Multi-target simultaneous detection method based on proximity ligation

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a multi-target simultaneous detection method based on ortho-position ligation, which comprises the following steps: simultaneously enriching multiple target molecules by adopting multiple microbeads coated with antibodies for identifying different targets, combining with an ortho-position probe, and then connecting the ortho-position probe by using a splint probe, so as to obtain the multi-target simultaneous detection method based on ortho-position ligation. And finally, the detection of various different target molecules is realized through qPCR (Quantitative Polymerase Chain Reaction) detection. The method provided by the invention not only can be used for simultaneously detecting various different target molecules, but also has higher detection accuracy compared with a conventional method.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for simultaneous detection of multiple targets based on proximity ligation. Background Art

[0002] Proximity Ligation Assay (PLA) is a highly sensitive in vitro protein analysis technology based on an enzyme-linked mechanism and a linker (an artificially designed linear oligonucleotide single chain, whose two ends can be complementary to a specific target sequence. When the linker hybridizes with the target sequence, they will be close to each other in space, and the two ends of the probe can be connected into a chain under the action of DNA ligase). This method first uses a pair of proximity probes to dual-recognize the target molecule to generate an amplifiable detection signal, and then realizes timed quantitative detection through quantitative PCR, converting the detection of protein into the detection of DNA, with extremely high sensitivity and specificity. PLA can analyze the signal of low-abundance proteins by amplifying antibody-antigen binding events, making it suitable for the development of multiplex detection for simultaneous quantification of protein and nucleic acid targets.

[0003] PLA detection includes many forms, such as homogeneous assay, solid phase assay, etc. Among them, solid phase PLA detection is favored because it can enrich target molecules with low abundance and eliminate the influence of other substances in complex biological samples on the determination. However, solid phase PLA detection still has the problem that the probe relies on random collision and is difficult to effectively connect in low-abundance targets, resulting in low PLA detection efficiency.

[0004] In the fields of biomedical research, early disease diagnosis, drug screening, and environmental monitoring, the sensitive, specific, and multiplex detection of target molecules has always been a core issue in analytical science. Especially in clinical diagnosis, the occurrence and development of many diseases are often accompanied by the abnormal expression of multiple biomarkers. Therefore, the development of a method that can simultaneously, accurately, and quantitatively detect multiple targets is of great significance for early screening, typing diagnosis, and personalized treatment of diseases. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for simultaneous detection of multiple targets based on microbead enrichment PLA assay. Specifically, the target molecules are enriched in the form of microbeads coated with antibodies. Since each microbead can load up to hundreds of antibodies, multiple target molecules can be enriched on a single microbead, thereby obtaining a significantly higher degree of enrichment, which facilitates subsequent probe connection and can significantly improve the connection efficiency. On this basis, the present invention uses a variety of microbeads coated with antibodies that recognize different targets to simultaneously enrich multiple target molecules, thereby achieving simultaneous detection of multiple targets. In addition, the present invention also found that there is a problem of signal loss when using conventionally designed linkers in this process, which is overcome by using the splint probe designed by the present invention.

[0006] Specifically, the present invention provides a method for simultaneous detection of multiple targets based on proximity ligation, comprising the following steps: (a) providing a plurality of microbead complexes, each of which comprises a microbead carrier and a first antibody immobilized on the surface of the microbead carrier, wherein the first antibodies specifically bind to different target molecules; (b) incubating the plurality of microbead complexes with a sample to be tested, so that the first antibodies bind to their respective target molecules; (c) The incubated complexes are separated to obtain a variety of microbead complexes bound to different target molecules.

[0007] Furthermore, the first antibody is fixed on the surface of the microbead carrier by chemical cross-linking or biotin-streptavidin binding, and the microbead carrier is a magnetic bead with surface carboxylation, aminoation or streptavidin modification.

[0008] The method further comprises: (d) incubating the plurality of microbead complexes bound to different target molecules with a plurality of first proximity probes and second proximity probe pairs, wherein the first proximity probe comprises a second antibody coupled to the 5' end of the first oligonucleotide, and the second proximity probe comprises a third antibody coupled to the 3' end of the second oligonucleotide, wherein the second antibody and the third antibody respectively bind to different epitopes of their respective target molecules.

[0009] Furthermore, the first oligonucleotide and the second oligonucleotide comprise a unified first adapter sequence and a unified second adapter sequence at the 3' end and the 5' end, respectively, and the remaining sequences of the oligonucleotides in different probe pairs are different.

