An electrochemical nucleic acid detection sensor element and detection method based on RPA and CRISPR / Cas

Abstract

The present invention relates to the field of electrochemical analysis and detection, and specifically to an electrochemical nucleic acid detection sensor element and detection method based on RPA and CRISPR / Cas. The present invention provides an electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas, which breaks the design thinking of traditional electrochemical sensor elements, combines RPA technology, CRISPR / Cas tools and electrochemical detection technology, and connects electrochemically active molecules and modifying groups to the reporter molecules in the CRISPR / Cas reaction system, realizing the construction of a non-immobilized, homogeneous electrochemical nucleic acid detection method, which can realize simple, sensitive, rapid, accurate and low-cost detection of target nucleic acids, and has a wide range of applications and strong portability.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical sequence analysis and detection, and in particular to an electrochemical nucleic acid detection sensor element and a detection method based on RPA and CRISPR / Cas. Background Art

[0002] In recent years, polymerase chain reaction (PCR) technology has been widely used as the main detection method, but it still has significant defects such as cumbersome operation steps, low instrument portability, detection sensitivity needs to be improved, and high dependence on professional environment. The organic integration of recombinase polymerase amplification (RPA) technology, clustered regularly interspaced short palindromic repeats (CRISPR) recognition technology, and electrochemical detection technology is expected to lay the foundation for the widespread application of non-professionals.

[0003] Recombinase Polymerase Amplification (RPA) is different from conventional fluorescent quantitative PCR. It does not require repeated cycles of denaturation-annealing-extension at 56°C, 65°C, and 95°C. Instead, it relies on a commonly used constant temperature amplification technology with the advantages of rapid response, high detection sensitivity, and low temperature requirements. It can complete the amplification of target nucleic acid at a constant temperature of 37°C.

[0004] As a nucleic acid shearing tool, CRISPR / Cas has attracted wide attention since its discovery. CRISPR / Cas tools have significant advantages such as high sensitivity, strong specificity, rapid reaction, simple operation, and high efficiency. In addition to its unique application in gene editing, it also brings new possibilities for nucleic acid detection. CRISPR / Cas nucleic acid detection tools are composed of Cas protein, crRNA, and reporter molecules. Cas protein and crRNA can form a stable binary complex. According to the different properties of Cas protein (such as Cas9, Cas12, Cas13 and Cas14, etc.), crRNA can selectively bind to RNA or ssDNA in the sample according to sequence matching, thereby forming a Cas / crRNA / RNA or Cas / crRNA / ssDNA ternary complex, thereby making the Cas protein conformational changes have the trans-cutting activity of RNA shearing enzyme or ssDNA shearing enzyme. The reporter molecule is composed of a DNA or RNA sequence coupled to a signal molecule. The Cas ternary complex shears the DNA or RNA sequence in the reporter molecule, releasing the free signal molecule, thereby generating detectable optical signals, color signals, etc. As a nucleic acid cutting tool, CRISPR / Cas tool has the advantages of high sensitivity, high specificity, rapid response, simple operation and high efficiency. It has been widely used in the field of nucleic acid detection in recent years.

[0005] The combination of RPA technology and CRISPR / Cas tools can further improve the detection sensitivity and achieve detection of target nucleic acids at the single-molecule level. Existing nucleic acid detection systems based on RPA and CRISPR / Cas mostly output fluorescent signals, and the supporting instruments are expensive and have poor portability. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide an electrochemical nucleic acid detection sensor element and detection method based on RPA and CRISPR / Cas.

[0007] To this end, the present invention provides the following technical solutions:

[0008] An electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas, comprising: a CRISPR / Cas reaction system and an RPA reaction system;

[0009] The CRISPR / Cas reaction system includes: a non-immobilized reporter molecule, wherein the reporter molecule is a non-specific nucleic acid molecule;

[0010] The reporter molecule comprises:

[0011] at least one electrochemically active molecule; and

[0012] At least one nucleotide.

