Nucleic acid detection method and use thereof

Abstract

Provided are a nucleic acid detection primer combination or kit, a nucleic acid hybridization method, a method for detecting a target nucleic acid, and a method for joint detection of a nucleic acid and a protein. By using the primer combination and the detection method, the incubation time for fluorescence reading / detecting a hybridization signal for a nucleic acid (such as RNA) can be shortened, ensuring that after hybridization of a nucleic acid molecular signal, multiple rounds of protein staining can still be carried out on a sample, i.e. an RNA-protein joint detection method, which provides two types of evidence for staining a target site by means of detection results.

Description

Nucleic acid detection methods and their applications Technical Field

[0001] The present invention relates to the field of nucleic acid hybridization technology, and in particular to a nucleic acid detection method and a combined detection method thereof with protein detection. Background Art

[0002] Fluorescence digital pathology microscopy is a key technology in the life sciences. By combining state-of-the-art omics methods with fluorescence microscopy and digitized parameters, biological samples (such as tissue sections) can be visualized digitally in millions of pixels, displaying tens of thousands of features.

[0003] A rapid, multiplexed digital pathology immunofluorescence instrument has been developed. Based on a desktop series of sequencers, it now supports tissue protein fluorescence staining and imaging, in addition to basic sequencing capabilities. Currently, this instrument focuses on multiple rounds of protein staining and photography on tissue sections. However, in potential future digital pathology diagnostic applications, multiple rounds of protein staining can only be considered a single diagnostic basis, leaving significant room for improvement. Therefore, adding the capture and identification of RNA hybridization signals at the target site to multiple rounds of protein staining can further enhance the accuracy of digital pathology diagnoses.

[0004] However, the existing multiple immunofluorescence technology needs to block the sample at room temperature, and each round of antibody elution needs to be re-blocked, which increases the staining time. Traditional immunofluorescence staining takes a long time, about 210 minutes, and the traditional staining method lacks a multiple immunofluorescence detection method for a single slice. The iterative indirect immunofluorescence imaging method is an improved version of the traditional method. After imaging, an antibody eluent (containing TCEP, urea, guanidine hydrochloride, Tris-HCl) is added for 30 minutes of antibody elution and then the staining step is repeated. However, the viscosity of the antibody eluent is too high, which can easily cause tissue shedding and serious damage. And due to the signal-to-noise ratio problem, the existing technology needs to be stained for a long time to reduce the signal-to-noise ratio in order to achieve the desired effect.

[0005] Therefore, there is an urgent need to develop a method that can significantly shorten the incubation time for fluorescent reading / detection of RNA hybridization signals, and ensure that after the RNA molecular signal hybridization, the RNA probe fluorescent substance is eluted to continue to achieve multiple rounds of protein staining on the tissue, that is, the RNA-protein combined detection method, so as to provide double evidence for the staining of the target through the detection results.

[0006] Summary of the Invention

[0007] The present invention is intended to solve one of the technical problems in the related art at least to a certain extent.For this reason, an object of the present invention is to provide a nucleic acid detection primer combination or suit, a nucleic acid hybridization method, a method for detecting a target nucleic acid, and a method for nucleic acid, protein joint detection (joint detection) etc. Utilizing the primer combination and detection method of the present invention, the incubation time of fluorescence reading / detection nucleic acid (such as RNA) hybridization signal can be greatly shortened, and it is ensured that after the nucleic acid molecule signal hybridization, multiple rounds of protein staining are continuously realized on the sample (such as tissue), i.e., a nucleic acid (such as RNA)-protein joint detection method, which can provide dual evidence of nucleic acid level and protein level to the target target staining by the detection result.

[0008] To this end, the first aspect of the present invention provides a primer combination, the primer combination comprising:

[0009] (1) one or more first primers, wherein the first primers include: a fourth region that pairs and binds to a fragment of a target nucleic acid, and at least one first region that does not pair and bind to the target nucleic acid;

[0010] (2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid;

[0011] (3) A third primer, comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid.

[0012] The present invention utilizes the described tertiary primer combination to ensure that the three primers have complementary binding domains. Furthermore, under the action of a ligase, the primer sequences at each level can be looped in a timely manner, further enabling tight binding and exponential amplification of the three primers. This unique primer sequence design, based on the tertiary primer hybridization logic, allows for the targeted capture of hybridization signals, significantly shortening the incubation time for fluorescent reading / detection of target nucleic acid hybridization signals. For example, whereas traditional fluorescence in situ hybridization techniques require overnight incubation at 37°C in a humid environment, a similarly efficient reaction can now be completed in only about 90 minutes.

[0013] The primer combination of the present invention can be applied to in situ hybridization. After the probe fluorescent substance is eluted, multiple rounds of protein staining can be continued on the tissue to provide double evidence for the staining of the target point.

[0014] According to an embodiment of the present invention, the first primer includes: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that do not pair and bind to a sequence of the target nucleic acid.

[0015] According to an embodiment of the present invention, the second region does not pair-bind with the first region.

[0016] According to an embodiment of the present invention, the third region does not pair-bind with the second region.

[0017] According to an embodiment of the present invention, the fourth region includes a first fragment and a second fragment, the first fragment and the second fragment are respectively located at the two ends of the first primer, and the first region is located between the first fragment and the second fragment; each first region includes at least a third fragment and a fourth fragment; the fifth region includes a fifth fragment and a sixth fragment, the fifth fragment and the sixth fragment are respectively located at the two ends of the second primer, and the second region is located between the fifth fragment and the sixth fragment; each second region includes at least a seventh fragment and an eighth fragment; the sixth region includes a ninth fragment and a tenth fragment, the ninth fragment and the tenth fragment are respectively located at the two ends of the third primer, and the third region is located between the ninth fragment and the tenth fragment; each third region includes at least the third fragment and the fourth fragment.

[0018] According to an embodiment of the present invention, the primer combination includes a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid.

[0019] According to an embodiment of the present invention, the first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the target nucleic acid fragment to which they are paired and bound.

[0020] According to an embodiment of the present invention, the lengths of the first fragment and the second fragment are each independently 15-20 nt.

[0021] According to an embodiment of the present invention, the lengths of the third fragment and the fourth fragment are each independently 8-20 nt, preferably 10-16 nt.

[0022] According to an embodiment of the present invention, the lengths of the fifth fragment and the sixth fragment are each independently 8-20 nt, preferably 10-16 nt.

[0023] According to an embodiment of the present invention, the lengths of the seventh fragment and the eighth fragment are each independently 8-20 nt, preferably 10-16 nt.

[0024] According to an embodiment of the present invention, the lengths of the ninth fragment and the tenth fragment are each independently 8-20 nt, preferably 10-16 nt.

[0025] According to an embodiment of the present invention, the first primer includes more than one first region, and the first regions are connected by a first spacer sequence.

[0026] According to an embodiment of the present invention, in the second primer, each of the second regions is connected by a second spacer sequence.

[0027] According to an embodiment of the present invention, in the third primer, each of the third regions is connected by a third spacer sequence.

