Multiplex nucleic acid in situ detection method and kit without fluid

Abstract

The application discloses a fluid-free multiplex nucleic acid in-situ detection method and a kit. The fluid-free multiplex nucleic acid in-situ detection method comprises the following steps: using pre-configured several types of fluorescent decoding probes to stain a target sample; wherein the fluorescence modification states of the several types of fluorescent decoding probes are different, and at least one type of the fluorescent decoding probes has a photosensitive group; performing first fluorescence imaging on the stained target sample to obtain a first imaging graph; irradiating the target sample after the staining to make the photosensitive group respond and change the fluorescence properties; performing second fluorescence imaging on the target sample after the irradiation to obtain a second imaging graph; determining the intensity information of all signal points according to the first imaging graph and the second imaging graph; and decoding the intensity information to obtain the gene distribution information of the sample to be detected. The method can encode dozens or even hundreds of genes and perform nucleic acid in-situ detection without relying on special equipment and fluid.

Description

Technical Field

[0001] This disclosure relates to the field of gene detection technology, and in particular to a fluid-free method and kit for in situ detection of multiplex nucleic acids. Background Technology

[0002] The spatial distribution of RNA transcripts is crucial for revealing the complex functions of biological tissues. In recent years, with the increasing demand for spatial omics technologies from biologists, existing methods, especially imaging-based high-resolution spatial omics technologies, have been developing rapidly.

[0003] Currently, mainstream space omics imaging methods face significant challenges in terms of 3D compatibility and accessibility. Existing high-resolution space omics methods, due to channel limitations, can detect a maximum of five genes at a time using conventional fluorescence microscopy. Therefore, increasing the number of genes detected generally requires a fluid-based multi-round hybridization label-imaging-elution cycle. This multi-round fluid strategy can encode genes exponentially.

[0004] However, the introduction of multiple-round fluids necessitates the use of complex fluid-imaging equipment, which not only increases experimental costs but also limits detection throughput, severely restricting the widespread adoption of this method in biological laboratories. Furthermore, multiple-round fluid flushing and fluid exchange are particularly challenging in thick tissues, where actual diffusion and exchange are extremely slow, significantly increasing the time required for multiple-round reactions. Prolonged reagent penetration and flushing can also cause minute tissue deformations, making signal alignment difficult. Summary of the Invention

[0005] In view of this, the present disclosure provides a fluidless multiplex nucleic acid in situ detection method and kit, which can solve the problems of existing spatial transcriptome imaging technology where single-round fluorescence detection is limited by optical channels and can only detect a few genes, and the few coding schemes that break through channels still have limited detection throughput and rely on special instruments. At the same time, although the mainstream multi-round fluid circulation labeling method can detect hundreds of genes, it has problems such as complex equipment integration and difficulty in popularization, difficulty in reagent penetration and washing of thick tissues, easy to induce tissue deformation leading to difficulty in signal point alignment, and poor adaptability to three-dimensional in situ imaging.

[0006] In a first aspect, embodiments of this disclosure provide a fluidless in-situ multiplex nucleic acid detection method, comprising: The target sample is stained using several pre-configured types of fluorescent decoding probes; wherein the fluorescent modification states of the several types of fluorescent decoding probes are different and at least one type of fluorescent decoding probe has a photosensitive group; the target sample is obtained by amplifying the sample to be tested. The stained target sample was subjected to a first fluorescence imaging to obtain a primary imaging image. The stained target sample is irradiated with light, causing the photosensitive group to respond and its fluorescence properties to change. A second fluorescence imaging process is performed on the irradiated target sample to obtain a secondary imaging image; Based on the primary imaging image and the secondary imaging image, determine the intensity information of all signal points; The intensity information is decoded to obtain the gene distribution information of the sample to be tested.

[0007] Secondly, this disclosure also provides a fluidless multiplex nucleic acid in situ detection kit, including a padlock probe set and several types of fluorescent decoding probes, wherein the padlock probe set includes multiple padlock probes; The padlock probe includes two end sequences and a coding sequence located between the two end sequences. The coding sequence includes N segment sequences, each segment sequence corresponding to a fluorescence channel. Each fluorescence channel is used to bind M fluorescent decoding probes with different fluorescent modification states; where N≥3 and M≥3. All of the fluorescent decoding probes have different fluorescent modification states, and at least one type of fluorescent decoding probe has a photosensitive group.

[0008] The fluidless in-situ multiplex nucleic acid detection method disclosed in this embodiment first stains the target sample with several pre-configured fluorescent decoding probes. Each of the fluorescent decoding probes has a different fluorescence modification state, and at least one probe has a photosensitive group. The target sample is obtained by amplifying the test sample. The stained target sample undergoes a first fluorescence imaging to obtain a primary image. The stained target sample is then irradiated with light to change the fluorescence properties of the photosensitive group. A second fluorescence imaging is performed on the irradiated target sample to obtain a secondary image. The intensity information of all signal points is determined based on the primary and secondary images. The intensity information is then decoded to obtain the gene distribution information of the test sample. This method performs two rounds of imaging, with light irradiation altering some signal states from the first fluorescence imaging between the two rounds. During decoding, the encoding of each channel is determined by the signals from both rounds of imaging. A pseudo-round is introduced through fluorescence resonance energy transfer and light-controlled conversion, enabling the encoding of hundreds of genes without relying on special equipment, fluids, or color intensity grading.

[0009] The above description is merely an overview of the technical solution disclosed herein. In order to better understand the technical means of this disclosure and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this disclosure more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 This is a schematic flowchart of a fluidless in-situ multiplex nucleic acid detection method provided in the embodiments of this disclosure.

[0012] Figure 2 This is a test schematic diagram provided for an embodiment of the present disclosure.

[0013] Figure 3 This is a schematic diagram of fluorescence intensity value extraction provided in an embodiment of this disclosure.

[0014] Figure 4 This is a schematic diagram of the encoding rules provided in the embodiments of this disclosure.

[0015] Figure 5 A schematic diagram of a barcode provided for an embodiment of this disclosure.

[0016] Figure 6 This is a schematic diagram comparing the encoding provided in the embodiments of this disclosure with general in situ sequencing encoding.

[0017] Figure 7 This is a schematic diagram of the signals for two-round imaging in four channels for the four encoding states provided in the embodiments of this disclosure.

[0018] Figure 8 This is a schematic diagram of the arrangement of the decoding fluorescent probes in the amplification products provided in the embodiments of this disclosure.