[0013] The reporter molecule is a non-specific nucleic acid molecule that does not undergo pairing recognition.

[0014] Optionally, the reporter molecule comprises at least one modification group:

[0015] The modifying group is modified on one end, the middle or both ends of the reporter molecule; and / or

[0016] The modifying group is a macromolecular substance with certain steric hindrance and does not generate interference peaks near the redox peaks of the electroactive substance.

[0017] The CRISPR / Cas reaction system also includes CRISPR / Cas tools and reaction buffer.

[0018] Optionally, the CRISPR / Cas reaction system further comprises T7 polymerase and rNTP; and / or

[0019] The modification group is at least one of carboxyfluorescein, biotin and digoxigenin.

[0020] Optionally, the CRISPR / Cas tool includes any one of CRISPR / Cas9, CRISPR / Cas12, CRISPR / Cas13 and CRISPR / Cas14; the CRISPR / Cas tool includes Cas protein and crRNA.

[0021] Optionally, the molar concentration ratio of the Cas protein to crRNA is in the range of 4:1 to 1:4; and / or

[0022] The concentration of the reporter molecule ranges from 1 to 50 μM; and / or

[0023] The concentration of the T7 polymerase is in the range of 1 to 10 U / μL; and / or

[0024] The concentration range of the rNTP is 0.1-2 mM; and / or

[0025] The concentration range of the reaction buffer is 0.2-1.5×.

[0026] Optionally, the molar concentration ratio of the Cas protein to crRNA is in the range of 2:1; and / or

[0027] The concentration of the reporter molecule ranges from 5 to 10 μM; and / or

[0028] The concentration of the T7 polymerase ranges from 4 to 8 U / μL; and / or

[0029] The concentration range of the rNTP is 0.5-1 mM; and / or

[0030] The concentration range of the reaction buffer is 0.4-1×.

[0031] Optionally, the non-specific nucleic acid molecule is a DNA chain, an RNA chain or a DNA / RNA doped chain, with a length of 5 to 20 bases; and / or

[0032] The electrochemically active molecule is at least one of methylene blue, ferrocene and thionine; and / or

[0033] The electrochemically active molecule is modified on one end, in the middle or at both ends of the reporter molecule; and / or

[0034] The RPA reaction system comprises an amplification primer pair designed for the target, an enzyme mixture, a reaction buffer, and magnesium acetate; and / or

[0035] The concentration of each primer in the amplification primer pair is 150-600 nM; and / or

[0036] The concentration of the enzyme mixture ranges from 0.8 to 1.2×; and / or

[0037] The concentration range of the reaction buffer is 0.5-1.1×; and / or

[0038] The concentration range of the magnesium acetate is 20-35 mM.

[0039] An electrochemical nucleic acid detection method based on RPA and CRISPR / Cas, comprising:

[0040] Method a:

[0041] Using the electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas, the sample to be tested is added to a mixed reaction system of the CRISPR / Cas reaction system and the RPA reaction system to react, thereby achieving amplification and electrochemical detection; or

[0042] Method b:

[0043] Using the electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas, the sample to be tested is first added to the RPA reaction system for amplification, and then the obtained reaction product is added to the CRISPR / Cas reaction system for electrochemical detection.

[0044] Optionally, in the method a, the reaction time is ≥10 min, and the reaction temperature is 30-42° C.; and / or

[0045] In the method b, the amplification reaction time of the RPA reaction system is ≥5 min, and the reaction temperature range is 30-42° C.; and / or

[0046] In the method b, the reaction time of the CRISPR / Cas reaction system is ≥5 min, and the reaction temperature ranges from 5 to 50° C.; and / or

[0047] In the method a or method b, the volume of the sample to be tested is 1 to 10 μL; and / or

[0048] In the method b, the volume of the amplification product is 1 to 50 μL; and / or

[0049] In the method a or method b, in the electrochemical detection, voltammetry is used for electrochemical detection.