[0028] According to an embodiment of the present invention, the length of the first spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.

[0029] According to an embodiment of the present invention, the length of the second spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.

[0030] According to an embodiment of the present invention, the length of the third spacer sequence is at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt.

[0031] The second aspect of the present invention provides a kit, which comprises: the primer combination described in the first aspect, and a first detection probe and / or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of the region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.

[0032] According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.

[0033] According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.

[0034] According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.

[0035] According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

[0036] According to an embodiment of the present invention, the label comprises a radioactive label or a non-radioactive label.

[0037] According to an embodiment of the present invention, the non-radioactive label comprises a fluorescent group.

[0038] A third aspect of the present invention provides a nucleic acid hybridization method, comprising the following steps:

[0039] S1. contacting a biological sample containing a target nucleic acid with one or more first primers in the primer combination described in the first aspect, wherein the fourth region of the one or more first primers pairs and binds to a fragment of the target nucleic acid to obtain a target nucleic acid-first primer hybrid 1;

[0040] S2. contacting the product obtained in step S1 with the second primer in the primer combination described in the first aspect, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain a target nucleic acid-first primer-second primer hybrid 2;

[0041] Optionally, S3. the product obtained in step S2 is contacted with the third primer in the primer combination described in the first aspect, and the sixth region of the third primer is paired and bound with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3.

[0042] According to an embodiment of the present invention, the method further comprises: the contacting in steps S1-S3 is performed in a hybridization reaction solution containing ethylene carbonate, and the concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v / v.

[0043] According to an embodiment of the present invention, the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

[0044] According to an embodiment of the present invention, the method further includes: before performing step S2, using a ligase to connect the first primer present in the system after the reaction of step S1 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess first primers that are not bound to the target nucleic acid.

[0045] According to an embodiment of the present invention, the method further includes: before performing step S3, using a ligase to connect the second primer present in the system after the reaction of step S2 is completed to form a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess second primers that are not bound to the first primer.

[0046] According to an embodiment of the present invention, the method further includes: after performing step S3, using a ligase to connect the third primer present in the system after the reaction of step S3 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess third primers that are not bound to the second primer.

[0047] According to an embodiment of the present invention, the method further comprises: performing a ligation reaction using the ligase at a reaction temperature of 30-40° C. and a reaction time of 5-30 min.

[0048] According to an embodiment of the present invention, the ligase is T4 ligase.

[0049] According to an embodiment of the present invention, the cleaning solution comprises ethylene carbonate, and the concentration of ethylene carbonate in the cleaning solution is 5-40% v / v.

[0050] According to an embodiment of the present invention, the cleaning solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

[0051] According to an embodiment of the present invention, the reaction temperature for pairing and binding in step S1 is 30-40° C., and the reaction time is 30-120 min.

[0052] According to an embodiment of the present invention, the reaction temperature for the pairing binding in step S2 is 30-40° C., and the reaction time is 20-40 min.

[0053] According to an embodiment of the present invention, the reaction temperature for the pairing binding in step S3 is 30-40° C., and the reaction time is 20-40 min.

[0054] According to an embodiment of the present invention, the method further comprises:

[0055] Ⅰ. After step S3, continue to repeat steps S2-S3 for at least N rounds, where N≥1 and N is an integer; or,

[0056] II. After step S3, continue to repeat steps S2-S3 for at least N' rounds, N'≥0, N' is an integer, and only repeat step S2 in the last round.

[0057] The fourth aspect of the present invention provides a method for detecting a target nucleic acid, the method comprising: using the nucleic acid hybridization method described in the third aspect to obtain a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer, and detecting the hybrid using a first detection probe and / or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of a region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of a region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.

[0058] According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.

[0059] According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.

[0060] According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.

[0061] According to an embodiment of the present invention, the second detection probe comprises a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer. According to an embodiment of the present invention, the target nucleic acid is single-stranded DNA or RNA.

[0062] According to an embodiment of the present invention, the method further comprises: after step S2, detecting a hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe.

[0063] According to an embodiment of the present invention, the method further includes: after step S3, continuing to repeat steps S2-S3 for at least N rounds, detecting the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer with the second detection probe, and optionally, using the first detection probe for auxiliary detection, where N is a natural number.

[0064] According to another embodiment of the present invention, after step S3, steps S2-S3 are repeated for at least N' rounds, and in the last round, only step S2 is repeated, then the first detection probe is used to detect the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer, and optionally, the second detection probe is used for auxiliary detection, wherein N' is a positive integer.

[0065] According to an embodiment of the present invention, the lengths of the first and second detection probes are independently 13-23 nt.

[0066] A fifth aspect of the present invention provides a method for combined detection of nucleic acids and proteins, comprising the following steps:

[0067] 1) performing nucleic acid detection on a biological sample containing nucleic acid using the method for detecting target nucleic acid described in the fourth aspect; and

[0068] 2) performing protein fluorescence staining and imaging on the biological sample.

[0069] According to an embodiment of the present invention, the protein fluorescent staining comprises performing multiple rounds of protein staining using protein markers (eg, antibodies).

[0070] The sixth aspect of the present invention provides the use of the primer combination described in the first aspect and the kit described in the second aspect in the following:

[0071] a. Nucleic acid in situ hybridization;

[0072] b. Combined detection of nucleic acid and protein;

[0073] c. Organizational map construction;

[0074] d. Determine the distribution of cells in tissues;

[0075] e. Digital pathology imaging and analysis;

[0076] f. Biomarker screening and discovery.

[0077] According to an embodiment of the present invention, in the combined detection of nucleic acids and proteins, the biological sample to be tested is preferably fixed on a solid support. According to an embodiment of the present invention, the biological sample to be tested is a tissue / cell section sample.

[0078] This invention improves upon traditional in situ hybridization staining methods, significantly increasing staining efficiency and shortening staining time. It is also applicable to conventional manual staining systems, improving tissue section staining efficiency, reducing operator workload, and shortening working hours. This method has a wide range of potential applications, including tissue mapping, RNA in situ hybridization, and aptamer staining. It can be used to construct maps of plant and animal cell distribution, tumors, and diseases, as well as for digital pathology and biomarker screening and discovery.

[0079] The inventors used the primer combination described above to perform fluorescence in situ hybridization, aiming to increase the capture and identification of nucleic acid molecule hybridization signals of the target target under the premise of multiple rounds of protein staining in the fast and multiple digital pathology immunofluorescence instrument, so as to realize the joint detection of nucleic acids and proteins on the machine. The experimental design includes tissue section preparation (dewaxing and hydration antigen repair), nucleic acid hybridization molecule sequence design and preparation, nucleic acid hybridization process reagent preparation, protein multiple rounds of staining reagent preparation, and integrated automatic staining and shooting of the entire process. Before the original multiple rounds of protein staining, on the basis of the automatic multiple rounds of staining of the fast and multiple digital pathology immunofluorescence instrument, the multiple rounds of hybridization of nucleic acid molecules in tissue sections, amplification, fluorescence signal reading, and fluorescence signal elution are added to achieve the effect of joint detection of nucleic acids and proteins.