[0019] Figure 9 This is a schematic diagram of the decoded gene corresponding to Example 1.

[0020] Figure 10 This is a schematic diagram of the decoded gene corresponding to Example 2.

[0021] Figure 11 This is a schematic diagram of the decoded gene corresponding to Example 3.

[0022] Figure 12 This is a schematic diagram of the decoded gene corresponding to Example 4. Detailed Implementation

[0023] The embodiments of this disclosure will now be described in detail with reference to the accompanying drawings.

[0024] It should be understood that the following specific examples illustrate the implementation of this disclosure, and those skilled in the art can easily understand other advantages and effects of this disclosure from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. This disclosure can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this disclosure. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0025] It should be noted that various aspects of embodiments within the scope of the appended claims are described below. It will be apparent that the aspects described herein can be embodied in a wide variety of forms, and any particular structure and / or function described herein is merely illustrative. Based on this disclosure, those skilled in the art will understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number of aspects set forth herein can be used to implement the device and / or practice the method. Additionally, this device and / or method can be implemented using structures and / or functionalities other than one or more of the aspects set forth herein.

[0026] It should also be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of this disclosure. The drawings only show the components related to this disclosure and are not drawn according to the number, shape and size of the components in actual implementation. In actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0027] Furthermore, specific details are provided in the following description to facilitate a thorough understanding of the examples. However, those skilled in the art will understand that the described aspects can be practiced without these specific details.

[0028] Reference Figure 1 and Figure 2 This application discloses a fluid-free in-situ detection method for multiplex nucleic acids, comprising: S100 uses several pre-configured fluorescent decoding probes to stain the target sample; wherein, the fluorescent modification states of the several fluorescent decoding probes are different and at least one fluorescent decoding probe has a photosensitive group, and the target sample is obtained by amplifying the test sample.

[0029] Specifically, the number of fluorescent decoding probes and the sequence information of the padlock probes are determined first.

[0030] In this embodiment, the number of fluorescent decoding probes is M, where M≥3, meaning that at least one of the M types of fluorescent decoding probes has a photosensitive group.

[0031] The padlock probe consists of two terminal sequences and a coding sequence located between the two terminal sequences. The terminal sequences are binding sequences that are adjacent to and complementary to the target nucleic acid sequence, and each terminal sequence corresponds to a gene.

[0032] The coding sequence consists of N segment sequences, each segment sequence corresponds to a fluorescent channel, each fluorescent channel has a corresponding fluorescent group, and each fluorescent channel is used to bind M fluorescent decoding probes with different fluorescent modification states, where N≥3.

[0033] In this embodiment, each channel can encode four preset pieces of information. Since this encoding method only relies on the readout of 0 or 1 and does not need to consider the amplification difference of RCA, and the different channels are completely orthogonal, the total number of codewords increases exponentially when multiple channels are encoded simultaneously, that is, N channels can encode... indivual.

[0034] Secondly, based on the number of species and the number of segment sequences, the encoding capacity of the padlock probe set and the number of fluorescent decoding probes to be synthesized are determined.

[0035] In this embodiment, the encoding capacity is The number of fluorescent decoding probes to be synthesized is M×N.

[0036] Next, padlock probe sets are synthesized according to the encoding capacity to obtain padlock probe set liquid; fluorescent decoding probe liquid is prepared according to the quantity to be synthesized.

[0037] Furthermore, each type of padlock probe is preferably prepared into a single liquid of a preset concentration according to preset requirements before mixing to obtain the desired padlock probe set liquid. Similarly, each type of fluorescent decoding probe is preferably prepared into a single liquid of a preset concentration according to preset requirements before mixing to obtain the desired fluorescent decoding probe liquid.

[0038] Then, the test samples were pretreated, and in situ hybridization was performed on the test samples using padlock probe collection liquid. After amplification, signal amplification samples were obtained. Finally, the signal amplification samples were stained with fluorescent decoding probe liquid. After staining, the samples were mounted and used as target samples.

[0039] Specifically, padlock probes are used to hybridize the target gene's mRNA, ligase is used for ligation, and rolling circle amplification is performed to amplify the signal. The barcode region on each padlock probe is divided into multiple segments, each segment corresponding to a channel's decoding fluorescent probe, which presents the encoded information through hybridization; there are several types of decoding fluorescent probes for each channel, distinguished by different sequences.

[0040] In this application, a padlock probe with a barcode is used to target and bind to the sample. After binding, the barcode sequence is amplified by rolling circle amplification to generate hundreds of times the barcode sequence. Subsequently, it is stained by a mixed decoding probe and presented to facilitate subsequent in-situ reading.

[0041] Furthermore, when the number of fluorescent decoding probes M is 3, the M-type fluorescent decoding probes preferably include probes modified with fluorescent groups, probes without fluorescent group modification, and probes modified with fluorescent groups linked by photo-cleaving bonds.

[0042] When the number of fluorescent decoding probes M is 4, the M-type fluorescent decoding probes preferably include probes modified with fluorescent groups, probes without fluorescent group modification, probes modified with fluorescent groups linked by photolytic bonds, and probes with fluorescent groups modified with FRET groups and photolytic bonds.

[0043] When the number of fluorescent decoding probes M is greater than 4, the M-type fluorescent decoding probes preferably include probes modified with fluorescent groups, probes without fluorescent group modification, probes modified with fluorescent groups linked by photolytic bonds, and probes with fluorescent groups modified with FRET groups and photolytic bonds. The fluorescent groups can be divided according to a custom intensity range, thereby obtaining fluorescent decoding probes of many states.

[0044] Among them, fluorescently modified probes refer to those fluorescent decoding probes that are chemically modified with DNA containing a complementary sequence to the corresponding fragment of the amplification product, directly modifying the corresponding fluorescent group, and remain unchanged before and after ultraviolet irradiation.

[0045] Among them, the probe without fluorescent group modification is only the DNA of the complementary sequence of the corresponding fragment of the amplification product, without fluorescent group or other types of modification.

[0046] The preparation method of the probe modified with a fluorescent group linked by photolytic cleavage bond includes: 1) Determine the type of probe body based on the number of fluorescent decoding probe types.

[0047] 2) Prepare corresponding probe bodies respectively, with each type of probe body having 15-30 nt bases, preferably 20; furthermore, the corresponding probe bodies can be synthesized by cyclic synthesis.

