WO2017181339A1 - 蛋白配体和基因同时检测方法及试剂盒 - Google Patents

蛋白配体和基因同时检测方法及试剂盒 Download PDF

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WO2017181339A1
WO2017181339A1 PCT/CN2016/079646 CN2016079646W WO2017181339A1 WO 2017181339 A1 WO2017181339 A1 WO 2017181339A1 CN 2016079646 W CN2016079646 W CN 2016079646W WO 2017181339 A1 WO2017181339 A1 WO 2017181339A1
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nucleic acid
detection
ligand
pcr
streptavidin
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PCT/CN2016/079646
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French (fr)
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廖世奇
廖正宇
曾家豫
袁红霞
王小琦
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廖世奇
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to a nucleic acid and protein simultaneous detection technology and a detection kit, in particular to a method for converting a protein signal into a nucleic acid signal by a nucleic acid beacon ligand to complete the unification of the protein signal and the gene detection signal by real-time quantitative PCR.
  • the antibody capture method is a simple and convenient screening method.
  • the antigen is coated on a solid phase support, then the antibody is used to bind the antigen, the unbound antibody is removed by washing, and the labeled antibody specifically recognized by the binding antibody is used to detect the bound antibody.
  • Many antibody capture methods are utilized. Indirect method to detect antibodies.
  • the detection antibody is a murine antibody and the detection molecule may be a rabbit anti-mouse antibody with a detection label.
  • Conventional detection labels include radioisotopes, dyes, and enzymes that act on substrates to produce detectable molecules such as chromogens.
  • the antigen capture assay is to detect the presence or absence of an antigen in a sample. First, the antibody is first bound to the support, then the antigen is added to react with the antibody to form a complex, and finally the complex is detected. The antigen and antibody may also be reacted to form a complex, then bound to a solid support, and then the complex is detected.
  • ELISA is a well-known immunoassay. When it was first published in 1971, it initiated a revolution in diagnostic methods.
  • the traditional ELISA technique is like a sandwich sandwich method, in which two antibodies bind to an antigen.
  • the capture antibody binds to the antigen in the sample, and then reacts with the detection antibody of the conjugated enzyme that binds to the antigen to form a capture antibody-antigen-detection antibody "sandwich” complex, and finally the splicing enzyme activity shows the detection result.
  • the antibody detection method has great application value, but its detection range is limited by the Kd value of the capture antibody and the antigen reaction.
  • the detection bottom line is about 1% of the Kd value, when the analyte concentration is lowered to this possible detection.
  • the detection limit of the antibody detection method using a fluorescent or chemiluminescent detection system is about 1 pg/ml (10-4 M for a protein having an average molecular weight of 50,000 Daltons).
  • PCR polymerase chain reaction
  • Tag polymerase which is produced by thermophilic bacteria in hot springs. It has thermal stability and is hardly affected by high temperature in PCR denaturation. Therefore, it is not necessary to apply Klenow polymerization which is not heat resistant. Enzyme I, supplemented with polymerase after each denaturation.
  • an immuno-PCR method is generated in which a specific analyte is attached to a microplate and then detected by PCR amplification amplification, and the result of this method cannot be quantified.
  • Calf serum albumin BSA
  • a specific antibody against BSA with protein A-chain avidin fusion protein and biotin-labeled reporter amplicon
  • Electrophoretic analysis reports amplicons that can detect hundreds of BSA molecules.
  • this method cannot be used for biological sample detection because of the lack of specific capture molecules of the analyte.
  • Sandwich immuno-PCR is a modification of the traditional ELISA method, that is, the detection antibody is linked with a DNA marker, and then applied to the analysis of the biological sample, the early antibody immuno-PCR detection form: the primary antibody is immobilized on the plate, and then Samples were added and biotinylated to detect antibodies, streptavidin and biotinylated DNA. It was later modified to directly link DNA to antibodies and to generate PCR products using labeled primers. ELISE assays can be used instead of gel electrophoresis. PCR amplification produces a large number of DNA markers that can be detected by various methods: such as typical gel electrophoresis. , staining analysis.
  • PCR amplification of DNA markers carried by antibodies can increase sensitivity to antigen detection (this method lacks detection by the gene chip method). Therefore, immuno-PCR technology has been used to detect a variety of analytes. Although immuno-PCR improves sensitivity and is more sensitive than traditional ELISA methods, purification products are purified by gel electrophoresis. A large number of manual operations are required, which is time consuming, and primers for PCR amplification can be dimerized to produce by-products upon annealing, and the presence or contamination of contaminated ribose will also be amplified.
  • the immuno-PCR method is similar to the direct ELISA sandwich sandwich method, except that the detection method is selected.
  • the immuno-PCR method has been successfully used for detection, and some sensitivity is attomol level (including detection of the following substances: tumor necrosis factor, ⁇ -galactosidase, human thyroid stimulating hormone, murine soluble T-cell receptor, recombination Hepatitis B surface antigen, different human atrial natriuretic peptide, ⁇ -glucoside kinase, chorionic gonadotropin).
  • the antigen concentration is usually determined by post-PCR product analysis, either gel electrophoresis or PCR-ELISA. Quantitative analysis of the DNA marker of the PCR endpoint product is prone to erroneous results, as the product formation rate decreases after several logarithmic growth cycles, and PCR sample processing can cause laboratory contamination. In addition, these experiments require multiple steps and require rinsing, during which the antigen-antibody complex may dissociate.
  • Real-time quantitative PCR is a more advanced PCR technique that has been used for nucleic acid analysis.
  • PCR amplification of DNA is carried out in the presence of a non-linearly labeled double fluorescent hybridization probe, one of which is used as a reporter molecule whose emission spectrum is quenched by a second fluorescent dye.
  • Real-time PCR cleaves hybridization probes with Taq polymerase 5' nucleic acid activity during chain extension, resulting in the release of the reporter fluorescent dye from the quencher dye, resulting in an increase in the emission peak of the reporter molecule.
  • the entire reaction is monitored in real time.
  • Reverse transcription-PCR can also be applied.
  • the series detection system uses a 96-well thermal cycler to continuously detect the fluorescence spectrum of the PCR reaction in each well, thus eliminating the contamination of the replicon laboratory.
  • the reporter dye is located at the 5' end of the probe (FAM) and the quencher dye is located at the 3' end (TAMRA).
  • FAM 5' end of the probe
  • TAMRA 3' end
  • This probe binds to the specific target sequence amplified by PCR. When not bound, the fluorescence emitted by the 5' end FAM is 3 'TAMRA is quenched, but as the PCR cycle increases, the amplicon increases, the hybridization probe is cleaved by the polymerase 5'-3' exonuclease activity, and the reporter dye is separated from the quencher dye.
  • the sequence detection system activated fluorescence (488 nm) with an argon atom laser, and the laser device camera monitored the PCR reaction to collect fluorescence from 500 nm to 660 nm emitted from all 96 wells. Then use the corresponding principle, set up the internal reference and analyze the quantification directly through certain software.
  • nucleic acids and proteins in cells are a common phenomenon. Nucleic acids can fold to form secondary and tertiary structures, which is important for their interaction with proteins.