[0080] The technical solution provided by the present invention has the following beneficial effects:

[0081] (1) Compared with traditional fluorescence in situ hybridization technology, which requires overnight incubation at 37°C in a humid environment for primers to hybridize with target sequences, the first primer in the primer combination of the present invention (hereinafter referred to as primer #1) can complete a similar efficient reaction in just 90 minutes;

[0082] (2) In traditional hybridization technology, fluorescent probes need to be customized for each target. This method only requires changing the #1 hybridization primer, without the need to customize the fluorescent probe. For different target sequences, the second primer (hereinafter also referred to as the #2 primer) and the third primer (hereinafter also referred to as the #3 primer) are universal, which can greatly reduce costs.

[0083] (3) Traditional fluorescence in situ hybridization technology is limited to capturing hybridization signals at the RNA / DNA molecular level, and cannot increase the capture of target gene protein signals on a molecular basis;

[0084] (4) The primer combination provided in the present invention is applied to the joint detection of nucleic acids and proteins in biological samples (such as tissue sections). The nucleic acid and protein automated dyeing and shooting machine ensures the repeatability of position information for background correction and rapid antibody incubation. The nucleic acid and protein joint detection in the present invention can be completely formed into an automated dyeing and shooting platform through a sequencer.

[0085] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0087] FIG1 is a schematic diagram showing the amplification process when performing RNA fluorescence detection using a first primer, a second primer, and a third primer;

[0088] FIG2 shows the comparative results of fluorescence in situ hybridization of the target CD8 mRNA under different reaction conditions in Example 1, wherein A. fluorescence in situ hybridization results under different read probe concentrations; B. statistical results of fluorescence in situ hybridization under different read probe concentrations;

[0089] FIG3 shows the comparative results of fluorescence in situ hybridization of target CD8 mRNA in Example 2 in a 10% ethylene carbonate reaction system (left) and a 10% polyvinylamine reaction system (right);

[0090] FIG4 shows the results of tissue protein fluorescence staining of the target proteins PD-L1 and CD8 protein in Example 3.

[0091] Detailed Description of the Invention

[0092] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0093] Conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F M Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL METHOD. APPROACH) (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), ANTIBODIES: A LABORATORY MANUAL (Harlow and Lane, eds. (1988)), and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0094] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0095] The endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0096] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0097] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0098] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0099] Herein, the term "primer" refers to an artificially synthesized oligonucleotide sequence that is complementary to at least a portion of a template strand of a target region. The primer may be a single-stranded DNA primer or an RNA primer.

[0100] According to a specific embodiment of the present invention, the present invention provides a primer combination comprising:

[0101] (1) one or more first primers, wherein the first primers include: a fourth region that pairs and binds to a fragment of a target nucleic acid, and at least one first region that does not pair and bind to the target nucleic acid;

[0102] (2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid;

[0103] (3) A third primer, comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid.

[0104] It should be noted that the first primer in the primer combination of the present invention can be designed as a plurality of different first primer sequences, and these different sequences can be complementary paired with different segments of the target nucleic acid, respectively. After the first primer binds to the target nucleic acid, it further hybridizes with the second primer and the third primer to form multiple target nucleic acid-primer hybrids.

[0105] The primer combination of the present invention can be applied to in situ hybridization. After the probe fluorescent substance is eluted, multiple rounds of protein staining can be continued on the tissue to provide double evidence for the staining of the target point.

[0106] According to a specific embodiment of the present invention, the first primer includes: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that do not pair and bind to a sequence of the target nucleic acid.

[0107] According to a specific embodiment of the present invention, the second region does not pair with the first region; and the third region does not pair with the second region.

[0108] According to a specific embodiment of the present invention, in the primer combination of the present invention, it is ensured that the second region does not pair and bind with the first region. Such primer design can ensure that the fifth region of the second primer and the second region do not competitively bind with the first region of the first primer.

[0109] According to a specific embodiment of the present invention, in the primer combination of the present invention, it is ensured that the third region does not pair and bind with the second region. Such primer design can ensure that the third region and the sixth region of the third primer do not competitively bind with the second region of the second primer.

[0110] According to a specific embodiment of the present invention, the fourth region includes a first fragment and a second fragment, the first fragment and the second fragment are respectively located at the two ends of the first primer, and the first region is located between the first fragment and the second fragment; each first region includes at least a third fragment and a fourth fragment; the fifth region includes a fifth fragment and a sixth fragment, the fifth fragment and the sixth fragment are respectively located at the two ends of the second primer, and the second region is located between the fifth fragment and the sixth fragment; each second region includes at least a seventh fragment and an eighth fragment; the sixth region includes a ninth fragment and a tenth fragment, the ninth fragment and the tenth fragment are respectively located at the two ends of the third primer, and the third region is located between the ninth fragment and the tenth fragment; each third region includes at least the third fragment and the fourth fragment.

[0111] According to a specific embodiment of the present invention, the primer combination includes a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid.

[0112] The primer combination provided by the present invention, when designed to include: a first segment and a second segment complementary to segments of different target nucleic acids, and at least two first regions that do not bind to the sequence of the target nucleic acids, can be used to simultaneously detect multiple target nucleic acids. According to an embodiment of the present invention, the first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the target nucleic acid segments to which they bind.

[0113] The first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the target nucleic acid fragment with which they are paired and bound. This means that when the first primer is paired and bound to the target nucleic acid fragment, the 5' end and 3' end of the first primer are paired and bound to the target nucleic acid fragment respectively, and the middle segment (first region) is not bound. The first primer forms a ring structure, and the first base at the 5' end of the first primer is adjacent to the last base at the 3' end, which facilitates subsequent ligation into a ring by ligase.

[0114] It should be noted that the "at least two" mentioned in the present invention refers to more than two, including three, four, five and more. According to a specific embodiment of the present invention, the primer combination provided by the present invention includes:

[0115] (1) one or more first primers, wherein the first primers include: a fourth region that binds to a target nucleic acid fragment, and two first regions that do not bind to the target nucleic acid;

[0116] (2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and two second regions that do not pair and bind to the target nucleic acid;

[0117] (3) A third primer, comprising: a sixth region that pairs and binds to the second region, and two third regions that do not pair and bind to the target nucleic acid.

[0118] According to an embodiment of the present invention, the length of the first fragment and the second fragment are each independently 15-20 nt. For example, the length of the first fragment and the second fragment are each independently 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt.

[0119] According to an embodiment of the present invention, the length of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment is each independently 8-20 nt, preferably 10-16 nt. For example, the length of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment is each independently 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt or 16 nt.

[0120] According to an embodiment of the present invention, the first primer includes more than one first region, and the first regions are connected by a first spacer sequence. The provision of the first spacer sequence makes it easier for the second primer to bind to the first primer.

[0121] According to an embodiment of the present invention, in the second primer, each of the second regions is connected by a second spacer sequence. The provision of the second spacer sequence makes it easier for the third primer to bind to the second primer.