[0048] 3) Determine the photolytic bonds and use the photolytic bonds to connect each type of probe body to the corresponding fluorescent group of the fluorescent channel to obtain probes modified with fluorescent groups connected by photolytic bonds.

[0049] Furthermore, the photosensitive group is preferably a photosensitive linker based on a 2-nitrophenyl skeleton, and the corresponding chemical bond can be broken by ultraviolet light irradiation. That is, when ultraviolet light irradiates, the chemical bond breaks, the fluorescent group leaves the decoding probe and the amplification product, and the signal at the corresponding signal point disappears.

[0050] The preparation method of the probe with a fluorescent group modified by the FRET group and the photolytic bond includes: 1) Determine the type of probe body based on the number of fluorescent decoding probe types.

[0051] 2) Prepare corresponding probe bodies respectively, with each type of probe body having 15-30 nt bases, preferably 20; furthermore, the corresponding probe bodies can be synthesized by cyclic synthesis.

[0052] 3) Determine the photo-cleavage bond. In this embodiment, the photo-cleavage bond is preferably of the o-nitrobenzyl type, p-methoxybenzoyl type, carbon-halogen type, etc.

[0053] 4) Determine the FRET group (fluorescence resonance energy transfer group) corresponding to each fluorescence channel, i.e., the quenching group.

[0054] In this embodiment, the FRET group is preferably BHQ1, BHQ2, BHQ3, etc.

[0055] 5) Connect the fluorescent group corresponding to the fluorescent channel to the base at a preset position on the probe body (preferably near the end).

[0056] 6) Use photolytic bonds to connect the FRET group to the end of the probe body to obtain a probe with a fluorescent group modified by the FRET group and photolytic bonds.

[0057] In this embodiment, the chemical modification of this type of fluorescent decoding probe is as follows: the DNA of the complementary sequence of the corresponding fragment of the amplification product is linked to the corresponding fluorescent group by chemical bonds that can change the fluorescence properties after ultraviolet light irradiation, that is, the fluorescence only appears after ultraviolet irradiation.

[0058] Furthermore, for detecting different samples / different gene detection sets, as long as the selected channel N is fixed, the same set of coding sequences can be used, only the end sequences need to be replaced. The corresponding fluorescent decoding probes can also use the same set, without the need for reconfiguration, effectively shortening the overall detection cycle. It should be noted that whether the fluorescent decoding probes are reconfigured for each staining or not reconfigured according to actual needs, it is within the scope of protection of this application.

[0059] S200 performs the first fluorescence imaging on the stained target sample to obtain a single image.

[0060] During the first round of imaging, the fluorescent group and the decoding probe were normally connected, and the signal appeared in the amplification product.

[0061] S300 involves irradiating the stained target sample with light to cause a change in the fluorescence properties of the photosensitive groups in response.

[0062] Furthermore, the photosensitive group preferably contains a photolytic bond, and this step preferably includes: irradiating the stained target sample with ultraviolet light to break the photolytic bond so as to cause a change in the fluorescence intensity of the photosensitive group, thereby changing part of the signal state during the first fluorescence imaging.

[0063] It should be noted that other light irradiation methods can also be used in this application, as long as they can cause a change in the fluorescence properties of the photosensitive group after response, they are all within the scope of protection of this application. In addition to the photosensitive group containing photolytic bonds, that is, in addition to light irradiation to break the photolytic bonds and cause a change in the fluorescence intensity of the photosensitive group, other response methods can also be used, such as photo-induced isomerization (e.g., azobenzene), other photo-induced cyclization addition, proton transfer, etc., as long as the corresponding response can lead to a change in fluorescence properties, they are also within the scope of protection of this application.

[0064] After the first fluorescence imaging, move the stage to expose the tissue as much as possible directly above it. UV irradiation can be performed using a 10W 365nm UV flashlight, positioned 1-2 cm above the tissue for 1-40 seconds, preferably no less than 10 seconds, ensuring the light spot covers the entire tissue. Furthermore, avoid exposing the tissue to UV radiation as much as possible before the first fluorescence imaging is complete; therefore, avoid imaging the DAPI channel during the first round of imaging.

[0065] During irradiation, the ultraviolet dose is excessive, so there is no need to remove the tissue from the stage, and the angle of the light can be slightly tilted.

[0066] S400 performs a second fluorescence imaging on the irradiated target sample to obtain a secondary imaging image.

[0067] When exposed to ultraviolet light, the fluorescence properties change after the chemical bonds respond. The quenching group leaves the fluorescent group, the decoding probe, and the amplification products, and the fluorescent group emits fluorescence again, presenting a signal in the second round of imaging.

[0068] S500 determines the intensity information of all signal points based on the primary and secondary imaging images.

[0069] The following example uses M=4 and N=4, specifically M-type fluorescent decoding probes, which include probes modified with fluorescent groups, probes without fluorescent group modification, probes modified with fluorescent groups linked by photolytic bonds, and probes with fluorescent groups modified with FRET groups and photolytic bonds. The coding sequence includes 4 segment sequences, corresponding to 4 types of fluorescence channels. In other words, a fluorescence microscope with 4 channels is used to perform two imaging results, i.e., 8 images, for detailed analysis.

[0070] Specifically, all secondary imaging images are aligned with all primary imaging images. A preset enhancement algorithm is used to enhance the signal points in all aligned imaging images. A local extremum algorithm is used to extract the positions of the enhanced signal points to obtain the positions of all signal points and the intensity information of the signals (such as 8 fluorescence intensity values). Based on the position of each signal point, the intensity information of the signals corresponding to the same points in all imaging images is merged to obtain the intensity information of all signal points.

[0071] Reference Figure 3 The sample to be tested is a coronal section of a mouse brain. Taking eight images obtained from two rounds of imaging as an example, the left side of the figure shows the intensity value matrix of each signal point in the example, and the right side shows the original image of one of the signal points.

[0072] S600 decodes the intensity information to obtain the gene distribution information of the sample to be tested.

[0073] Taking M=4 as an example, the decoding steps will be explained in detail.

[0074] In the first embodiment, the method for obtaining gene distribution information of the sample to be tested includes: 1) Analyze the intensity information using an intensity threshold to determine the first barcode sequence corresponding to the location of each signal point.

[0075] The purpose of this step is to quickly and with low precision assign a preliminary barcode to each signal point.

