A logic gate detection method and kit based on cyclic exponential amplification combined with CRISPR / Cas12a

Abstract

The present invention belongs to the field of biological detection technology, and in particular to a logic gate detection method and kit based on cyclic exponential amplification combined with CRISPR / Cas12a. The main process of the detection method of the present invention is as follows: miRNA-210 binds to a template to activate cyclic exponential amplification, and the obtained product P1 can be used as a catalyst for Cas12a / crRNA1. When miRNA-21 is present, miRNA-21 can be used as crRNA2, and crRNA1 and crRNA2 collaborate to form a complete crRNA spacer sequence, activate Cas12a trans-cleavage activity, and produce a detectable fluorescent signal. The present invention utilizes miRNA-21 as the spacer sequence in the crRNA structure, demonstrates the universal applicability of miRNA as a functional element, and establishes a kind of AND logic gate to realize the simultaneous detection of multiple miRNAs and the logical judgment of the results.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a logic gate detection method and kit based on cyclic exponential amplification combined with CRISPR / Cas12a. Background Art

[0002] miRNAs, a class of endogenous, non-coding small RNA molecules approximately 20-24 nucleotides in length, play a variety of important regulatory roles within cells, regulating the expression of one-third of human genes. This regulatory role is crucial for biological processes such as cell growth, differentiation, and metabolism. In the development of cancer, abnormal miRNA expression is closely associated with tumor development, progression, and prognosis. Therefore, miRNAs are widely used as biomarkers for cancer diagnosis. Studies have shown that miRNA expression levels show significant changes in certain early-stage cancers, such as colorectal cancer, liver cancer, cervical cancer, and pancreatic cancer. Measuring the levels of these miRNAs in blood and tissue samples can help physicians determine whether a patient is at risk for cancer. Abnormal miRNA levels in a patient often indicate the presence of a malignant tumor. miRNA-21 and miRNA-210 are among the first miRNAs to be associated with breast cancer. Therefore, measuring blood levels of miRNA-21 and miRNA-210 can enable early screening and diagnosis of breast cancer, which is crucial for improving tumor detection and reducing mortality.

[0003] Although traditional miRNA detection methods occupy an important position in scientific research and clinical practice, their complicated operation procedures and high costs have become a major challenge for large-scale application. For example, Northern blotting, as a classic molecular biology technique, can intuitively display the size and abundance of miRNA, but its operation is cumbersome and time-consuming, and requires a large amount of high-quality RNA samples, which limits its ability to respond quickly and be widely used. In addition, qPCR, as one of the most widely used miRNA quantification technologies, its high sensitivity and specificity are unquestionable. However, this technology also relies on sophisticated instruments and professional operators, and each test can only target a limited number of miRNAs. When large-scale screening is required, the cost-effectiveness ratio is not ideal. Therefore, traditional miRNA detection methods still face challenges in efficiently detecting miRNA in biological specimens.

[0004] As a versatile platform technology, CRISPR technology is gradually becoming a core tool in the new generation of biosensors. Numerous analytical platforms based on the CRISPR-Cas system have been reported, such as the DETECTR platform for DNA detection and the SHERLOCK platform for RNA virus detection. With the rapid development of isothermal nucleic acid amplification technology, the CRISPR-Cas system is increasingly being combined with diverse amplification strategies to construct efficient and sensitive bioassay systems. This technological integration not only fully leverages the high specificity and precision of the CRISPR-Cas system, but also leverages isothermal amplification techniques such as rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) to achieve rapid and specific enrichment of target nucleic acid molecules, thereby improving detection sensitivity and efficiency. However, most current CRISPR-Cas systems combined with isothermal amplification-mediated detection methods focus primarily on a single target, which is quite limited when dealing with complex biological samples requiring simultaneous screening of multiple targets, making it difficult to meet the needs of comprehensive and efficient detection.

[0005] Therefore, developing a detection technology that can simultaneously detect multiple miRNA targets plays an important role in the diagnosis and screening of diseases. Summary of the Invention

[0006] To solve the above problems, the present invention provides a logic gate detection method and kit based on cyclic exponential amplification combined with CRISPR / Cas12. The method established by the present invention can realize the simultaneous detection of multiple miRNAs and logical judgment of the results. It has high sensitivity and high specificity and has broad application prospects in disease screening.

