Nucleic acid in-situ detection method based on double ligation probes and rolling circle amplification

Abstract

The invention relates to the technical field of nucleic acid in-situ detection, in particular to a nucleic acid in-situ detection method based on double ligation probes and rolling circle amplification. In a double-connection probe cyclization step of the in-situ detection method, under the action of ligase, target nucleic acid and a cyclization clamping plate are respectively used as connection templates of two DNA (deoxyribonucleic acid) gaps to perform cyclization reaction, and then the cyclization clamping plate is used as a primer to perform subsequent rolling circle amplification. Compared with the prior art, the method provided by the invention has higher detection efficiency and signal intensity, and the sensitivity of the method is improved; meanwhile, the original two different enzyme reactions are combined into one step, so that the operation is simpler and more convenient; in addition, according to the method, the time required by the detection process and the use of part of enzyme are reduced, the detection time is saved, and the reagent cost is reduced.

Description

Technical Field

[0001] The present application relates to the technical field of in situ detection of nucleic acids, and in particular to an in situ detection method of nucleic acids based on double-ligated probes and rolling circle amplification. Background Art

[0002] In situ detection of nucleic acids can visualize nucleic acid molecules in tissues or cells in situ, thereby achieving the purpose of positioning and quantification. As the earliest technology for in situ detection of nucleic acids, traditional in situ hybridization technology has reaction products of small molecules or precipitates, which are very easy to diffuse in the environment or even detach from the detection probe bound to the target molecule, and cannot accurately locate the specific position of the nucleic acid molecule to be detected. Although the in situ detection method of directly labeling the probe with a fluorescent group can achieve a certain spatial accuracy, the sensitivity is limited. The single-molecule fluorescence in situ hybridization technology, which was later regarded as the gold standard, uses multiple-band single-fluorescent-labeled probes or single-band multiple-fluorescent-labeled probes. Although it can achieve a certain resolution and sensitivity, it is necessary to design fluorescent-labeled detection probes for different target molecules. The synthesis cost is high, and the signal amplification factor is limited by the accumulation of a limited number of fluorescent group signal values. It is also susceptible to interference from background fluorescence. Therefore, efficient and specific targeted probes and signal amplification pathways with higher amplification factors can help achieve higher-performance in situ detection of nucleic acids.

[0003] The related technology discloses an RNA in situ detection scheme based on double-ligated probes and rolling circle amplification, and its specific technical route is: first, the double-ligated probes hybridize with the target nucleic acid, and after the first ligation with the target nucleic acid as the ligation template and the second ligation with the ring-forming splint as the ligation template, the double-ligated probes form a closed DNA circle, and then subsequent rolling circle amplification is carried out.

[0004] Although this method has advantages in specificity and signal strength, it still has the problem of low sensitivity, which leads to low detection efficiency. At the same time, this method is relatively complicated to operate, the detection process is time-consuming, and it also requires the rental of multiple enzymes, and the reagent cost is high. Summary of the invention

[0005] To this end, the present application provides a method for in situ detection of nucleic acids based on dual-ligation probes and rolling circle amplification. Compared with the prior art, the method provided by the present application has higher detection efficiency and signal strength, and improves the sensitivity of the method; at the same time, the two different enzyme reactions that were originally required are combined into one step, which is easier to operate; in addition, the method also reduces the time required for the detection process and the use of some enzymes, saving detection time and reducing reagent costs.

[0006] The principle of the nucleic acid in situ detection method in the related art is: the double-ligated probe hybridizes with the target nucleic acid, and after the first ligation with the target nucleic acid as the ligation template and the second ligation with the ring-forming splint as the ligation template, the double-ligated probe forms a closed DNA ring, and then subsequent rolling circle amplification is carried out.

[0007] The realization of the signal method requires two conditions, namely primers (circular splints) and circular DNA.

[0008] The principle of the nucleic acid in situ detection method provided in this application is as follows:

[0009] (a) If Figure 1 As shown, (step 1) the double ligation probe hybridizes with the target nucleic acid (depending on the principle of base complementary pairing), and (step 2) under the action of a specific ligase, the target nucleic acid and the ring-forming splint are used as the connection templates of the two DNA gaps to carry out a ring-forming reaction, and then the ring-forming splint is used as a primer for subsequent rolling circle amplification to achieve the process of signal amplification.

