛋白进行切割，所述的切割为旁路单链核酸的反式切割；
其中，所述向导RNA-报告核酸复合探针被Cas蛋白切割，则表示所述样本中存在对应的靶标核酸分子；而所述向导RNA-报告核酸复合探针不被Cas蛋白切割，则表示所述样本中不存在对应的靶标核酸分子。
在本发明中，所述的待检测的样本包括未经扩增的样本以及经过扩增(或核酸 扩增)的样本，还可包括未经转录的样本以及经过转录的样本。
在本发明的一个实施方式中，所述核酸扩增的方法选自下组：PCR扩增、LAMP扩增、RPA扩增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增、滚环扩增、HDA、SPIA、NEAR、TMA和SMAP2。
在一个优选的实施方式中，所述的PCR包括高温PCR、常温PCR、和/或低温PCR。
在本发明一个优选的实施方式中，在步骤(ii)中的检测包括荧光检测法。优选地，所述荧光检测法采用酶标仪或者荧光分光光度计进行检测。
在本发明的一个实施方式中，所述的方法是体外检测方法。优选地，所述的样本是体外的或离体的样本。
在本发明的另一个实施方式中，所述的方法是非诊断性的和非治疗性的。
本发明的主要优点包括：
1)高效：本发明多靶标检测方法实现了在同一检测体系中，以极高的灵敏度检测出多种同时存在的靶标核酸分子，可以检测到10 -17M浓度的核酸分子(如DNA)。
2)低成本：由于可以在很小的样本中同时检测多种靶标核酸分子，本发明方法可以极大地节约检测成本。实验中没有特殊的材料或酶，而且涉及到的材料、试剂等较少，可以进行微量化的测试分析
3)快速：在测试条件准备好的情况下，从拿到样品，到拿到检测结果只需约1小时。
4)多用途：可检测不同的核酸样本，包括DNA样本和RNA样本。
5)简单：没有特殊复杂的步骤，如果制成试剂盒以及设定好程序，只需简单的加入样品等操作。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：Cas12a多靶标检测测试
1.1向导RNA-报告核酸复合探针的结构
以Cas12a型为例，向导RNA-报告核酸复合探针的结构如图1所示。在本实施例中，设计并合成2种探针，序列如下：
表1 示例性向导RNA-报告核酸复合探针序列
Figure PCTCN2020104588-appb-000015
*BHQ1、BHQ2为淬灭基团；FAM和ROX分别为不同的荧光基团。
1.2靶标DNA的获得
首先，需要对目的序列进行扩增，可以使用PCR或其他任何扩增方法，在本实施例中，使用等温LAMP扩增。
LAMP扩增反应：将男性唾液95℃加热10min后，作为模板。每个反应体系总体积为20μL，分别加入两类引物，分别扩增常染色体上的基因DNMT1-3和男性Y染色体特有的基因sry(序列见引物表2)。具体引物量是1.6μM的FIP与BIP、0.2μM的F3与B3、0.4μM的LoopF与LoopB，LAMP反应所用的试剂盒为
Figure PCTCN2020104588-appb-000016
LAMP Kit(NEB)。LAMP反应程序为65℃ 40min。上述产物称为DNM和sry。
另外，还以结核分枝杆菌的基因组为模板扩增了IS6110-1片段，其扩增产物称为IS-1。
1.3 Cas12a反应
在20μL反应体系中，加入
10*NEB buffer 3.1 2μL
FnCas12a   1.5μL(添加后终浓度1.5μM)
探针       各加0.5μL(10μM的3种探针)
模板       0.5μL(LAMP扩增之后的产物)
ddH 2O     加至20μL混匀。
加完之后，在荧光定量PCR仪中检测，采用恒温40℃的反应条件。
共8个样品，其他都一样，仅加入的模板不同，分别是1.ddH 2O；2.DNM；3.IS-1；4.sry；5.DNM+IS-1；6.DNM+sry；7.IS-1+sry；8.DNM+IS-1+sry。