[0007] The technical solution adopted in the present invention is as follows:

[0008] A logic gate detection method based on cyclic exponential amplification combined with CRISPR / Cas12a includes the following steps:

[0009] S1. Extraction of miRNA-21 and miRNA-210 nucleic acids from test samples;

[0010] S2. Preparation of DMCas premix: Mix DNA polymerase, nicking endonuclease, dNTP, template, probe, LbCas12a, crRNA1, 10× reaction buffer 1, 10× reaction buffer 2, and sterile ultrapure water;

[0011] S3. Detect miRNA-21 and miRNA-210 using cyclic exponential amplification combined with CRISPR / Cas12a: Add the nucleic acid sample to be tested extracted in step S1 to the DMCas mixture prepared in step S2, perform a constant temperature timed reaction, detect the fluorescence signal and analyze the results.

[0012] Preferably, the template sequence described in step S2 is shown as SEQ ID NO.4; the nucleotide sequence of the crRNA1 is shown as SEQ ID NO.1; and the sequence of the probe is 5'-ROX-CCCCCC-BHQ2-3'.

[0013] 5'-TCAGCCGCTGTCACACGCACAGCCTCAGCGGTCTCTTTGTAG CTTATCACCTCAGCTCAGCCGCTGTCACACGCACAG-3' (SEQ ID NO. 4);

[0014] 5'-UAAUUUCUACUAAGUGUAGAUGGUCUCUUUG-3' (SEQ ID NO. 1);

[0015] Preferably, the 10× reaction buffer 1 in step S2 is made of sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, and dithiothreitol; and the 10× reaction buffer 2 is made of potassium acetate, triacetate, magnesium acetate, and recombinant albumin.

[0016] Preferably, the working concentration range of the LbCas12a enzyme in step S2 is 0.001-1 μM, the nicking endonuclease is Nb.BbvCI, the working concentration range is 0.125-0.5 U / μL, the working concentration range of crRNA1 is 0.001-1 μM, and the working concentration range of Template is 10-500 nM; the constant temperature timing reaction conditions in step S3 are 28-42 ° C reaction, and the reaction time is 60-90 min.

[0017] Preferably, in step S2, the working concentration of the LbCas12a enzyme is 100 nM, the nicking endonuclease is Nb.BbvCI, the working concentration is 0.25 U / μL, the working concentration of crRNA1 is 50 nM, and the working concentration of Template is 50 nM; the constant temperature timing reaction condition in step S3 is 37 ° C reaction, and the reaction time is 60 min.

[0018] Preferably, the constant temperature and timed reaction conditions in step S3 are 28-42° C. and 60-90 min.

[0019] The present invention also provides a kit for detecting target nucleic acid in a sample, comprising a DMCas premix and sterile ultrapure water; the DMCas premix comprises DNA polymerase, nicking endonuclease, dNTP, template, probe, LbCas12a, crRNA1, 10×reaction buffer 1, and 10×reaction buffer 2.

[0020] Preferably, the template sequence is shown as SEQ ID NO.4; the nucleotide sequence of the crRNA1 is shown as SEQ ID NO.1; and the sequence of the probe is 5'-ROX-CCCCCC-BHQ2-3'.

[0021] Preferably, the 10× reaction buffer 1 comprises sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, and dithiothreitol; and the 10× reaction buffer 2 comprises potassium acetate, triacetate, magnesium acetate, and recombinant albumin.

[0022] Preferably, the kit is used to detect miRNA-21 and miRNA-210, and further comprises a P1 sequence having a nucleotide sequence as shown in SEQ ID NO.3.

[0023] 5'-TGATAAGCTACAAAGAGACC-3' (SEQ ID NO.3);

[0024] In the detection method of the present invention, crRNA-1 targets the 10 bases at the 5' end of P1. The combination of crRNA-1 and P1 alone cannot activate the trans-cleavage activity of Cas12a. The probe is a CRISPR / Cas12a trans-cleavage reporter sequence, with a 5' end labeled with a ROX group and a 3' end labeled with a BHQ2 group. Under natural conditions, the ROX group and the BHQ2 group of the probe are close, and the fluorescence is quenched. When CRISPR / Cas12a activates the trans-cleavage activity, it can cut the probe, and the ROX group and the BHQ2 group move away, generating a detectable fluorescent signal.