[0076] Specifically, intensity information is analyzed using intensity thresholds to determine the binary state of each signal point in the first fluorescence imaging and the binary state of each signal point in the second imaging. The binary state includes "signal present" and "no signal present." Preferably, when the intensity of a signal point is not less than the intensity threshold, it is determined to have a signal, which can be recorded as 1; when the intensity of a signal point is less than the intensity threshold, it is determined to have no signal, which can be recorded as 0.

[0077] The barcode sequence is determined based on the binary states of the two images, as detailed in the following reference. Figure 4When there is a signal in both rounds of imaging (i.e., when it is 11), it is defined as the first type of encoded information (such as α); when there is no signal in both rounds of imaging (i.e., when it is 00), it is defined as the second type of encoded information (such as γ); when there is a signal only in the first round (i.e., when it is 10), it is defined as the third type of encoded information (such as τ); when there is a signal only in the second round (i.e., when it is 01), it is defined as the fourth type of encoded information (such as χ). In this way, the barcode sequence corresponding to the position of each signal point can be obtained, which is denoted as the first barcode sequence.

[0078] Furthermore, the coding state type corresponding to each bit can be determined by the two values ​​R1 and R2 for each channel. If the larger absolute strength value of R1 and R2 is above the corresponding given threshold, it is one of α, τ, or χ; based on this, if log((R2+0.01) / (R1+0.01)) is greater than the corresponding given threshold, it is χ; if it is less than the corresponding given threshold, it is τ. If they are within a certain range, the two values ​​are considered similar and are determined to be α. If the larger absolute strength value of R1 and R2 is below the corresponding given threshold, and log((R2+0.01) / (R1+0.01)) is also within a certain range, it is determined to be γ; signal points outside the above range are determined to be ν (indicating undetermined) and the signal points in that row are discarded in subsequent analyses.

[0079] By mapping each signal point to its barcode using eight-dimensional information, we extract the first-round intensity value information for each channel. After crosstalk correction and scaling, we calculate log((second-round intensity value + 0.01) / (first-round intensity value + 0.01)). Combining these two values, we determine the state type of each channel. We manually set a background threshold and a threshold for intensity change between two rounds. Based on preset recognition conditions, we decode to obtain the corresponding state. This state recognition method can initially determine the four-digit barcode of the signal point. This method considers both the intensity value and the change information after ultraviolet irradiation between two rounds, making it more comprehensive than decoding methods that only consider the intensity values ​​of the two rounds individually.

[0080] 2) Vector projection is used to analyze the signal strength information of each signal point to obtain the second barcode sequence and the corresponding confidence level.

[0081] In this step, barcode verification is performed using vector projection. A 4-bit (α / τ / χ / γ) standard barcode is equivalent to an 8-bit binary barcode consisting of 0s and 1s. The decoding process essentially maps the measured eight-bit actual fluorescence intensity data to an 8-bit standard barcode of 0s and 1s.

[0082] Each signal point's eight-bit actual fluorescence intensity value is treated as an eight-dimensional vector, and all standard barcodes can also be considered as eight-dimensional vectors, with their magnitudes set to 1. The eight-dimensional vector of each signal point is projected (dot product) onto the eight-dimensional vectors of all standard barcodes to obtain the projection length of this signal point across all standard barcodes. The standard barcode with the longest projection length is the barcode result obtained by vector projection of this signal point. Additionally, the second most similar barcode can be found. To ensure accuracy, the values ​​of the longest and second longest projections for each signal point can be calculated to determine its specificity (confidence). If this value is large, the confidence is high, and the decoding result is relatively reliable; if the value is very close to 1, the confidence is low, the result is unreliable, and the signal point should be discarded.

[0083] In this embodiment, the ratio of the maximum projected length to the second largest projected length obtained by projecting the signal point is defined as the confidence level of the signal point. The higher the confidence level, the more accurate the standard barcode result obtained by vector projection of the signal point is, and vice versa.

[0084] It should be noted that the analysis processes of the first barcode sequence and the second barcode sequence can be performed in parallel. This embodiment is only a preferred method and does not limit the scope of protection of this application.

[0085] 3) Determine the sequence difference information between the first barcode sequence and the second barcode sequence corresponding to the position of each signal point.

[0086] Specifically, for each signal point, its corresponding first barcode sequence and second barcode sequence are compared bit by bit to determine the consistency of each bit. Based on the bit-by-bit comparison results, the sequence difference information between the first and second barcode sequences is statistically analyzed. The sequence difference information includes at least the number of differing bits and the difference rate, where the number of differing bits is the total number of bits that are inconsistent in the bit-by-bit comparison between the first and second barcode sequences (the difference rate is the ratio of the number of differing bits to the total length of the barcode sequence). The sequence difference information is then associated one-to-one with each signal point to obtain the sequence difference information corresponding to each signal point.

[0087] The maximum number of differences is preferably 1 (the maximum difference rate is preferably 0.125).

[0088] 4) Obtain all signal points whose sequence difference information is within a preset range and whose confidence level is not less than a preset threshold, and record them as valid signal points.

[0089] Specifically, a preset sequence difference range and a confidence threshold are defined. The sequence difference range is the preset maximum number of difference bits or the preset maximum difference rate, and the confidence threshold is the preset minimum reliable confidence level corresponding to the second barcode sequence. For each signal point, it is determined whether its corresponding sequence difference information is within the preset sequence difference range and whether the confidence level of its corresponding second barcode sequence is not less than the preset confidence threshold. Signal points that simultaneously satisfy the condition that the sequence difference information is within the preset sequence difference range and the confidence level is not less than the preset confidence threshold are marked as valid signal points. The preset confidence threshold is preferably the 50th percentile of the confidence levels of all signal points corresponding to each barcode.

[0090] 5) Use the gene type information corresponding to the second barcode sequence as the gene type information of the corresponding valid signal point.

[0091] In this embodiment, N adjacent status information in the barcode sequence correspond to one gene type.

[0092] 6) Based on the gene type information of all valid signal points, obtain the gene distribution information of the sample to be tested.

[0093] In the second embodiment, the method for obtaining gene distribution information of the sample to be tested includes: 1) Analyze the intensity information by intensity threshold to determine the binary state of each signal point in the first fluorescence imaging and the binary state of each signal point in the second imaging. The binary state includes signal presence and no signal presence.

[0094] Preferably, when the intensity of a signal point is not less than the intensity threshold, it is determined that there is a signal, which can be recorded as 1; when the intensity of a signal point is less than the intensity threshold, it is determined that there is no signal, which can be recorded as 0.