[0010] Furthermore, the coupling of the second antibody and the third antibody to the oligonucleotide is achieved by EDC / NHS coupling, click chemistry or maleimide-thiol reaction.

[0011] The method further comprises: (e) incubating the above-mentioned microbead complex bound to the proximity probe with a splint probe, wherein the splint probe consists of short hairpins at both ends and a hybridization region in the middle, and the hybridization region contains sequences complementary to the first adapter sequence and the second adapter sequence, respectively, so that the first and second oligonucleotides are adjacent to each other.

[0012] Furthermore, the short hairpins at both ends of the splint probe each have a loop with a length of 4-6 bases and a reverse complementary double strand with a length of 4-6 base pairs.

[0013] The method further comprises: (f) Incubating the multiple microbead complexes obtained in step S5 with DNA ligase, so that the adjacent 3' end of the first oligonucleotide is ligated to the 5' end of the second oligonucleotide to form a complete ligation product.

[0014] The method further comprises: (g) mixing the complex obtained in step (f) with a qPCR reaction system comprising a plurality of primer pairs and performing amplification detection, wherein the primer pairs are complementary to the first and second oligonucleotides in each probe.

[0015] Furthermore, the qPCR reaction system includes Taq DNA polymerase, buffer, ATP, dNTP, multiple detection probes and nuclease-free water. The two ends of the detection probe are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups of each probe are different to distinguish the target molecules.

[0016] In addition, the present invention also provides a method for simultaneous detection of multiple targets based on proximity ligation, which comprises the following steps: (a) providing a plurality of microbead complexes, each of the plurality of microbead complexes comprising: (i) a microbead carrier; and (ii) a first antibody that specifically binds to a target molecule and is immobilized on the surface of the microbead carrier; The first antibodies immobilized on different microbead complexes each specifically bind to a different target molecule; (b) incubating the plurality of microbead complexes with a sample to be tested, so that the first antibodies immobilized on different microbead complexes bind to their respective target molecules; (c) separating the multiple microbead complexes from the sample to be tested to obtain multiple microbead complexes bound to different target molecules; (d) incubating a plurality of microbead complexes bound to different target molecules with a plurality of first proximity probes and second proximity probe pairs, wherein the plurality of first proximity probes and second proximity probe pairs correspond to different target molecules, respectively, wherein the first proximity probe comprises a second antibody coupled to the 5' end of a first oligonucleotide, the second proximity probe comprises a third antibody coupled to the 3' end of a second oligonucleotide, the sequences of the first oligonucleotide and the second oligonucleotide in each pair of first proximity probes and second proximity probes are different among the plurality of first proximity probes and second proximity probe pairs, but the 3' ends of all first oligonucleotides have the same first adaptor sequence and the 5' ends of all second oligonucleotides have the same second adaptor sequence, and the second antibody and the third antibody in each pair of first proximity probes and second proximity probes each bind to a different epitope on the target molecule corresponding to the first proximity probe and the second proximity probe pair, thereby obtaining a plurality of microbead complexes bound to the plurality of first proximity probes and second proximity probe pairs; (e) incubating the plurality of microbead complexes of step (d) with a splint probe, wherein the splint probe is composed of short hairpins located at both ends and a hybridization region located in the middle, wherein the hybridization region comprises complementary sequences complementary to the first adapter sequence and the second adapter sequence, respectively, so that the 3' end of the first oligonucleotide and the 5' end of the second oligonucleotide are adjacent to each other by hybridization with the splint probe; (f) incubating the plurality of microbead complexes of step (e) with DNA ligase, so that the 3' ends of the adjacent first oligonucleotides are ligated to the 5' ends of the second oligonucleotides; (g) mixing the multiple microbead complexes of step (f) with a qPCR reaction system comprising multiple primer pairs, and performing a qPCR reaction to determine the presence of the target molecule or two interacting target molecules, wherein the multiple primer pairs correspond to multiple first proximity probes and second proximity probe pairs, respectively, and each pair of primers consists of an upstream primer and a downstream primer that are complementary to the first oligonucleotide and the second oligonucleotide in the corresponding first proximity probe and second proximity probe pairs.

[0017] As used herein, a plurality of microbead complexes may include at least two microbead complexes that specifically recognize different target molecules, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more microbead complexes. Wherein the first antibody that specifically binds to the same target molecule is fixed on the microbeads of each microbead complex, that is, each microbead complex specifically recognizes the same target molecule, and each microbead complex may contain at least one or more microbead complexes that specifically recognize the same target molecule, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more microbead complexes.