[0025] Among them, the P1 sequence can simulate the fragment cut out after the miRNA-210 binds to the template (Template) and is amplified. The first 10 bases at the 5' end of the P1 sequence match the partial sequence of Cas12a crRNA-1, and the last 10 bases match the partial sequence of miRNA-21; after the 22 bases at the 5' end of the template sequence and the target miRNA-210 are complementary, the middle contains the nicking endonuclease Nb.BbvCI recognition cleavage sequence (5'-CCTCAGC-3'), which can start the cyclic exponential amplification reaction and amplify the P1 fragment and P2 fragment. The P1 fragment is used as an activator of crRNA-1 and crRNA-2 (ie, miRNA-21), and the P2 fragment is used for the operation of the cyclic exponential amplification reaction.

[0026] Therefore, in the present invention, miRNA-210 is first combined with Template (template) to activate cyclic exponential amplification, and the product P1 obtained can be used as the activator (catalyst) of Cas12a / crRNA. When miRNA-21 is present, miRNA-21 can be used as crRNA2, and crRNA1 and crRNA2 collaborate to form the complete spacer sequence of crRNA, activate Cas12a trans-cleavage activity, and produce a detectable fluorescent signal; When miRNA-21 is missing, independent crRNA1 cannot activate Cas12a trans-cleavage activity, so no fluorescent signal is produced. MiRNA-21 is cleverly used as the spacer sequence in the crRNA structure, showing the universal applicability of miRNA as a functional element. This invention uses miRNA-210 and miRNA-21 as input signals, and the AND logic gate established can realize the logical judgment of simultaneous detection and results of multiple miRNAs.

[0027] This invention aims to efficiently and accurately detect and analyze multiple miRNA targets in parallel. This method, through a carefully designed logic gate architecture, not only improves detection sensitivity and specificity but also greatly simplifies the process of simultaneously identifying multiple miRNAs in complex samples, broadening its application prospects in biomedical research, early disease diagnosis, and personalized medicine.

[0028] Compared with the prior art, the technical advantages of the present invention are as follows:

[0029] (1) miRNA functionalization: The miRNA used in the present invention is directly used as the spacer region in crRNA. This strategy has wide applicability and can be flexibly expanded to the detection of other miRNA targets.

[0030] (2) Dual-target combined detection: In the designed detection system, there is a specific logical association, that is, when and only when miRNA-210 and miRNA-21 appear at the same time, a series of reactions will be triggered, and finally a detectable fluorescent signal will be generated. This provides more comprehensive and reliable biomarker information for disease diagnosis, thereby helping to improve the efficiency and accuracy of diagnosis.

[0031] (3) High sensitivity: Simply add miRNA-210 to the pre-configured reaction mixture to quickly initiate an efficient cyclic exponential amplification reaction. This process can produce a large number of activator molecules that activate the trans-cleavage activity of Cas12a in a very short time. Subsequently, through the fluorescence detection step, the fluorescence results can be obtained within 1 hour.

[0032] (4) Good specificity: The specific binding ability between the activator and miRNA-21 based on base complementary pairing is utilized. This design ensures that the activator can be accurately recognized and bound only when the target miRNA-21 is present, effectively reducing nonspecific signal interference;

[0033] (5) Low contamination: The design excludes operations such as amplification and opening the lid, while avoiding direct amplification of the target, thereby effectively reducing the risk of aerosol contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown is a schematic diagram of the detection method of the present invention;

[0035] Figure 2 shows the results of the design and screening of logic gate detection sequences for cyclic exponential amplification combined with CRISPR / Cas12a;

[0036] Figure 3 shows the optimization of the system for joint recognition of miRNA-21 and miRNA-210 by cyclic exponential amplification combined with CRISPR / Cas12a;

[0037] Figure 4 Shown is the sensitivity analysis of the combined recognition of miRNA-21 and miRNA-210 by cyclic exponential amplification combined with CRISPR / Cas12a;

[0038] Figure 5 Shown is the specific analysis of the combined recognition of miRNA-21 and miRNA-210 by cyclic exponential amplification combined with CRISPR / Cas12a;

[0039] Figure 6 Shown is the detection of miRNA-210 and miRNA-21 in plasma by cyclic exponential amplification combined with CRISPR / Cas12a. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the raw materials, equipment, etc. used in the following embodiments can be purchased through conventional channels.

