Logical gate detection method and kit based on cycle index amplification combined with CRISPR / Cas12a (clustered regularly interspaced short palindromic repeats / CRISPR / Cas12a)

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a logic gate detection method and kit based on cycle index amplification combined with CRISPR / Cas12a. The main process of the detection method is as follows: miRNA-210 is combined with a template to activate cycle index amplification, and the obtained product P1 can be used as a catalyst of Cas12a / crRNA1. When the miRNA-21 exists, the miRNA-21 can be used as crRNA2, the crRNA1 and the crRNA2 cooperate to form a crRNA complete spacer region sequence, the Cas12a trans-cleavage activity is activated, and a detectable fluorescence signal is generated. According to the invention, miRNA-21 is used as a spacer sequence in a crRNA structure, the general applicability of miRNA as a functional element is shown, and an AND logic gate is established to realize simultaneous detection and result logic judgment of multiple miRNAs.

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 a kit based on cyclic exponential amplification combined with CRISPR / Cas12a. Background Art

[0002] As a class of endogenous, non-coding small RNA molecules with a length of about 20-24 nucleotides, miRNA has a variety of important regulatory functions in cells and can regulate the expression of one-third of human genes. This regulatory function is crucial to biological processes such as cell growth, differentiation, and metabolism. In terms of cancer occurrence, abnormal expression of miRNA is closely related to the occurrence, development, and prognosis of tumors, so miRNA is widely used as a biomarker for cancer diagnosis. Studies have shown that the expression level of miRNA shows significant changes in certain types of early cancers, such as colorectal cancer, liver cancer, cervical cancer, pancreatic cancer, etc. By detecting the content of these miRNAs in samples such as blood and tissues, doctors can assist in determining whether patients have tumor risks. When abnormal miRNA levels are found in patients, it often indicates the possibility of malignant tumors. miRNA-21 and miRNA-210 are miRNAs that were discovered to be associated with breast cancer earlier. Therefore, by detecting the levels of miRNA-21 and miRNA-210 in the blood, early screening and diagnosis of breast cancer can be achieved, which is of great significance for improving the detection rate of tumors and reducing mortality.

[0003] Although traditional miRNA detection methods occupy an important position in scientific research and clinical practice, their operation procedures are complicated and costly, which has 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, time-consuming, and requires a large number of high-quality RNA samples, which limits its ability to respond quickly and be widely used. In addition, qPCR is one of the most widely used miRNA quantification technologies, and 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. A variety of 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 combined with a variety of amplification strategies to jointly build an efficient and sensitive biological detection system. This technology fusion not only gives full play to the high specificity and accuracy of the CRISPR-Cas system, but also uses isothermal amplification 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 its detection sensitivity and efficiency. However, most of the current CRISPR-Cas systems combined with isothermal amplification-mediated detection methods mainly focus on a single target, which is quite limited when dealing with scenarios where multiple targets need to be screened simultaneously in complex biological samples, and it is 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 simultaneous detection of multiple miRNAs and logical judgment of the results, and has high sensitivity and high specificity, and has broad application prospects in disease screening.

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

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

[0009] S1. Extraction of nucleic acid miRNA-21 and miRNA-210 from the 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 mixed solution 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, in step S2, the working concentration range of the LbCas12a enzyme is 0.001-1 μM, the nicking endonuclease is Nb.BbvCI, the working concentration range 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.

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

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

[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, and 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 labeling group ROX and a 3'-end labeling group BHQ2. 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, the probe can be cut, and the ROX group and the BHQ2 group are far away, generating a detectable fluorescence signal;

[0025] Among them, the P1 sequence can simulate the fragment cut out after the miRNA-210 binds to the template (Template) and amplifies. 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 are complementary to the target miRNA-210, the nicking endonuclease Nb.BbvCI recognizes the nicking sequence (5'-CCTCAGC-3') in the middle, which can start the cyclic exponential amplification reaction and amplify the P1 fragment and the 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 obtained product P1 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 cooperate to form a complete crRNA spacer sequence, activate Cas12a trans-cutting activity, and produce a detectable fluorescent signal; When miRNA-21 is missing, a single crRNA1 cannot activate Cas12a trans-cutting activity, so no fluorescent signal is generated. 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] The present invention aims to efficiently and accurately achieve parallel detection and analysis of multiple miRNA targets. This method not only improves the detection sensitivity and specificity through a carefully designed logic gate architecture, 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 extended 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, ultimately generating a detectable fluorescent signal. 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 start 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 period of time, and then through the fluorescence detection step, the fluorescence results can be obtained within 1 hour.