[0122] According to an embodiment of the present invention, in the third primer, each of the third regions is connected by a third spacer sequence. The provision of the third spacer sequence makes it easier for the second primer in the next round to bind to the third primer.

[0123] According to an embodiment of the present invention, the length of the first spacer sequence, the second spacer sequence and the third spacer sequence is independently at least 1 nt, preferably 1-30 nt, and more preferably 2-5 nt. For example, the length of the first spacer sequence, the second spacer sequence and the third spacer sequence is each independently 2 nt, 3 nt, 4 nt or 5 nt.

[0124] The present invention also provides a kit comprising: the primer combination described above, and a first detection probe and / or a second detection probe; wherein the first detection probe includes a sequence that pairs and binds to at least a portion of the region of the second primer that is not paired with the first primer, the second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that is not paired with the second primer, and the first detection probe and the second detection probe contain a marker that can detect a signal.

[0125] According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer.

[0126] According to an embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.

[0127] According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer.

[0128] According to an embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

[0129] According to an embodiment of the present invention, the label comprises a radioactive label or a non-radioactive label. According to an embodiment of the present invention, the non-radioactive label comprises a fluorescent group.

[0130] The present invention also provides a nucleic acid hybridization method, comprising:

[0131] S1. contacting a biological sample containing a target nucleic acid with one or more first primers from the primer combination described above, wherein the fourth region of the one or more first primers pairs and binds to a fragment of the target nucleic acid to obtain a target nucleic acid-first primer hybrid 1;

[0132] S2. contacting the product obtained in step S1 with the second primer in the aforementioned primer combination, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain target nucleic acid-first primer-second primer hybrid 2;

[0133] Optionally, S3. the product obtained in step S2 is contacted with the third primer in the primer combination described above, and the sixth region of the third primer is paired and bound with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3.

[0134] According to a specific embodiment of the present invention, the nucleic acid hybridization method further comprises: the contacting in steps S1-S3 is carried out in a hybridization reaction solution containing ethylene carbonate, the concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v / v, preferably, the concentration of ethylene carbonate in the hybridization reaction solution is 10% v / v.

[0135] According to a specific embodiment of the present invention, the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

[0136] According to a specific embodiment of the present invention, the hybridization reaction solution is 10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, and the solvent is 1X SSC.

[0137] According to a specific embodiment of the present invention, the nucleic acid hybridization method further includes: before performing step S2, using a ligase to connect the first primer present in the system after the reaction of step S1 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess first primers that are not bound to the target nucleic acid.

[0138] According to an embodiment of the present invention, the nucleic acid hybridization method further includes: before performing step S3, using a ligase to connect the second primer present in the system after the reaction of step S2 is completed into a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess second primers that are not bound to the first primer.

[0139] According to a specific embodiment of the present invention, the nucleic acid hybridization method further includes: after performing step S3, using a ligase to connect the third primer present in the system after the reaction of step S3 is completed to form a ring, and after the ring formation reaction is completed, using a cleaning solution to perform an elution treatment to remove excess third primers that are not bound to the second primer.

[0140] It should be noted that when ligating primers into a ring using a ligase, there are no particular restrictions on the temperature and time of the ligation reaction. The reaction is preferably carried out at a temperature suitable for the ligase, which may fluctuate above or below the optimal temperature for ligation. The ligation time only needs to ensure that all single-stranded primers are ligated into a ring.

[0141] According to a specific embodiment of the present invention, the nucleic acid hybridization method further comprises: using the ligase to perform a ligation reaction at a reaction temperature of 30-40° C. and a reaction time of 5-30 minutes. Preferably, the reaction temperature is 37° C. and the reaction time is 20 minutes.

[0142] According to a specific embodiment of the present invention, the ligase is T4 ligase. It should be noted that the type of ligase used in the nucleic acid hybridization method of the present invention is not limited to T4 ligase, and other types of ligases can also be used as long as they can ensure end-to-end ligation of the 5' and 3' ends of the primers.

[0143] According to a specific embodiment of the present invention, the cleaning solution comprises ethylene carbonate, and the concentration of the ethylene carbonate in the cleaning solution is 5-40% v / v.

[0144] According to a specific embodiment of the present invention, the cleaning solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

[0145] According to a specific embodiment of the present invention, the reaction temperature for the pairing binding in step S1 is 30-40° C., and the reaction time is 30-120 min.

[0146] The first hybridization primer of the traditional fluorescence in situ hybridization technique needs to be incubated overnight in a humid environment at 37°C, while the hybridization method of the present invention only takes about 30-120 minutes to complete a high-efficiency reaction with similar effects.

[0147] According to a specific embodiment of the present invention, the reaction temperature for the pairing binding in step S2 is 30-40° C., and the reaction time is 20-40 min.

[0148] According to a specific embodiment of the present invention, the reaction temperature for the pairing binding in step S3 is 30-40° C., and the reaction time is 20-40 min.

[0149] According to a specific embodiment of the present invention, the nucleic acid hybridization method further comprises:

[0150] Ⅰ. After step S3, continue to repeat steps S2-S3 for at least N rounds, where N≥1 and N is an integer; or,

[0151] II. After step S3, continue to repeat steps S2-S3 for at least N' rounds, N'≥0, N' is an integer, and only repeat step S2 in the last round.

[0152] The present invention provides a method for detecting a target nucleic acid, comprising: using the aforementioned nucleic acid hybridization method to obtain a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer; and detecting the hybrid using a first detection probe and / or a second detection probe, wherein the first detection probe includes a sequence that pairs and binds to at least a portion of a region of the second primer that is not paired with the first primer; the second detection probe includes a sequence that pairs and binds to at least a portion of a region of the third primer that is not paired with the second primer; and the first detection probe and the second detection probe contain markers that can detect signals.

[0153] According to a specific embodiment of the present invention, the first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer; the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.

[0154] According to a specific embodiment of the present invention, the second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer; the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

[0155] According to a specific embodiment of the present invention, the method further comprises:

[0156] i. After step S2, detecting the hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe; or

[0157] ii. if steps S2-S3 are repeated for at least N rounds after step S3, the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer is detected with the second detection probe, and optionally, auxiliary detection is performed using the first detection probe, where N is a natural number; or

[0158] ii. After step S3, continue to repeat steps S2-S3 for at least N' rounds, and in the last round, only repeat step S2, then use the first detection probe to detect the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer, and optionally, use the second detection probe for auxiliary detection, wherein N' is a positive integer.

[0159] According to a specific embodiment of the present invention, the lengths of the first and second detection probes are independently 13-23 nt.

[0160] The present invention also provides a method for combined detection of nucleic acids and proteins, comprising:

[0161] 1) performing nucleic acid detection on a biological sample containing nucleic acid using the aforementioned method for detecting target nucleic acid; and

[0162] 2) performing protein fluorescence staining and imaging on the biological sample.

[0163] According to a specific embodiment of the present invention, the protein fluorescent staining comprises multiple rounds of protein staining using antibodies.

[0164] According to an embodiment of the present invention, when performing a combined nucleic acid and protein detection, the biological sample containing nucleic acid is preferably fixed on a solid support. According to a specific embodiment of the present invention, the biological sample is a tissue / cell section sample.