[0041] The formula of 10× reaction buffer 1 (pH 7.9) in the present invention is as follows: 500 mM NaCl, 100 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 100 mM MgCl 2 , and 10 mM DTT (dimercaptothreitol).

[0042] The formula of 10× reaction buffer 2 (pH 7.9) is as follows: 500 mM Potassium Acetate, 200 mM Tris-acetate, 100 mM Magnesium Acetate, and 1000 μg / mL Recombinant Albumin.

[0043] Example 1 Logic gate detection sequence design for cyclic exponential amplification combined with CRISPR / Cas12a

[0044] First, crRNA1 sequences of different lengths were designed as the first half of the complete crRNA, and crRNA3 of different lengths (i.e., a variety of different sequences during crRNA2 screening, named here to distinguish crRNA3 from crRNA2) were designed as the second half of the complete crRNA. An activator-1 was designed as an activator recognized by crRNA1 and crRNA3 to ensure that crRNA1 alone would not activate Cas12a, while crRNA1 and crRNA3 could efficiently activate Cas12a after synergistic action;

[0045] crRNA1-1:

[0046] 5'-UAAUUUCUACUAAGUGUAGAUGGUC-3' (SEQ ID NO. 5);

[0047] crRNA1-2:

[0048] 5'-UAAUUUCUACUAAGUGUAGAUGGUCUC-3' (SEQ ID NO. 6);

[0049] crRNA1-3:

[0050] 5’-UAAUUUCUACUAAGUGUAGAUGGUCUCUU-3’(SEQ ID NO.7);

[0051] crRNA1-4:

[0052] 5’-UAAUUUCUACUAAGUGUAGAUGGUCUCUUUG-3’(SEQ ID NO.1);

[0053] crRNA1-5:

[0054] 5’-UAAUUUCUACUAAGUGUAGAUGGUCUCUUUGGU-3’(SEQ ID NO.8);

[0055] crRNA1-6:

[0056] 5’-UAAUUUCUACUAAGUGUAGAUGGUCUCUUUGGUCA-3’(SEQ ID NO.9);

[0057] crRNA3-1:

[0058] 5’-UCUUUGGUCAAUCCCG-3’(SEQ ID NO.10);

[0059] crRNA3-2:

[0060] 5’-UUUGGUCAAUCCCG-3’(SEQ ID NO.11);

[0061] crRNA3-3:

[0062] 5’-UGGUCAAUCCCG-3’(SEQ ID NO.12);

[0063] crRNA3-4:

[0064] 5’-GUCAAUCCCG-3’(SEQ ID NO.13);

[0065] crRNA3-5:

[0066] 5’-CAAUCCCG-3’(SEQ ID NO.14);

[0067] crRNA3-6:

[0068] 5’-AUCCCG-3’(SEQ ID NO.15);

[0069] activator-1:

[0070] 5'-CGGGATTGACCAAAGAGACC-3' (SEQ ID NO. 16);

[0071] Then, a sequence consisting of C bases preferentially cut by CRISPR / Cas12a was designed as a reporter probe, with a ROX group modified at the 5' end and a BHQ2 group modified at the 3' end to generate a fluorescent signal.

[0072] Probe: 5'-ROX-CCCCCC-BHQ2-3'(SEQ ID NO.2);

[0073] Secondly, the nucleic acid sequence of miRNA-21 was downloaded from the NCBI database, and a partial sequence of miRNA-21 was used as crRNA2. crRNA1 and crRNA2 can be used as complete crRNA to activate P1. According to the different positions of miRNA-21 at the front, middle and back, crRNA2 was used as the following P1-head, mid and tail.

[0074] miRNA-21: 5'-UAGCUUAUCAGACUGAUGUUGA-3' (SEQ ID NO. 17);

[0075] P1-head:5'-TGATAAGCTACAAAGAGACC-3'(SEQ ID NO.3);

[0076] P1-mid:5'-TCAGTCTGATCAAAGAGACC-3' (SEQ ID NO.18);

[0077] P1-tail:5'-TCAACATCAGCAAAGAGACC-3'(SEQ ID NO.19);

[0078] Finally, the nucleic acid sequence of miRNA-210 was downloaded from the NCBI database, and miRNA-210 was used as the amplification primer. The template sequence was designed so that the front and back ends of the template matched miRNA-210, and the middle part was complementary to P1. The nicking endonuclease Nb.BbvCI recognition cleavage sequence (5'-CCTCAGC-3') was inserted between the P1 complementary sequence and the miRNA-210 complementary sequence. When miRNA-210 binds to the template sequence, the cyclic exponential amplification reaction can be initiated.