[0050] Optionally, in the method a, the reaction time is ≥10 min, and the reaction temperature is 35-40° C.; and / or

[0051] In the method b, the amplification reaction time of the RPA reaction system is 15 to 40 minutes, and the reaction temperature ranges from 35 to 40° C.; and / or

[0052] In the method b, the reaction time of the CRISPR / Cas reaction system is 15 to 30 minutes, and the reaction temperature ranges from 20 to 40° C.; and / or

[0053] In the method a or method b, the volume of the sample to be tested is 3 to 8 μL; and / or

[0054] In the method b, the volume of the amplification product is 5 to 20 μL; and / or

[0055] In the method a or method b, the voltammetry includes cyclic voltammetry, square wave voltammetry or differential pulse voltammetry.

[0056] The technical solution of the present invention has the following advantages:

[0057] 1. The present invention provides an electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas, comprising: a CRISPR / Cas reaction system and an RPA reaction system; the CRISPR / Cas reaction system comprises: a non-immobilized reporter molecule; the reporter molecule comprises: at least one electrochemically active molecule; and at least one nucleotide; the reporter molecule is a non-specific nucleic acid molecule; the present invention has found in the research process that in electrochemical nucleic acid detection based on CRISPR / Cas, one end of the reporter molecule is generally connected to the electrochemically active molecule, and the other end is fixed to the electrode surface. As the reaction proceeds, the reporter molecule is sheared to release the electroactive substance, so that the electroactive substance is away from the electrode surface, the electrical signal decreases, and electrochemical detection is achieved. The research idea is summarized It is limited to the method of fixing the reporter molecule on the electrode and keeping it away from the electrode. However, the present invention finds that the above-mentioned method of fixing the electrode and keeping it away from the electrode has the following problems in terms of "precision and repeatability": the electrode surface needs to be modified with the reporter molecule, which makes the electrode preparation process complicated, the batch difference is large, and it cannot be reused; in terms of "sensitivity and specificity", the solid-liquid reaction mode limits the efficiency of Brownian motion molecular collisions and cannot prevent collisions from occurring, resulting in low reporter molecule shearing efficiency during sensitive identification and interference signals generated by free active molecules colliding with the electrode during specific detection; in terms of "quantitativeness and detection limit", the detection signal is inversely proportional to the target concentration, making it impossible to eliminate the high noise of the detection result, resulting in poor linear response and difficult extraction of weak signals, which together lead to serious restrictions on the improvement of detection performance. The present invention finds that when the reporter molecule is free in the reaction system without immobilization, since the reporter molecule and the CRISPR / Cas ternary complex are both homogeneous, the high shear efficiency brought by the improvement of the collision efficiency of the two is greatly improved. At the same time, when the reporter molecule is sheared, the released electroactive substance molecules are smaller and have a greater diffusion rate, and have a more advantageous ability to generate signals by colliding with the electrode than the reporter molecule, thereby presenting a clear electrical signal enhancement that is positively correlated with the detection target concentration, thereby achieving an improvement in sensitivity and the construction of a positively correlated response. In summary, the present invention constructs an electrochemical nucleic acid detection sensor element for the combination of RPA and CRISPR / Cas, which has a fast signal response speed and a simple structure, and can achieve simple, sensitive, rapid, accurate, and low-cost detection of target nucleic acids, and has a wide range of applications and strong portability.

[0058] 2. The present invention provides an electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas, wherein the reporter molecule contains at least one modification group: the modification group is modified at one end, the middle or both ends of the reporter molecule; and / or the modification group is a variety of macromolecular substances with certain steric hindrance, which do not produce interference peaks near the redox peaks of electroactive substances; during the research process, the present invention found that after the modification group on the reporter molecule connected to the electrochemically active molecule is added, the steric hindrance of the reporter molecule will increase, and the larger steric hindrance, the wrapping and entanglement of the modification group and the nucleic acid molecule will hinder the electron transfer of the electrochemically active substance, thereby basically eliminating the background signal of the free reporter molecule, thereby realizing the extraction of weak signals with low detection limits. Based on this, the mechanism discovered above can be used to break the traditional design thinking of electrochemical sensor elements, combine RPA technology, CRISPR / Cas tools and electrochemical detection technology, and connect electrochemically active molecules and modification groups to the reporter molecules in the CRISPR / Cas reaction system. Before the reporter molecules are sheared, the electrical signal is extremely weak due to the modification groups on the reporter molecules, reducing the background signal; after the reporter molecules are sheared, the electrochemically active molecules are released, the steric hindrance becomes smaller, and they are fully free, which greatly increases the diffusion coefficient and electrode contact probability of the electrochemically active molecules, greatly enhances the electrical signal, and the specific recognition signal is strong.