  • the in vitro method for detecting nucleic acid protein interactions is matured by diversifying the nucleotide sequences. SELEX technology is used to separate nucleotide ligands from selected targets. These ligands are called ligands or aptamers, meaning that nucleic acids can form a knot. Aligned into the pocket of the target molecule, SELEX technology is a method to screen the target molecule ligand using this principle.
  • Gold et al. (Gold L, et al. Annu Rev Biochem, 64: 763--797) used SELEX to screen the RNA and ssDNA ligands of systemic lupus erythematosus-specific antibodies in 1995, not only for the diagnosis of systemic lupus erythematosus, And to carry out disease monitoring and efficacy testing.
  • Gold et al. (Gold L, et al. Diagn Dec; 4(4):381-8) studied the resolution of ligand microarrays in the molecular diagnostic applications of ligand microarrays. Both show that nucleic acid ligand detection has great application prospects.
  • the current ligand detection method is a direct amplification method for ligand PCR amplification. This method is complicated to operate, and it is necessary to separate the ligand and the ligand, and the sensitivity is low (due to the separation of the ligand and the ligand, the purity of the ligand and the recombination of the residual ligand and the ligand block DNA replication) and the accuracy is poor.
  • RNA molecules After the start of SELEX technology, many target nucleotide ligands have been screened.
  • many proteins known to bind to nucleic acids can be used as suitable targets for SELEX technology, such as T4 DNA polymerase, phage R17 envelope protein, Escherichia coli rho Factor, Escherichia coli ribosomal protein S1, phenylalanine-tRNA synthetase, autoimmune antibody recognizing RNA, E2F transcription factor, different HIV-associated proteins.
  • T4 DNA polymerase phage R17 envelope protein
  • Escherichia coli rho Factor Escherichia coli ribosomal protein S1
  • phenylalanine-tRNA synthetase autoimmune antibody recognizing RNA
  • E2F transcription factor different HIV-associated proteins.
  • ligands can be obtained by in vitro screening by SELEX technology, see U.S. Patent Nos. WO 96/40991 and WD 97/38,134, which are incorporated herein by reference.
  • the sandwich method detects a capture molecule-antigen complex using a conventional enzyme-linked detection antibody, whereas labeling the oligonucleotide with a reporter enzyme molecule requires a chemical synthesis step and additional labor. The above method of using an antibody reagent itself is also difficult.
  • diagnostic methods still require increased sensitivity, reduced manual handling, and improved dynamic monitoring to quickly analyze and quantify the presence or absence of targets in a sample.
  • the object of the present invention is to provide a method and a kit for simultaneously detecting a protein ligand and a gene, which can convert a non-nucleic acid molecule such as a protein ligand into a nucleic acid signal by using a nucleic acid beacon ligand, and pass (multiple) real-time quantitative PCR. (or rolling circle replication) Amplification of nucleic acid beacons and gene nucleic acid molecules, simultaneous detection of ligands and genes.
  • the present invention provides a protein ligand and gene simultaneous detection kit for simultaneous real-time quantitative-PCR detection of a target molecule and a gene, the kit comprising:
  • At least a first reagent comprising a plurality of solid phase separation carriers coated with streptavidin agar, the solid phase separation carrier capable of separating the composite molecules;
  • At least a second reagent comprising a plurality of linked capture molecules, wherein the link capture molecule is capable of capturing a target molecule, and the solid phase separation carrier is capable of binding and binding to the linked capture molecule;
  • At least a third reagent comprising a plurality of detection molecules capable of specifically binding to the target molecule or capable of immunologically binding to the target antigen
  • At least a fourth reagent comprising a nucleic acid detection reagent comprising a beacon detection primer and a probe, a gene detection primer and a probe, and a nucleic acid polymerase.
  • the solid phase separation carrier coated with streptavidin agar is streptavidin agar magnetic bead particles, streptavidin magnetic bead particles, streptavidin agar test paper or streptavidin Agar chip, chemical group (carboxyl and amino groups, etc.) chips.
  • the ligated capture molecule is a biotinylated single (poly) cloning antibody, a biotinylated aptamer or a biotinylated nucleic acid ligand or an antibody with a chemical group (carboxyl and amino groups, etc.) Ligand.
  • the detection molecule is a target molecule nucleic acid beacon ligand, a nucleic acid beacon ligand detection molecule or a nucleic acid beacon ligand immunodetection molecule.
  • the nucleic acid detecting reagent is a primer and a probe comprising a beacon detection, a primer for detecting the gene, and Reverse transcription multiplex real-time quantitative-PCR reaction solution (rolling ring replication reaction solution) of probe and nucleic acid polymerase.
  • the solid phase separation carrier of the first reagent is streptavidin agar magnetic bead particles, and the streptavidin agar magnetic bead particles are suspended in a phosphate buffer.
  • streptavidin agar magnetic bead particles in the first reagent are dissolved in 5-10 mL of a 0.01-0.1 M phosphate buffer having a pH of 7.4.
  • the volume ratio of the streptavidin agar magnetic beads particles dissolved in the phosphate buffer was 50%.
  • the streptavidin agar magnetic bead particles have a particle diameter of 5 to 50 nm.
  • the first reagent contains 0.01% by weight of a sodium azide preservative.
  • each of the first reagents comprises 5-10 mL of a 0.01-0.1 M first phosphate buffer having a pH of 7.4, and the first phosphate buffer has a volume ratio dissolved therein. 50% of streptavidin agar magnetic beads having a particle diameter of 5 to 50 nm, and the first phosphate buffer contains 0.01% by weight of a sodium azide preservative.
  • the ligated capture molecule biotinylated monoclonal antibody, biotinylated aptamer or biotinylated nucleic acid ligand
  • a phosphate buffer a phosphate buffer
  • the linked capture molecule in the second reagent is dissolved in 5-10 mL of a 0.01-0.1 M phosphate buffer having a pH of 7.4.
  • concentration of the linked capture molecule in the second reagent is 0.003-3 ⁇ g/L.
  • the second reagent contains 0.01% by weight of a sodium azide preservative.
  • each of the second reagents comprises 5-10 mL of a 0.01-0.1 M second phosphate buffer having a pH of 7.4, and the second phosphate buffer is dissolved in biotin.
  • the monoclonal antibody is 0.003-3 ⁇ g/L, and the second phosphate buffer contains 0.01% by weight of a sodium azide preservative;
  • the detection molecule in the third reagent is a target molecule nucleic acid beacon ligand, and the target molecule nucleic acid beacon ligand is dissolved in a binding buffer.
  • the target molecule nucleic acid beacon ligand in the third reagent is dissolved in 5-10 mL of a binding buffer solution having a pH of 7.4 and a phosphate concentration of 0.01-0.1 M.
  • the binding buffer comprises at least 138 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na 2 HPO 4 , 1.1 mmol/L KH 2 PO 4 and 1 mmol/L MgCl 2 , pH value. Is 7.4. After preparation, it was sterilized by autoclaving for 20 min and stored at room temperature.
  • concentration of the detection molecule in the third reagent is 0.001-4 ⁇ g/L.