[0010] (ii) Figure 2 As shown, (step 1) the double ligation probe hybridizes with the target nucleic acid (depending on the principle of base complementary pairing), (step 2) the circular splint then hybridizes with the double ligation probe (depending on the principle of base complementary pairing), and (step 3) the two DNA gaps are then connected under the action of ligase to form a closed circle for rolling circle amplification.

[0011] (III) Figure 3 As shown, (step 1) the double-ligation probe and the circular splint are hybridized synchronously (depending on the principle of base complementary pairing), and then (step 2) the two DNA gaps are connected under the action of ligase to form a closed circle for rolling circle amplification.

[0012] The present application provides a method for in situ detection of nucleic acid based on double-ligated probes and rolling circle amplification, which adopts the following technical scheme:

[0013] A method for in situ detection of nucleic acids based on double-ligated probes and rolling circle amplification. In the double-ligated probe circularization step of the in situ detection method, under the action of a ligase, a circularization reaction is carried out using a target nucleic acid and a circularization splint as connection templates for two DNA gaps, respectively, and then subsequent rolling circle amplification is carried out using the circularization splint as a primer.

[0014] Optionally, the ligase is selected from T4 DNA ligase, DNA ligase I, DNA ligase III and DNA ligase IV.

[0015] Optionally, the ligase is T4 DNA ligase.

[0016] Alternatively, a dual ligation probe is hybridized to the target nucleic acid and subsequently the looping splint is hybridized to the dual ligation probe.

[0017] Optionally, dual ligation probes and looping splints are hybridized simultaneously.

[0018] Optionally, the target nucleic acid is DNA and / or RNA.

[0019] Optionally, the test sample is selected from a single cell, a cell population, a tissue, an organ or any combination thereof.

[0020] Optionally, the cell is selected from a eukaryotic cell, a prokaryotic cell, an archaeal cell, an artificial cell or any combination thereof.

[0021] In summary, the present application includes at least one of the following beneficial technical effects:

[0022] Compared with the prior art, the method provided in the present application has higher detection efficiency and signal strength, and improves the sensitivity of the method; at the same time, it combines the two different enzyme reactions that originally required into one step, which is easier to operate; in addition, the method also reduces the time required for the detection process and the use of some enzymes, saving detection time and reducing reagent costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Principle of in situ detection using dual ligation probes for this application Figure 1 .

[0024] Figure 2 Principle of in situ detection using dual ligation probes for this application Figure 2 .

[0025] Figure 3 Principle of in situ detection using dual ligation probes for this application Figure 3 .

[0026] Figure 4 The figures are the results of microscopic examination of fresh frozen sections of mouse heart under three ringing conditions (the left side is Example 1; the middle is Example 2; the right side is Comparative Example 1; wherein the white solid circle is the cell nucleus, and the surrounding white bright spots are the signal detection amounts of the test gene).

[0027] Figure 5 The counting results of the signal detection amount of fresh frozen sections of mouse heart under three ring-forming conditions (1 is Example 1; 2 is Example 2; 3 is Comparative Example 1).

[0028] Figure 6 The counting results of the signal detection amount of A549 paraffin sections under three ring-forming conditions (1 is Example 4; 2 is Example 5; 3 is Comparative Example 2).

[0029] Figure 7The results are the counting results of the signal detection amount of SKBR3 cell paraffin sections under three ringing conditions (1 is Example 5; 2 is Example 6; 3 is Comparative Example 3). DETAILED DESCRIPTION

[0030] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0031] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0032] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0033] In the present application, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application, but not excluding other contents.

[0034] The present application provides a method for in situ detection of nucleic acids based on double-ligated probes and rolling circle amplification. The method for in situ detection of nucleic acids is to optimize and improve the double-ligated probe circularization step. In the double-ligated probe circularization step of the in situ detection method, under the action of a ligase, a circularization reaction is performed with the target nucleic acid and the circularization splint as the connection templates of the two DNA gaps, and then the subsequent rolling circle amplification is performed with the circularization splint as a primer.

