Probe primer group for reducing cell nucleus background in in-situ sequencing method based on sequencing while connecting and application of probe primer group

Abstract

The invention relates to the technical field of space transcriptomics, in particular to a probe primer group for reducing a cell nucleus background in an in-situ sequencing method based on sequencing while connection and application of the probe primer group. The probe primer group comprises at least one ring forming probe for forming a rolling circle amplification product and a detection probe group capable of hybridizing with the rolling circle amplification product, the ring forming probe and the target sequence are subjected to specific hybridization complementation; 5'to 3 'are taken as forward directions, and the in-situ sequencing method sequentially comprises upstream forward sequencing, upstream reverse sequencing, downstream reverse sequencing and downstream forward sequencing; the detection probe group comprises an upstream anchor primer, a downstream anchor primer, a first fluorescent sequencing probe group and a second fluorescent sequencing probe group; wherein the upstream and downstream anchor primers and the fluorescent sequencing probe group have the same direction, and double-end homodromous sequencing is realized. By utilizing the probe primer group provided by the invention, the signal-to-noise ratio can be effectively improved, and meanwhile, the signal detection quantity and detectable target flux of the method are improved.

Description

Technical Field

[0001] The present application relates to the technical field of spatial transcriptomics, and in particular to a probe primer set and application thereof for reducing the cell nucleus background in an in situ sequencing method based on sequencing by ligation. Background Art

[0002] Organs are composed of different cell types and are highly structured in order to perform their corresponding biological functions correctly and efficiently. Traditional bulk RNA-seq (RNA sequencing technology) and scRNA-seq (single-cell RNA sequencing) destroy the structure of tissues, thus losing the original spatial information of cells or genes. In order to more fully understand the gene functions of specific cell types and the communication between spatially adjacent cells or genes, we need to understand the abundance of gene expression and the specific time and spatial location information of its expression. This urgent need has led to the rapid development of spatial transcriptomics technology in recent years.

[0003] Spatial transcriptomics technology can locate and distinguish the active expression of functional genes in specific tissue regions, thus providing important information for basic research and clinical diagnosis. As a breakthrough new technology in omics research, it can detect gene activity in different microenvironments in tissue samples and draw a spatial map of active gene expression.

[0004] At present, according to the spatial positioning and abundance determination methods of target genes, spatial transcriptomics technology is mainly divided into two categories: microscopic imaging-based methods and sequencing-based methods. The microscopic imaging-based method is to perform in situ sequencing or multiple rounds of single-molecule fluorescence in situ hybridization and imaging based on the original position of mRNA molecules in cells or tissues, thereby obtaining a string of signals encoded by different fluorescent colors, and using the fluorescent signal and its intensity to decode the target gene and its abundance, which can directly obtain the spatial location information of the target gene. The sequencing-based method is a method based on primer-encoded spatial information. By adding position information tags to cDNA, its abundance is determined by sequencing, and the spatial position of the target gene in the tissue is restored after sequencing, thereby obtaining the spatial location information of the target gene.

[0005] The related art discloses an in situ sequencing method, which is a method based on microscopic imaging. The in situ sequencing method is based on the amplification pathway of double-linked probes and rolling circle amplification, and the rolling circle amplification product is sequenced in 4 channels and 4 rounds, which can theoretically reach 256 (4 4 ) kinds of RNA detection throughput.

[0006] Although this method can achieve high resolution and specificity, it has the problem of serious background in the cell nuclei of upstream sequencing tissues, which interferes with the reading of fluorescence signals and can seriously lead to extremely low upstream sequencing signal-to-noise ratio, resulting in a low number of gene detections obtained after decoding after multiple rounds of sequencing imaging.

