One-pot type multi-target orthogonal detection system based on incision enzyme and application of one-pot type multi-target orthogonal detection system

Abstract

The invention discloses a one-pot multi-target orthogonal detection system based on nicking endonuclease and application of the one-pot multi-target orthogonal detection system, and belongs to the field of biological detection. The detection system comprises a recognition sequence of nicking endonuclease designed for a plurality of targets, a fluorescent report sequence capable of being completely complementarily paired with the recognition sequence, nicking endonuclease capable of recognizing double strands formed by the recognition sequence and the fluorescent report sequence and playing a nicking role, and a reaction buffer solution, wherein the recognition sequence is a single-stranded DNA fragment, and the single-stranded DNA fragment comprises a fixed region and a variable region required by the nicking effect of the nicking endonuclease. The invention proves that the nicking activity of the nicking endonuclease is influenced by the complementarity of the recognition sequence of the nicking endonuclease, and a one-pot multi-target orthogonal detection technology is developed by utilizing the characteristic.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a one-pot multi-target orthogonal detection system based on nicking endonuclease and application thereof. Background Art

[0002] Nicking enzyme is a type of site-specific endonuclease that can specifically recognize double-stranded DNA sequences and only nick one of the strands in the double-stranded DNA. Based on its characteristics, nicking endonucleases are an indispensable tool in the research of bioengineering fields such as the construction of recombinant DNA and synthetic biology. For example, nicking endonucleases can be used to prepare pre-nicked DNA substrates in DNA repair research. At the same time, nicking endonucleases can also be used in isothermal exponential amplification reactions through their specific nicking effect on DNA. At the incision formed by the nicking endonuclease, through the action of DNA polymerase, dNTPs are used as raw materials to polymerize and extend from the 3' end of the incision, displacing the DNA strand at the same position, thereby forming a new and complete DNA sequence containing the fixed region of the nicking endonuclease. This double-stranded DNA is recognized and nicked by the nucleic acid nicking endonuclease again, and then the "polymerization-nicking" cycle begins, producing a large number of displaced single-stranded DNA strands, thereby achieving isothermal amplification.

[0003] Although nicking endonucleases have been widely used in the fields of molecular biology and analytical chemistry, there are no reports on nicking endonucleases having orthogonal reaction properties, let alone technical reports on applying their orthogonal reaction properties to one-pot target multiplex detection. Summary of the invention

[0004] The purpose of the present invention is to provide a one-pot multi-target orthogonal detection system based on nicking endonuclease and its application to solve the problems existing in the above-mentioned prior art. The detection system is constructed based on the orthogonal reaction characteristics of nicking endonuclease and can realize simultaneous orthogonal detection of multiple targets.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a one-pot multi-target orthogonal detection system based on a nicking endonuclease, comprising a recognition sequence of a nicking endonuclease designed for multiple targets, a fluorescent reporter sequence that can be fully complementary to the recognition sequence, and a nicking endonuclease and a reaction buffer solution that can recognize a double strand formed by the recognition sequence and the fluorescent reporter sequence and exert a nicking effect; wherein the recognition sequence is a single-stranded DNA fragment, and the single-stranded DNA fragment includes a fixed region and a variable region required for the nicking endonuclease to exert a nicking effect.

[0007] Preferably, the 5' end of the fluorescent reporter sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a corresponding fluorescent quencher group.

[0008] Preferably, the target comprises double-stranded DNA, single-stranded DNA, RNA or protein.

[0009] The nicking endonucleases include Nt.BstNBI, One or more of Nt.BstNBI, Nt.BspQI, Nb.BsrDI, Nt.BsmAI, Nt.AlwI, Nb.BtsI, Nb.BsmI, Nt.CviPII, Nb.BbvCI, Nt.BbvCI and Nb.BssSI.