[0035] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0037] The present application is further described in detail below in conjunction with the embodiments, drawings and test results.

[0038] Four genes, Ubc, Ppib, Acta2 and the negative control gene dapB, were detected on fresh frozen mouse heart tissue using different in situ detection methods to compare the performance differences between different schemes.

[0039] Example 1

[0040] This embodiment provides a method for in situ detection of nucleic acid based on double-ligated probes and rolling circle amplification.

[0041] The method specifically comprises the following steps:

[0042] 1. Tissue pretreatment of frozen samples

[0043] (1) Fixation: After the frozen sections were taken out from -80°C, 4% PFA (Biyuntian) was added at room temperature and fixed for 5 min. The tissues were then washed three times with PBST.

[0044] (2) Dehydration and drying: The sections were sequentially immersed in 70%, 85% and 100% ethanol for gradient dehydration, each time for 1 min.

[0045] (3) Drawing a hydrophobic circle: After the slices are dried, use the ImmEdgeTM Pen to draw a hydrophobic circle with a diameter of 1 cm around the tissue. After the hydrophobic circle is dry, wash the tissue inside the hydrophobic circle three times with PBST.

[0046] (4) Permeabilization: Add 0.1 M hydrochloric acid to the tissue and permeabilize at 37°C for 5 min. Then remove the slices and wash the tissue three times with PBST.

[0047] (II) In-situ testing process

[0048] (1) Dual-ligation probe hybridization

[0049] Add 50 μL of hybridization mixture containing 0.1 μM dual-ligation probe (Shanghai Bioengineering, the same probe below), 6×SSC (Sigma), and 10% formamide (Sigma) to the tissue, and hybridize at 46°C for 4 h to allow the probes designed for different target genes to fully bind to their targets, then wash the tissue three times with PBST; then use a probe washing solution containing 2×SSC and 20% formamide to wash the tissue three times at 46°C for 5 min each time, and then wash the tissue three times with PBST.

[0050] The probe sequences used are shown in Table 1, specifically the nucleotide sequences shown in SEQ ID NO 1-41.