[0018] Furthermore, the microbead carrier is a magnetic bead with surface carboxylation, aminoation or streptavidin modification.

[0019] Furthermore, the first antibody is fixed on the surface of the microbeads by chemical cross-linking or biotin-streptavidin binding.

[0020] Furthermore, the coupling of the second antibody and the third antibody to the oligonucleotide is achieved by chemical cross-linking or biotin-streptavidin binding.

[0021] Furthermore, the chemical cross-linking is selected from EDC / NHS coupling, click chemistry (CuAAC) or maleimide-thiol reaction.

[0022] Furthermore, the antibody may be a monoclonal antibody or a polyclonal antibody.

[0023] As used herein, the first antibody of each microbead complex and the second and third antibodies of each corresponding first proximity probe and second proximity probe pair can specifically bind to the same target molecule, but each recognizes a different epitope on the target molecule, i.e., has different antigenic determinants.

[0024] Furthermore, the splint probe is a dumbbell-shaped oligonucleotide, which comprises short hairpins at both ends and a hybridization region in the middle.

[0025] Furthermore, the short hairpins at both ends of the splint probe each have a loop with a length of 4-6 bases and a reverse complementary double strand with a length of 4-6 base pairs.

[0026] Furthermore, the hybridization region in the middle of the splint probe is single-stranded and has a length of 8-12 bases, wherein the length of the complementary sequence complementary to the first adapter sequence is 4-6 bases, and the length of the complementary sequence complementary to the second adapter sequence is 4-6 bases.

[0027] Preferably, the short hairpins at both ends of the splint probe each have a loop with a length of 4 bases and a reverse complementary double strand with a length of 4 base pairs, and the length of the complementary sequence complementary to the first adapter sequence is 6 bases, and the length of the complementary sequence complementary to the second adapter sequence is 6 bases.

[0028] Furthermore, the method may combine steps eg together, for example, may include incubating the multiple microbead complexes of step (d) with a qPCR reaction system comprising a splint probe, a DNA ligase and multiple primer pairs and performing a qPCR reaction to determine the presence of multiple different target molecules.

[0029] Furthermore, the DNA ligase is T4 DNA ligase.

[0030] Furthermore, the qPCR reaction system also includes a reaction buffer, Taq DNA polymerase, ATP, dNTP, a plurality of detection probes and nuclease-free water.

[0031] Furthermore, each of the plurality of detection probes is used to detect a different target molecule, for example, is complementary to the first or second oligonucleotide in a plurality of first proximity probes and second proximity probe pairs corresponding to different target molecules.

[0032] Furthermore, both ends of the multiple detection probes are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups of the multiple detection probes are different from each other.

[0033] Furthermore, the fluorescent group may be selected from but not limited to FAM, HEX, VIC, TET, ROX, CY5, etc.; the quenching group may include at least one of TAMRA, MGB, BHQ1, BHQ2 and BHQ3.

[0034] Furthermore, the detection probe is a Taqman probe.

[0035] Advantageous Effects of the Invention The present invention first improves the existing solid phase PLA assay format by using microbeads coated with a large number of antibodies to highly enrich the target molecules, so that the proximity probes and target molecules can collide and bind more efficiently. As can be seen from the examples, the method of the present invention can detect target molecules as low as 0.001 ng / mL, with excellent detection sensitivity.

[0036] However, during this method, multiple proximity probes will bind to each target molecule simultaneously on the same microbead, and the presence of more proximity probes on the same microbead may result in the proximity of the proximity probes of each target molecule crossing over, resulting in the number of target molecules detected being lower than the actual number of target molecules.

[0037] For conventional linkers, since it is necessary to consider not only the strength and specificity of binding to the template, but also the length of the duplex that the ligase must meet to perform the connection, its length is usually longer, reaching about 30bp. The present invention found that when a linker of such a length is subjected to the microbead enrichment PLA assay of the present invention, the measurement value described above is low. In order to solve this problem, the present invention uses a unique splint probe to replace the conventional linker, and it is found that the presence of the target molecule can be detected more accurately. Compared with conventional linkers, the splint probe of the present invention has a short hairpin structure at both ends that allows the ligase to stably bind and perform the connection reaction, so it is shorter in length. In addition, the dumbbell-shaped splint probe has a higher rigidity and a lower flexibility of the molecular chain, which is more suitable for the microbead enrichment PLA method of the present invention, and reduces the cross-approaching between adjacent probes of multiple target molecules.