[0010] The present invention also provides a method for constructing a one-pot multi-target orthogonal detection system based on a nicking endonuclease, comprising the following steps:

[0011] (1) designing and introducing fixed regions containing recognition sequences of nicking endonuclease recognition sites according to multiple targets to be detected;

[0012] (2) according to the recognition sequence, adding a fluorescent reporter sequence that can be fully complementary to different recognition sequences to the reaction system, so that the recognition sequence and the fluorescent reporter sequence are combined to form a double-stranded DNA, while the fluorescence remains in a quenched state; the reaction system includes a reaction buffer solution and a nicking endonuclease;

[0013] The 5' end of the fluorescent reporter sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a corresponding quencher group, so that the fluorescent signal emitted by the reporter group is absorbed by the quencher group. The fluorescent reporter sequence cannot bind to the recognition sequence of incomplete complementary pairing or can only bind unstably.

[0014] (3) In the reaction system, the nicking endonuclease recognizes the fixed region of the orthogonal reaction sequence and performs nicking and signal amplification in the variable region, causing the fluorescent reporter sequence to break into two independent fragments, thereby restoring the fluorescent signal. Based on the orthogonal reactivity of the nicking endonuclease, different targets output different fluorescent signals, so that multiple targets can be detected simultaneously.

[0015] Preferably, the recognition sequence is selected from any one of the following sequences:

[0016] (a) a sequence of the target itself;

[0017] (b) A sequence possessed by the target is transformed through a molecular recognition pathway, such as recognition by an antibody labeled with antigen-ssDNA.

[0018] Preferably, the fixed regions of the recognition sequences are completely identical, and the base differences of the variable regions are ≥40%.

[0019] Preferably, the fluorescent reporter group labeled at the 5' end of the fluorescent reporter sequence includes FAM, TEXAS RED, JOE, TAMRA or ROX, and the fluorescent quencher group labeled at the 3' end includes BHQ1 or BHQ2.

[0020] The present invention discloses the following technical effects:

[0021] The present invention proves that the nicking activity of the nicking endonuclease is affected by the complementarity of its recognition sequence, and uses this property to develop a one-pot multi-target orthogonal detection technology. The nicking endonuclease can only exert its maximum nicking ability when the double-stranded DNA substrate is fully complementary. If the double-stranded DNA of the recognition sequence is not fully complementary, that is, there is a certain mismatch, its nicking activity will be greatly reduced or even disappear. Therefore, the present invention proves the orthogonal reaction characteristics of the nicking endonuclease, and constructs a multi-target orthogonal detection technology based on this characteristic, providing a new detection method for simultaneous detection of multiple targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Schematic diagram of one-pot multi-target orthogonal detection using nicking endonucleases, A: Schematic diagram of the structure of coding sequence and probe sequence; B: Orthogonal reaction regulation is achieved by changing the double-stranded complementary recognition ability of the probe sequence and the coding sequence;

[0024] Figure 2 Taking Nt.BstNBI nicking endonuclease as an example, the fixed region is fixed as the recognition site of Nt.BstNBI, while the variable region is any base and the coding sequence is completely complementary to the probe sequence.

[0025] Figure 3 Taking Nt.BstNBI nicking endonuclease as an example, the result diagram of the effect of single base mismatch in coding sequence on nicking endonuclease nicking effect;

[0026] Figure 4 Taking Nt.BstNBI nicking endonuclease as an example, the result diagram of the effect of double base mismatch in coding sequence on the nicking effect of nicking endonuclease;

[0027] Figure 5 Taking Nt.BstNBI nicking endonuclease as an example, the result diagram of the effect of three base mismatches in the coding sequence on the nicking effect of the nicking endonuclease;

[0028] Figure 6 Taking Nt.BstNBI nicking endonuclease as an example, the result diagram of the effect of four-base and five-base mismatches in the coding sequence on the nicking effect of the nicking endonuclease;

[0029] Figure 7 Taking Nt.BstNBI nicking endonuclease as an example, the effect of mispairing between position 1 and position 5 in the variable region on the coding ability;