[0032] (4) Good specificity: The specific binding ability between 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] FIG2 shows a result diagram of the design and screening of logic gate detection sequences for cyclic exponential amplification combined with CRISPR / Cas12a;

[0036] FIG3 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, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. The raw materials, equipment, etc. used in the following embodiments can be purchased through conventional channels unless otherwise specified.

[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 MgCl2, and 10 mM DTT (dithiothreitol).

[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 Design of logic gate detection sequence 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 composed of C bases that CRISPR / Cas12a preferentially cuts was designed as a reporter probe 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 front, middle and back positions of miRNA-21 as crRNA2, the following P1-head, mid and tail were designed.

[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 primer for amplification. The template sequence was designed so that the front and back ends of the template matched those of 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 complementary sequence of P1 and the complementary sequence of miRNA-210. 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 Figure 1 As shown, 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 logic relationship shown is (0,0,0)(1,0,0) or (0,1,0). Only when miRNA-21 and miRNA-210 exist at the same time can a fluorescent signal be generated, and the logic 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, that is, the best 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 and prevent the conformational change of the catalytic site. Therefore, the 5' end of miRNA-21 is selected as crRNA2 for the next study.

[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 the HPLC purity standard.

[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 of the system of the present invention.

[0090] (2) Optimize the amount of crRNA1, fix other parameters, use the 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, 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), and select 50nM for subsequent experiments.

[0091] (3) Optimization of Template dosage: Fix other parameters, use the synthesized miRNA-21 and miRNA-210 as templates, and adjust the final concentration of Template to 10nM, 20nM, 50nM, 80nM, and 100nM. Mix 2.5μL miRNA-21, 2.5μL miRNA-210 with 20μL DMCas premix, place in a fluorescence quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for 60min, observe the change of fluorescence curve, as shown in Figure 3(C), and select 50nM for subsequent experiments.

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

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

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

[0095]

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

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

[0098] S1. The nucleic acids of the samples to be tested, miRNA-21 and miRNA-210, 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 concentration and proportion 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 mixed solution 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 combined 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 dilute it 10-fold in sequence to obtain a mixed template of miRNA-21 and miRNA-210 with a concentration 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 DMCas premixed 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 per 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: Specific 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 premixed 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 per 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 superior 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 Reagent (100mL, Thermo Fisher Scientific) was used to extract free RNA in plasma, and then the optimal system described in Table 1 in Example 2 was used to prepare the solution. 5 μL of each concentration of plasma RNA extract was taken for detection, and the reaction tube was placed in a fluorescent quantitative PCR instrument (SLAN 96S / 96P, Shanghai Hongshi) for fluorescence detection. The reaction conditions were set as follows: heating at 37°C for 60 minutes and detecting fluorescence once per minute. ROX was selected as the channel. After the reaction was completed, 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 of 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, characterized in that: The process includes: S1. Extraction of the nucleic acids miRNA-21 and miRNA-210 to be tested; 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. Add the nucleic acids miRNA-21 and miRNA-210 to be tested extracted in step S1 to the DMCas premix prepared in step S2, perform a constant temperature and timed reaction, detect the fluorescence signal and analyze the results.

2. The logic gate detection method according to claim 1, characterized in that: 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'.

3. The logic gate detection method according to claim 1, characterized in that: The 10×reaction buffer 1 in step S2 includes sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, and dithiothreitol; and the 10×reaction buffer 2 includes potassium acetate, triacetate, magnesium acetate, and recombinant albumin.

4. The logic gate detection method according to claim 1, characterized in that: In step S2, the working concentration range of LbCas12a enzyme is 0.001-1 μM, the nicking endonuclease is Nb.BbvCI, the working concentration range 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.

5. The logic gate detection method according to claim 4, characterized in that: 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.

6. A kit for detecting a target nucleic acid in a sample, 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.

7. The kit according to claim 6, characterized in that The template sequence is shown in SEQ ID NO.4; the nucleotide sequence of the crRNA1 is shown in SEQ ID NO.1; and the sequence of the probe is 5'-ROX-CCCCCC-BHQ2-3'.

8. The kit according to claim 6, characterized in that The 10×reaction buffer 1 includes sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, and dithiothreitol; and the 10×reaction buffer 2 includes potassium acetate, triacetate, magnesium acetate, and recombinant albumin.

9. The kit according to claim 6, characterized in that The kit is used for detecting miRNA-21 and miRNA-210, and also includes a nucleotide sequence such as the P1 sequence shown in SEQ ID NO.3.

Images