[0007] In addition, under the premise of keeping the tissue in its original position, the tissue background will increase with the increase in sequencing rounds, limiting the number of rounds that can be detected by this method; and increasing the number of detection rounds is currently the most important idea for improving detection throughput. Therefore, this method currently still has the problem of relatively limited detection throughput. Summary of the invention

[0008] The present application provides a probe primer set for reducing the background of the nucleus in an in situ sequencing method based on sequencing by ligation and its application. In the in situ sequencing method based on sequencing by ligation (SBL), the probe primer set provided by the present application can effectively reduce the problem of deep background of the nucleus in the microscopic examination results, and can effectively improve the signal-to-noise ratio, thereby effectively improving the detection efficiency of the SBL in situ sequencing method, and improving the signal detection amount and detectable target flux of the method.

[0009] In a first aspect, the present application provides a probe primer set for reducing the nucleus background in an in situ sequencing method based on sequencing by ligation, using the following technical solution:

[0010] A probe primer set for reducing the cell nucleus background in an in situ sequencing method based on sequencing by ligation, the probe primer set comprising at least one circularization probe for forming a rolling circle amplification product and a detection probe set capable of hybridizing with the rolling circle amplification product;

[0011] The looping probe is specifically hybridized and complementary to the target sequence;

[0012] Taking 5' to 3' as the forward direction, the in situ sequencing method includes upstream forward sequencing, upstream reverse sequencing, downstream reverse sequencing and downstream forward sequencing in sequence;

[0013] The detection probe group includes an upstream anchor primer, a downstream anchor primer, a first fluorescent sequencing probe group, and a second fluorescent sequencing probe group; wherein the upstream and downstream anchor primers have the same direction as the fluorescent sequencing probe group, which is double-end unidirectional sequencing;

[0014] The upstream forward sequencing uses the upstream anchor primer and the first fluorescent sequencing probe set;

[0015] The upstream reverse sequencing uses the upstream anchor primer and the second fluorescent sequencing probe set;

[0016] The downstream reverse sequencing uses the downstream anchor primer and the first fluorescent sequencing probe set;

[0017] The downstream forward sequencing uses the downstream anchor primer and the second fluorescent sequencing probe set.

[0018] Optionally, the target sequence includes RNA of the cell itself and RNA expressed by an exogenous gene inside the cell.

[0019] Optionally, the cells are free cells to be tested or cells in tissues.

[0020] Optionally, the looping probe is a double ligation probe or a padlock probe.

[0021] In a second aspect, the present application provides a kit for an in situ sequencing method, wherein the kit comprises the above-mentioned probe primer set.

[0022] Optionally, the kit includes: a hybridization mixture including a circularization probe; a ligation reaction mixture; a circularization mixture; a rolling circle amplification reaction solution and a sequencing reaction mixture including a detection probe group.

[0023] Optionally, the looping probe is a double ligation probe or a padlock probe.

[0024] In a third aspect, the present application provides the use of the above-mentioned probe primer set and kit in in situ sequencing for non-diagnostic and therapeutic purposes.

[0025] In a fourth aspect, the present application provides a method for sequencing the in situ sequencing method using the above-mentioned probe primer set or the above-mentioned kit for sequencing.

[0026] In summary, the present application includes at least one of the following beneficial technical effects:

[0027] In the related art, the in situ sequencing method is based on the amplification pathway of double-ligated probes and rolling circle amplification. The rolling circle amplification product is sequenced in 4 channels and 4 rounds, which can theoretically reach 256 (4 4 ) kinds of RNA detection throughput. However, there is a serious problem of upstream sequencing tissue nucleus background, which interferes with the reading of fluorescence signals and can seriously lead to an extremely low upstream sequencing signal-to-noise ratio, resulting in a low gene detection amount obtained by decoding after multiple rounds of sequencing imaging. In addition, the tissue background will increase with the increase of sequencing rounds, limiting the number of rounds that can be detected by this method, so there is also the problem of limited detection throughput.

[0028] In the in situ sequencing method based on sequencing by ligation (SBL), the probe primer set provided in the present application can effectively reduce the problem of deep background of cell nuclei in microscopic examination results, and can effectively improve the signal-to-noise ratio, thereby effectively improving the detection efficiency of the SBL in situ sequencing method, and improving the signal detection amount and detectable target throughput of the method.