[0030] Figure 8 The relationship between the change in nicking efficiency of Nt.BstNBI and the mismatched bases;

[0031] Fig. 9 Taking Nt.BstNBI nicking endonuclease as an example, the orthogonal reaction ability results of 117 probe sequences and coding sequences are shown in the figure;

[0032] Fig.10 Taking Nt.BstNBI nicking endonuclease as an example, the sequence diagram of 117 probe sequences and coding sequences;

[0033] Fig.11 Taking Nt.BstNBI nicking endonuclease as an example, the three-dimensional fluorescence spectrum results of one-pot detection of five targets are as follows: A: three-dimensional fluorescence spectrum results of one-pot detection when five targets exist at the same time; B: background three-dimensional fluorescence spectrum results of the one-pot detection system when there is no target; C: three-dimensional fluorescence spectrum results when only target 1 exists in the one-pot detection system; D: three-dimensional fluorescence spectrum results when only target 2 exists in the one-pot detection system; E: three-dimensional fluorescence spectrum results when only target 3 exists in the one-pot detection system; F: three-dimensional fluorescence spectrum results when only target 4 exists in the one-pot detection system; G: three-dimensional fluorescence spectrum results when only target 5 exists in the one-pot detection system. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] This example uses Nt.BstNBI nicking endonuclease as an example to perform one-pot multi-target orthogonal detection using the nicking endonuclease.

[0041] The probe sequence that Nt.BstNBI can recognize is a sequence that contains a fixed region of 5 bases (5'→3': GAGTC) and a variable region of 5 bases (5'→3': NNNNN). Under the premise of containing a completely complementary coding sequence, Nt.BstNBI can recognize the fixed region and produce a single-stranded nick at the 3' end of the 4th base in the variable region. Figure 1 As shown in A, the coding region of the coding sequence and the probe sequence is mainly a recognition region of 5 bases (i.e., a fixed region) and a variable region of 5 bases. Based on this feature, the base difference between the sequences can be no less than 2 bases by changing the five bases in the variable region, so that only the sequence with complete complementary pairing and the probe sequence can be nicked, and the sequence with incomplete complementary pairing and the probe sequence cannot exert the nicking ability of the nicking endonuclease (see Figure 1(middle B).

[0042] To verify the above theory, urea-denatured polyacrylamide gel electrophoresis was used to verify the nicking ability of the nicking enzyme. The specific experimental steps of urea-denatured polyacrylamide gel electrophoresis (Urea-PAGE) are:

[0043] (1) Preparation of 10% ammonium persulfate (APS): Dissolve 1 g of APS in 10 mL of water and shake to fully dissolve.

[0044] (2) Prepare 8 M urea solution: Add 4.8 g urea to 10 mL of water until fully dissolved.

[0045] (3) Prepare 1× urea loading buffer: Mix 6× loading buffer and urea in a volume ratio of 1:5 for later use.

[0046] (4) Prepare 20× TBE solution: Dissolve 1× TBE powder (1 L) in 50 mL for later use (i.e., 20×).

[0047] (5) Prepare 15% Urea-PAGE premix: 240 g urea, 25 mL 20×TBE buffer, 250 mL acrylamide / methylenebisacrylamide 30% solution (29:1), and add water to make up to 500 mL. Dissolve the urea completely by ultrasound or by magnetic stirring at 50°C.

[0048] (6) Prepare the glue plate in advance according to the conventional glue making method, clamp the glass, and align the rear bayonet.

[0049] (7) Take a 15 mL centrifuge tube, add 10 mL of 15% Urea-PAGE premix, 30 μL of 10% APS, and 10 μL of tetramethylethylenediamine (TEMED), and gently invert for about ten times to mix.

[0050] (8) Use a 5 mL pipette to add the solution prepared in (5) above to the gelatin plate.

[0051] (9) After adding, insert the gel comb and wait for 20-30 minutes for the gel to solidify. During the waiting period, the sample preparation in step (11) can be completed.