  • the third reagent contains 0.01% by weight of a sodium azide preservative.
  • each of the third reagents comprises 5-10 mL of a binding buffer solution having a pH of 7.4 and a phosphate concentration of 0.01-0.1 M, and the binding buffer solution is dissolved in the target molecule nucleic acid signal.
  • the standard base is 0.001-4 ⁇ g / L, and the binding buffer solution contains 0.01% by weight of a sodium azide preservative;
  • each of the fourth reagents comprises 1-3 mL of reverse transcription multiplex real-time quantitative PCR reaction solution
  • each of the fourth reagents comprises 10-30 units of nucleic acid polymerase
  • the reverse transcription multiplex real-time quantitative PCR reaction The liquid contains multiple pairs of primers and probes.
  • the unit of the nucleic acid polymerase herein refers to a biological unit of activity, a unit of measurement of the catalytic ability of the enzyme.
  • the present invention also provides a method for simultaneous detection of protein ligands and genes for simultaneous real-time quantitative-PCR detection of target molecules and genes, the method comprising the steps of:
  • the detection specimen is 10-1000 ⁇ L
  • the biotinylated monoclonal antibody is 20-100 ⁇ L
  • the nucleic acid beacon ligand is 20-100 ⁇ L.
  • the incubation is incubation at 37 ° C for 30-60 minutes.
  • step (1) may adopt an embodiment in which 10-100 ⁇ L of the test specimen, 20-100 ⁇ L of the biotinylated monoclonal antibody, and 20-100 ⁇ L of the nucleic acid beacon ligand and 500 ⁇ L of the binding buffer solution are incubated at 37 ° C. - 60 minutes, an antigen-antibody nucleic acid aptamer sandwich complex was formed.
  • the binding buffer comprises at least 138 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na 2 HPO 4 , 1.1 mmol/L KH 2 PO 4 and 1 mmol/L MgCl 2 , and the pH is 7.4. . After preparation, it was sterilized by autoclaving for 20 min and stored at room temperature.
  • the streptavidin-added agar magnetic bead particles are 10-100 ⁇ L.
  • the incubation is performed at 37 ° C for 30-60 minutes.
  • step (2) may adopt an embodiment in which 10-100 ⁇ L of streptavidin-coated agar magnetic beads particles are added to the antigen-antibody nucleic acid aptamer sandwich complex, and incubated at 37 degrees for 30-60 minutes.
  • the antigen-antibody nucleic acid aptamer sandwich complex and the streptavidin-coated agar magnetic bead microparticles are combined by the action of biotin and streptavidin to obtain a conjugate.
  • the washing operation is to adsorb the conjugate on the magnetic pole by electromagnetic action, and wash the mixture with the TPBS buffer 3-12 times for 3-10 minutes each time to remove the unbound substance.
  • the step (3) may adopt an embodiment in which the conjugate is adsorbed on the magnetic pole by electromagnetic action, and washed with TPBS buffer for 3-12 times for 3-10 minutes each time, the unbound substance is removed, and the reverse is added.
  • the real-time quantitative-PCR was recorded by multiplex real-time quantitative PCR reaction, and the real-time quantitative-PCR instrument automatically calculated the calibration result through the 4-point calibration calibration curve.
  • the test specimen includes serum, urine and body fluid, and the serum, urine and body fluid contain pathogenic bacteria, pathogenic particles and ligand molecules (nucleic acids, proteins, polypeptides, organic dyes, ATP, metal ions). Any molecule).
  • biotinylated monoclonal antibody chemically couples the monoclonal antibody to biotin.
  • nucleic acid beacon ligand is composed of a single-stranded ligand and a double-stranded beacon.
  • the nucleic acid beacon ligand can be selected from the nucleic acid beacon ligand disclosed in Chinese Patent No. CN1521272, "New Ligand Detection Method".
  • the reverse transcription multiplex real-time quantitative-PCR reaction solution is composed of a reverse transcription and a real-time quantitative system, which can reverse-reverse RNA into DNA, and then quantify genes and beacon molecules by real-time quantitative-PCR reaction.
  • the detection kit of the invention converts the target molecule signal into a nucleic acid signal by binding the nucleic acid beacon ligand to the target molecule (non-nucleic acid molecule), completes the unification of the protein signal and the nucleic acid signal, and performs quantitative detection by real-time quantitative-PCR.
  • the detection method can convert signals of a plurality of non-nucleic acid ligands into nucleic acid signals through nucleic acid beacon ligands, and has the characteristics of rapidity, high sensitivity, strong specificity, multi-ligand microarray detection and mechanization completion.
  • the invention utilizes agar magnetic beads (equal magnetic beads) as a carrier for magnetic separation of the detection molecules, has attached target molecules, can be mechanically sampled, separated, eluted, incubated, etc., and does not need to separate and purify the target molecules.
  • agar magnetic beads equal magnetic beads
  • an artificially modified DNA or RNA sequence ligated to an antibody or a ligand is used as a signal molecule, and detection of an antigen or a ligand is achieved by PCR amplification of a signal modification sequence.
  • the repair The decorated DNA or RNA sequence has a pair of primers and an intermediate human marker sequence, and the pair of primers may be the same or different for the target molecule to be detected and the purpose of detection, and the intermediate marker sequence is a marker of the target molecule. For example, when a plurality of samples are simultaneously detected, a pair of the same primers can make a plurality of sample detection data more realistic, and the label sequence is in one-to-one correspondence with the detection target molecules.
  • the detection instrument of the invention can achieve the correctness of the data and effectively eliminate the pollution by transforming the DNA or RNA modification sequence.
  • the detection instrument of the present invention collects and processes data by performing amplification product detection or oligonucleotide chip detection using a real-time quantitative-PCR instrument. Facilitate the detection of a single sample or multiple samples.
  • the detection instrument of the invention collects and processes the data by performing amplification product detection by real-time quantitative-PCR instrument, the whole process is completed in the PCR tube at one time, without the need to convert the product, purify, detect and the like, and the PCR product It can be closed and destroyed in the PCR tube to avoid environmental pollution caused by the escaped product.
  • the process is to completely automate the PCR product into the oligonucleotide chip detection system by pipetting, and the process is completely closed. No manual purification, separation, detection, etc. are required.
  • the detection instrument of the invention can mechanize all the detection processes, including: sample loading, incubation, washing, real-time quantitative-PCR instrument (or microarray chip) data detection and data processing, and reporting.
  • the present invention also provides a method for simultaneous detection of protein ligands and genes for simultaneous real-time quantitative-PCR detection of target molecules and genes, the method comprising the steps of:
  • the method for simultaneously detecting protein ligands and genes of the present invention has the beneficial effects of high mechanization degree; and the apparatus of the invention can be used for loading, adding reagents, washing, PCR, detecting and data processing, etc., and can be fully mechanized; Artificially modified sequences store a large amount of information.
  • the modified nucleic acid sequence of the present invention is a nucleic acid sequence (which may be ssDNA, dsDNA and RNA) which does not affect the binding activity of the specific oligonucleotide ligand terminal, and the length of the modified sequence may be based on the detection purpose.