[0095] 2) Determine the barcode sequence based on the binary state of the two imaging rounds. Specifically, when there is a signal in both imaging rounds (i.e., when it is 11), it is defined as the first type of encoding information (e.g., α); when there is no signal in both imaging rounds (i.e., when it is 00), it is defined as the second type of encoding information (e.g., γ); when there is a signal only in the first round (i.e., when it is 10), it is defined as the third type of encoding information (e.g., τ); when there is a signal only in the second round (i.e., when it is 01), it is defined as the fourth type of encoding information (e.g., χ).

[0096] 3) Use the gene type information corresponding to the barcode sequence as the gene type information of the corresponding signal point.

[0097] Reference Figure 5 and Figure 6 In this application, optically controlled pseudo-cycles are used to encode each channel with 4 bits without the need for fluid. If there are N channels in total, then there are a total of barcodes ( Each padlock probe is divided into several segments, each segment corresponding to a channel, which can be combined with a type of decoding probe, meaning each segment can correspond to four types of preset information. This application transforms the longitudinal multi-round information encoding of general in situ sequencing into lateral multi-channel encoding, maintaining encoding capability without fluid, hence the name Virtual In-situ Sequencing Multiplexed by Optical Recoding (VISMORE). Since four-channel imaging is widely used in sequencing and in situ sequencing, four-channel encoding is preferred in this embodiment, meaning it can encode (…). (Number of genes)

[0098] The difference is that traditional in situ sequencing uses multiple rounds of fluidic reactions, while the sequencing chemistry of SBS or SBL ensures that one amplification product emits a fluorescent signal (fluorescent base) in one channel during each round. After N rounds of sequencing, the total number of codes is... (Where 4 represents the number of channels and N represents the number of sequencing epochs). The virtual in situ sequencing in this application consists of only one epoch. Light control conversion and two rounds of imaging enable each channel to generate four preset information values, presented across N channels. The total number of codes (where 4 represents the state information generated by the pseudo-rounds, and N is the number of channels) is equivalent to recoding the multi-round vertical information in in situ sequencing into single-round horizontal multi-channel information, improving the efficiency of in situ sequencing in acquiring imaging information (information entropy). Theoretically, the two encoding methods have the same encoding capacity. Due to the increased information entropy, the signal density of this method is doubled compared to traditional four-color sequencing, meaning the optical dilution capability is halved as a trade-off for eliminating the need for fluids. However, due to the 0 or 1 readout mode and the overall higher optical dilution capability, this method has a higher resistance to optical congestion than PRISM (Intensity-Dependent Sequencing). Currently, the maximum number of genes that can be detected by four-color in situ sequencing is around 3000-5000. Therefore, the upper limit of encoding this method due to optical congestion is at least 1500 genes.

[0099] Reference Figure 7 The signals from two rounds of imaging in four channels for four coding states are shown. The effects of two rounds of imaging for the four states in each channel are summarized. Signals from 16 different amplification products are extracted for demonstration. For example, the τ signal in each of the four channels is composed of two rounds of imaging from the corresponding channels of barcode τγγγ, γτγγ, γγτγ, and γγγτ. The γ signal determines the location of the amplification product by its position in other channels where signals are present. In the superimposed image below, pink represents the fluorescence signal from the first round of imaging, cyan represents the fluorescence signal from the second round of imaging, and therefore white represents signals from both rounds (pink + cyan).

[0100] In the N-channel embodiment, the channels corresponding to two adjacent segment sequences are staggered as much as possible in terms of wavelength to prevent crosstalk between fluorescent decoding probes of different segments (e.g., FRET effect between fluorescent groups of different segments). See reference. Figure 8 This demonstrates that such a design ensures that different information from different channels will not interfere with or affect each other.

[0101] Example 1: The sample to be tested was a coronal section of the mouse brain.

[0102] In this embodiment, the number of fluorescent decoding probes is determined to be 4. The two terminal sequences of the padlock probes in the padlock probe set are binding sequences complementary to a segment of the target RNA sequence, and the connecting sequence in the middle is a coding sequence used to encode the binding sequence. The target RNA sequence consists of 120 genes selected for cell classification and state identification. Each coding sequence includes 4 segment sequences, each segment sequence being used to bind to yellow, blue, red, and green fluorescent probes, respectively. All color channel segment sequences include four sub-segment sequences, each binding to one of the four types of decoding probes.

[0103] For the barcode area, each channel has a sequence of four preset information types, each 20 nt in length; there are a total of There are several different sequences (N is the number of channels). When arranging barcodes, the fluorescence bands of adjacent probes should be staggered as much as possible to prevent unintended fluorescence resonance energy transfer.

[0104] Table 1 Padlock Probe Set Referring to Table 1, it includes the padlock probe number, probe name (i.e., gene name 1, gene name 2, ... which are the names of the targeted genes), and the type of sequence targeting the gene. Multiple probes may be used to target the same gene to improve sensitivity; all probes corresponding to the same gene have the same barcode. In this embodiment, 120 barcodes are selected to encode the gene, and all padlock probe sequences are shown in the table.

[0105] In this embodiment, all available barcodes corresponding to the four modification state probe sequences of the four channels are: kind.

[0106] After obtaining the padlock probe set liquid and the fluorescent decoding probe liquid, the sample to be tested was pretreated. Specifically, a coronal frozen section of mouse brain was taken, fixed with 4% paraformaldehyde for 30 minutes, washed once with PBST (PBS, 0.05% Tween, the same below), and the tissue was digested (treated with 0.01% pepsin and 0.1M hydrochloric acid at 37°C for 2 minutes, then washed with PBST), dehydrated (treated with 80% ethanol for 10 minutes, treated with 100% ethanol for 2 minutes, then treated with PBST 3 times for 2 minutes each time), and blocked (100 nM oligo dT, 20% formamide, 50 mM KCl, 20 μg / mL bovine serum albumin, 20 μg / mL yeast tRNA and 1 U / μL RNase inhibitor were added to Ampligase buffer, and the mixture was treated at room temperature for 30 minutes).

[0107] In situ hybridization was performed using a designed padlock probe set. Padlock probes were added to Ampligase buffer [Lucigen] to achieve a final concentration of 200 nM / μL for each padlock probe, along with 20% formamide, 50 mM KCl, 20 μg / mL bovine serum albumin (BSA), 20 μg / mL yeast tRNA [AM7119, Invitrogen], and 1 U / μL RNase inhibitor [ThermoScientific]. The mixture was incubated at 55°C for 15 minutes, followed by incubation at 45°C for 2 hours. Washing was performed three times with 10% formamide and 2x SSC solution for 10 minutes, followed by three more washes with PBST. Ligation was then performed using SplintR buffer, 20 μg / mL BSA, 1 U / μL RNase inhibitor, and 2.5 U / μL SplintR ligase [New England Biolabs]. The ligation reaction was carried out at 37°C for 2 hours, followed by washing with PBST.