[0051] Table 1 Probe sequences used

[0052] SEQ ID NO: Name Sequence Information (5' to 3') 1 Mmu-DLP-Ubc-L1 TTTGCCTTGACATTCTTCTCGTTTTCTGAACTGCATCGGTATCAAG 2 Mmu-DLP-Ubc-R1 CTGTTTAAGAAGCAGCGAACCATACGTGACCTCCTTGTCCTGGATC 3 Mmu-DLP-Ubc-L2 AGTCTGACAGGGTGCGTCTCGTTTTCTGAACTGCATCGGTATCAAG 4 Mmu-DLP-Ubc-R2 CTGTTTAAGAAGCAGCGAACCATACGTGACTCTTTCTGGATGTTGT 5 Mmu-DLP-Ubc-L3 TCTGAGGCGAAGGACTTCTCGTTTTCTGAACTGCATCGGTATCAAG 6 Mmu-DLP-Ubc-R3 CTGTTTAAGAAGCAGCGAACCATACGTGACAGATCTGCATCCCACC 7 Mmu-DLP-Ubc-L4 ATGGTGTCACTGGGCTTCTCGTTTTCTGAACTGCATCGGTATCAAG 8 Mmu-DLP-Ubc-R4 CTGTTTAAGAAGCAGCGAACCATACGTGACTGCCTTGACATTCTCT 9 Mmu-DLP-Ubc-L5 TTCCTGTTTGACCTTCTCTCGTTTTCTGAACTGCATCGGTATCAAG 10 Mmu-DLP-Ubc-R5 CTGTTTAAGAAGCAGCGAACCATACGTGACGGAAGGTACGTCTGTC 11 Mmu-DLP-Ppib-L1 ATTGCGCTCCGAGAGGTCTCGTTTTCTGAACTGCTACGGTATCAAG 12 Mmu-DLP-Ppib-R1 CTGTTTAAGAAGTAGCCGTGACTATCGACTCGAAGAGCACCTTCAT 13 Mmu-DLP-Ppib-L2 TTCTTCTTATCGTTGGTCTCGTTTTCTGAACTGCTACGGTATCAAG 14 Mmu-DLP-Ppib-R2 CTGTTTAAGAAGTAGCCGTGACTATCGACTTGTGACTTTAGGTCCC 15 Mmu-DLP-Ppib-L3 TCCTGTAGCTAAGGCTTCTCGTTTTCTGAACTGCTACGGTATCAAG 16 Mmu-DLP-Ppib-R3 CTGTTTAAGAAGTAGCCGTGACTATCGACTAGCCAAATCCTTTCTC 17 Mmu-DLP-Ppib-L4 ACTCCTTGGCAATGGCTCTCGTTTTCTGAACTGCTACGGTATCAAG 18 Mmu-DLP-Ppib-R4 CTGTTTAAGAAGTAGCCGTGACTATCGACTGTCCCCCAGGCTCTCT 19 Mmu-DLP-Ppib-L5 AGGGCCAGGGTGGCACTCTCGTTTTCTGAACTGCTACGGTATCAAG 20 Mmu-DLP-Ppib-R5 CTGTTTAAGAAGTAGCCGTGACTATCGACTATCAGATGGAAGCACT 21 Mmu-DLP-Acta2-L1 ACTCCCTGATGTCTGGTCTCGTTTTCTGAACTATCGCGGTATCAAG 22 Mmu-DLP-Acta2-R1 CTGTTTAAGAGGGCCTTATTCCGGTGCTATGCCCATTCCAACCATT 23 Mmu-DLP-Acta2-L2 TCGGCCAGCCAAGTCCTCTCGTTTTCTGAACTATCGCGGTATCAAG 24 Mmu-DLP-Acta2-R2 CTGTTTAAGAGGGCCTTATTCCGGTGCTATGGTAGTCGGTGAGATC 25 Mmu-DLP-Acta2-L3 TTTGGGCAGGAATGATTCTCGTTTTCTGAACTATCGCGGTATCAAG 26 Mmu-DLP-Acta2-R3 CTGTTTAAGAGGGCCTTATTCCGGTGCTATAGTAACAAATCAAAGC 27 Mmu-DLP-Acta2-L4 TGCATATCCATGCACGTCTCGTTTTCTGAACTATCGCGGTATCAAG 28 Mmu-DLP-Acta2-R4 CTGTTTAAGAGGGCCTTATTCCGGTGCTATAGTTAAAAGTCACGCA 29 Mmu-DLP-Acta2-L5 TGATAGGCAAAGGAACTCTCGTTTTCTGAACTATCGCGGTATCAAG 30 Mmu-DLP-Acta2-R5 CTGTTTAAGAGGGCCTTATTCCGGTGCTATTCTGTAGTTCCCATTC 31 DLP-dapB-L1 TTCAGATACGCCATGATCTCGTTTTCTGAACTATCGCGGTATCAAG 32 DLP-dapB-R1 CTGTTTAAGAGGGCCTTATTCCGGTGCTATATGGATGGCCAAACGC 33 DLP-dapB-L2 TTGACGCACCAAGCGGTCTCGTTTTCTGAACTATCGCGGTATCAAG 34 DLP-dapB-R2 CTGTTTAAGAGGGCCTTATTCCGGTGCTATCATCATGACCAGAAGC 35 DLP-dapB-L3 AACAACCGGTTTCTCATCTCGTTTTCTGAACTATCGCGGTATCAAG 36 DLP-dapB-R3 CTGTTTAAGAGGGCCTTATTCCGGTGCTATTCCGTCCAGTTGTCGG 37 DLP-dapB-L4 TTGGCAGAACGAACACTCTCGTTTTCTGAACTATCGCGGTATCAAG 38 DLP-dapB-R4 CTGTTTAAGAGGGCCTTATTCCGGTGCTATGCAGGAAGCTGTTAAA 39 DLP-dapB-L5 TCCGTCTTCCGGGACTTCTCGTTTTCTGAACTATCGCGGTATCAAG 40 DLP-dapB-R5 CTGTTTAAGAGGGCCTTATTCCGGTGCTATATTCGCTTGCACAGCG 41 Splint TCTTAAACAGCTTGATACCG

[0053] (2) Double-ligation probe ring formation

[0054] 1) Dual ligation probe circularization-circularization splint hybridization

[0055] 50 μL of hybridization mixture containing 0.5 μM Splint, 6×SSC (Sigma) and 10% formamide (Sigma) was added to the tissue, hybridization was carried out at 37° C. for 30 min, and the tissue was then washed three times with PBST.