  • a method for detecting a ligand is to use a specific binding of an antigen antibody and a specific binding of a ligand ligand to enhance the specificity of detection; when a ligand and a ligand directly bind to form a stable complex
  • the ligand information can be directly transmitted and amplified by PCR, so the sensitivity of the ligand is obviously improved; the detection sample is flexible.
  • Real-time fluorescent quantitative PCR can be used to detect single or small samples, or micro-array can be used to detect multiple samples; accurate information acquisition and processing systems. Since the information acquisition and processing system of the instrument of the invention is detected by real-time quantitative-PCR detection and microarray chip, there is a calibrated data report; the use of a single vessel, the operation process is simple and safe.
  • the detection uses a single vessel, that is, a disposable PCR tube, the operation process is simple, the data collection and processing can be completed in a completely closed state, the amplification product can be closed and destroyed, and the environment is cleaned effectively; the operation of the invention is simple and easy to popularize. application.
  • the operation is simple and convenient, and is convenient for general application in a general laboratory or clinical laboratory; assembly by the present invention
  • the detection kit or the constructed biochip can be widely applied to basic research and clinical detection, and can bring certain economic benefits and social benefits.
  • the present invention provides a nucleic acid beacon ligand for converting non-nucleic acid molecules such as protein ligands into nucleic acid signals, and amplifying nucleic acid beacons and genes by (multiple) real-time quantitative-PCR (or rolling circle replication). Nucleic acid molecules, a technical method for simultaneous detection of ligands and genes.
  • a kit for simultaneously detecting proteins (such as non-nucleic acid target molecules) and genes utilizes a nucleic acid beacon ligand molecule (this molecule has been applied for a novel Ligand detection method "CN1521272 patent) ligand binds to protein ligand to form a ligand-nucleic acid beacon ligand molecular complex, which converts target molecule ligand information into nucleic acid beacon information, and makes target molecule information and nucleic acid The gene information is unified, and the target molecule and the nucleic acid gene are simultaneously detected by real-time quantitative PCR.
  • the kit not only improves the sensitivity of the ligand target molecule, but also enables the ligand and the gene to be molecularly detected at the same level.
  • the detection technology has the characteristics of rapid, high sensitivity, high specificity, multi-ligand microarray detection and mechanization completion. Research on the omics of pathogens, genes and proteins is of great significance.
  • FIG. 1 is a schematic flow chart of a method for detecting a mixed gene after immobilization of a target molecule according to an embodiment of the present invention
  • FIG. 2 is a schematic flow chart of a method for simultaneously immobilizing and separating target molecules and detecting a gene according to an embodiment of the present invention
  • FIG. 3 is a schematic flow chart of an integrated detection method of an immunonucleic acid beacon ligand target molecule to a target molecule gene according to an embodiment of the present invention
  • 4a is a standard real-time quantitative-PCR curve of immunological PCR for detecting HBsAg according to an embodiment of the present invention
  • FIG. 4b is a standard curve diagram of immunosPCR for detecting HBsAg (FIG. 4a) according to an embodiment of the present invention
  • 4c is a real-time quantitative-PCR curve diagram of 16 results of repeated detection of HBsAg by immuno-PCR in an embodiment of the present invention
  • 4d is a statistical analysis diagram of 16 results of repeated detection of HBsAg by immunoPCR according to an embodiment of the present invention.
  • the nucleic acid beacon and the gene nucleic acid molecule are amplified by (multiple) real-time quantitative-PCR (or rolling circle replication), and the ligand and the ligand are Simultaneous detection of genes
  • the present invention provides a simultaneous detection kit for protein ligands and genes for simultaneous real-time quantitative-PCR detection of target molecules and genes, the kit comprising:
  • At least a first reagent comprising a plurality of solid phase separation carriers coated with streptavidin agar, the solid phase separation carrier capable of separating the composite molecules;
  • At least a second reagent comprising a plurality of linked capture molecules, wherein the link capture molecule is capable of capturing a target molecule, and the solid phase separation carrier is capable of binding and binding to the linked capture molecule;
  • At least a third reagent comprising a plurality of detection molecules capable of specifically binding to the target molecule or capable of immunologically binding to the target antigen
  • At least a fourth reagent comprising a nucleic acid detection reagent comprising a beacon detection primer and a probe, a gene detection primer and a probe, and a nucleic acid polymerase.
  • the solid phase separation carrier coated with streptavidin agar is streptavidin agar magnetic bead particles, streptavidin magnetic bead particles, streptavidin test paper, streptavidin chip , chemical groups (carboxyl and amino groups, etc.) chips.
  • the ligated capture molecule is a biotinylated single (poly) cloning antibody, a biotinylated aptamer, a biotinylated nucleic acid ligand or an antibody with a chemical group (carboxyl and amino groups, etc.) and an adaptation body.
  • the detection molecule is a target molecule nucleic acid beacon ligand, a nucleic acid beacon ligand detection molecule or a nucleic acid beacon ligand immunodetection molecule.
  • the nucleic acid detection reagent is a primer and a probe comprising a beacon detection, a primer and a probe for gene detection, and a reverse transcription multiplex real-time quantitative PCR reaction solution (rolling ring replication reaction solution) of a nucleic acid polymerase.
  • the solid phase separation carrier of the first reagent is streptavidin agar magnetic bead particles, and the streptavidin agar magnetic bead particles are suspended in a phosphate buffer.
  • streptavidin agar magnetic bead particles in the first reagent are dissolved in 5-10 mL of a 0.01-0.1 M phosphate buffer having a pH of 7.4.
  • the volume ratio of the streptavidin agar magnetic beads particles dissolved in the phosphate buffer was 50%.
  • the streptavidin agar magnetic bead particles have a particle diameter of 5 to 50 nm.
  • the first reagent contains 0.01% by weight of a sodium azide preservative.
  • each of the first reagents comprises 5-10 mL of a 0.01-0.1 M first phosphate buffer having a pH of 7.4, and the first phosphate buffer has a volume ratio dissolved therein. 50% of streptavidin agar magnetic beads having a particle diameter of 5 to 50 nm, and the first phosphate buffer contains 0.01% by weight of a sodium azide preservative.
  • the ligated capture molecule biotinylated single (poly) cloning antibody, biotinylated aptamer or biotinylated nucleic acid ligand
  • the second reagent is dissolved in a phosphate buffer.
  • the linked capture molecule in the second reagent is dissolved in 5-10 mL of a 0.01-0.1 M phosphate buffer having a pH of 7.4.
  • concentration of the linked capture molecule in the second reagent is 0.003-3 ⁇ g/L.
  • the second reagent contains 0.01% by weight of a sodium azide preservative.
  • each of the second reagents comprises 5-10 mL of a 0.01-0.1 M second phosphate buffer having a pH of 7.4, and the second phosphate buffer is dissolved in biotin.
  • the monoclonal antibody is 0.003-3 ⁇ g/L, and the second phosphate buffer contains 0.01% by weight of a sodium azide preservative;
  • the detection molecule in the third reagent is a target molecule nucleic acid beacon ligand, and the target molecule nucleic acid beacon ligand is dissolved in a binding buffer.