[0165] According to a specific embodiment of the present invention, the present invention provides a nucleic acid detection primer combination, which includes:

[0166] (1) A first primer comprising: a. two pairing regions complementary to the target nucleic acid sequence to be detected; and b. a non-pairing region non-complementary to the target nucleic acid sequence to be detected, wherein the two pairing regions in a. are located at both ends of the first primer, and the non-pairing region comprises two groups of identical sequences, the two groups of identical sequences are respectively connected to the pairing regions at both ends of the first primer, and the two groups of identical sequences are connected by a first spacer sequence;

[0167] (2) a second primer, comprising: c. a pairing region complementary to any one of the two identical sequences in the non-pairing region of the first primer; and d. a non-pairing region non-complementary to the first primer, wherein the pairing region in c. comprises two sequences and is located at both ends of the second primer, respectively; and the non-pairing region in d. comprises two identical sequences and is connected to the pairing regions at both ends of the second primer, respectively, and the two identical sequences are connected by a second spacer sequence;

[0168] (3) a third primer, comprising: e. a pairing region complementary to any one of the two identical sequences in the non-pairing region of the second primer; and f. a non-pairing region non-complementary to the second primer, wherein the pairing region in e. comprises two sequences and is located at both ends of the third primer, respectively; the non-pairing region in f. comprises two identical sequences and is connected to the pairing regions at both ends of the third primer, respectively; and the two identical sequences are connected by a third spacer sequence.

[0169] Among them, the two groups of identical sequences in the non-paired region in b. are identical to the two groups of identical sequences in the non-paired region in f.

[0170] According to a specific embodiment of the present invention, in the first primer, the lengths of the two pairing regions in a. are independently 15-20 nt. For example, in the first primer, the lengths of the two pairing regions in a. are 15 nt and 16 nt respectively. "Independent of each other" means that the lengths do not affect each other. The lengths of the two pairing regions can be the same or different. For different target gene mRNA or DNA, multiple target sites can be set according to the sequence length of the gene, for example, 10-30 sites can be set to achieve the binding of multiple first primers on the same target nucleic acid, that is, to achieve signal amplification of multiple sites.

[0171] According to a specific embodiment of the present invention, the primer combination provided by the present invention is complementary to the target mRNA, and the structure is as shown in Figure 1. The first segment and the second segment of the #1 primer (the first primer) (the two segments constitute the fourth (IV) region) bind to the mRNA on the glass slide to form a primary hybridization signal; and in addition to containing the first segment (marked with the number "①" in the figure, the other segments are represented in the same way, only the numbers are different) and the second segment, the #1 primer also contains two groups of respectively connected third segments and fourth segments (the two segments constitute the first (I) region), and they respectively bind to the second segments corresponding to the two #2 primers. The fifth and sixth segments (together forming the fifth (V) region) complement each other; each #2 primer contains not only the fifth and sixth segments, but also two sets of linked seventh and eighth segments (together forming the second (II) region), which complement each other with the corresponding ninth and tenth segments of the two #3 primers. In addition to the ninth and tenth segments, the #3 primer also contains two sets of sequences identical to the third and fourth segments of the #1 primer. The two linked third and fourth segments contained in the #3 primer can be used to complementarily bind with the fifth and sixth segments of the second primer in the next round. The present invention achieves exponential amplification through the complementary binding of multiple #2 primers and multiple #3 primers. Fluorescent signal capture is achieved by targeting a 15-bp readout sequence (fluorescent probe) of the #2 or #3 primers that exhibits a specific sequence pattern, enabling easier and faster detection of the target nucleic acid. For each primer sequence, T4 DNA ligase is used to link the head and tail ends of this sequence into a loop at the end of each amplification stage.

[0172] This primer sequence design ensures targeted binding between primer sequences at each level, making capture easier; the setting of the double binding region facilitates further amplification, forming an exponential amplification effect; at the same time, the use of T4 DNA ligase instead of click chemistry to form a ring greatly reduces costs.

[0173] It should be noted that, according to a specific embodiment of the present invention, as shown in Figure 1, the first fragment and the adjacent third fragment, the second fragment and the adjacent fourth fragment, the fifth fragment and the adjacent seventh fragment, the sixth fragment and the adjacent eighth fragment, the ninth fragment and the adjacent third fragment, and the tenth fragment and the adjacent fourth fragment can each independently have a spacer sequence or be directly connected. When there is a spacer sequence, the length of the spacer sequence is preferably 2-5nt.

[0174] According to a specific embodiment of the present invention, the target nucleic acid to be detected is single-stranded DNA or RNA. The RNA can be mRNA directly transcribed in cells in a tissue section, and the single-stranded DNA can be single-stranded DNA derived from some tumor cells.

[0175] According to a specific embodiment of the present invention, the nucleic acid primer combination and kit provided by the present invention can be used for the following purposes:

[0176] a. Nucleic acid in situ hybridization;

[0177] b. Combined detection of nucleic acid and protein;

[0178] c. Organizational map construction;

[0179] d. Determine the distribution of cells in tissues;

[0180] e. Digital pathology imaging and analysis;

[0181] f. Biomarker screening and discovery.

[0182] According to a specific embodiment of the present invention, combined detection of proteins and nucleic acids (such as RNA) is preferably performed in tissues. By locating cells expressing specific markers, tissue maps can be constructed, cell distribution within tissues can be determined, and biomarkers can be screened. The nucleic acid primer combinations and kits of the present invention can also be used for digital medical imaging.

[0183] According to a specific embodiment of the present invention, a method for in situ hybridization of a target nucleic acid is provided, comprising using the aforementioned nucleic acid detection primer combination to complementarily pair with a target nucleic acid and amplify the target nucleic acid signal, and detecting the target nucleic acid using a fluorescently labeled probe complementary to an unpaired region of the second or third primer. The target nucleic acid is single-stranded DNA or RNA, and the fluorescently labeled probe has a probe length of 13-23 nt.

[0184] According to a specific embodiment of the present invention, the present invention provides a fluorescence in situ hybridization method, which comprises the following steps:

[0185] 1) Preparing tissue sections to be tested;

[0186] 2) contacting the first primer in the aforementioned nucleic acid detection primer combination with the tissue in the tissue section to be tested, and performing a complementary pairing reaction between the first primer and the target RNA in the tissue to be tested;

[0187] 3) using a ligase to link the first primers in 2) into a ring, removing excess first primers that have not bound to the target RNA, thereby obtaining a tissue section to be tested containing the target RNA-circularized first primer;

[0188] 4) contacting the second primer in the aforementioned nucleic acid detection primer combination with the tissue to be tested treated in step 3) to allow for a complementary pairing reaction between the second primer and the first primer that has been circularized in the tissue to be tested treated in step 3);

[0189] 5) ligating the second primer in 4) into a ring using a ligase, and removing excess second primer not bound to the first primer, thereby obtaining a tissue section to be tested containing target RNA-circularized first primer-circularized second primer;

[0190] 6) contacting the third primer in the aforementioned nucleic acid detection primer combination with the tissue to be tested treated in step 5) to allow for a complementary pairing reaction between the third primer and the looped second primer in the tissue to be tested treated in step 5);

[0191] 7) using a ligase to link the third primer in step 6) into a ring, removing excess third primer not bound to the second primer, thereby obtaining a tissue section to be tested containing target RNA-circularized first primer-circularized second primer-circularized third primer; and

[0192] 8) contacting the tissue to be tested treated in step 7) with the fluorescently labeled probe to allow for a complementary pairing reaction between the fluorescently labeled probe and the third primer that has been circularized in the tissue to be tested treated in step 7), removing excess unbound fluorescently labeled probe, and imaging the tissue section to be tested.