[0052] (10) After the gel solidifies, it can be transferred to an electrophoresis tank for later use or stored in 1×TBE buffer at 4°C. If used immediately, pre-electrophoresis at 120V for 30 minutes is required. During the waiting period, the sample preparation in step (11) can be completed.

[0053] (11) Sample preparation: In a total volume of 50 μL ddH2O, add the coding sequence to a final concentration of 500 nM, 5 μL NEBuffer r3.1 and 0.5 U / μL Nt.BstNBI. React in a metal bath at 37°C for 2 h and then heat to 85°C to inactivate the enzyme. Generally, 10 μL sample + 10 μL 1× urea loading buffer is mixed; if a marker is required, 2 μL marker + 10 μL 1× urea loading buffer is mixed.

[0054] (12) After pre-electrophoresis, clean the sample wells again with a pipette or syringe before loading the sample, and load the sample immediately after cleaning.

[0055] (13) Sample loading: Add 10 μL of the sample or marker obtained in (11) above into the sample loading well according to the predetermined order.

[0056] (14) Electrophoresis was performed at 120 V for 60 min.

[0057] (15) Prepare 1× GelRed solution: dilute 2 μL GelRed (10000×) with ultrapure water to 1× for later use.

[0058] (16) After electrophoresis, the gel was removed and placed in 1× GelRed solution. Incubated at room temperature with shaking for 20 min to allow the DNA bands to be fully colored.

[0059] (17) The images were taken under a gel imaging system. Fluorescence analysis was performed based on the bands. (Wherein, the fluorescence intensity of the band is the fluorescence intensity after removing the background.) The stronger the fluorescence intensity of the sequence band after being cut, the weaker the fluorescence intensity of the sequence band that has not been cut, indicating that the cutting enzyme has more cutting products. Therefore, the cutting efficiency of the cutting enzyme for the pair of sequences can be calculated by the fluorescence intensity.

[0060] The results are as follows Figure 2 As shown, when the fixed region is fixed as the recognition site of Nt.BstNBI, and the variable region is any base and the coding sequence is completely complementary to the probe sequence, Nt.BstNBI can efficiently cleave each pair of DNA sequences regardless of the bases and arrangement of the variable sequence. When the probe sequence is fixed and the coding sequence base is changed to cause a mismatch between the two, there are two situations: ① When the mismatch site is in the fixed region, Nt.BstNBI can hardly cleave the pair of sequences; ② When the mismatch site is in the variable region and the number of mismatched bases is one, the cleavage ability of the cleavage endonuclease is weakened to varying degrees (see Figure 3 ). It is worth noting that the change in the nicking efficiency of Nt.BstNBI has nothing to do with the type of mismatch (see Figure 8 ). Then, when the mismatch site is in the variable region and is a double base mismatch or above, except for the mutation of 1 / 5 position in the variable region, the performance of the nicking endonuclease almost completely disappears (see Figure 4-Figure 6 ). The above results show that nicking endonucleases are expected to be used for orthogonal probe sequence cleavage and signal release. It was also proved that the mismatches at positions 1 and 5 in the variable region are not suitable for coding, but when the mismatches at positions 1 and 5 are combined with mismatches at positions 2-4, they still have good coding performance (see Figure 7 ). Figure 2-Figure 7 In the sequences involved, the probe sequence is based on the sequence in the figure, with 3 additional T bases "TTT" at the 5' end and 27 additional T bases "TTTTTTTTTTTTTTTTTTTTTTTTTT" at the 3' end; the coding sequence is based on the sequence in the figure, with 1 additional T base "T" at the 3' end; in addition Figure 3-Figure 7 The probe sequences in are labeled with a FAM modification at the 3' end. Figure 7 The sequences involved do not change the probe sequence, and only a single base is changed in the non-mismatch coding sequence as shown in the figure. The 1st and 5th bases close to the fixed region are defined as the 1st and 5th positions, respectively.