[0029] Meanwhile, the related art requires the use of 4 rounds of 4 different sets of fluorescent sequencing probes, while the present application reduces it to 4 rounds requiring only 2 different sets of fluorescent sequencing probes, thereby reducing the reagent cost of the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the principle of the in situ sequencing method of ligation and sequencing in the related art.

[0031] Figure 2 This is the technical roadmap for the in situ sequencing method of ligation-while-sequencing in this application.

[0032] Figure 3 Figure 2 is a diagram of the design strategy of the dual ligation probes in this application.

[0033] Figure 4 The principle of using the double ligation probe of the present application to perform ligation and sequencing on the rolling circle amplification product.

[0034] Figure 5 It is a microscopic image of the first round of upstream sequencing results (the left side is the sequencing result of the comparative example; the right side is the sequencing result of the embodiment).

[0035] Figure 6 It is a microscopic image of the second round of upstream sequencing results (the left side is the sequencing result of the comparative example; the right side is the sequencing result of the embodiment).

[0036] Figure 7 It is a microscopic image of the downstream third round sequencing results (the left side is the sequencing result of the comparative example; the right side is the sequencing result of the embodiment).

[0037] Figure 8 It is a microscopic image of the fourth round of downstream sequencing results (the left side is the sequencing result of the comparative example; the right side is the sequencing result of the embodiment).

[0038] Fig. 9 This is the final decoding result after 4 rounds of sequencing in the example of this application.

[0039] in, Figure 5-Figure 8 The DAPI cell nucleus channel is not included in the figure. The bright spots in the figure are real signals, and the large gray solid area near the signals is the cell nucleus background. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0044] like Figure 1 As shown, in the related art, when performing the in situ sequencing method of ligation and sequencing, in terms of the upstream and downstream relative positional relationship of the anchor primer and the fluorescent sequencing probe, it is necessary to perform four rounds of upstream and downstream double-end reverse sequencing, so the fluorescent sequencing probes required for the upstream and downstream are different, and the two rounds of sequencing in the upstream are very likely to cause serious background in the tissue cell nuclei, interfere with signal reading, and reduce the number of detectable rounds.

[0045] like Figure 2 As shown, in the present application, the technical route of the in situ sequencing method by ligation and sequencing is: first, the double-ligation probe is hybridized with the target RNA, and rolling circle amplification is performed after ligation into a circle; then the generated rolling circle amplification product is used as a sequencing template, and after multiple rounds of fluorescence imaging and decoding, the expression level and spatial position information of each target RNA are obtained.

[0046] like Figure 3As shown, in the present application, the double-ligated probe is a linear single-stranded DNA probe composed of two segments of a target recognition sequence and a non-target-dependent intermediate sequence. In the order from 5' to 3', the upstream probe sequence sequentially includes an upstream target recognition sequence, an upstream anchor primer site, an upstream fluorescent sequencing probe site, and a site complementary to the splint sequence (a part of which serves as a downstream anchor primer site); the downstream probe sequence includes a downstream anchor primer site, a downstream fluorescent sequencing probe site, other intermediate sequences, and a downstream target recognition site.

[0047] like Figure 4 As shown, the principle of ligating and sequencing the rolling circle amplification products using the dual ligation probe of the present application is as follows: taking 4 rounds of sequencing, 2 rounds upstream and 2 rounds downstream, as an example, when performing ligation and sequencing, the 2 rounds of upstream sequencing need to add the upstream anchor primer and the upstream fluorescent sequencing probe of the 1st round or the 2nd round, respectively, and the 1st round and the 2nd round have different sequencing interrogation sites; the 2 rounds of downstream sequencing need to add the downstream anchor primer and the downstream fluorescent sequencing probe of the 3rd round or the 4th round, respectively, and the 3rd round and the 4th round have different sequencing interrogation sites; because the upstream and downstream anchor primers and the fluorescent sequencing probe have the same direction, that is, double-end unidirectional sequencing, the same fluorescent sequencing probe can be used in the 1st and 3rd rounds, and the 2nd and 4th rounds.