[0059] 3. The electrochemical nucleic acid detection method based on RPA and CRISPR / Cas provided by the present invention breaks the traditional design thinking of electrochemical sensor elements, combines RPA technology, CRISPR / Cas tools and electrochemical detection technology, and connects electrochemically active molecules and modifying groups to the reporter molecules in the CRISPR / Cas reaction system, thereby realizing the construction of a non-immobilized, homogeneous electrochemical nucleic acid detection method, which can realize simple, sensitive, rapid, accurate and low-cost detection of target nucleic acids, and has a wide range of applications and strong portability. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0061] Figure 1 is the electrical signal detection result using the methods of Example 1 and Example 7 in Experimental Example 1 of the present invention;

[0062] Figure 2 This is the electrical signal detection result using the methods of Example 4 and Example 8 in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0063] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0064] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0065] In the following embodiments:

[0066] 1× CRISPR / Cas12a reaction buffer is NEB buffer 3.0 or 3.1;

[0067] 1× CRISPR / Cas13a reaction buffer consists of 20mM HEPES-Na (pH 6.8), 50mM KCl, 10mM MgCl 2 , and 5% glycerol;

[0068] 1× RPA reaction buffer consisted of 50 mM Tris (pH 8.4), 70 mM potassium acetate, 15 mM magnesium acetate, 2 mM DTT, 5% polyethylene glycol, 200 μM dNTPs, 3 mM ATP, 20 mM creatine phosphate, and 100 ng / μL creatine kinase;

[0069] The 1× enzyme mixture consisted of 125 ng / μL UvsX recombinase, 600 ng / μL Gp32 single-stranded binding protein, 30 ng / mL UvsY recombinase, and 20 ng / μL strand displacement DNA polymerase.

[0070] Example 1

[0071] This embodiment provides an electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas13a, including:

[0072] Construct RPA reaction system and CRISPR / Cas13a reaction system:

[0073] RPA reaction system: 400 nM (each) amplification primer pair, 1× enzyme mixture, 1× RPA reaction buffer, 28 mM magnesium acetate, and the balance is water.

[0074] CRISPR / Cas13a reaction system: 7 μM reporter molecule, 60 nM Cas13a protein, 30 nM crRNA, 1× CRISPR / Cas13a reaction buffer, 5 U / μL T7 polymerase, 1 mM rNTP, and the balance is water.

[0075] The reporter molecule is composed of RNA chain, methylene blue (MB) and biotin, and the sequence of RNA chain is consistent with the shear tendency of Cas13a protein, with a length of 6 bases. The nucleotide sequence of the reporter molecule is Cas13a-SHERLOCK (Ding R, Shen Y, Yuan M, Zheng X, Chen S, Duan G. Rapid and facile detection of HBV with CRISPR / Cas13a. New Journal of Chemistry. 2022; 46 (41): 19997-20004.) The nucleotide sequence of the biotin reporter molecule (see the biotin reporter in the table under the ssRNA reporter item in Table S1), Biotin is modified at 5' end, and MB is modified at 3' end.