[0108] RCA amplification was then performed using the following reaction system: Phi29 polymerase buffer, 0.25 U / μL Phi29 polymerase, 250 μM dNTPs, 50 μM aminodUTP (Thermo Scientific), 10% glycerol, 20 μg / mL bovine serum albumin, and 600 nM amplification primers. Amplification was performed overnight at 30°C. The sample was washed twice with PBST, fixed with 10 μg / μL BS(PEG)9 (Thermo Scientific), and washed three times with 65% formamide.

[0109] Table 2 16 fluorescent decoding probe sequences Referring to Table 2, in this embodiment, probes modified with fluorescent groups can be denoted as (1,1), probes without fluorescent group modification can be denoted as (0,0), probes modified with fluorescent groups linked by photolytic bonds can be denoted as (1,0), and probes with fluorescent groups modified by quenching groups (FRET groups) and photolytic bonds can be denoted as (0,1).

[0110] The amplification products were stained with 16 pre-mixed decoding probes (4 channels × 4 states) (20% formamide, 2X SSC, final concentration of 100 nM for each decoding probe), followed by the first round of imaging. Each target mRNA sequence in the sample formed a signal point.

[0111] After the first round of imaging is completed, ultraviolet (UV) irradiation is performed using a 10W 365nm UV flashlight positioned 1-2 cm above the sample, ensuring the light spot completely covers the tissue for approximately 20 seconds. The tissue is not moved during UV irradiation; it remains on the stage.

[0112] A second round of imaging was performed, including four signal point encoding channels and a nuclear staining DAPI channel.

[0113] Reference Figure 9 The method employs state decoding with four channels for each signal point, and obtains the in situ distribution information of target RNA in coronal sections of the mouse brain based on the decoding results. The figure shows the results of detecting 120 genes in mouse brain tissue using this method. The left image displays the results of all 120 decoded genes simultaneously, with each gene represented by a false color. The right image shows the detected genes and their corresponding false color types.

[0114] Example 2: The sample to be tested is a pathological section of human colorectal cancer.

[0115] The padlock probes in the padlock probe set have two terminal sequences that are complementary to a segment of the target RNA sequence, and a connecting sequence in the middle that is a coding sequence used to encode the binding sequence. The target RNA sequence consists of 120 genes selected for cell classification and state identification. Each coding sequence includes four segment sequences, each segment sequence being used to bind to yellow, blue, red, and green fluorescent probes, respectively. All color channel segment sequences include four sub-segment sequences, each binding to one of four types of decoding probes. The four modified probe sequences and modifications for the four channels are consistent with those in Example 1, and all usable barcodes are... kind.

[0116] Table 3 Padlock Probe Set Table 3 includes the padlock probe number, probe name (i.e., gene name 1, gene name 2, ... which are the target gene names), and the sequence types targeting the gene. Multiple probes may be used to target the same gene to improve sensitivity; all probes corresponding to the same gene have the same barcode; in this embodiment, 120 types are selected to encode the gene.

[0117] In this embodiment, pathological sections of human colorectal cancer tumor samples were taken, and the fixation conditions were the same as in Example 1. In addition, the digestion time was extended to 4 minutes; the dehydration, blocking, probe hybridization and ligation conditions, and other conditions were the same as in Example 1.

[0118] Reference Figure 10 The state decoding of each signal point is performed using four channels. Based on the decoding results, the in situ distribution information of the target RNA in the pathological section is obtained. This figure shows the results of all 120 decoded genes being displayed simultaneously, with each gene represented by a false color.

[0119] Furthermore, the results showed that different cell types and structures were marked by multiple marker genes, such as tumor cells and stromal cells. The distribution of tumor cell marker genes such as EPCAM, KRT18, and KRT10 also differed. Similarly, COL1A1, MMP14, and ACTA2 marked different types of fibroblasts, all of which suggest tissue heterogeneity. The region on the right side of the tissue with high expression of MS4A1 and CD79A was a tertiary lymphoid structure, and the presence of some B cells nearby suggested immune enrichment near the intestinal tissue and possible changes in response to tumors.

[0120] Example 3: The sample to be tested is a pathological section of human liver cancer.

[0121] The padlock probes in the padlock probe set have two terminal sequences that are complementary to a segment of the target RNA sequence, and a connecting sequence in the middle that is a coding sequence used to encode the binding sequence. The target RNA sequence consists of 120 genes selected for cell classification and state identification. Each coding sequence includes four segment sequences, each segment sequence being used to bind to yellow, blue, red, and green fluorescent probes, respectively. All color channel segment sequences include four sub-segment sequences, each binding to one of four types of decoding probes. The four modified probe sequences and modifications for the four channels are consistent with those described above, resulting in 255 available barcodes. In this example, 120 of these are selected to encode genes, and all padlock probe sequences are consistent with those in Example 2.

[0122] Pathological sections of human colorectal cancer tumor samples were taken. Except for the digestion time being extended to 4 minutes, the fixation conditions, dehydration and sealing conditions, probe hybridization and ligation conditions, and other conditions were the same as in Example 1.

[0123] Reference Figure 11 For each signal point, state decoding using four channels was employed to obtain the in-situ distribution information of target RNA in the pathological section based on the decoding results. These results also demonstrate the high heterogeneity of gene expression and immune cell penetration in different regions within the tumor. Examples 2 and 3 showcase the detection capability of this method for different types of pathological sections.

[0124] Example 4 The sample to be tested was a three-dimensional mouse brain sample: a tissue section 120 micrometers thick.

[0125] The padlock probes in the padlock probe set have two terminal sequences that are complementary to a segment of the target RNA sequence, and a connecting sequence in the middle that is a coding sequence used to encode the binding sequence. The target RNA sequence consists of 120 genes selected for cell classification and state identification. Each coding sequence includes four segment sequences, each segment binding to a yellow, blue, red, or green fluorescent probe, respectively. All color channel segment sequences include four sub-segment sequences, each binding to one of four types of decoding probes. The four modified state probe sequences and modifications for the four channels are as described above, resulting in 255 possible barcodes.