[0079] miRNA-210: 5'-CUGUGCGUGUGACAGCGGCUGA-3' (SEQ ID NO. 20);

[0080] template:

[0081] 5'-TCAGCCGCTGTCACACGCACAGCCTCAGCGGTCTCTTTGTAGCTTATCACCTCAGCTCAGCCGCTGTCACACGCACAG-3' (SEQ ID NO. 4);

[0082] Detection principle diagram as shown Figure 1 As shown in the figure, the AND logic gate relationship established by miRNA-210 and miRNA-21, when miRNA-201 and miRNA-21 are absent or only one exists, cannot activate the trans-cleavage activity of Cas12a, and the logical relationship shown is (0,0,0)(1,0,0) or (0,1,0). Only when miRNA-21 and miRNA-210 are present at the same time can a fluorescent signal be generated, and the logical relationship shown is (1,1,1).

[0083] As shown in Figure 2(A), when crRNA1 has 10 bases and crRNA3 has 10 bases, the ratio of the positive fluorescence value to the negative fluorescence value (F-positive / F-negative, i.e., F P / F N ) is the largest, therefore, the base composition of crRNA1 was selected as 10 for the following experiments.

[0084] As shown in Figure 2(B), P1-1 (P1-head) acts as an activator to activate the ratio of positive fluorescence value to negative fluorescence value (F-positive / F-negative, i.e., F P / F N ) is the largest, indicating that the optimal result is obtained when the 5' end of miRNA-21 is used as crRNA2. This may be because the redundant sequence between crRNA1 and crRNA2 may cause steric hindrance, preventing conformational changes at the catalytic site. Therefore, the 5' end of miRNA-21 was selected as crRNA2 for further research.

[0085] As shown in Figure 2(C), only when miRNA21 and miRNA210 are present at the same time can a fluorescent signal be generated. When miRNA-21 and miRNA-210 are not present or only one exists, the trans-cleavage activity of Cas12a cannot be activated to generate a fluorescent signal.

[0086] To ensure the quality of primers and molecular beacon probes, each sequence was commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis according to HPLC purity standards.

[0087] Example 2 Optimization of the system for combined recognition of miRNA-21 and miRNA-210 by cyclic exponential amplification combined with CRISPR / Cas12a

[0088] When preparing the DMCas premix solution of the present invention, except for the changes in parameters (1)-(4), the rest are prepared according to Table 1.

[0089] (1) Temperature optimization: other parameters were fixed, and the synthesized miRNA21 and miRNA210 were used as templates. 2.5 μL miRNA21, 2.5 μL miRNA210 and 20 μL DMCas premix were mixed and placed in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for 60 min. The system temperature was adjusted to 27°C, 32°C, 37°C, and 42°C, respectively, and the changes in the fluorescence curve were observed. As shown in Figure 3(A), too high or too low temperature was not conducive to the enzyme reaction. According to F P / F N value, and 37°C was selected as the optimal reaction temperature for the system of the present invention.

[0090] (2) Optimize the amount of crRNA1. Keep other parameters fixed. Use synthetic miRNA-21 and miRNA-210 as templates and adjust the final concentration of crRNA1 to 10nM, 20nM, 50nM, 80nM, and 100nM. Mix 2.5μL miRNA-21, 2.5μL miRNA-210 with 20μL DMCas premix and place in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for 60min. Observe the changes in the fluorescence curve, as shown in Figure 3(B). Select 50nM for subsequent experiments.

[0091] (3) Optimization of Template Dosage: With other parameters fixed, the final concentrations of the templates were adjusted to 10 nM, 20 nM, 50 nM, 80 nM, and 100 nM using the synthesized miRNA-21 and miRNA-210 as templates. 2.5 μL miRNA-21, 2.5 μL miRNA-210, and 20 μL DMCas premix were mixed and placed in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for 60 min. The changes in the fluorescence curve were observed, as shown in Figure 3(C). 50 nM was selected for subsequent experiments.