[0076] Preparation method of the reporter molecule:

[0077] ① Load the solid phase carrier CPG (commercially available) labeled with 5' biotin into an automatic synthesizer (ABI, model 394), synthesize the oligonucleotide chain in the 5'→3' direction, and take out the CPG powder after the sequence is completed;

[0078] ② Add 0.5 mL of 25% ammonia water and perform aminolysis at 55°C for 8 h to release the oligonucleotide chain from the solid carrier CPG;

[0079] ③ Add 2eq of 1mol / LTBAF in THF solution and incubate at room temperature for 5h to expose the silanol group at the 2' position;

[0080] ④ After separating the impurities, the obtained product and 5eq of 0.5mol / L MB-NHS DMSO solution were placed in a sodium bicarbonate solution at pH = 9 and incubated at 50°C for 8h to obtain the predetermined sequence;

[0081] ⑤ After ultrafiltration, the sample was subjected to preparative HPLC to prepare the reporter molecule.

[0082] This embodiment provides an electrochemical nucleic acid detection method using the electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas13a, comprising:

[0083] S1. Add 3 μL of the sample to be tested (the sample to be tested can be an extraction-free product or a nucleic acid after extraction and purification) into the RPA reaction system (volume 50 μL) and react at 37°C for 20 minutes.

[0084] S2. Connect the gold electrode to the electrochemical workstation and immerse it in the CRISPR / Cas13a reaction system.

[0085] S3. Take 6 μL of the amplified product of the RPA reaction system and add it to the CRISPR / Cas13a reaction system (volume 30 μL) and react at 37°C for 25 min.

[0086] S4. After the reaction is completed, the methylene blue electrical signal in the CRISPR / Cas13a reaction system is detected by square wave voltammetry.

[0087] The detection principle of the above method is: when there is a target DNA in the sample to be tested, the target DNA can be amplified in large quantities in the RPA reaction system, and the amplified product can be transcribed into a target RNA with the help of T7 polymerase and rNTP in the CRISPR / Cas13a reaction system. The target RNA can form a ternary complex with the Cas13a protein and crRNA, activate the trans-cleavage activity of the Cas13a protein, shear the RNA chain in the reporter molecule, and release methylene blue. When there is no target DNA in the sample to be tested, neither amplification nor transcription will be started, the trans-cleavage activity of the Cas13a protein will not be activated, the RNA chain in the reporter molecule will not be sheared, and the methylene blue modified therein will not be released. Since the free complete reporter molecule is modified with a group, the steric hindrance is large and is wrapped by nucleic acid and a modified group, the methylene blue modified therein cannot transfer electrons with the electrode, so when there is no target DNA in the sample to be tested, the electrical signal of methylene blue in the CRISPR / Cas13a reaction system is extremely weak, and the background signal is low. Methylene blue itself is small in size, has a fast diffusion rate, and has a small steric hindrance. Therefore, when there is target DNA in the sample to be tested, the electrical signal of methylene blue in the CRISPR / Cas13a reaction system is strong. In summary, the detection of target nucleic acids is simple, sensitive, rapid, accurate, and inexpensive, and has a wide range of applications and strong portability.

[0088] Example 2

[0089] The difference between this embodiment and embodiment 1 is that the CRISPR / Cas13a reaction system: 1 μM reporter molecule, 2 μM Cas13a protein, 0.5 μM crRNA, 1.5× CRISPR / Cas13a reaction buffer, 10 U / μL T7 polymerase, 2 mM rNTP, and the balance is water;

[0090] In step S1, the reaction conditions are 30°C for 5 min;

[0091] In step S3, the mixture was reacted at 5°C for 10 min.

[0092] Example 3

[0093] The difference between this embodiment and embodiment 1 is that the CRISPR / Cas13a reaction system: 10 μM reporter molecule, 0.5 μM Cas13a protein, 2 μM crRNA, 0.2×CRISPR / Cas13a reaction buffer, 6 U / μL T7 polymerase, 0.8 mM rNTP, and the balance is water;

[0094] In step S1, the reaction conditions are 42°C for 20 min;

[0095] In step S3, the reaction was carried out at 50°C for 5 minutes.