[0126] Table 4 Padlock Probe Set A 120-micrometer-thick mouse brain tissue sample was taken (the mouse brain was perfused with pre-cooled PBS and PFA, then fixed with PFA overnight, washed with PBS, and cut into 120-micrometer coronal brain slices using a vibrating microtome, and then stored in 70% ethanol for long-term preservation). It was treated with PBST for 20 minutes, repeated three times, and then blocked (100 nM oligo dT, 20% formamide, 50 mM KCl, 20 μg / mL bovine serum albumin, 20 μg / mL yeast tRNA and 1 U / μL RNase inhibitor were added to Ampligase buffer, and the mixture was treated at room temperature for 30 minutes). In situ hybridization was performed using the designed padlock probe set. Padlock probes were added to Ampligase buffer to achieve a final concentration of 200 nM / μL for each padlock probe, along with 20% formamide, 50 mM KCl, 20 μg / mL bovine serum albumin, 20 μg / mL yeast tRNA, and 1 U / μL RNase inhibitor. The mixture was then treated at 45°C for 24 hours (using a shaker).

[0127] The sample was washed with 10% formamide and 2×SSC solution for 20 minutes, repeated three times, followed by three washes with PBST. The ligation reaction was then performed using the following reaction mixture: SplintR buffer, 20 μg / mL bovine serum albumin, 1 U / μL RNase inhibitor, and 2.5 U / μL SplintR ligase. The ligation reaction was completed overnight at 37°C, followed by two washes with PBST.

[0128] RCA amplification was then performed using the following reaction system: Phi29 polymerase buffer, 0.25 U / μL Phi29 polymerase, 250 μM dNTPs, 50 μM aminodUTPs, 10% glycerol, 20 μg / mL bovine serum albumin, and 600 nM amplification primers. Amplification was carried out at 30°C for 24 hours. After amplification, the sample was washed twice with PBST, fixed with 10 μg / μL BS(PEG)9, and washed three times with 65% formamide.

[0129] The amplification products were stained and the first round of imaging was performed using 16 pre-mixed decoding probes (4 channels x 4 states). Each target mRNA sequence in the test sample formed a signal spot. The fluorescent probe design was the same as in Example 1.

[0130] The above experiments were performed on the entire brain slice. During three-dimensional imaging using confocal or other methods, the brain region of interest was located and imaged via pre-scanning. The subsequent imaging-UV processing-imaging and decoding process remained the same as in Example 1. After decoding, the three-dimensional gene distribution was obtained. (Refer to...) Figure 12 This image shows a portion of the gene decoding results from a 3D thick brain slice; each image is a single gene decoding map.

[0131] This application discloses a fluidless multiplex nucleic acid in situ detection method that can encode hundreds of genetic diseases for in situ nucleic acid detection without relying on special equipment or fluids. By introducing sham rounds through fluorescence resonance energy transfer (FRET) and light-controlled conversion, hundreds of genes can be encoded without fluids or color intensity grading, achieving a gene detection capacity similar to in situ sequencing. This method addresses the challenge of simultaneously achieving high throughput and high gene detection capacity in three dimensions, further expanding the application scope and scenarios of high-resolution nucleic acid in situ imaging technology.

[0132] Furthermore, the fluidless virtual in situ sequencing technology provided in this application can encode hundreds of different genes using only four optical channels of a conventional fluorescence microscope without relying on fluid, which can effectively solve the problems of accessibility and three-dimensional compatibility caused by multi-round fluid.

[0133] Secondly, this application discloses a fluidless multiplex nucleic acid in situ detection kit, including a padlock probe set and several types of fluorescent decoding probes, wherein the padlock probe set includes multiple padlock probes.

[0134] The padlock probe includes two terminal sequences and a coding sequence located between the two terminal sequences. The terminal sequences correspond to a type of gene information, and the coding sequence includes N segment sequences. Each segment sequence corresponds to a fluorescence channel, and each fluorescence channel is used to bind M fluorescent decoding probes with different fluorescent modification states; N≥3, M≥3.

[0135] Among them, all fluorescent decoding probes have different fluorescent modification states, and at least one type of fluorescent decoding probe has a photosensitive group.

[0136] It should be noted that the specific applications of the padlock probe set and several types of fluorescent decoding probes in this embodiment are detailed in the fluidless multiplex nucleic acid in situ detection method disclosed in the first aspect of this application, and will not be repeated here.

[0137] For a detailed description of this embodiment, please refer to the corresponding descriptions in the foregoing embodiments, which will not be repeated here.

[0138] The basic principles of this disclosure have been described above with reference to specific embodiments. However, it should be noted that the advantages, benefits, and effects mentioned in this disclosure are merely examples and not limitations, and should not be considered as essential features of each embodiment of this disclosure. Furthermore, the specific details disclosed above are for illustrative and facilitative purposes only, and are not limitations. These details do not limit the scope of this disclosure to the necessity of employing the aforementioned specific details for implementation.

[0139] In this disclosure, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. The block diagrams of devices, apparatuses, devices, and systems involved in this disclosure are merely illustrative examples and are not intended to require or imply that they must be connected, arranged, or configured in the manner shown in the block diagrams. As those skilled in the art will recognize, these devices, apparatuses, devices, and systems can be connected, arranged, and configured in any manner. Words such as "comprising," "including," "having," etc., are open-ended terms meaning "including but not limited to," and are used interchangeably with them. The terms "or" and "and" as used herein refer to the terms "and / or," and are used interchangeably with them unless the context clearly indicates otherwise. The term "such as" as used herein refers to the phrase "such as but not limited to," and is used interchangeably with it.

[0140] It should also be noted that in the systems and methods of this disclosure, the components or steps can be decomposed and / or recombined. These decompositions and / or recombinations should be considered as equivalent solutions to this disclosure.

[0141] Various changes, substitutions, and modifications can be made to the technology described herein without departing from the teachings defined by the appended claims. Furthermore, the scope of the claims of this disclosure is not limited to the specific aspects of the processes, machines, manufactures, events, means, methods, and actions described above. Currently existing or later-developed processes, machines, manufactures, events, means, methods, or actions that perform substantially the same function or achieve substantially the same result as the corresponding aspects described herein can be utilized. Therefore, the appended claims include such processes, machines, manufactures, events, means, methods, or actions within their scope.