[0092] (4) The dosage of Nb.BbvCI (nicking endonuclease) was optimized. Other parameters were fixed. Using the synthesized miRNA-21 and miRNA-210 as templates, the final concentrations of Nb.BbvCI were adjusted to 0.125 U / μL, 0.25 U / μL, 0.375 U / μL, and 0.5 U / μL. 2.5 μL of miRNA-21 and 2.5 μL of miRNA-210 were mixed with 20 μL of DMCas premix and placed in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for 60 min. The changes in the fluorescence curve were observed, as shown in Figure 3(D). 0.25 U / μL was selected for subsequent experiments.

[0093] The optimized DMCas premix system for cyclic exponential amplification combined with CRISPR / Cas12a to jointly recognize miRNA-210 and miRNA-21 is shown in Table 1:

[0094] Table 1 CRISPR / Cas12a combined with targeted cyclic exponential amplification detection system for miRNA-210 and miRNA-21

[0095]

[0096] Example 3 A logic gate detection method based on CRISPR / Cas12a combined with cyclic exponential amplification

[0097] The logic gate detection method includes the following processes:

[0098] S1, the nucleic acid miRNA-21 and miRNA-210 of the test samples are used Reagent (100 mL, ThermoFisher Scientific);

[0099] S2. Preparation of DMCas premix: Mix DNA polymerase, nicking endonuclease, dNTP, template, probe, LbCas12a, crRNA1, 10× reaction buffer 1, 10× reaction buffer 2, and sterile ultrapure water according to the concentrations and proportions in Table 1;

[0100] S3. Detect miRNA-21 and miRNA-210 using cyclic exponential amplification combined with CRISPR / Cas12a: Add the nucleic acid sample to be tested extracted in step S1 to the DMCas mixture prepared in step S2, react at 37°C for 60 minutes, detect the fluorescence signal and analyze the results.

[0101] Experimental Example 1 Sensitivity Analysis of Joint Recognition of miRNA-21 and miRNA-210 by Cyclic Exponential Amplification Combined with CRISPR / Cas12a

[0102] 1. Detection method: The specific process is as follows:

[0103] (1) Sample processing: Take a high concentration of 100 μM premixed template of miRNA-21 and miRNA-210, and perform a 10-fold serial dilution to obtain a mixed template of miRNA-21 and miRNA-210 with the concentrations of 1000 nM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, and 100 fM;

[0104] (2) The optimal system described in Table 1 of Example 2 was used to prepare the solution. 5 μL of the mixed template of step (1) was mixed with 20 μL of the DMCas premix, and the mixture was placed in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for reaction at 37°C for 60 min. The fluorescence was detected once every minute to observe the changes in the fluorescence curve.

[0105] 2. Test results: Test results are as follows: Figure 4 As shown, it can be seen that the detection limit of the combined recognition of miRNA-21 and miRNA-210 by cyclic exponential amplification combined with CRISPR / Cas12a in the present invention is 1 pM.

[0106] Experimental Example 2: Specificity Analysis of Combined Recognition of miRNA-21 and miRNA-210 by Cyclic Exponential Amplification Combined with CRISPR / Cas12a

[0107] 1. Detection method: (1) Sample processing: miRNA-210 was cross-mixed with miRNA-21 and miRNA-141, miRNA-155, and let-7a synthetic RNA and diluted to the same concentration (100 nM) as the target for specific analysis. The targets after cross-mixing were miRNA-210 + miRNA-21, miRNA-210 + miRNA141, miRNA-210 + miRNA-155, miRNA-210 + let-7a, miRNA-141 + miRNA-155, and miRNA-155 + let-7a;

[0108] miRNA-141: 5'-UAACACUGUCUGGUAAAGAUGG-3' (SEQ ID NO. 21);

[0109] miRNA-155: 5'-UUAAUGCUAAUCGUGAUAGGGGU-3' (SEQ ID NO. 22);

[0110] let-7a: 5'-UGAGGUAGUAGGUUGUAUAGUU-3' (SEQ ID NO. 23);

[0111] (2) The optimal system described in Table 1 of Example 2 was used to prepare the solution. 5 μL of the mixed template was mixed with 20 μL of the DMCas premix solution, and the mixture was placed in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for reaction at 37°C for 60 min. The fluorescence was detected once every minute to observe the changes in the fluorescence curve.