[0061] Based on the above-discovered coding characteristics, taking Nt.BstNBI nicking endonuclease as an example, the present invention designed a total of 117 pairs of highly orthogonal coding sequences and probe sequences ( Fig. 9 ), the paired sequences are completely complementary and the number of mismatches between the unpaired sequences is not less than 2 bases (inclusive). Fig. 9 Among the sequences involved, the probe sequence is based on the illustrated sequence, with an additional T base "T" at the 5' end and 19 additional T bases "TTTTTTTTTTTTTTTTTT" at the 3' end; the coding sequence is based on the illustrated sequence, with an additional T base "T" at the 3' end.

[0062] According to the sample preparation method in step (11) above, 117 pairs of completely complementary sequence pairs and mismatched sequence pairs with mismatches of not less than 2 bases were cross-incubated, and the cleavage efficiency was verified by urea-denatured polyacrylamide gel electrophoresis. When incubating the mismatched sequences, ten coding sequences were co-incubated with a probe as a group, where the probe was mismatched with the ten coding sequences. For all completely complementary sequence pairs, they were incubated separately to verify the cleavage ability. After analysis by the aforementioned electrophoresis experimental scheme, the following results were obtained: Fig.10 The heat map shown shows that the nicking endonuclease has extremely strong orthogonal encoding performance.

[0063] Example 2

[0064] Taking the simultaneous detection of five target models using the nicking endonuclease Nt.BstNBI as an example, 5 pairs of probe sequences modified with different fluorescent molecules and quenching groups were designed for each target. The 5 groups of target sequences and probe sequences are shown in Table 1:

[0065] Table 1 Five groups of target sequences and probe sequences

[0066] name Sequence (5'-3') Sequence number Coding sequence 1 (target 1) GCATCGACTCT SEQ ID NO:1 Coding sequence 2 (target 2) GGGAGGACTCT SEQ ID NO:2 Coding sequence 3 (target 3) GATCAGACTCT SEQ ID NO:3 Coding sequence 4 (target 4) ATTACGACTCT SEQ ID NO:4 Coding sequence 5 (target 5) TGCCCGACTCT SEQ ID NO:5 Probe sequence 1 BHQ1-TGAGTCGATGCT-FAM SEQ ID NO:6 Probe sequence 2 BHQ2-TGAGTCCTCCCT-TEXAS RED SEQ ID NO:7 Probe sequence 3 BHQ1-TGAGTCTGATCT-JOE SEQ ID NO:8 Probe sequence 4 BHQ2-TGAGTCGTAATT-TAMRA SEQ ID NO:9 Probe sequence 5 BHQ2-TGAGTCGGGCAT-ROX SEQ ID NO:10

[0067] In a total volume of 50 μL of ddH2O, add any one coding sequence or all five coding sequences (SEQ ID NO: 1-5) to a final concentration of 100 nM; add the probe sequence (SEQ ID NO: 6-10) to a final concentration of 500 nM; 5 μL NEBuffer r3.1; 0.5 U / μL Nt.BstNBI. After reacting at 37°C for 2 hours in a metal bath, heat it to 85°C to inactivate the enzyme. It contains 7 samples, and except for the different contents of the added coding sequences, the other components are completely consistent. The sequence contents in the 7 samples include: a negative control without adding any coding sequence, a positive control with all five coding sequences added, and a case where one of the five coding sequences is added respectively. After complete incubation, a fluorescence spectrometer is used to detect the three-dimensional fluorescence spectrum, with the excitation light wavelength set to 350 nm to 700 nm, the emission light wavelength set to 400 nm to 900 nm, the scanning speed 6000 nm / min, the excitation bandwidth 3.0 nm, and the emission bandwidth 3.0 nm. The results are as follows Fig.11 As shown, the results show that nicking endonucleases allow for one-pot orthogonal detection.