[0048] In short, the sequencing method in the related art is double-end reverse ligation sequencing, and two sets of fluorescent sequencing probes are required for the upstream and downstream respectively, and a total of four sets of fluorescent sequencing probes are required; the sequencing method of the present application is double-end unidirectional ligation sequencing, and two sets of fluorescent sequencing probes can be shared by the upstream and downstream, that is, a total of two sets of fluorescent sequencing probes are required.

[0049] The present application provides a probe primer set for reducing the background of the nucleus in an in situ sequencing method based on sequencing by ligation and its application. In the in situ sequencing method based on sequencing by ligation (SBL), the probe primer set provided by the present application can effectively reduce the problem of deep background of the nucleus in the microscopic examination results, and can effectively improve the signal-to-noise ratio, thereby effectively improving the detection efficiency of the SBL in situ sequencing method, and improving the signal detection amount and detectable target flux of the method.

[0050] 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.

[0051] 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.

[0052] The present application is further described in detail below in conjunction with the embodiments, drawings and test results.

[0053] The present application compares the sequencing results of the same gene by the sequencing method of the present application and the original sequencing method in the related art on the continuous paraffin sections of breast cancer. In the present application, the scheme results are described by taking 4 rounds of sequencing, 2 rounds each in the upstream and downstream, and 4 rounds of sequencing imaging are performed in the order from upstream to downstream.

[0054] The sequences used in the double ligation probes in the present application and related technologies are shown in Table 1. Among them, the sequences used in the double ligation probes in the scheme of the comparative example of the related technology are the nucleotide sequences shown in SEQ ID NO: 1-62 in Table 1, and the sequences used in the double ligation probes in the scheme of the embodiment of the present application are the nucleotide sequences shown in SEQ ID NO: 63-124 in Table 1.

[0055] Table 1 Sequences used in dual ligation probes

[0056]

[0057]

[0058]

[0059]

[0060] The sequences used in the anchor primers and fluorescent sequencing probes in each round in this application and related technologies are shown in Table 2.

[0061] Among them, in the comparative example of the related art, the sequence used by the anchor primers for the first and second rounds of sequencing is the nucleotide sequence shown in SEQ ID NO: 126 in Table 2, the sequence used by the fluorescent sequencing probes for the first round is the nucleotide sequence shown in SEQ ID NO: 129-132 in Table 2, and the sequence used by the fluorescent sequencing probes for the second round is the nucleotide sequence shown in SEQ ID NO: 133-136 in Table 2; the sequence used by the anchor primers for the third and fourth rounds of sequencing is the nucleotide sequence shown in SEQ ID NO: 127 in Table 2, the sequence used by the fluorescent sequencing probes for the third round is the nucleotide sequence shown in SEQ ID NO: 137-140 in Table 2, and the sequence used by the fluorescent sequencing probes for the fourth round is the nucleotide sequence shown in SEQ ID NO: 141-144 in Table 2.

[0062] Among them, in the examples of the present application, the sequence used by the anchor primers for the first and second rounds of sequencing is the nucleotide sequence shown in SEQ ID NO: 128 in Table 2; the sequence used by the anchor primers for the third and fourth rounds of sequencing is the nucleotide sequence shown in SEQ ID NO: 127 in Table 2; the sequences used by the fluorescent sequencing probes for the first and third rounds are all the nucleotide sequences shown in SEQ ID NO: 137-140 in Table 2, and the sequences used by the fluorescent sequencing probes for the second and fourth rounds are all the nucleotide sequences shown in SEQ ID NO: 141-144 in Table 2.

[0063] In contrast, the present application reduces the use of fluorescent sequencing probes shown in SEQ ID NOs: 129-136, thereby reducing the reagent costs of the reaction.