[0056] 2) Double ligation probe ring-ligation ring

[0057] 50 μL of a ligation reaction mixture containing 25% glycerol (Sigma), 0.2 μg / μL BSA, 1× SplintR buffer (NEB), 0.25 U / μL SplintR ligase (NEB), and 1 U / μL RiboLock RNase inhibitor (Thermo) was added to the tissue and incubated at 37°C for 30 min, followed by washing the tissue three times with PBST.

[0058] (3) Rolling circle amplification

[0059] Add 50 μL of rolling circle amplification reaction solution containing 1 mM dNTP (Thermo), 5% glycerol, 0.2 μg / μL BSA, 1×phi29 buffer, 1 U / μL phi29 DNA polymerase and 1 U / μL RiboLock RNase inhibitor to the tissue, incubate at 30°C for 12-16 hours, and then wash the tissue three times with PBST.

[0060] (4) Fluorescent probe detection

[0061] Add 50 μL of the detection reaction solution containing 2×SSC, 20% formamide, and 0.1 μM of each fluorescent detection probe to the tissue and incubate at room temperature for 30 min; then wash the tissue three times with PBST. After the tissue is naturally air-dried, add Slowfade anti-fluorescence quencher (Thermo) containing 0.5 μg / mL DAPI and incubate at room temperature for about 5 min before fluorescence microscopy imaging.

[0062] The fluorescence detection probe sequences are shown in Table 2. Specifically, they are nucleotide sequences shown in SEQ ID NOs: 42-45.

[0063] Table 2 Fluorescence detection probe sequences

[0064] SEQ ID NO: Name Sequence Information (5' to 3') 5' Modification 42 DP-1-Cy3 AGTAGCCGTGACTATCGACT Cy3 43 DP-2-TXR GGGCCTTATTCCGGTGCTAT TXR 44 DP-3-Cy5 AGCAGCGAACCATACGTGAC Cy5 45 DP-4-Cy7 TGCGTCTATTTAGTGGAGCC Cy7

[0065] Example 2

[0066] This embodiment provides a method for in situ detection of nucleic acid based on dual ligation probes and rolling circle amplification. The difference from embodiment 1 is the step of "double ligation probe circularization" in the in situ detection process, and the rest of the steps are consistent with embodiment 1.

[0067] In this example, "double ligation probe circularization" is as follows:

[0068] 50 μL of a ligation reaction mixture containing 0.5 μM Splint, 25% glycerol (Sigma), 0.2 μg / μL BSA, 1× SplintR buffer (NEB), 0.25 U / μL SplintR ligase (NEB) and 1 U / μL RiboLock RNase inhibitor (Thermo) was added to the tissue and incubated at 37°C for 30 min, followed by washing the tissue three times with PBST.

[0069] Comparative Example

[0070] Comparative Example 1

[0071] This comparative example provides a method for in situ detection of nucleic acid based on dual-ligation probes and rolling circle amplification. The difference from Example 1 is the step of "double-ligation probe circularization" in the in situ detection process, and the rest of the steps are consistent with Example 1.

[0072] In this comparative example, the "double ligation probe looping" is as follows:

[0073] (1) Double ligation probe circularization-ligation

[0074] 50 μL of a ligation reaction mixture containing 25% glycerol (Sigma), 0.2 μg / μL BSA, 1× SplintR buffer (NEB), 0.25 U / μL SplintR ligase (NEB), and 1 U / μL RiboLock RNase inhibitor (Thermo) was added to the tissue and incubated at 37°C for 30 min, followed by washing the tissue three times with PBST.