结果如图2所示，图2B是Green通道，即检测针对DNM靶标的FAM荧光，其中能检测到显著上升曲线的分别是2、5、6和8号样品；图2C是Orange通道，也就是检测针对sry靶标的ROX荧光，其中能检测到显著上升曲线的分别是4、6、7和8号样品。
以上结果表明，一旦所述检测体系中存在相应的靶标即可检测到对应的荧光信号，与预期的结果相符；反之，可以根据所检测到的荧光信号种类来判断某种靶标是否存在。
表2 引物序列
Figure PCTCN2020104588-appb-000017
实施例2：Cas12b多靶标检测测试
2.1 Cas12b报告核酸复合探针的结构
2.1.1向导RNA-报告核酸复合探针的合成
以AacCas12b型为例，向导RNA-报告核酸复合探针的结构如图6所示，由tracrRNA和crRNA报告核酸探针组成。在本实施例中，设计并合成2种探针，序列如下：
表3 示例性向导RNA-报告核酸复合探针序列
Figure PCTCN2020104588-appb-000018
*BHQ1、BHQ2为淬灭基团；FAM和ROX分别为不同的荧光基团。
2.1.2 Cas12b tracrRNA的制备：
首先，通过用T7-crRNA-F与合成的寡核苷酸Cas12b_tracrRNA(表3)进行退火来制备转录模板。具体是，将配对的寡核苷酸(4μM)在1×PCR缓冲液(Transgen Biotech)中退火，总体积为50μL，然后进行退火程序：在95℃初始变性5分钟，然后从95℃冷却至20℃，使用热循环仪每分钟降低1℃。使用T7高产量转录试剂盒合成tracrRNA，并且反应在37℃下进行过夜(约16h)。使用DNase I处理模板DNA，然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，保存到-80℃冰箱中。
注：Cas12b_tracrRNA(SEQ ID NO:23)：
5’-TTTTTGAGAAGCTCAACGGGCTTTGCCACCTGGAAAGTGGCCATTGGCACACCCGTTGAAAAATTCTGTCCTCTAGACCCTATAGTGAGTCGTATTA-3’；
T7-crRNA-F(SEQ ID NO:24):5’-GAAATTAATACGACTCACTATAGGG-3’
2.1.3 Cas12b tracrRNA:crRNA-报告核酸复合探针退火反应：
在Tris缓冲液条件下(50mM Tris-HCl[pH 8.3]、75mM KCl、3mM MgCl 2)，将tracrRNA:crRNA-报告核酸复合探针以2：1的摩尔浓度混合(终浓度分别为10μM和5μM)，在PCR仪上进行退火反应。在85℃初始变性5分钟，然后从85℃冷却至25℃，使用热循环仪每分钟降低3℃。退火完成后的复合物探针可以用于Cas12b的切割反应以检测靶标核酸。
2.2靶标DNA的获得
首先，需要对目的序列进行扩增，可以使用PCR或其他任何扩增方法，在本实施例中，使用PCR扩增方法。
PCR反应：分别抽提沙门氏菌基因组DNA和大肠杆菌O157基因组DNA作为PCR扩增的模板。每个反应体系总体积为20μL，分别加入两类引物，分别扩增沙门氏菌中的特异片段(产物命名为SE)和大肠杆菌O157中的特异片段(产物命名为O157)(序列见引物表4)。PCR反应程序为，95℃ 2min，然后开始35个循环98℃ 10s，60℃ 15s，72℃ 10s。PCR完成后，其产物直接用于Cas12b反应。
2.3 Cas12b反应
在20μL反应体系中，加入
10*NEB buffer 3.1 2μL
AacCas12b     1.5μL(添加后终浓度1.5μM)
复合物探针    1μL(每种探针的终浓度为500nM)
ddH 2O         加至19.5μL混匀。
加完之后，先在48℃放置5min，然后向反应体系中添加0.5μL的模板(PCR扩增之后的产物)，并将反应体系放置于荧光定量PCR仪中进行检测，采用恒温48℃的反应条件，检测FAM荧光。