  • the target molecule nucleic acid beacon ligand in the third reagent is dissolved in 5-10 mL of a binding buffer solution having a pH of 7.4 and a phosphate concentration of 0.01-0.1 M.
  • the binding buffer comprises at least 138 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na 2 HPO 4 , 1.1 mmol/L KH 2 PO 4 and 1 mmol/L MgCl 2 , pH value. Is 7.4. After preparation, it was sterilized by autoclaving for 20 min and stored at room temperature.
  • concentration of the detection molecule in the third reagent is 0.001-4 ⁇ g/L.
  • the third reagent contains 0.01% by weight of a sodium azide preservative.
  • each of the third reagents comprises 5-10 mL of a binding buffer solution having a pH of 7.4 and a phosphate concentration of 0.01-0.1 M, and the binding buffer solution is dissolved in the target molecule nucleic acid signal.
  • the standard base is 0.001-4 ⁇ g / L, and the binding buffer solution contains 0.01% by weight of a sodium azide preservative;
  • Each of the fourth reagents comprises 1-3 mL of reverse transcription multiplex real-time quantitative PCR reaction solution, 10-30 units of nucleic acid polymerase, and the reverse transcription multiplex real-time quantitative PCR reaction solution contains a plurality of pairs of primers and Probe.
  • the unit of the nucleic acid polymerase herein refers to a biological unit of activity, a unit of measurement of the catalytic ability of the enzyme.
  • the invention also provides a simultaneous detection method of protein ligands and genes for simultaneous real-time quantitative-PCR detection of target molecules and genes, the method comprising the following steps:
  • the detection specimen is 10-1000 ⁇ L
  • the biotinylated monoclonal antibody is 20-100 ⁇ L
  • the nucleic acid beacon ligand is 20-100 ⁇ L.
  • the incubation is incubation at 37 ° C for 30-60 minutes.
  • step (1) may adopt an embodiment in which 10-100 ⁇ L of the test specimen, 20-100 ⁇ L of the biotinylated monoclonal antibody, and 20-100 ⁇ L of the nucleic acid beacon ligand and 500 ⁇ L of the binding buffer solution are incubated at 37 ° C. - 60 minutes, an antigen-antibody nucleic acid aptamer sandwich complex was formed.
  • the binding buffer comprises at least 138 mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na 2 HPO 4 , 1.1 mmol/L KH 2 PO 4 and 1 mmol/L MgCl 2 , and the pH is 7.4. . After preparation, it was sterilized by autoclaving for 20 min and stored at room temperature.
  • the streptavidin-added agar magnetic bead particles are 10-100 ⁇ L.
  • the incubation is performed at 37 ° C for 30-60 minutes.
  • step (2) may adopt an embodiment in which 10-100 ⁇ L of streptavidin-coated agar magnetic beads particles are added to the antigen-antibody nucleic acid aptamer sandwich complex, and incubated at 37 degrees for 30-60 minutes.
  • the antigen-antibody nucleic acid aptamer sandwich complex and the streptavidin-coated agar magnetic bead microparticles are combined by the action of biotin and streptavidin to obtain a conjugate.
  • the washing operation is to adsorb the conjugate on the magnetic pole by electromagnetic action, and wash the mixture with the TPBS buffer 3-12 times for 3-10 minutes each time to remove the unbound substance.
  • the step (3) may adopt an embodiment in which the conjugate is adsorbed on the magnetic pole by electromagnetic action, and washed with TPBS buffer for 3-12 times for 3-10 minutes each time, the unbound substance is removed, and the reverse is added.
  • the real-time quantitative-PCR was recorded by multiplex real-time quantitative PCR reaction, and the real-time quantitative-PCR instrument automatically calculated the calibration result through the 4-point calibration calibration curve.
  • the test specimen includes serum, urine and body fluid, and the serum, urine and body fluid contain pathogenic bacteria, pathogenic particles and ligand molecules (nucleic acids, proteins, polypeptides, organic dyes, ATP, metal ions). Any molecule).
  • biotinylated monoclonal antibody chemically couples the monoclonal antibody to biotin.
  • nucleic acid beacon ligand is composed of a single-stranded ligand and a double-stranded beacon.
  • the nucleic acid beacon ligand can be selected from the nucleic acid beacon ligand disclosed in Chinese Patent No. CN1521272, "New Ligand Detection Method".
  • the reverse transcription multiplex real-time quantitative-PCR reaction solution is composed of a reverse transcription and a real-time quantitative system, which can reverse-reverse RNA into DNA, and then quantify genes and beacon molecules by real-time quantitative-PCR reaction.
  • the detection kit of the invention converts the target molecule signal into a nucleic acid signal by binding the nucleic acid beacon ligand to the target molecule (non-nucleic acid molecule), completes the unification of the protein signal and the nucleic acid signal, and performs quantitative detection by real-time quantitative-PCR.
  • the detection method can convert signals of a plurality of non-nucleic acid ligands into nucleic acid signals through nucleic acid beacon ligands, and has rapid, high sensitivity, specificity, multi-ligand microarray detection and Mechanized completion and other characteristics.
  • the invention utilizes agar magnetic beads (equal magnetic beads) as a carrier for magnetic separation of the detection molecules, has attached target molecules, can be mechanically sampled, separated, eluted, incubated, etc., and does not need to separate and purify the target molecules.
  • agar magnetic beads equal magnetic beads
  • an artificially modified DNA or RNA sequence ligated to an antibody or a ligand is used as a signal molecule, and detection of an antigen or a ligand is achieved by PCR amplification of a signal modification sequence.
  • the modified DNA or RNA sequence has a pair of primers and an intermediate human marker sequence, and the pair of primers may be the same or different for the target molecule to be detected and the purpose of detection, and the intermediate marker sequence is a marker of the target molecule.
  • the pair of the same primers can make a plurality of sample detection data more realistic, and the label sequence is in one-to-one correspondence with the detection target molecules.
  • the detection instrument of the invention can achieve the correctness of the data and effectively eliminate the pollution by transforming the DNA or RNA modification sequence.
  • the detection instrument of the present invention collects and processes data by performing amplification product detection or oligonucleotide chip detection using a real-time quantitative-PCR instrument. Facilitate the detection of a single sample or multiple samples.
  • the detection instrument of the invention collects and processes the data by performing amplification product detection by real-time quantitative-PCR instrument, the whole process is completed in the PCR tube at one time, without the need to convert the product, purify, detect and the like, and the PCR product It can be closed and destroyed in the PCR tube to avoid environmental pollution caused by the escaped product.
  • the process is to completely automate the PCR product into the oligonucleotide chip detection system by pipetting, and the process is completely closed. No manual purification, separation, detection, etc. are required.
  • the detection instrument of the invention can mechanize all the detection processes, including: sample loading, incubation, washing, real-time quantitative-PCR instrument (or microarray chip) data detection and data processing, and reporting.