[0075] (2) Double-ligation probe ring-ring

[0076] Add 50 μL of a circularization mixture containing 0.5 μM of the circularization splint sequence Splint, 2 mM ATP, 0.2 μg / μL BSA, 1×T4 DNA Ligase buffer, 1 U / μL T4 DNA Ligase, and 1 U / μL RiboLock RNase inhibitor to the tissue, incubate at 37°C for 30 min, and then wash the tissue three times with PBST.

[0077] Test result 1

[0078] The test results of the above examples 1-2 and comparative example 1 are as follows Figure 4-5 As shown. Among them, Figure 4 These are the results of microscopic examination of fresh frozen sections of mouse heart under three ringing conditions, with the left side showing the test results of Example 1, the middle showing the test results of Example 2, and the right side showing the test results of Comparative Example 1, wherein the white solid circle is the cell nucleus, and the surrounding white bright spots are the signal detection amounts of the test gene. Figure 5 The results are shown in the following table for counting the signal detected in fresh frozen sections of mouse heart under three ringing conditions.

[0079] In the method of Example 1, "double ligation probe circularization" only requires one ligase, SplintR; in the method of Example 2, "double ligation probe circularization" is combined into one step circularization, which only requires one ligase, SplintR, and is easier to operate; in the method of Comparative Example 1, "double ligation probe circularization" requires two ligases, SplintR and T4 DNA ligase.

[0080] Depend on Figure 4 It can be seen that the control gene dapB was not detected under the three ring-forming conditions, but the signal detection amounts of the other three positive genes were higher in the solution of the embodiment of the present application. Figure 5 It can be seen that the positive gene signal detection efficiency is increased by 26.4% compared with Comparative Example 1.

[0081] On A549 paraffin sections, three genes, MET, EGFR, and PPIB, were detected using different in situ detection methods to compare the performance differences between different schemes.

[0082] Example 3

[0083] This embodiment provides a method for in situ detection of nucleic acid based on dual-ligated probes and rolling circle amplification. The difference between this method and embodiment 1 is that the samples are different, and the probe sequences used in (1) "dual-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are different. The remaining steps are consistent with embodiment 1.

[0084] The probe sequences of (1) "double-ligated probe hybridization" in the in situ detection process are shown in Table 3, specifically the nucleotide sequences shown in SEQ ID NOs: 46-75. The probe sequences of (4) "fluorescent probe detection" in the in situ detection process are specifically the nucleotide sequences shown in SEQ ID NOs: 42-44.

[0085] Table 3 Probe sequences used

[0086]

[0087]

[0088] Example 4

[0089] This embodiment provides a method for in situ detection of nucleic acid based on dual-ligated probes and rolling circle amplification. The difference between this method and embodiment 2 is that the samples are different, and the probe sequences used in (1) "dual-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are different. The remaining steps are consistent with embodiment 3.

[0090] Among them, the probe sequence of (1) "double-ligation probe hybridization" and (4) "fluorescence probe detection" in the in situ detection process are consistent with those in Example 3.

[0091] Comparative Example 2

[0092] This comparative example provides a method for in situ detection of nucleic acid based on dual-ligated probes and rolling circle amplification. The difference between this method and comparative example 1 is that the samples are different, and the probe sequences used in (1) "dual-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are different. The remaining steps are consistent with comparative example 1.

[0093] Among them, the probe sequence of (1) "double-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are consistent with those in Comparative Example 1.

[0094] Test result 2

[0095] The test results of the above-mentioned Examples 3-4 and Comparative Example 2 are as follows Figure 6 As shown. Among them, Figure 6 The results are shown in Figure 2. The signal detection results of A549 paraffin sections under three ring formation conditions.

[0096] Depend on Figure 6 It can be seen that all genes were detected under the method of the present application, especially the method of Example 4 had the highest signal detection, and the overall signal detection amount of the example of the present application was increased by 50.7% compared with that of Comparative Example 2.

[0097] On paraffin sections of SKBR3 cells, HER2, IFI6, and PPIB genes were detected using different in situ detection methods to compare the performance differences between different schemes.

[0098] Example 5

[0099] This embodiment provides a method for in situ detection of nucleic acid based on dual-ligated probes and rolling circle amplification. The difference between this method and embodiment 1 is that the samples are different, and the probe sequences used in (1) "dual-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are different. The remaining steps are consistent with embodiment 1.