[0096] Example 4

[0097] This embodiment provides an electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas12a, including:

[0098] Constructing a hybrid system of RPA and CRISPR / Cas12a reaction system:

[0099] 400nM (each primer) amplification primer pair, 1× enzyme mix, 0.6× RPA reaction buffer, 25mM magnesium acetate, 10μM reporter molecule, 500nM Cas12a protein, 250nM crRNA, 0.8× CRISPR / Cas12a reaction buffer, and the balance is water.

[0100] The reporter molecule is composed of a DNA chain, methylene blue (MB) and carboxyfluorescein (FAM), and the sequence of the DNA chain is consistent with the shearing tendency of the CRISPR / Cas12a protein, and the length is 10 bases. The nucleotide sequence of the reporter molecule is the nucleotide sequence of the surface probe in E-CRISPR (Dai Y, Somoza RA, Wang L, Welter JF, Li Y, Caplan AI, et al. Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor. Angew Chem Int Ed Engl. 2019; 58 (48): 17399-405.) (see surface probe-10nt in the table under the material item), FAM is modified at the 5' end, and MB is modified at the 3' end;

[0101] Preparation method of the reporter molecule:

[0102] ① Load the 3' end MB-labeled solid phase carrier CPG into an automatic synthesizer (ABI, 394 model), synthesize the oligonucleotide chain in the 3'→5' direction on the solid phase carrier, and finally couple with FAM phosphoramidite reagent (commercially available) (incubate at 50℃ for 8h) to obtain the predetermined sequence;

[0103] ② Use tert-butylamine: methanol: water (1:1:2, v / v / v) for release and deprotection, and place in a water bath at 65°C for 3 hours.

[0104] ③ After ultrafiltration, the sample was subjected to preparative HPLC to prepare the reporter molecule.

[0105] This embodiment also provides a method for electrochemical nucleic acid detection using the above-mentioned electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas12a, including:

[0106] S1. Connect the gold electrode to the electrochemical workstation and immerse it in the RPA and CRISPR / Cas12a reaction system.

[0107] S2. Add 3 μL of the sample to be tested into the RPA and CRISPR reaction system (50 μL) and react at 37°C for 25 min. The sample to be tested can be an extraction-free product or an extracted and purified nucleic acid.

[0108] S3. After the reaction is completed, the methylene blue electrical signal in the RPA and CRISPR / Cas12a reaction system is detected by differential pulse voltammetry.

[0109] Example 5

[0110] The difference between this embodiment and embodiment 4 is that a 50 μL mixed system of RPA and CRISPR / Cas12a reaction system is constructed:

[0111] 150nM (each primer) amplification primer pair, 0.8× enzyme mix, 0.8× RPA reaction buffer, 20mM magnesium acetate, 1μM reporter molecule, 50nM Cas12a protein, 50nM crRNA, 0.6× CRISPR / Cas12a reaction buffer, and the balance is water.

[0112] In step S2, the reaction conditions are: react at 30°C for 30 minutes.

[0113] Example 6

[0114] The difference between this embodiment and embodiment 4 is that a 50 μL mixed system of RPA and CRISPR / Cas12a reaction system is constructed:

[0115] 600nM (each) amplification primer pair, 1× enzyme mix, 0.5× reaction buffer, 35mM magnesium acetate, 15μM reporter molecule, 1μM Cas12a protein, 1μM crRNA, 1.5× CRISPR / Cas12a reaction buffer, and the balance is water.

[0116] In step S2, the reaction conditions are: reacting at 42°C for 10 minutes.

[0117] Example 7

[0118] The difference between this embodiment and embodiment 1 is that the reporter molecule does not contain the modification group biotin.

[0119] In step ① of the reporter molecule preparation method, a common solid phase carrier CPG is used, rather than a solid phase carrier CPG labeled with biotin at the 5' end.

[0120] Example 8

[0121] The difference between this embodiment and embodiment 4 is that the reporter molecule does not contain the modification group carboxyfluorescein (FAM).

[0122] In step ① of the reporter molecule preparation method, carboxyfluorescein (FAM), a synthetic raw material, is not added.