  • the invention also provides a simultaneous detection method of protein ligands and genes for simultaneous real-time quantitative-PCR detection of target molecules and genes, the method comprising the following steps:
  • the method for simultaneously detecting protein ligands and genes of the present invention has the beneficial effects of high mechanization degree; and the apparatus of the invention can be used for loading, adding reagents, washing, PCR, detecting and data processing, etc., and can be fully mechanized; Artificially modified sequences store a large amount of information.
  • the modified nucleic acid sequence of the present invention is a nucleic acid sequence (which may be ssDNA, dsDNA and RNA) which does not affect the binding activity of the specific oligonucleotide ligand terminal, and the length of the modified sequence may be based on the detection purpose.
  • a method for detecting a ligand is to use a specific binding of an antigen antibody and a specific binding of a ligand ligand to enhance the specificity of detection; when a ligand and a ligand directly bind to form a stable complex
  • the ligand information can be directly transmitted and amplified by PCR, so the sensitivity of the ligand is obviously improved; the detection sample is flexible.
  • Real-time fluorescent quantitative PCR can be used to detect single or small samples, or micro-array can be used to detect multiple samples; accurate information acquisition and processing systems. Since the information acquisition and processing system of the instrument of the invention is detected by real-time quantitative-PCR detection and microarray chip, there is a calibrated data report; the use of a single vessel, the operation process is simple and safe.
  • the detection uses a single vessel, that is, a disposable PCR tube, the operation process is simple, the data collection and processing can be completed in a completely closed state, the amplification product can be closed and destroyed, and the environment is cleaned effectively; the operation of the invention is simple and easy to popularize. application.
  • the operation is simple and convenient, and is convenient for general application in a general laboratory or clinical laboratory; assembly by the present invention
  • the detection kit or the constructed biochip can be widely applied to basic research and clinical detection, and can bring certain economic benefits and social benefits.
  • the present invention proposes to use a nucleic acid beacon ligand to transfer non-nucleic acid molecules such as protein ligands.
  • the nucleic acid signal is exchanged, and the nucleic acid beacon and the gene nucleic acid molecule are amplified by (multiple) real-time quantitative-PCR (or rolling circle replication), and a technical method for simultaneous detection of the ligand and the gene is carried out.
  • a kit for simultaneously detecting a protein (such as a non-nucleic acid target molecule) and a gene is a ligand that binds to a protein ligand using a ligand of a nucleic acid beacon ligand molecule (which has been applied for the "New Ligand Detection Method" CN1521272 patent) to form a ligand.
  • - Nucleic acid beacon ligand complex which converts the target molecule ligand information into nucleic acid beacon information, unifies the information of the target molecule and the nucleic acid gene information, and simultaneously detects the target molecule and the nucleic acid gene by real-time quantitative PCR.
  • the kit not only improves the sensitivity of the ligand target molecule, but also enables the ligand and the gene to be molecularly detected at the same level.
  • the detection technology has the characteristics of rapid, high sensitivity, high specificity, multi-ligand microarray detection and mechanization completion. Research on the omics of pathogens, genes and proteins is of great significance.
  • FIG. 1 is a schematic flowchart of a method for detecting a mixed gene after immobilizing a target molecule according to an embodiment of the present invention, the method comprising the following steps:
  • the electromagnetic pole uses the electromagnetic pole to adsorb the magnetic beads, separate and remove the unbound nucleic acid beacon ligand, add the washing solution 0.01-0.1M PBS, containing Tween-20 0.01-0.1M 400 ⁇ L, wash 3-12 times, 3-6min/time .
  • the aspirated serum is then added to the magnetic beads.
  • the lysate was added, chloroform phenol was extracted, and ethanol was precipitated to extract HIV-RNA 8 of HIV virus 5.
  • FIG. 2 is a schematic flowchart of a method for simultaneously fixing and separating a target molecule and a gene detection method according to an embodiment of the present invention, where the method includes the following steps:
  • Electromagnetically adsorbing magnetic beads separating and removing unbound biotinylated monoclonal antibody and nucleic acid beacon ligand, adding washing solution 0.01-0.1M PBS, containing Tween-20 0.01-0.1M 400-1000 ⁇ L, washing 3 -12 times, 3-6min/time.
  • FIG. 3 is a schematic flowchart of a method for integrally detecting a target molecule gene by using an immunonucleic acid beacon ligand target molecule according to an embodiment of the present invention, the method comprising the following steps:
  • mouse anti-human HBs-IgG monoclonal antibody 13 and mouse IgG Fc fragment-specific nucleic acid beacon ligand 12 are incubated together to form an IgG-nucleic acid beacon ligand complex; 1000 ⁇ L of the specimen, 20-100 ⁇ L of biotinylated goat anti-human-McAnti-HBs antibody 11 and 20-100 ⁇ L of mouse anti-human HBs-IgG antibody-nucleic acid beacon ligand complex were incubated to form McAnti-HBs antibody-HBV-small Mouse anti-human HBs-IgG antibody-nucleic acid beacon ligand complex.
  • the unpolarized biotinylated monoclonal antibody and the nucleic acid beacon ligand are separated and removed by the electromagnetic pole adsorption magnetic beads, and the washing solution is added to 0.01-0.1M PBS, containing Tween-20 0.01-0.1M 400 ⁇ L, and washed 3-12. Times, 3-6min/time.
  • This example is a detection experiment of HBsAg (hepatitis B virus surface antigen) by carboxy agar magnetic beads nucleic acid beacon ligand immuno-PCR.
  • the carboxyagarose magnetic beads were used as a carrier to coat a 100-fold dilution of 0.02 mg/ml of hepatitis B goat anti-human polyclonal antibody, and four 10 5 -10 8 fold dilution concentrations of 1000 pg, 100 pg, 10 pg, 1 pg were detected.
  • Figure 4a shows the quantitative curve of HBsAg detected by immunoPCR: the abscissa is the cycle number (Cycle), the ordinate is the fluorescence value (Norm Fluoro), and the threshold value (Threshold), the detection antigen concentration is (a): 1000pg (b) :100pg(c): 10pg(d): 1pg, the results show that the Ct value detected by real-time quantitative-PCR increases with the decrease of antigen concentration;
  • Figure 4b shows the standard curve for detecting HBsAg: the abscissa is the antigen concentration, the ordinate It is the Cts regression coefficient of real-time quantitative-PCR. As shown in Fig. 4a and Fig. 4b, it can be seen that HBsAg has a linear relationship in the quantitative domain of 10 5 - 10 8 , and the amount of protein to be detected can be detected.
  • the agarose beads carboxyl groups as the carrier, coated 100-fold diluted concentration of 0.02mg / ml polyclonal goat anti-human hepatitis B antibody, hole 16 is provided under the same reaction conditions as was diluted 107-fold repeat testing samples of HBsAg 5mg/ml and hepatitis B surface antigen to be tested, and then add 1000 dilutions of mouse anti-human hepatitis B surface antigen IgG monoclonal antibody and mouse IgG Fc nucleic acid beacon ligand, after incubation, washing, real-time quantitative PCR Detection, the stability of the sample test has been verified.
  • Figure 4c is a quantitative curve of repeated detection of HBsAg by magnetic bead immunoPCR.