[0112] 2. Test results: Test results are as follows: Figure 5 As shown, only the miRNA-210+miRNA-21 mixed template showed obvious changes in fluorescence intensity, indicating that the proposed cyclic exponential amplification combined with CRISPR / Cas12a joint recognition detection method has excellent specificity.

[0113] Experimental Example 3: Detection of miRNA-210 and miRNA-21 in plasma by cyclic exponential amplification combined with CRISPR / Cas12a

[0114] 1. Detection method:

[0115] (1) Sample processing: Different concentrations of miRNA-210 and miRNA-21 mixed templates (1 nM, 10 nM, 100 nM) were added to plasma extracted from mice to simulate the free miRNA-210 and miRNA-21 in the plasma of cancer patients.

[0116] (2) Use first Plasma-free RNA was extracted using 100 mL of Reagent (Thermo Fisher Scientific). Solutions were then prepared using the optimized system described in Table 1 of Example 2. 5 μL of each plasma RNA extract was collected for analysis. The reaction tubes were placed in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for fluorescence detection. The reaction conditions were: heating at 37°C for 60 min, with fluorescence measured every minute. The ROX channel was selected. After completion of the reaction, the fluorescence curve was observed.

[0117] 2. Test results: The results are as follows Figure 6 As shown, it can be seen that plasma miRNA extracts with different concentrations were successfully detected, indicating that the proposed cyclic exponential amplification combined with CRISPR / Cas12a joint identification of miRNA-210 and miRNA-21 detection method has practical application value.

Claims

1. A logic gate detection method based on cyclic exponential amplification combined with CRISPR / Cas12a, wherein the logic gate detection method is used for non-diagnostic or therapeutic purposes, characterized in that: The following processes are included: S1. Extraction of test nucleic acids miRNA-21 and miRNA-210; S2. Preparation of DMCas premix: Mix DNA polymerase, nicking endonuclease, dNTP, template, probe, LbCas12a enzyme, crRNA1, 10× reaction buffer 1, 10× reaction buffer 2, and sterile ultrapure water; S3, adding the nucleic acids to be tested, miRNA-21 and miRNA-210, extracted in step S1, to the DMCas premix prepared in step S2, performing a constant temperature and timed reaction, detecting the fluorescence signal and analyzing the results, to obtain; The template sequence described in step S2 is shown in SEQ ID NO.4; the nucleotide sequence of the crRNA1 is shown in SEQ ID NO.1; the sequence of the probe is 5'-ROX-CCCCCC-BHQ2-3'; the 10× reaction buffer 1 includes sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, and dithiothreitol; the 10× reaction buffer 2 includes potassium acetate, triacetate, magnesium acetate, and recombinant albumin; and the nicking endonuclease is Nb.BbvCI.

2. The logic gate detection method according to claim 1, wherein: In step S2, the working concentration range of LbCas12a enzyme is 0.001-1 μM, the working concentration range of nicking endonuclease is 0.125~0.5U / μL, the working concentration range of crRNA1 is 0.001-1 μM, and the working concentration range of template is 10-500nM; the constant temperature timing reaction conditions in step S3 are 28-42 ° C reaction, and the reaction time is 60-90min.

3. The logic gate detection method according to claim 2, wherein: In step S2, the working concentration of LbCas12a enzyme is 100 nM, the nicking endonuclease is Nb.BbvCI, the working concentration is 0.25 U / μL, the working concentration of crRNA1 is 50 nM, and the working concentration of the template is 50 nM; the constant temperature timing reaction condition in step S3 is 37 ° C reaction, and the reaction time is 60 min.

4. A kit for detecting target nucleic acids in a sample, wherein the target nucleic acids are miRNA21 and miRNA210, characterized in that: It includes DMCas premix and sterilized ultrapure water; the DMCas premix includes DNA polymerase, nicking endonuclease, dNTP, template, probe, LbCas12a enzyme, crRNA1, 10×reaction buffer 1, and 10×reaction buffer 2; the nicking endonuclease is Nb.BbvCI; the template sequence is shown in SEQ ID NO.4; the nucleotide sequence of the crRNA1 is shown in SEQ ID NO.1; the sequence of the probe is 5'-ROX-CCCCCC-BHQ2-3'; the 10×reaction buffer 1 includes sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, and dithiothreitol; the 10×reaction buffer 2 includes potassium acetate, triacetate, magnesium acetate, and recombinant albumin.

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