[0100] Among them, the probe sequences of (1) "double-ligated probe hybridization" in the in situ detection process are shown in Table 4, specifically the nucleotide sequences shown in SEQ ID NOs: 66-104, specifically see Table 3 (SEQ ID NOs: 66-75) and Table 4 (SEQ ID NOs: 76-104). The probe sequences of (4) "fluorescent probe detection" in the in situ detection process are specifically the nucleotide sequences shown in SEQ ID NOs: 42-44.

[0101] Table 4 Probe sequences used

[0102] SEQ ID NO: Name Sequence Information (5' to 3') 76 Hsa-DLP-HER2-L1 TGACAATCCTCAGAACTCTCGTTTTCTGAACTTAGCCGGTATCAAG 77 Hsa-DLP-HER2-R1 CTGTTTAAGAGAGAGGCCGTCGCTATACATAGTGCGCGTCAGGCTC 78 Hsa-DLP-HER2-L2 AGATCTCTGTGAGGCTTCTCGTTTTCTGAACTTAGCCGGTATCAAG 79 Hsa-DLP-HER2-R2 CTGTTTAAGAGAGAGGCCGTCGCTATACATAAGACCCCTCCTTTCA 80 Hsa-DLP-HER2-L3 TCCCACGTCCGTAGAATCTCGTTTTCTGAACTTAGCCGGTATCAAG 81 Hsa-DLP-HER2-R3 CTGTTTAAGAGAGAGGCCGTCGCTATACATAGACGAGGGTGCAGGA 82 Hsa-DLP-HER2-L4 ATTACTTGCAGGTTCTTCTCGTTTTCTGAACTTAGCCGGTATCAAG 83 Hsa-DLP-HER2-R4 CTGTTTAAGAGAGAGGCCGTCGCTATACATCAGAATTCGTCCCCGG 84 Hsa-DLP-HER2-L5 TGACACACTGGGTGGGTCTCGTTTTCTGAACTTAGCCGGTATCAAG 85 Hsa-DLP-IFI6-L1 TGCAAGTGAAGAGCAGTCTCGTTTTCTGAACTATCGCGGTATCAAG 86 Hsa-DLP-IFI6-R1 CTGTTTAAGAGGGCCTTATTCCGGTGCTATCCTGCCTCCACCCCAC 87 Hsa-DLP-IFI6-L2 ATGACGACGCTGCTGCTCTCGTTTTCTGAACTATCGCGGTATCAAG 88 Hsa-DLP-IFI6-R2 CTGTTTAAGAGGGCCTTATTCCGGTGCTATGGCACCAATATTACCT 89 Hsa-DLP-IFI6-L3 TTGTGGGTGGCGTAGCTCTCGTTTTCTGAACTATCGCGGTATCAAG 90 Hsa-DLP-IFI6-R3 CTGTTTAAGAGGGCCTTATTCCGGTGCTATCTCACTATCGAGATAC 91 Hsa-DLP-IFI6-L4 TTGGAGGAGAGAAGAGTCTCGTTTTCTGAACTATCGCGGTATCAAG 92 Hsa-DLP-IFI6-R4 CTGTTTAAGAGGGCCTTATTCCGGTGCTATGCTCCGTCACTAGACC 93 Hsa-DLP-IFI6-L5 TTCCAGAACCCGGAGCTCTCGTTTTCTGAACTATCGCGGTATCAAG 94 Hsa-DLP-IFI6-R5 CTGTTTAAGAGGGCCTTATTCCGGTGCTATCATGAAGGTCAGGGCC 95 Hsa-DLP-IFI6-L6 TCATCAGCGAGGCAGCTCTCGTTTTCTGAACTATCGCGGTATCAAG 96 Hsa-DLP-IFI6-R6 CTGTTTAAGAGGGCCTTATTCCGGTGCTATAGGATCGCAGACCAGC 97 Hsa-DLP-IFI6-L7 TGATAGATGGGCACAGCAGCTCTCGTTTTCTGAACTATCGCGGTATCAAG 98 Hsa-DLP-IFI6-R7 CTGTTTAAGAGGGCCTTATTCCGGTGCTATATCTTCAGCCCGGAGCCTGC 99 Hsa-DLP-IFI6-L8 ACCGCCTTCTGCCGCATGGTTCTCGTTTTCTGAACTATCGCGGTATCAAG 100 Hsa-DLP-IFI6-R8 CTGTTTAAGAGGGCCTTATTCCGGTGCTATGGTAGCACAAGAAAAGCGAT 101 Hsa-DLP-IFI6-L9 TGGAAGAGTTAGGCCAAGAATCTCGTTTTCTGAACTATCGCGGTATCAAG 102 Hsa-DLP-IFI6-R9 CTGTTTAAGAGGGCCTTATTCCGGTGCTATGCAAAGTTCTAGATCCTAAC 103 Hsa-DLP-IFI6-L10 TGGACAATATAGTGAGAACCTCTCGTTTTCTGAACTATCGCGGTATCAAG 104 Hsa-DLP-IFI6-R10 CTGTTTAAGAGGGCCTTATTCCGGTGCTATATAGCCACTGCACTCTAGCC