[0193] According to a specific embodiment of the present invention, in steps 2), 4), and 6), the primers and the tissue to be tested are contacted in a hybridization reaction solution containing ethylene carbonate. The concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v / v. The addition of ethylene carbonate to the buffer further shortens the incubation time between #1 and the target mRNA.

[0194] According to a specific embodiment of the present invention, the present invention provides a method for joint detection of RNA and protein in tissue sections, comprising the following steps:

[0195] (I) performing RNA fluorescence in situ hybridization on the tissue sections using the aforementioned fluorescence in situ hybridization method, and photographing and recording the processed tissue sections; and

[0196] (II) Directly perform tissue protein fluorescence staining and imaging on the tissue sections after photographing.

[0197] According to a specific embodiment of the present invention, the tissue protein fluorescent staining includes multiple rounds of protein staining using antibodies.

[0198] The nucleic acid in situ hybridization method provided by the present invention is efficient, time-saving, inexpensive, and capable of combined RNA and protein detection. RNA fluorescence detection can amplify the signal without the need for excessive use of fluorescent probes, thus shortening the incubation time. The nucleic acid in situ hybridization method provided by the present invention uses a primer combination that only requires changing the sequence of the first primer, while the second and third primers are universal primers, and the same fluorescent probe can be used for detection of different RNA targets, greatly reducing costs. After RNA detection, protein staining can be performed directly, and repeated positioning can be used to achieve combined detection.

[0199] Those skilled in the art will understand that the following examples are merely illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications were used. Where the manufacturer of the reagents or instruments used is not specified, they are all commercially available conventional products.

[0200] Example 1: RNA in situ hybridization

[0201] RNA in situ hybridization was performed on mouse tumor tissue, which was an MDA-MB231 tumor model. The paraffin blocks were purchased from Wuhan Saiweier.

[0202] 1. Pretreatment of paraffin sections:

[0203] a. Slice the tumor tissue paraffin block using a paraffin microtome and place it in a 40-45°C water bath.

[0204] b. Use a surface-treated (Poly-l-lysine, APTS, Gelatin, Gelatin Chrome Alum, etc.) slide to fish out the slide and let it stand upright overnight;

[0205] c. Dewaxing with xylene for 3 hours to overnight;

[0206] d. Add anhydrous ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and ultrapure water in sequence (10-30 minutes for each solution) for hydration;

[0207] e. Place the sections in antigen retrieval solution and perform antigen retrieval at high temperature (95°C) for 10-20 minutes.

[0208] 2. RNA fluorescence in situ hybridization detection:

[0209] a. Dissolve and dilute the reaction primers #1, #2, and #3 (probe primers are all diluted to 100 μM using TE) and the read primer: Dissolve each primer in varying amounts of TE buffer until completely dissolved. Then, dilute each primer in an EP tube at a 1:100 concentration (or the desired concentration, determined by calculation) and label it as the diluted primer tube.

[0210] b. Prepare conventional wash buffer and read wash buffer: dilute 20X SSC to 1X SSC, using ultrapure water as solvent; prepare conventional wash buffer (10% ethylene carbonate, using 1X SSC as solvent); read wash buffer = 1X SSC + 0.1% Triton-X-100;

[0211] c. Preparation of hybridization reaction solution and read buffer: 10% hybridization reaction solution (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC) for primer #1-tissue hybridization and for primer #2 and #3 amplification; 10% hybridization reaction solution (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC) for eluting non-specifically bound primers; read buffer: 10% dextran sulfate + 0.1% Tx-100 + 5% ethylene carbonate; T4 DNA ligase reaction solution: 1 μl T4 DNA ligase + 99 μl 1X enzyme buffer;

[0212] d. Calculate #1, #2, #3, read, and prepare the total amount of each raw material in the T4 ligase reaction system. The formula is as shown in c, and each reaction solution is about 200 μL.

[0213] e. Main reaction: Open the hot plate and set it to 37°C. Place the creased sealing film in the center of the hot plate. Add 1 ml of regular washing buffer to the center of the sealing film. Place the tissue section upside down in the center of the sealing film and start the reaction timer.

[0214] f. After the timer expires, discard the wash solution and carefully transfer the inverted slide to the center of another drop of sealing film containing 100 μL of the total reaction system #1 (10% ethylene carbonate, 10% dextran sulfate, solvent: 1X SSC, and 3.6 nM of each #1 probe). Be careful not to scrape the tissue when transferring the slide. Continue the reaction at 37°C for 90 or 150 minutes.

[0215] g. Add T4 DNA ligase reaction solution to the center of the sealing membrane to form a ring. Continue the reaction at 37°C for 20 minutes.

[0216] h. Wash the slides with 3 ml of conventional wash buffer, add to the same sealing film, and wash at 37°C for 10 minutes;

[0217] i. Transfer the inverted slide to the center of another drop of sealing film containing the total reaction system #2 (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC, and 20nM probe #2) and amplify on a hot plate at 37°C for 30 minutes.

[0218] j. Add T4 DNA ligase reaction solution to the center of the sealing membrane to form a ring. Continue the reaction at 37°C for 20 minutes.

[0219] k. Wash the slides with 3 ml of conventional cleaning solution, add it to the same sealing film, and wash at 37°C for 10 minutes;

[0220] Transfer the inverted slide to the center of another drop of sealing film containing the total reaction system #3 (10% ethylene carbonate, 10% dextran sulfate, 0.1% Triton-X-100, solvent: 1X SSC, and 20 nM probe #3) and amplify on a hot plate at 37°C for 30 minutes.

[0221] Add T4 DNA ligase reaction solution to the center of the sealing membrane to form a ring. Continue the reaction at 37°C for at least 20 minutes.

[0222] Wash the slides with 3 ml of conventional cleaning solution, then apply to the same sealing film and wash at 37°C for 10 minutes. If multiple rounds of amplification #2 and #3 are required, repeat steps i.-n. several times until the fluorescent markers can be read.

[0223] After calculating and preparing the read buffer system, transfer the inverted slide to the center of another drop of sealing film containing the read buffer reaction system and incubate at room temperature for 20 minutes. Adjust the concentration of the read buffer reaction system according to the number of amplification rounds;

[0224] p. Read the rinsing buffer and wash the slides twice at room temperature for 1 min. Store the slides in 1X SSC at 4°C or add imaging buffer to the slides and take photos of the staining status.