[0123] Experimental Example 1

[0124] The sequences of the amplification primer pair and crRNA are respectively RPA-F / R and crRNA in the published article (Ding R, Shen Y, Yuan M, Zheng X, Chen S, Duan G. Rapid and facile detection of HBV with CRISPR / Cas13a. New Journal of Chemistry. 2022; 46(41): 19997-20004.) (see crRNA1 in Table S1, and F2 and R2 in RAA primer), and then implemented according to Example 1 and Example 7, as follows:

[0125] S1. Build an RPA response system, including:

[0126] 400 nM (each) amplification primer pair, 1× enzyme mix, 1× RPA reaction buffer, 28 mM magnesium acetate, the balance is water.

[0127] S2. Construction of CRISPR / Cas13a reaction system, including:

[0128] 7 μM reporter, 60 nM Cas13a protein, 30 nM crRNA, 1× CRISPR / Cas13a reaction buffer, 5 U / μL T7 polymerase, 1 mM rNTP, and the balance is water.

[0129] S3. Add 3 μL of extracted and purified nucleic acid (concentration is about 10 copies / μL) into the RPA reaction system (volume 50 μL) and react at 37°C for 20 minutes.

[0130] S4. Connect the gold electrode to the electrochemical workstation and immerse it in the CRISPR / Cas13a reaction system.

[0131] S5. Take 6 μL of the amplified product of the RPA reaction system and add it to the CRISPR / Cas13a reaction system (volume 30 μL) and react at 37°C for 25 min.

[0132] S6. After the reaction is completed, the methylene blue electrical signal in the CRISPR / Cas13a reaction system is detected by square wave voltammetry.

[0133] The results are as follows Figure 1As shown, the biotin-RNA-MB in the figure is the reporter molecule in Example 1, and RNA-MB is the reporter molecule in Example 7. It can be seen from the figure that the reporter molecules in Example 1 and Example 7 both obtained high positive signals and low background signals. Further comparison of Example 1 and Example 7 found that the background signal of Example 1 was lower and the positive signal was higher, indicating that the biotin-RNA-MB in Example 1 has a lower background signal and a better signal-to-noise ratio.

[0134] Experimental Example 2

[0135] The sequences of the amplification primer pair and crRNA are respectively B1 FL-RPA and crRNA in the published article (Lei R, Li L, Wu P, Fei X, Zhang Y, Wang J, et al. RPA / CRISPR / Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment. Acs Synth Biol. 2022; 11(5): 1772-81) (see crRNA in Table S1, and primer B1-RPA-F / B1-RPA-R), and then implemented according to Example 4 and Example 8, respectively, as follows:

[0136] S1. Construction of a hybrid system of RPA and CRISPR / Cas12a reaction system:

[0137] 400nM (each) amplification primer pair, 1× enzyme mix, 0.6× RPA reaction buffer, 25mM magnesium acetate, 10μM reporter molecule, 500nM Cas12a protein, 250nM crRNA, 0.8× CRISPR / Cas12a reaction buffer, and the balance is water.

[0138] S2. Connect the gold electrode to the electrochemical workstation and immerse it in the RPA and CRISPR / Cas12a reaction system.

[0139] S3. Add 3 μL of extracted and purified nucleic acid (concentration is about 10 copies / μL) into the RPA and CRISPR reaction system (50 μL) and react at 37°C for 25 minutes.

[0140] S4. After the reaction is completed, the methylene blue electrical signal in the RPA and CRISPR / Cas12a reaction system is detected by differential pulse voltammetry.

[0141] The results are as follows Figure 2As shown in the figure, FAM-DNA-MB is the reporter molecule in Example 4, and DNA-MB is the reporter molecule in Example 8. It can be seen from the figure that the reporter molecules in Example 4 and Example 8 both obtained high positive signals and low background signals. Further comparison of Example 4 and Example 8 found that the background signal of Example 4 was lower and the positive signal was higher, indicating that the FAM-DNA-MB of Example 4 has a lower background signal and a better signal-to-noise ratio.