[0103] Example 6

[0104] This embodiment provides a method for in situ detection of nucleic acid based on dual-ligated probes and rolling circle amplification. The difference between this method and embodiment 2 is that the samples are different, and the probe sequences used in (1) "dual-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are different. The remaining steps are consistent with embodiment 3.

[0105] Among them, the probe sequence of (1) "double-ligation probe hybridization" and (4) "fluorescence probe detection" in the in situ detection process are consistent with Example 5.

[0106] Comparative Example 3

[0107] This comparative example provides a method for in situ detection of nucleic acid based on dual-ligated probes and rolling circle amplification. The difference between this method and comparative example 1 is that the samples are different, and the probe sequences used in (1) "dual-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are different. The remaining steps are consistent with comparative example 1.

[0108] Among them, the probe sequence of (1) "double-ligated probe hybridization" and (4) "fluorescent probe detection" in the in situ detection process are consistent with those in Comparative Example 1.

[0109] Test result three

[0110] The test results of the above examples 5-6 and comparative example 3 are as follows Figure 7 As shown. Among them, Figure 7 The results are shown in Figure 2. The signal detection results of SKBR3 cell paraffin sections under three ring formation conditions.

[0111] Depend on Figure 7 It can be seen that each gene has a high signal detection amount under the method of the present application, especially the method of Example 6 has the highest signal detection amount, and the overall signal detection amount of the example of the present application is increased by 7.9% compared with that of Comparative Example 3.

[0112] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for in situ detection of nucleic acid based on double-ligated probes and rolling circle amplification, characterized in that: In the double-ligated probe circularization step of the in situ detection method, under the action of ligase, the target nucleic acid and the circularization splint are used as the connection templates of the two DNA gaps to carry out circularization reaction, and then the circularization splint is used as a primer for subsequent rolling circle amplification.

2. The method for in situ detection of nucleic acid according to claim 1, characterized in that: The ligase is selected from the group consisting of T4 DNA ligase, DNA ligase I, DNA ligase III and DNA ligase IV.

3. The method for in situ detection of nucleic acid according to claim 1, characterized in that: The ligase is T4 DNA ligase.

4. The method for in situ detection of nucleic acid according to claim 1, characterized in that: The dual ligation probe is hybridized to the target nucleic acid, and then the looping splint is hybridized to the dual ligation probe.

5. The method for in situ detection of nucleic acid according to claim 1, characterized in that: Simultaneous hybridization of dual ligation probes and looping splints.

6. The method for in situ detection of nucleic acid according to claim 1, characterized in that: The target nucleic acid is DNA and / or RNA.

7. The method for in situ detection of nucleic acid according to claim 1, characterized in that: The test sample is selected from a single cell, a cell group, a tissue, an organ or any combination thereof.

8. The method for in situ detection of nucleic acid according to claim 7, characterized in that: The cell is selected from the group consisting of a eukaryotic cell, a prokaryotic cell, an archaeal cell, an artificial cell, or any combination thereof.

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