[0225] The primers used for RNA fluorescence in situ hybridization are shown in Table 1 below. Multiple primer combinations #1-1 to #1-16 were designed using CD8 mRNA as the detection target. Primers #2 and #3 were used for signal amplification, and read probes #2 and #3 were used for signal detection:

[0226] Table 1 Primer and probe sequences

[0227] The results of RNA fluorescence in situ hybridization detection are shown in FIG2 , indicating that the use of the primer combination #1-#3 of the present invention can greatly shorten the incubation time for fluorescence reading and detecting RNA hybridization signals, and can quickly detect the in situ expression of target RNA.

[0228] The inventors conducted a comparative experiment on the reaction time of primer #1 and the probe concentration in the reading buffer. As shown in Figure 2, the three experimental groups were divided into: 90-minute incubation time of the primary probe + 10 nM detection probe; 90-minute incubation time of the primary probe + 20 nM detection probe; and 150-minute incubation time of the primary probe + 20 nM detection probe. All other conditions of the three experimental schemes were the same. The results showed that the optimal reading probe concentration was 20 nM and the incubation time of the #1 reaction system was 90 minutes.

[0229] Furthermore, the inventors conducted a comparative experiment comparing the hybridization reaction solution components in "2. RNA Fluorescence In Situ Hybridization, Step c" (see "2. RNA Fluorescence In Situ Hybridization Detection"). One reaction solution contained 10% ethylene carbonate, while the other replaced 10% ethylene carbonate with 10% polyvinylamine. The probe concentration was 20 nM, and the incubation time for reaction system #1 was 90 minutes. As shown in Figure 3, hybridization experiments using 10% polyvinylamine under the same conditions failed to produce corresponding punctate FISH signals, displaying only nonspecific autofluorescence signals, indicating that polyvinylamine staining is less effective than ethylene carbonate.

[0230] Example 2 RNA in situ hybridization and protein fluorescence staining

[0231] RNA in situ hybridization and tissue protein fluorescence staining were performed on mouse tumor tissue. The tumor tissue was an MDA-MB231 tumor model, and the paraffin blocks were purchased from Wuhan Saiweier.

[0232] 1. Pretreatment of paraffin sections: The operation steps are the same as those in “1. Pretreatment of paraffin sections” in Example 1.

[0233] 2. RNA fluorescence in situ hybridization detection: The operation steps are the same as those in "2. RNA fluorescence in situ hybridization detection" of Example 1, wherein the reaction time in step f is 90 minutes.

[0234] The primers used for RNA fluorescence in situ hybridization are shown in Table 1 of Example 1. Multiple primer combinations #1-1 to #1-16 were designed using CD8 mRNA as the detection target. Primers #2 and #3 were used for signal amplification, and read probes #2 and #3 were used for signal detection.

[0235] The results of RNA fluorescence in situ hybridization detection are shown in FIG2 , indicating that the use of the primer combination #1-#3 of the present invention can greatly shorten the incubation time for fluorescence reading and detecting RNA hybridization signals, and can quickly detect the in situ expression of target RNA.

[0236] 3. After taking a photo of the slide after in situ hybridization, perform fully automated protein staining directly. Specifically, this includes making a flow channel and continuing to perform immunofluorescence protein staining on the slide after in situ hybridization. The steps are as follows:

[0237] a. Add 0.5% TritonX-100 and perform punching for 15 minutes;

[0238] b. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl per minute);

[0239] c. Add imaging buffer (0.2 mg / ml trolox) and capture the original background image;

[0240] d. Wash five times with PBS (time: 60 seconds, flow rate: 1200 μl / min);

[0241] e. Add antibody elution buffer (0.8% Beta-ME, 62.5 mM Tris-HCl, 1% SDS) (56°C, 10 minutes);

[0242] f. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl per minute);

[0243] g. Add Sino-Biological (#10084-T24) PD-L1 primary antibody (37°C, 10 minutes);

[0244] h. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl per minute);

[0245] i. Add Thermo Fisher Scientific (#A11009) AF532 goat anti-rabbit secondary antibody (37°C, 10 minutes);

[0246] j. Wash 5 times with cleaning solution (time: 60 seconds, flow rate: 1200 μl / min)

[0247] k. Repeat steps 3-10 for multiple rounds of immunofluorescence staining with antibodies.

[0248] The results of immunofluorescence protein staining are shown in FIG4 , indicating that after completing the mRNA hybridization experiment, antibodies can be used to further perform immunofluorescence staining on proteins on the tissue, indicating that the use of the specific primer combination of the present invention and the optimized reaction system can successfully perform nucleic acid and protein joint detection.

[0249] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean 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 invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0250] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A primer combination comprising: (1) one or more first primers, each of which comprises: a fourth region that binds to a target nucleic acid fragment and at least one first region that does not bind to the target nucleic acid; (2) a second primer, the second primer comprising: a fifth region that pairs and binds to the first region, and at least two second regions that do not pair and bind to the target nucleic acid; (3) A third primer, comprising: a sixth region that pairs and binds to the second region, and at least two third regions that do not pair and bind to the target nucleic acid.

2. The primer combination according to claim 1, wherein The first primer includes: a fourth region that pairs and binds to a fragment of the target nucleic acid, and at least two first regions that do not pair and bind to a sequence of the target nucleic acid.

3. The primer combination according to claim 1 or 2, wherein The second region does not pair with the first region; and the third region does not pair with the second region.

4. The primer combination according to any one of claims 1 to 3, wherein The fourth region includes a first fragment and a second fragment, wherein the first fragment and the second fragment are respectively located at two ends of the first primer, and the first region is located between the first fragment and the second fragment; Each of the first regions includes at least a third segment and a fourth segment; The fifth region includes a fifth segment and a sixth segment, the fifth segment and the sixth segment are respectively located at two ends of the second primer, and the second region is located between the fifth segment and the sixth segment; Each of the second regions includes at least a seventh segment and an eighth segment; The sixth region includes a ninth segment and a tenth segment, the ninth segment and the tenth segment are located at both ends of the third primer, respectively, and the third region is located between the ninth segment and the tenth segment; Each of the third regions includes at least the third segment and the fourth segment. 5 . The primer combination according to claim 1 , comprising a plurality of first primers, wherein the fourth region of each of the first primers pairs and binds to fragments of different target nucleic acids or different fragments of the same target nucleic acid.

6. The primer combination according to any one of claims 1 to 5, wherein The first base at the 5' end and the last base at the 3' end of the first primer correspond to adjacent bases on the target nucleic acid fragment to which they are paired and bound.

7. The primer combination according to any one of claims 4 to 6, wherein The lengths of the first fragment and the second fragment are each independently 15-20 nt; Optionally, the lengths of the third fragment, the fourth fragment, the fifth fragment, the sixth fragment, the seventh fragment, the eighth fragment, the ninth fragment and the tenth fragment are each independently 8-20 nt, preferably 10-16 nt.