共2个反应样品，区别在于加入的模板不同，分别是SE和O157的PCR扩增产物，以添加无菌水的反应体系为负对照。
该反应使用的荧光定量PCR仪器为ABI StepOne Plus，检测的信号是FAM荧光。结果如图7所示，扣除了背景信号(即添加了无菌水为模板)后，添加了SE模板的反应组FAM荧光信号很低，而添加了O157模板的反应组FAM荧光信号增长很快，与SE组的信号强度形成了显著差异。根据以上结果可以推断：SE模板不能激活O157的报告核酸探针的切割，而O157模板可以激活O157的报告核酸探针的切割。反之，利用酶标仪的检测ROX荧光的结果显示，O157模板不能激活SE的报告核酸探针的切割，而SE模板可以激活SE的报告核酸探针的切割。以上结果表明，一旦所述检测体系中存在相应的靶标即可检测到对应的荧光信号，与预期的结果相符；反之，可以根据所检测到的荧光信号种类来判断某种靶标是否存在。
表4 引物序列
引物名称 序列(5’-3’) SEQ ID NO:
SE-F TGTCACCGTGGTCCAGTTTA 25
SE-R CGACAAGACCATCACCAATG 26
O157-F3 gatgggaacgattatatcgaagg 27
O157-R2 cctgacagaatattataagctccg 28
实施例3：Cas14多靶标检测测试
3.1 Cas14报告核酸复合探针的结构
3.1.1向导RNA-报告核酸复合探针的合成
以Cas14a1为例，向导RNA-报告核酸复合探针的结构如图6所示，由tracrRNA和crRNA报告核酸探针组成。在本实施例中，设计并合成2种探针，序列如下：
表5 示例性向导RNA-报告核酸复合探针序列
Figure PCTCN2020104588-appb-000019
*BHQ1、BHQ2为淬灭基团；FAM和ROX分别为不同的荧光基团。
3.1.2 Cas14a1 tracrRNA的制备：
首先，合成带有Cas14a1的tracrRNA的片段，并克隆至pUC57载体上。再用T7-crRNA-F和Cas14a-tracr-R引物(表6)对进行扩增，并将扩增的产物纯化后用来转录tracrRNA。使用200ng的转录模板和T7高产量转录试剂盒合成tracrRNA，并且反应在37℃下进行过夜(约16h)。使用DNase I处理模板DNA，然后使用RNA纯化与浓缩试剂盒纯化RNA，并用NanoDrop 2000C定量，保存到-80℃冰箱中。
注：Cas14a-tracr-R(SEQ ID NO:31)：
5’-AAATGAATTTGTTTCGAGGGTTAC-3’
合成的带有T7启动子的Cas14a1 tracrRNA序列(SEQ ID NO:32)：
5’-GAAATTAATACGACTCACTATAGGGCTTCACTGATAAAGTGGAGAACCGCTTCACCAAAAGCTGTCCCTTAGGGGATTAGAACTTGAGTGAAGGTGGGCTGCTTGCATCAGCCTAATGTCGAGAAGTGCTTTCTTCGGAAAGTAACCCTCGAAACAAATTCATTTTTC-3’，黑体标记的序列为T7启动子序列，其余序列为Cas14a1tracrRNA序列。
3.1.3 Cas14tracrRNA:crRNA-报告核酸复合探针退火反应：
在Tris缓冲液条件下(50mM Tris-HCl[pH 8.3],75mM KCl,3mM MgCl 2)，将tracrRNA:crRNA-报告核酸复合探针以2：1的摩尔浓度混合(终浓度分别为10μM和5μM)，在PCR仪上进行退火反应。在85℃初始变性5分钟，然后从85℃冷却至25℃，使用热循环仪每分钟降低3℃。退火完成后的复合物探针可以用于Cas14的切割反应以检测靶标核酸。
3.2靶标DNA的获得
首先，需要对目的序列进行扩增，可以使用PCR或其他任何扩增方法，在本实施例中，使用PCR扩增方法。