[0064] Table 2 Sequences used in anchor primers and fluorescent probes in each round

[0065] SEQ ID NO: name Sequence information (5' to 3') 5' modification 3' modification 126 Anchor-1 TGCGTCTATTTAGTG PHO - 127 Anchor-2 CTATTTAGTGGAGCC - - 128 Anchor-3 CTGGACCTTAATCGT - - 129 L1-A ACTCANNAG CY5 - 130 L1-C ACTCANNCT TXR - 131 L1-G ACTCANNGA CY3 - 132 L1-T ACTCANNTC 750 - 133 L2-A ACTCAAGNN CY5 - 134 L2-C ACTCACTNN TXR - 135 L2-G ACTCAGANN CY3 - 136 L2-T ACTCATCNN 750 - 137 R1-A AGNNACTCA PHO CY5 138 R1-C CTNNACTCA PHO TXR 139 R1-G GANNACTCA PHO CY3 140 R1-T TCNNACTCA PHO 750 141 R2-A NNAGACTCA PHO CY5 142 R2-C NNCTACTCA PHO TXR 143 R2-G NNGAACTCA PHO CY3 144 R2-T NNTCACTCA PHO 750

[0066] Example

[0067] This embodiment provides an in situ sequencing method based on the double-end same-direction principle.

[0068] The specific steps include:

[0069] 1. Tissue pretreatment of paraffin samples

[0070] (1) Baking: Bake FFPE tissue sections (breast cancer paraffin serial sections, hereinafter referred to as sections) in an oven at 60°C for 30 min.

[0071] (2) Dewaxing and rehydration: Immerse the sections in two cylinders of xylene at room temperature to ensure that the tissue is fully covered, and dewax for 15 min and 10 min respectively; then immerse the sections in two cylinders of 100% alcohol in turn, rehydrate for 2 min each, and wash the tissue three times with PBST (1× PBS containing 0.1% Tween-20).

[0072] (3) Fixation: Add 4% PFA (Biyuntian) to the tissue, discard the liquid after fixation for 10 minutes, and wash the tissue three times with PBST.

[0073] (4) Permeabilization: 0.1 M hydrochloric acid containing 0.3 mg / mL pepsin (Sigma) was added to the tissue and permeabilized at 37°C for 30 min. The tissue was then removed and washed three times with PBST.

[0074] (5) Dehydration and drying: The sections were sequentially immersed in 70%, 85% and 100% ethanol for gradient dehydration, each time for 1 min.

[0075] (6) 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.

[0076] (II) Sequencing process

[0077] (1) Dual-ligation probe hybridization

[0078] Add 50 μL of hybridization mixture containing 10 nM dual-linked probe (Shanghai Bioengineering, the same as the probe below), 6×SSC (Sigma), and 10% formamide (Sigma) to the tissue, hybridize at 46°C for 4 h to allow the probes designed for different target genes to fully bind to their targets, and then wash the tissue three times with PBST. After that, wash the tissue with probe washing solution containing 2×SSC and 20% formamide at 46°C for 3 times, each time for 5 min, and then wash the tissue three times with PBST.

[0079] (2) First step of ligation of dual ligation probes

[0080] 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.

[0081] (3) Circularization of dual-ligation probes

[0082] Add 50 μL of a circularization mixture containing 0.5 Μ circularization template sequence ST, 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. Wherein, the circularization template sequence is the nucleotide sequence shown in SEQ ID NO: 125, specifically: GGCTCCACTAAATAGACGCA.

[0083] (4) Rolling circle amplification

[0084] 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.

[0085] (5) Four-wheel probe detection and imaging

[0086] Add 50 μL of sequencing reaction mixture containing 0.2 μM anchor primer, 0.1 μM of each fluorescent probe, 1×T4 DNA Ligase buffer, 1 mM ATP and 0.1 U / μL T4 DNA Ligase to the tissue and incubate at 30°C for 45 min; then wash the tissue three times with PBST.