  • the abscissa is the cycle number (Cycle), the ordinate is the fluorescence value (Norm Fluoro), and the threshold value (Threshold), and Figure 4d is the immuno-PCR for HBsAg.
  • the statistical analysis of 16 results was repeated, and the abscissa was 10 7 dilutions of the standard sample at 10 pg and the hepatitis B surface antigen to be detected, and the ordinate was the Ct value.
  • Figure 4c shows that the HBsAg test results are reproducible.
  • the statistical results in Fig. 4d show that the C value of the 16 test values to be tested is less than 0.05, and the advantage is significant and the repeatability is high.
  • the carboxy agarose magnetic beads were used as the isolation vector, and the magnetic bead-polyantibody-antigen-monoclonal-ligand beacon complex sandwich model was combined with Real Time based on the antigen-antibody specific reaction of the nucleic acid beacon ligand.
  • HBsAg hepatitis B surface antigen protein
  • the invention Compared with the ELISA method, the invention has the advantages that the nucleic acid beacon ligand is used instead of the secondary antibody, and the Real time-PCR detection technology has the characteristics of simple operation, intuitiveness, high repetition rate and high sensitivity, and the sensitivity reaches 10 3 -10. 4 times can be easily extended to the detection of trace proteins based on other aptamers.
  • the invention provides a method and a kit for simultaneously detecting protein ligands and genes, and belongs to the field of molecular biology. It includes molecular recognition of protein ligands and nucleic acid ligands, and converts protein signals into nucleic acid information through nucleic acid beacon ligands to achieve uniformity with gene detection information, and simultaneous detection of proteins and genes by real-time quantitative PCR.
  • the invention has the advantages that the target protein signal is converted into a uniform nucleic acid signal by using a nucleic acid beacon ligand; the detection of the target protein is enhanced by real-time quantitative PCR for the detection of the target protein; the conversion of the target protein protein nucleic acid information realizes the protein and The unification of the gene in the detection information; the conversion of the target molecule protein nucleic acid information realizes the uniformity of the detection sensitivity of the protein and the gene. Therefore, the invention is of great significance for proteomics and genomics research and clinical molecular detection.

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Abstract

一种蛋白配体和基因同时检测方法及试剂盒，属于分子生物学领域。包括蛋白配体和核酸配基的分子识别，通过核酸信标配基将蛋白信号转化成核酸信息，实现与基因检测信息的统一，利用实时定量PCR完成对蛋白和基因的同时检测。其优点是利用核酸信标配基将靶蛋白信号转换成统一核酸信号；利用实时定量PCR对信标扩增检测提高靶分子蛋白的检测灵敏的；靶分子蛋白核酸信息的转换实现蛋白和基因在检测信息的统一；靶分子蛋白核酸信息的转换实现蛋白和基因在检测灵敏度的统一。因此，对蛋白质组学和基因组学研究与临床分子检测具有重要意义。

Description

蛋白配体和基因同时检测方法及试剂盒 技术领域
本发明涉及一种核酸和蛋白同时检测技术和检测试剂盒的应用，尤其是涉及一种通过核酸信标配基将蛋白信号转换成核酸信号完成蛋白信号和基因检测信号统一，通过实时定量-PCR对蛋白配体和基因进行同时检测的检测方法及试剂盒。
背景技术
早期蛋白质检测是以抗体同特定待检物蛋白质的低解离常数和一定的特异性为基础，抗体捕捉方法是简易方便的筛选方法。抗体捕捉法中，抗原包被于固相支持物，然后用抗体去结合抗原，洗板去掉未结合的抗体，最后用和结合抗体特异识别的标记分子去检测结合抗体，许多抗体捕捉法是利用间接法来检测抗体。例如，检测抗体是鼠抗体，检测分子可能是带检测标记的兔抗鼠抗体。传统的检测标记包括放射性同位素，染料和作用于底物产生可检测分子如色原的酶。
抗原捕捉检测法是检测样品中有无抗原。首先抗体先结合到支持物上，然后抗原加入和抗体反应形成复合物，最后检测复合物。也可以抗原抗体先反应形成复合物后，再结合到固相支持物上，然后检测复合物。
ELISA是广为人知的免疫检测法，1971年一问世，就启动了诊断方法的革命，传统的ELISA技术似三明治夹心法，即两抗体共同结合到某一抗原。捕捉抗体同样品中的抗原结合，再加入同抗原结合的偶连酶的检测抗体反应形成捕捉抗体-抗原-检测抗体“三明治”复合物，最后测偶连酶活性显示检测结果。