[0038] On this basis, in order to achieve simultaneous detection of multiple targets, the present invention will simultaneously incubate the sample to be tested with microbead complexes that recognize different target molecules, and improve the sequence of the adjacent probe pairs used to introduce a universal adapter sequence, so that a single splint probe can be used to achieve simultaneous detection of multiple different targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic structural diagram of the clamping probe of the present invention is shown.

[0040] Figure 2 The amplification curve of the TNF-α content in the sample was determined by microbead-enriched multiplex PLA in the example.

[0041] Figure 3 The amplification curve of the IL-6 content in the sample of the microbead-enriched multiplex PLA assay in the example is shown.

[0042] Figure 4 The Ct value standard curve of the TNF-α content in the sample of the microbead-enriched multiple PLA assay in the example is shown.

[0043] Figure 5 The Ct value standard curve of the IL-6 content in the microbead-enriched multiple PLA assay samples in the example is shown.

[0044] Figure 6 The Ct value standard curve of TNF-α content in the microbead-enriched multiple PLA assay samples in the comparative example is shown.

[0045] Figure 7 The Ct value standard curve of IL-6 content in the microbead-enriched multiple PLA assay samples in the comparative example is shown. DETAILED DESCRIPTION

[0046] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0047] The first antibody against TNF-α used in the example is a biotinylated TNF-α antibody (Fabgennix, TNFA-BIOTIN), the second antibody is a TNF-α antibody (Invitrogen, PA5-120124), and the third antibody is a TNF-α antibody (Invitrogen, PA5-19810); the first antibody against IL-6 is a biotinylated IL-6 antibody (Invitrogen, 13-7068-81), the second antibody is an IL-6 antibody (Invitrogen, MA5-44642), and the third antibody is an IL-6 antibody (Invitrogen, 701028), wherein the first, second and third antibodies each have a different antigenic determinant; human TNF-α recombinant protein was purchased from MedChemExpress (HY-P700291); and human IL-6 recombinant protein was purchased from Gibco (PHC0061).

[0048] The sequences of the oligonucleotides, splint probes and primers used in the examples and the conventional linkers used in the comparative examples are shown in Table 1.

[0049] Table 1: Sequences of oligonucleotides, splint probes and primers and conventional linkers of comparative examples

[0050] Example: Bead-enriched multiplex PLA assay for TNF-α and IL-6 content in samples (1) Prepare a mixture of microbead complexes targeting TNF-α and IL-6: incubate 1 μL of Dynabeads™ MyOne™ Streptavidin T1 magnetic beads with 100 μL of TNF-α or IL-6 primary antibody dilution (concentration of 100 μg / mL) at room temperature for 30 min under stirring, collect the magnetic beads by magnetic separation, wash them three times with 10 mM PBS, incubate the magnetic beads in 10 mM PBS containing 0.1% BSA with shaking for 15 min, collect the magnetic beads targeting TNF-α and IL-6, mix them together and resuspend them in 10 mM PBS; (2) TNF-α and IL-6 proteins were dissolved in 10 mM PBS to prepare mixed sample solutions of different concentrations (TNF-α and IL-6 protein concentrations were 0 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1000 ng / mL, respectively), and incubated with the microbead complex mixture at room temperature for 1 h. (3) Magnetic separation of the microbead complex, washing three times and resuspending in 50 μL 10 mM PBS; (4) Preparation of proximity probes for TNF-α and IL-6, respectively: 10 μL of 2 μg / μL of the second antibody and the third antibody, respectively, were added with 1 μL of 4 mM dibenzocyclooctyne-PEG4-N-hydroxysuccinimide ester (DBCO) for reaction at room temperature for 30 minutes to add an NHS group to the N-terminus, and 1 μL of 1 M Tris-HCl was added for incubation at room temperature for 5 minutes to terminate the above reaction; 20 μg of the second antibody and the third antibody modified with the NHS group were incubated with 40 μM of the first oligonucleotide modified with an azide group at the 5' end and the second oligonucleotide modified with an azide group at the 3' end dissolved in 10 mM PBS at 4°C overnight to couple the second antibody to the 5' end of the first oligonucleotide and the third antibody to the 3' end of the second oligonucleotide, thereby obtaining the first and second proximity probes; (5) Dilute the first and second proximity probes for TNF-α and IL-6, respectively, to 1 μg / mL with PBS, mix the four probe solutions in equal volumes, and then mix them with equal volumes of the microbead complex separated in step (3), and incubate them at room temperature with rotation for 90 min to obtain an incubation solution; (6) Prepare the PCR reaction system according to Table 2 Table 2: PCR reaction system