[0142] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas, characterized in that: It consists of CRISPR / Cas reaction system and RPA reaction system; The CRISPR / Cas reaction system consists of a non-immobilized reporter molecule, Cas protein, crRNA, reaction buffer, T7 polymerase, rNTP, and water; the reporter molecule consists of an electrochemically active molecule, a DNA / RNA chain, and a modification group; The modifying group is a macromolecular substance that has a certain steric hindrance and does not produce an interfering peak near the redox peak of the electroactive substance; The modifying group is modified at one end, the middle or both ends of the reporter molecule; The modification group is at least one of carboxyfluorescein, biotin and digoxigenin; The electrochemically active molecule is at least one of methylene blue, ferrocene and thionine; The RPA reaction system is composed of an amplification primer pair designed for the target, an enzyme mixture, a reaction buffer, magnesium acetate, and water.

2. The electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas according to claim 1, characterized in that: The Cas protein is Cas12a or Cas13a.

3. The electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas according to claim 2, characterized in that: The molar concentration ratio of the Cas protein to crRNA is in the range of 4:1 to 1:4; The concentration range of the reporter molecule is 1 to 50 μM; The concentration range of the T7 polymerase is 1 to 10 U / μL; The concentration range of the rNTP is 0.1 ~ 2 mM.

4. The electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas according to claim 3, characterized in that: The molar concentration ratio of the Cas protein to crRNA is 2:1; The concentration range of the reporter molecule is 5 to 10 μM; The concentration range of the T7 polymerase is 4 to 8 U / μL; The concentration range of the rNTP is 0.5 ~ 1 mM.

5. The electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas according to any one of claims 1 to 4, characterized in that: The DNA / RNA chain length is 5 to 20 bases; The electrochemically active molecule is modified on one end, in the middle or at both ends of the reporter molecule; The concentration of each primer in the amplification primer pair is 150 to 600 nM; The concentration range of the magnesium acetate is 20-35 mM.

6. An electrochemical nucleic acid detection method based on RPA and CRISPR / Cas, characterized in that: include: Method a: Using the electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas according to any one of claims 1 to 5, adding the sample to be tested into a mixed reaction system of the CRISPR / Cas reaction system and the RPA reaction system for reaction to achieve amplification and electrochemical detection; or Method b: Using the electrochemical nucleic acid detection sensor element based on RPA and CRISPR / Cas according to any one of claims 1 to 5, the sample to be tested is first added to the RPA reaction system for amplification, and then the obtained reaction product is added to the CRISPR / Cas reaction system for electrochemical detection.

7. The electrochemical nucleic acid detection method based on RPA and CRISPR / Cas according to claim 6, characterized in that: In the method a, the reaction time is ≥10 min and the reaction temperature is 30 ~ 42 ° C; In the method b, the amplification reaction time of the RPA reaction system is ≥5 min, and the reaction temperature ranges from 30 to 42°C; In the method b, the reaction time of the CRISPR / Cas reaction system is ≥5 min, and the reaction temperature ranges from 5 to 50°C; In the method a or method b, the volume of the sample to be tested is 1 to 10 μL; In the method b, the volume of the amplified product is 1 to 50 μL; In the method a or method b, in the electrochemical detection, voltammetry is used for electrochemical detection.

8. The electrochemical nucleic acid detection method based on RPA and CRISPR / Cas according to claim 7, characterized in that: In the method a, the reaction time is ≥10 min, and the reaction temperature is 35-40° C.; In the method b, the amplification reaction time of the RPA reaction system is 15 to 40 minutes, and the reaction temperature ranges from 35 to 40°C; In the method b, the reaction time of the CRISPR / Cas reaction system is 15 to 30 min, and the reaction temperature ranges from 20 to 40°C; In method a or method b, the volume of the sample to be tested is 3 to 8 μL; In the method b, the volume of the amplified product is 5 to 20 μL; In the method a or method b, the voltammetry includes cyclic voltammetry, square wave voltammetry or differential pulse voltammetry.

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