PCR反应：分别抽提沙门氏菌基因组DNA和大肠杆菌O157基因组DNA作为PCR扩增的模板。每个反应体系总体积为20μL，分别加入两类引物进行扩增。每对引物中，其中一条的5’端为磷硫酰化修饰。用上述引物对分别扩增沙门氏菌中的特异片段(产物命名为SE-ps)和大肠杆菌O157中的特异片段(产物命名为O157-ps)(序列见引物表6)。PCR反应程序为，95℃ 2min，然后开始35个循环98℃ 10s，60℃ 15s，72℃ 10s。PCR完成后，其产物直接用于Cas14反应。
3.3 Cas14反应
在20μL反应体系中，加入：
10*Cas14buffer 2μL
Cas14a1        1.5μL(添加后终浓度1.5μM)
复合物探针     1μL(每种探针的终浓度为500nM)
ddH 2O          加至19μL混匀。
加完之后，先在37℃放置5min，然后向反应体系中添加0.5μL的模板(PCR扩增之后的产物)和0.5μL的T7核酸外切酶(10Units/μL)，并将反应体系放置于荧光定量PCR仪中进行检测，采用恒温37℃的反应条件，检测FAM荧光。10*Cas14的反应缓冲液为：250mM NaCl、200mM HEPES、pH 7.5、10mM DTT、50％glycerol和50mM MgCl 2。
共2个反应样品，区别在于加入的模板不同，分别是SE-ps和O157-ps的PCR扩增产物，以添加无菌水的反应体系为负对照。
该反应使用的荧光定量PCR仪器为ABI StepOne Plus，检测的信号是FAM荧 光。结果如图8所示，扣除了背景信号(即添加了无菌水为模板)后，添加了SE-ps模板的反应组FAM荧光信号较低，而添加了O157-ps模板的反应组FAM荧光信号增长很快，与SE-ps组的信号强度形成了显著差异。根据以上结果可以推断：SE-ps模板不能激活O157-ps的报告核酸探针的切割，而O157-ps模板可以激活O157-ps的报告核酸探针的切割。反之，利用酶标仪的检测ROX荧光的结果显示，O157-ps模板不能激活SE-ps的报告核酸探针的切割，而SE-ps模板可以激活SE-ps的报告核酸探针的切割。以上结果表明，一旦所述检测体系中存在相应的靶标即可检测到对应的荧光信号，与预期的结果相符；反之，可以根据所检测到的荧光信号种类来判断某种靶标是否存在。
表6 引物序列
引物名称 序列(5’-3’) SEQ ID NO:
SE-F tgtcaccgtggtccagttta 25
SE-R-ps c*g*a*c*aagaccatcaccaatg 33
O157-F-ps c*a*g*t*agggaagcgaacagag 34
O157-R2 cctgacagaatattataagctccg 28
注：除了磷硫酰化修饰(以*标示)外，其余序列与Cas12b的扩增引物一致。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于检测靶标核酸分子的检测体系，其特征在于，所述检测体系包含：
    (a)n种向导RNA-报告核酸复合探针，所述的向导RNA-报告核酸复合探针具有如式Ia、Ib、Ic或Id所示结构，
    Z1-Z2-Z3-Z4-Z5  (式Ia)
    Figure PCTCN2020104588-appb-100001
    Z5-Z4-Z3-Z2-Z1  (式Ic)
    Figure PCTCN2020104588-appb-100002
    其中，
    Z1为第一茎环结构区；
    Z2为无或核酸连接区；
    Z3为向导RNA区；
    Z5为带可检测标记的单链待切割核酸，其中，所述的可检测标记在所述单链待切割核酸被切割和未被切割的情况下，呈现不同的检测状态从而被检测出；
    其中，当靶标核酸不存在于所述检测体系时，Z3与Z5形成一互补配对的双链结构区；而当靶标核酸存在于所述检测体系时，Z3与Z5不形成所述的互补配对的双链结构区；
    Z4为无、或用于连接Z3和Z5的化学键或连接区；