8. The primer combination according to any one of claims 1 to 7, wherein The first primer includes more than one first region, and each first region is connected by a first spacer sequence; Optionally, in the second primer, each of the second regions is connected by a second spacer sequence; Optionally, in the third primer, each of the third regions is connected by a third spacer sequence; Optionally, the sequence length of the first spacer sequence, the second spacer sequence and the third spacer sequence is at least 1 nt, preferably 1-30 nt, and further preferably 2-5 nt.

9. A kit comprising: The primer combination according to any one of claims 1 to 8, and the first detection probe and / or the second detection probe, wherein the first detection probe comprises a sequence that pairs and binds to at least a portion of the region of the second primer that does not pair with the first primer; The second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that does not pair with the second primer; The first detection probe and the second detection probe contain a label capable of generating a detectable signal.

10. The kit according to claim 9, wherein: The first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer; Optionally, the first detection probe comprises at least a portion of the seventh segment and the eighth segment of the second primer. at least a portion of the paired binding sequence.

11. The kit according to claim 9 or 10, wherein: The second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer; Optionally, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

12. The kit according to any one of claims 9 to 11, wherein: The label includes a radioactive label or a non-radioactive label; Optionally, the non-radioactive label comprises a fluorescent group.

13. A nucleic acid hybridization method comprising: S1. contacting a biological sample containing a target nucleic acid with one or more first primers of the primer combination of any one of claims 1 to 8, wherein the fourth region of the one or more first primers pairs and binds to a fragment of the target nucleic acid to obtain a target nucleic acid-first primer hybrid 1; S2. contacting the product obtained in step S1 with the second primer in the primer combination of any one of claims 1 to 8, wherein the fifth region of the second primer pairs and binds with the first region of the first primer to obtain target nucleic acid-first primer-second primer hybrid 2; Optionally, S3. contacting the product obtained in step S2 with the third primer in the primer combination of any one of claims 1 to 8, and pairing and binding the sixth region of the third primer with the second region of the second primer to obtain a target nucleic acid-first primer-second primer-third primer hybrid 3.

14. The nucleic acid hybridization method according to claim 13, wherein The method further comprises: the contacting in steps S1-S3 is performed in a hybridization reaction solution containing ethylene carbonate, wherein the concentration of ethylene carbonate in the hybridization reaction solution is 5-40% v / v; Optionally, the hybridization reaction solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

15. The nucleic acid hybridization method according to claim 13 or 14, wherein The method further comprises: before performing step S2, using a ligase to ligate the first primers present in the system after the reaction in step S1 into a ring, and after the ring formation reaction is completed, performing an elution treatment with a washing solution to remove excess first primers that are not bound to the target nucleic acid; Optionally, the method further comprises: before performing step S3, using a ligase to ligate the second primer present in the system after the reaction in step S2 into a ring, and after the ring formation reaction is completed, performing an elution treatment using a washing solution to remove excess second primer that is not bound to the first primer; Optionally, the method further comprises: after performing step S3, using a ligase to ligate the third primer present in the system after the reaction in step S3 into a ring, and after the ring formation reaction is completed, performing an elution treatment using a washing solution to remove excess third primer that is not bound to the second primer; Optionally, the reaction temperature for the ligation reaction using the ligase is 30-40° C., and the reaction time is 5-30 min.

16. The nucleic acid hybridization method according to any one of claims 13 to 15, wherein The ligase is T4 ligase; Optionally, the cleaning solution comprises ethylene carbonate, and the concentration of ethylene carbonate in the cleaning solution is 5-40% v / v; Optionally, the cleaning solution further comprises dextran sulfate and Triton-X-100, wherein the concentration of the dextran sulfate is 5-20%, and the concentration of the Triton-X-100 is 0.05-0.5%.

17. The nucleic acid hybridization method according to any one of claims 13 to 16, wherein The reaction temperature for pairing in step S1 is 30-40°C, and the reaction time is 30-120 min; Optionally, the reaction temperature for pairing in step S2 is 30-40°C, and the reaction time is 20-40 min; Optionally, the reaction temperature for the pairing binding in step S3 is 30-40° C., and the reaction time is 20-40 min.

18. The nucleic acid hybridization method according to any one of claims 13 to 17, wherein The method further comprises: Ⅰ. After step S3, continue to repeat steps S2-S3 for at least N rounds, where N≥1 and N is an integer; or, II. After step S3, continue to repeat steps S2-S3 for at least N' rounds, N'≥0, N' is an integer, and only repeat step S2 in the last round.

19. A method for detecting a target nucleic acid, comprising: obtaining a hybrid consisting of a target nucleic acid, a first primer, a second primer, and an optional third primer using the nucleic acid hybridization method according to any one of claims 13 to 18, and detecting the hybrid using the first detection probe and / or the second detection probe; wherein the first detection probe comprises a sequence that pairs and binds to at least a portion of the region of the second primer that does not pair with the first primer; The second detection probe includes a sequence that pairs and binds to at least a portion of the region of the third primer that does not pair with the second primer; The first detection probe and the second detection probe contain a label capable of generating a detectable signal.

20. The method according to claim 19, wherein The first detection probe includes a sequence that pairs and binds to at least a portion of the second region in the second primer; Optionally, the first detection probe includes a sequence that pairs and binds to at least a portion of the seventh fragment and at least a portion of the eighth fragment in the second primer.

21. The method according to claim 19 or 20, wherein The second detection probe includes a sequence that pairs and binds to at least a portion of the third region in the third primer; Optionally, the second detection probe includes a sequence that pairs and binds to at least a portion of the third fragment and at least a portion of the fourth fragment in the third primer.

22. The method according to any one of claims 19 to 21, wherein: The method further comprises: i. After step S2, detecting the hybrid consisting of the target nucleic acid, the first primer and the second primer with the first detection probe; or i. after step S3, continue to repeat steps S2-S3 for at least N rounds, detecting the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer with the second detection probe, optionally using the first detection probe for auxiliary detection, wherein N is a natural number; or ii. After step S3, continue to repeat steps S2-S3 for at least N' rounds, and in the last round, only repeat step S2, then use the first detection probe to detect the hybrid consisting of the target nucleic acid, the first primer, the second primer and the third primer, and optionally, use the second detection probe for auxiliary detection, wherein N' is a positive integer.

23. The method according to any one of claims 19 to 22, wherein: The lengths of the first and second detection probes are independently 13-23 nt.

24. A method for combined detection of nucleic acids and proteins, comprising the following steps: 1) performing nucleic acid detection on a biological sample containing nucleic acid using the method for detecting a target nucleic acid according to any one of claims 19 to 23; and 2) performing protein fluorescence staining and imaging on the biological sample.

25. The method according to claim 24, wherein The protein fluorescent staining includes multiple rounds of protein staining using antibodies.

26. Use of the primer combination according to any one of claims 1 to 8 or the kit according to any one of claims 9 to 12 in the following: a. Nucleic acid in situ hybridization; b. Combined detection of nucleic acid and protein; c. Organizational map construction; d. Determine the distribution of cells in tissues; e. Digital pathology imaging and analysis; f. Biomarker screening and discovery.

Images