[0051] (7) Add 5 μL of the incubation solution to the PCR reaction system, perform qPCR reaction according to the procedure in Table 3 and output the Ct value. The amplification curves of TNF-α and IL-6 (three replicates) are as follows: Figure 2 and 3 As shown, the linear analysis is as follows: Figure 4 and 5 shown.

[0052] Table 3: qPCR reaction procedure

[0053] Comparative Example: Determination of TNF-α and IL-6 in samples using conventional linkers in microbead-enriched multiple PLA Steps (1) to (5) are the same as in the embodiment; Step (6): replace the splint probe in Table 2 with a conventional linker, and keep the other components unchanged; Step (7) is the same as in the example, detecting the relationship between the output Ct value and the concentration of TNF-α and IL-6 ( Figure 3 ) found that compared with the splint probe used in the present invention, the linear relationship of microbead-enriched PLA multiplex target detection using conventional linkers was poor, and a higher Ct value was required to detect the same concentration of target molecules, indicating that the detection accuracy using conventional linkers was poor.

[0054] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A method for simultaneous detection of multiple targets based on proximity ligation, characterized in that: The following steps are involved: (a) providing a plurality of microbead complexes, each of which comprises a microbead carrier and a first antibody immobilized on the surface of the microbead carrier, wherein the first antibodies specifically bind to different target molecules; (b) incubating the plurality of microbead complexes with a sample to be tested, so that the first antibodies bind to their respective target molecules; (c) The incubated complexes are separated to obtain a variety of microbead complexes bound to different target molecules.

2. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 1, characterized in that: The first antibody is fixed on the surface of the microbead carrier by chemical cross-linking or biotin-streptavidin binding, and the microbead carrier is a magnetic bead with surface carboxylation, aminoation or streptavidin modification.

3. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 1, characterized in that: The method further comprises: (d) incubating the plurality of microbead complexes bound to different target molecules with a plurality of first proximity probes and second proximity probe pairs, wherein the first proximity probe comprises a second antibody coupled to the 5' end of the first oligonucleotide, and the second proximity probe comprises a third antibody coupled to the 3' end of the second oligonucleotide, wherein the second antibody and the third antibody respectively bind to different epitopes of their respective target molecules.

4. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 3, characterized in that: The first oligonucleotide and the second oligonucleotide comprise a uniform first adapter sequence and a uniform second adapter sequence at the 3' end and the 5' end, respectively, and the remaining sequences of the oligonucleotides in different probe pairs are different.

5. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 3, characterized in that: The coupling of the second antibody and the third antibody to the oligonucleotide is achieved by EDC / NHS coupling, click chemistry or maleimide-thiol reaction.

6. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 3, further comprising: (e) incubating the above-mentioned microbead complex bound to the proximity probe with a splint probe, wherein the splint probe consists of short hairpins at both ends and a hybridization region in the middle, and the hybridization region contains sequences complementary to the first adapter sequence and the second adapter sequence, respectively, so that the first and second oligonucleotides are adjacent to each other.

7. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 6, characterized in that: The short hairpins at both ends of the splint probe each have a loop with a length of 4-6 bases and a reverse complementary double strand with a length of 4-6 base pairs.

8. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 6, further comprising: (f) Incubating the multiple microbead complexes obtained in step S5 with DNA ligase, so that the adjacent 3' end of the first oligonucleotide is ligated to the 5' end of the second oligonucleotide to form a complete ligation product.

9. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 8, characterized in that: Further including: (g) mixing the complex obtained in step (f) with a qPCR reaction system comprising a plurality of primer pairs and performing amplification detection, wherein the primer pairs are complementary to the first and second oligonucleotides in each probe.

10. The method for simultaneous detection of multiple targets based on proximity ligation according to claim 9, characterized in that: The qPCR reaction system includes Taq DNA polymerase, buffer, ATP, dNTP, multiple detection probes and nuclease-free water. The two ends of the detection probe are respectively labeled with a fluorescent group and a quenching group, and the fluorescent groups of each probe are different to distinguish target molecules.

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