[0087] After the tissue was naturally air-dried, Slowfade anti-fluorescence quencher (Thermo) containing 0.5 μg / mL DAPI was added and incubated at room temperature for about 5 min before fluorescence microscopy imaging.

[0088] Probe elution operation needs to be introduced between each round of sequencing imaging. First, the glass slide is placed in 1×PBS and soaked until the coverslip falls off. After washing off the sealing medium, 100% formamide is added and washed three times at 37°C, each time for 10 minutes. After the end, confirm that there is no residual fluorescence under a fluorescence microscope before proceeding to the next round of detection. This cycle completes 4 rounds of sequencing imaging.

[0089] Comparative Example

[0090] This comparative example provides an in situ sequencing method in the related art.

[0091] The difference between the present invention and the embodiment lies in the differences in the dual ligation probes, anchor primers and fluorescent sequencing probes used in each round, and the other operation steps are consistent with the embodiment.

[0092] Test results

[0093] The microscopic examination results of the four rounds of the embodiments and comparative examples are as follows: Figure 5-8 As shown. Among them, Figure 5-8 The comparison diagrams of the microscopic examination results of rounds 1 to 4 are shown respectively. In addition, the gene distribution results obtained by imaging and finally decoding using the sequencing method of the present application are as follows: Fig. 9 shown.

[0094] By comparison Figure 5 and Figure 6It can be seen from the microscopic examination results that the signal detection amounts of the two upstream rounds (the first round and the second round) in the embodiment of the present application are both higher than the signal detection amounts in the comparative example of the related technology, indicating that the scheme of the present application can effectively improve the problem of serious nuclear background.

[0095] At the same time, by comparing Figure 1-Figure 4 From the microscopic examination results, it can be seen that after 4 rounds of sequencing, the scheme of the present application showed no trend of increasing nuclear background.

[0096] 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.

[0097] 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 probe primer set for reducing the cell nucleus background in an in situ sequencing method based on sequencing by ligation, characterized in that: The probe primer set includes at least one circular probe for forming a rolling circle amplification product and a detection probe set capable of hybridizing with the rolling circle amplification product; The looping probe is specifically hybridized and complementary to the target sequence; Taking 5' to 3' as the forward direction, the in situ sequencing method includes upstream forward sequencing, upstream reverse sequencing, downstream reverse sequencing and downstream forward sequencing in sequence; The detection probe group includes an upstream anchor primer, a downstream anchor primer, a first fluorescent sequencing probe group, and a second fluorescent sequencing probe group; wherein the upstream and downstream anchor primers have the same direction as the fluorescent sequencing probe group, which is double-end unidirectional sequencing; The upstream forward sequencing uses the upstream anchor primer and the first fluorescent sequencing probe set; The upstream reverse sequencing uses the upstream anchor primer and the second fluorescent sequencing probe set; The downstream reverse sequencing uses the downstream anchor primer and the first fluorescent sequencing probe set; The downstream forward sequencing uses the downstream anchor primer and the second fluorescent sequencing probe set.

2. The probe primer set according to claim 1, characterized in that: The target sequence includes the cell's own RNA and the RNA expressed by exogenous genes inside the cell.

3. The probe primer set according to claim 1, characterized in that: The cells are free cells to be detected or cells in tissues.

4. The probe primer set according to claim 1, characterized in that: The looping probe is a double ligation probe or a padlock probe.

5. A kit for in situ sequencing method, characterized in that: The kit comprises the probe primer set according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The kit comprises: a hybridization mixture including a ring-forming probe; a ligation reaction mixture; a ring-forming mixture; a rolling circle amplification reaction solution and a sequencing reaction mixture including a detection probe group.

7. The kit according to claim 5, characterized in that The looping probe is a double ligation probe or a padlock probe.

8. Use of the probe primer set according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 7 in in situ sequencing for non-diagnostic and therapeutic purposes.

9. An in situ sequencing method, characterized in that: The in situ sequencing method uses the probe primer set described in any one of claims 1 to 4 or the kit described in any one of claims 5 to 7 to perform sequencing.

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