[0068] As can be seen from the above embodiments, the present invention utilizes the orthogonal reaction performance of the nicking endonuclease. A part of the nicking endonuclease recognition sequence is a fixed region, and the other part is a variable region, and the orthogonal reaction performance is achieved by adjusting the double-stranded complementary recognition ability of the variable region. Among them, the nicking endonuclease recognition sequence is a nicking endonuclease recognition sequence inherent in the target molecule, or a single target or multiple targets are converted into a nicking endonuclease recognition sequence through molecular recognition technology.

[0069] The above embodiment is only described by taking the nicking endonuclease Nt.BstNBI as an example. The orthogonal detection system based on the nicking endonuclease of the present invention can also be used to perform one-pot multi-target orthogonal detection simply by changing the molecular label of the detection probe to an enzyme label; wherein the detection probe is a molecular beacon and the output signal is a fluorescent signal. Alternatively, one-pot multi-target orthogonal detection can be performed by simply changing the type of nicking endonuclease or by combining multiple nicking endonucleases.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A one-pot multi-target orthogonal detection system based on nicking endonuclease, characterized in that: It includes a recognition sequence of a nicking endonuclease designed for multiple targets, a fluorescent reporter sequence that can be fully complementary to the recognition sequence, and a nicking endonuclease and a reaction buffer solution that can recognize the double strand formed by the recognition sequence and the fluorescent reporter sequence and exert a nicking effect; wherein the recognition sequence is a single-stranded DNA fragment, and the single-stranded DNA fragment includes a fixed region and a variable region required for the nicking endonuclease to exert a nicking effect.

2. The one-pot multi-target orthogonal detection system based on nicking endonuclease according to claim 1, characterized in that: The 5' end of the fluorescent reporter sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a corresponding fluorescent quencher group.

3. The one-pot multi-target orthogonal detection system based on nicking endonuclease according to claim 1, characterized in that: The target includes double-stranded DNA, single-stranded DNA, RNA or protein.

4. The one-pot multi-target orthogonal detection system based on nicking endonuclease according to claim 1, characterized in that: The nicking endonucleases include Nt.BstNBI, One or more of Nt.BstNBI, Nt.BspQI, Nb.BsrDI, Nt.BsmAI, Nt.AlwI, Nb.BtsI, Nb.BsmI, Nt.CviPII, Nb.BbvCI, Nt.BbvCI and Nb.BssSI.

5. A method for constructing a one-pot multi-target orthogonal detection system based on a nicking endonuclease according to any one of claims 1 to 4, characterized in that: The steps include: (1) designing and introducing fixed regions of recognition sequences containing nicking endonuclease recognition sites according to multiple targets to be detected; (2) according to the recognition sequence, adding a fluorescent reporter sequence that can be fully complementary to different recognition sequences to the reaction system, so that the recognition sequence and the fluorescent reporter sequence are combined to form a double-stranded DNA, while the fluorescence remains in a quenched state; the reaction system includes a reaction buffer solution and a nicking endonuclease; (3) In the reaction system, the nicking endonuclease recognizes the fixed region of the orthogonal reaction sequence and performs nicking and signal amplification in the variable region, causing the fluorescent reporter sequence to break into two independent fragments, thereby restoring the fluorescent signal. Based on the orthogonal reactivity of the nicking endonuclease, different targets output different fluorescent signals, so that multiple targets can be detected simultaneously.

6. The construction method according to claim 5, characterized in that: The recognition sequence is selected from any one of the following sequences: (a) a sequence of the target itself; (b) A sequence possessed by the target through a molecular recognition conversion pathway.

7. The construction method according to claim 5, characterized in that: The fixed regions of the recognition sequences are completely identical, and the base differences of the variable regions are ≥40%.

8. The construction method according to claim 5, characterized in that: The fluorescent reporter group labeled at the 5' end of the fluorescent reporter sequence includes FAM, TEXAS RED, JOE, TAMRA or ROX, and the fluorescent quencher group labeled at the 3' end includes BHQ1 or BHQ2.

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