[0142] The above description of the disclosed aspects is provided to enable any person skilled in the art to make or use this disclosure. Various modifications to these aspects will be readily apparent to those skilled in the art, and the general principles defined herein can be applied to other aspects without departing from the scope of this disclosure. Therefore, this disclosure is not intended to be limited to the aspects shown herein, but rather to be carried out within the widest scope consistent with the principles and novel features disclosed herein.

[0143] The above description has been given for purposes of illustration and description. Furthermore, this description is not intended to limit the embodiments of this disclosure to the forms disclosed herein. Although numerous exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize certain variations, modifications, alterations, additions, and sub-combinations therein.

Claims

1. A fluidless in-situ multiplex nucleic acid detection method, characterized in that, include: The target sample is stained using several pre-configured types of fluorescent decoding probes; wherein the fluorescent modification states of the several types of fluorescent decoding probes are different and at least one type of fluorescent decoding probe has a photosensitive group; the target sample is obtained by amplifying the sample to be tested. The stained target sample was subjected to a first fluorescence imaging to obtain a primary imaging image. The stained target sample is irradiated with light, causing the photosensitive group to respond and its fluorescence properties to change. A second fluorescence imaging process is performed on the irradiated target sample to obtain a secondary imaging image; Based on the primary imaging image and the secondary imaging image, determine the intensity information of all signal points; The intensity information is decoded to obtain the gene distribution information of the sample to be tested.

2. The fluidless in-situ multiplex nucleic acid detection method according to claim 1, characterized in that, The method of staining the target sample using several pre-configured types of fluorescent decoding probes includes: Determine the number of fluorescent decoding probe types and the sequence information of the padlock probe; The padlock probe includes two end sequences and a coding sequence located between the two end sequences. The coding sequence includes N segment sequences, each segment sequence corresponding to a fluorescence channel. Each fluorescence channel is used to bind M fluorescent decoding probes with different fluorescent modification states; where N≥3 and M≥3. Based on the number of species and the number of segment sequences, determine the encoding capacity of the padlock probe set and the number of fluorescent decoding probes to be synthesized; The padlock probe set is synthesized based on the encoded capacity; Prepare fluorescent decoding probes according to the quantity to be synthesized; The sample to be tested is pretreated, and the sample is subjected to in situ hybridization using the padlock probe set and amplification reaction to obtain the target sample. The target sample is then stained using the fluorescent decoding probe.

3. The fluidless in-situ multiplex nucleic acid detection method according to claim 2, characterized in that, The photosensitive group has a photolytic bond; The process of irradiating the stained target sample with light to cause a change in the fluorescence properties of the photosensitive group after the photosensitive group responds includes: The stained target sample is irradiated with ultraviolet light to break the photolytic bonds, thereby altering the fluorescence imaging state of the photosensitive group.

4. The fluidless in-situ multiplex nucleic acid detection method according to claim 3, characterized in that, The pre-configured types of fluorescent decoding probes include probes modified with fluorescent groups, probes without fluorescent group modification, probes modified with fluorescent groups linked by photolytic bonds, and probes with fluorescent groups modified with FRET groups and photolytic bonds.

5. The fluidless in-situ multiplex nucleic acid detection method according to claim 4, characterized in that, The method for preparing the probe modified with a fluorescent group linked by the photolytically cleaved bond includes: The type of probe body is determined based on the number of types of fluorescent decoding probes; Prepare probe bodies of the corresponding types respectively; Each type of probe body is connected to a fluorophore of the corresponding fluorescence channel using a selected photolytic bond to obtain a probe modified with a fluorophore connected by a photolytic bond.

6. The fluidless in-situ multiplex nucleic acid detection method according to claim 4, characterized in that, The method for preparing the FRET group and photolytic bond modified probe with a fluorescent group includes: The type of probe body is determined based on the number of types of fluorescent decoding probes; Prepare probe bodies of the corresponding types respectively; Identify the photolytic bonds and the FRET groups corresponding to each fluorescence channel; A fluorescent group corresponding to the fluorescent channel is attached to a base at a predetermined position on the probe body; The FRET group is attached to the end of the probe body using the photolytic bond to obtain a probe with a fluorescent group modified by the FRET group and the photolytic bond.

7. The fluidless in-situ multiplex nucleic acid detection method according to claim 1, characterized in that, The step of determining the intensity information of all signal points based on the primary imaging image and the secondary imaging image includes: Align all the secondary imaging images with all the primary imaging images; Signal point enhancement is performed on all aligned images; The locations of the enhanced signal points are extracted to obtain the location and intensity information of all signal points; Based on the location of each signal point, the intensity information of signals corresponding to the same location in all imaging images is merged to obtain the intensity information of all signal points.

8. The fluidless in-situ multiplex nucleic acid detection method according to claim 1, characterized in that, Decoding the intensity information to obtain the gene distribution information of the sample to be tested includes: The intensity information is analyzed for the first time using an intensity threshold to determine the first barcode sequence corresponding to the position of each signal point; The intensity information of each signal point is analyzed a second time using vector projection to obtain the second barcode sequence; Determine the sequence difference information between the first barcode sequence and the second barcode sequence corresponding to the position of each signal point; If the sequence difference information is within a preset range, it is recorded as a valid signal point. The gene type information corresponding to the second barcode sequence is used as the gene type information corresponding to the effective signal point; Based on the gene type information of all the effective signal points, the gene distribution information of the sample to be tested is obtained.

9. The fluidless in-situ multiplex nucleic acid detection method according to claim 1, characterized in that, Decoding the intensity information to obtain the gene distribution information of the sample to be tested includes: The intensity information is analyzed by intensity thresholds to determine the binary state of each signal point in the first fluorescence imaging and the binary state of each signal point in the second imaging. The barcode sequence is determined based on the binary state of the two images; The gene type information corresponding to the barcode sequence is used as the gene type information of the corresponding signal point; Based on the gene type information of all signal points, the gene distribution information of the sample to be tested is obtained.

10. A fluidless multiplex nucleic acid in situ detection kit, characterized in that, It includes a padlock probe set and several types of fluorescent decoding probes, wherein the padlock probe set includes multiple padlock probes; The padlock probe includes two end sequences and a coding sequence located between the two end sequences. The coding sequence includes N segment sequences, each segment sequence corresponding to a fluorescence channel. Each fluorescence channel is used to bind M fluorescent decoding probes with different fluorescent modification states; where N≥3 and M≥3. All of the fluorescent decoding probes have different fluorescent modification states, and at least one type of fluorescent decoding probe has a photosensitive group.

Images