    Figure PCTCN2020104588-appb-100003
    为碱基互补配对的氢键；
    并且，n为n≥1的正整数；和
    (b)Cas蛋白，所述Cas蛋白是具有旁路单链核酸切割活性的Cas蛋白。
  2. 如权利要求1所述的检测体系，其特征在于，所述的Cas蛋白选自下组：Cas12型、Cas13a型、Cas13b型、Cas14型、或其组合。
  3. 如权利要求1所述的检测体系，其特征在于，所述体系中：
    (i)n≥2，并且n为正整数；
    (ii)所述的可检测标记为荧光基团，并且Z5带有荧光基团，而所述Z4和/或Z5中还带有淬灭基团，当且仅当所述的单链待切割核酸被切割时，可检测到荧光基团所发出的荧光信号；和/或
    (iii)在所述的n种向导RNA-报告核酸复合探针中，所述的荧光基团两两不同，从而可被区分。
  4. 如权利要求1所述的检测体系，其特征在于，所述Z3中包含可以引导所述Cas蛋白特异性结合靶标核酸分子的核酸序列。
  5. 如权利要求1所述的检测体系，其特征在于，所述Z5为DNA单链核酸序列，或者RNA单链核酸序列，或同时具有RNA和DNA的核酸序列。
  6. 如权利要求1所述的检测体系，其特征在于，所述的检测包括：定性检测或定量检测。
  7. 一种用于检测靶标核酸分子的试剂盒，其特征在于，所述试剂盒包括：
    i)第一容器以及位于第一容器内的n种具有如式Ia、Ib、Ic或Id所示结构的向导RNA-报告核酸复合探针，
    Z1-Z2-Z3-Z4-Z5  (式Ia)
    Figure PCTCN2020104588-appb-100004
    Z5-Z4-Z3-Z2-Z1  (式Ic)
    Figure PCTCN2020104588-appb-100005
    其中，
    Z1为第一茎环结构区；
    Z2为无或核酸连接区；
    Z3为向导RNA区；
    Z5为带可检测标记的单链待切割核酸，其中，所述的可检测标记在所述单链待切割核酸被切割和未被切割的情况下，呈现不同的检测状态从而被检测出；
    其中，当靶标核酸不存在于所述检测体系时，Z3与Z5形成一互补配对的双链结构区；而当靶标核酸存在于所述检测体系时，Z3与Z5不形成所述的互补配对的双链结构区；
    Z4为无、或用于连接Z3和Z5的化学键或连接区；
    Figure PCTCN2020104588-appb-100006
    为碱基互补配对的氢键；
    并且，n为n≥1的正整数；
    ii)第二容器以及位于第二容器内的Cas蛋白，所述Cas蛋白是具有旁路单链核酸切割活性的Cas蛋白；
    iii)任选的第三容器以及位于第三容器中的缓冲液。
  8. 如权利要求7所述的试剂盒，其特征在于，所述试剂盒还包括：
    iv)第四容器以及位于第四容器内的用于扩增靶标DNA的聚合酶；
    v)任选的第五容器以及位于第五容器内的用于反转录的反转录酶和/或用于转录的转录酶；
    vii)第六容器以及位于第六容器内的用于扩增反应和/或反转录反应的dNTPs和/或用于转录反应的NTPs。
  9. 一种检测样本中的靶标核酸分子的方法，其特征在于，包括以下步骤：
    (i)提供如权利要求2所述的用于同时检测多种靶标核酸分子的检测体系，并且所述的检测体系还含有待检测的样本；和
    (ii)检测所述检测体系中的向导RNA-报告核酸复合探针是否被Cas蛋白进行切割，所述的切割为针对单链核酸的旁路(或反式)切割；
    其中，所述向导RNA-报告核酸复合探针被Cas蛋白切割，则表示所述样本中存在对应的靶标核酸分子；而所述向导RNA-报告核酸复合探针不被Cas蛋白切割，则表示所述样本中不存在对应的靶标核酸分子。
  10. 如权利要求9所述的方法，其特征在于，所述的待检测的样本是经过扩增而获得的样本。
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