抗体检测法具有较大的应用价值，但是他的检测范围受捕捉抗体和抗原反应的Kd值限制，在实践中，检测底线大约是Kd值的1％，当待分析物浓度降低到这个可能检测限时，捕捉到待分析物抗体百分率不足以产生相对于信噪比的可检测信号。因此，用荧光或化学发光检测系的抗体检测法的检测下限约1pg/ml(10-4M对平均分子量50,000道尔顿的蛋白质)
随着基因技术应用的快速发展，在抗原抗体检测方面已有较多的基因检测 技术。
核酸待测物的检测要求不同于抗体检测的技术。在二十世纪八十年代中期，DNA技术的研究发现了通过酶重复扩增过程可扩增DNA的方法，后来叫多聚酶链反应(PCR)：首先加入两互补的寡核苷酸序列(叫引物)，它将结合在所要扩增的区域的两侧，然后加热变性，在降温退火让引物同互补序列结合，再加入Klenow片段DNA聚合酶I以延伸引物。通过重复变性、退火、延伸所希望的片段，目标DNA就会以指数方式扩增。PCR曾经过许多改进，一个重要的变化是Tag聚合酶的应用，它产于温泉中嗜热菌，具有热稳定性，几乎不被PCR变性中高温影响，所以不必象应用不耐热的Klenow聚合酶I，每一次变性后得补加聚合酶。
以PCR作为扩增系统的运用中，产生了免疫-PCR方法，它是把特定待测物连接到微孔板上，然后用PCR扩增放大能力来检测，这个方法结果也不能定量。小牛血清白蛋白(BSA)被动吸附到一免疫检测板上，加入对BSA的特异抗体(连有蛋白A链亲和素融合蛋白及生物素标记的报告扩增子)，PCR后用琼脂糖电泳分析报告扩增子，可检测到几百个BSA分子。然而，这种方法不能用于生物样品检测因为缺乏待测物的特异捕捉分子。人们曾对该方法做了改进：用生物素化第二抗体和一个连接生物素化报告扩增子的链亲和素融合蛋白。然而5种试剂的加入、洗板、扩增、检测很费时费力，而且解离复杂。所以产生了新的改进：把报告扩增子共价连到第二抗体上，直接连接减少了试剂数目和解离复杂性，然而仍需要PCR操作，增加了劳动和试验室污染的可能性。
三明治式免疫-PCR是由传统ELISA方式的改编而来，即检测抗体连有DNA标记，再运用到分析检测生物样品，早期的抗体免疫-PCR检测形式：初级抗体被固定在平板上，然后依次加入样品，再生物素化检测抗体，链亲和素和生物素化的DNA。后来改进为直接连接DNA到抗体上和用标记引物产生PCR产物，可ELISE检测分析代替凝胶电泳分析，PCR的扩增能力产生大量DNA标记，可以用各种方法检测：如典型的凝胶电泳、染色分析。PCR扩增抗体携带的DNA标记可实现提高对抗原检测的灵敏度(该法缺少用基因芯片法来检测)。所以免疫-PCR技术已被用于检测多种待检物。虽然免疫-PCR提高了灵敏度，比传统ELISA方法的灵敏度还高，但用凝胶电泳纯化扩增产物 需要大量人工操作，因此耗时，而且用于PCR扩增的引物在退火时可二聚体扩增产生副产品，另外，它污染核糖的存在或污染也将同样被扩增。
采用一个捕捉抗体，免疫PCR方法就类似于直接ELISA三明治夹心法，不同点在于选择检测方法。免疫-PCR方法已被成功用于检测，有的敏感度达attomol水平(包括检测下列物质：肿瘤坏死因子、β-半乳糖甘酶、人甲状腺刺激激素、鼠可溶T-细胞受体、重组乙肝表面抗原、不同的人心钠素、β-葡糖苷激酶、绒毛膜促性腺激素)。
在免疫PCR中，抗原浓度通常由PCR后产物分析决定，可以是凝胶电泳分析，也可以是PCR-ELISA分析。定量分析PCR终点产物的DNA标记易产生错误结果，因为产物形成率在几个对数增长循环后即下降，再者PCR样品处理可致实验室污染。另外，这些实验需多步骤且需冲洗，这个过程中抗原抗体复合物可能解离。
实时定量PCR是更先进的PCR技术，已用于核酸分析。在实时PCR中，PCR扩增DNA是在非线性标记双荧光杂交探针存在条件下进行，其中一种荧光染料用作报告分子，它的发射光谱被第二种荧光染料淬灭。实时PCR在链延伸时，利用Taq多聚酶5’核酸活性切割杂交探针，结果使报告荧光染料从淬灭染料中释放出来，致报告分子发射峰值增加。整个反应实时监控。逆转录-PCR也可应用。系列检测系统用96孔热循环仪可连续检测每孔中PCR反应时的荧光谱，因此，可排除复制子试验室污染。
报告染料位于探针5’端(FAM)，淬灭染料位于3’端(TAMRA)，此探针和PCR扩增的特殊靶序列结合，当未结合时，5’端FAM发射的荧光被3’TAMRA淬灭，但随着PCR循环增加，扩增子增多，杂交探针被多聚酶5’-3’外切酶活性切割，报告染料就从淬灭染料上分离出来。序列检测系统用氩原子激光激活荧光(488nm)，激光装置照相机监控PCR反应，收集从所有96孔发出的500nm-660nm荧光。然后利用相应原理、设立内参照直接通过一定的软件分析定量。
核酸与蛋白质在细胞内相互作用是普遍现象。核酸能折叠形成二级结构和三级结构，这对其与蛋白质相互结合作用非常重要。通过使核苷酸序列多样化而使体外检测核酸蛋白质相互作用方法成熟。SELEX技术被用来分离所选靶目标的核苷酸配体，这些配体被称为配基或适配体，意即核酸可以形成一定结 构并适配入靶分子的口袋，SELEX技术就是利用该原理筛选靶分子配基的方法。
Gold等在1995年(Gold L，et al.Annu Rev Biochem，64：763--797)应用SELEX筛选出的系统性红斑狼疮特异抗体的RNA和ssDNA配基，不仅对系统性红斑狼疮进行诊断，而且进行病情监测和疗效检验。Gold等又在1999年(Gold L，et al.Diagn Dec；4(4)：381-8)在配基微阵分子诊断应用研究中，对配基微阵分辨率进行研究。均显出核酸配基检测有巨大的应用前景。但是目前的配基检测都是采用直接对配基PCR扩增放大检测的方法。这种方法操作复杂，需要将配体和配基分离，且灵敏度低(由于配体和配基分离后，配基纯度及残留配体和配基的再结合阻断DNA复制)和准确性差。
SELEX技术开始后，许多靶标的核苷酸配基已被筛选出，尤其是已知能和核酸结合的许多蛋白质可作为SELEX技术的较合适靶标，如T4DNA聚合酶、噬菌体R17被膜蛋白，大肠杆菌rho因子，大肠杆菌核糖体蛋白S1，苯丙氨酸-tRNA合成酶，识别RNA的自身免疫抗体，E2F转录因子，不同的HIV相关蛋白。
SELEX技术可筛选出许多不同蛋白配基的事实引发了配基应用的拓展，可作为单抗和多抗产品的替代品应用于诊断和治疗。配基代替抗体应用于诊断已显示其价值。DNA聚合酶的配基已被用于热启动PCR来诊断低拷贝的复制子，提高PCR敏感性和保真性。配基也被用于促进实验方法。中性弹性蛋白的酶配体荧光标记后用于流式细胞仪检测弹性酶浓度，中性弹性蛋白酶配基尚用于鼠肺炎症诊断模型体内诊断。
在酶联寡核苷酸方法(ELDNA)中，一个或多个抗体被对抗原有高亲和力高特异性的配基取代。这样的配基可通过SELEX技术体外筛选获得，见美国专利WO96/40991和WD97/38134描述了酶联寡核苷酸方法，其中用核苷酸配基代替夹心法中的检测抗体或捕捉抗体。通常，夹心法用传统的酶联检测抗体检测捕捉分子-抗原复合物，然而用报告酶分子标记寡核苷酸，这需要化学合成步骤和额外的劳动。上述用抗体试剂的方法本身也存在着困难。
上两个专利中也提到捕捉分子-靶分子-检测分子复合物中核酸配基PCR扩增检测系统；利用常用报告分子如酶、生物素等的PCR引物来扩增扩增子，这样做是提高了配体的数量，但是也带来了不利：需进一步把扩增的配基和不 纯的核苷酸引物二聚体分离的步骤。传统的凝胶分离需大量人工劳作。DNA和RNA配基都存在着这样的问题。用标记的引物不能解决不纯问题和引物二聚体扩增问题，因此，无法定量。
尽管上述进步显著，但诊断方法仍需提高灵敏度，减少人工操作，改进动态监测以快速分析样品中靶标的存在与否及对其定量。
发明公开
本发明的目的在于提出一种蛋白配体和基因同时检测方法及试剂盒，使其可以利用核酸信标配基将蛋白配体等非核酸分子转换成核酸信号，通过(多重)实时定量-PCR(或滚环复制)扩增核酸信标和基因核酸分子，实施对配体和基因同时检测。
为实现上述目的，本发明提供一种蛋白配体和基因同时检测试剂盒，用于靶分子和基因同时实时定量-PCR检测，该试剂盒包括：
至少一第一试剂，该第一试剂内包含多个包覆有链霉亲合素琼脂的固相分离载体，该固相分离载体能够分离复合分子；
至少一第二试剂，该第二试剂内包含多个连接捕捉分子，该连接捕捉分子能够捕捉靶分子，该固相分离载体与该连接捕捉分子之间能够连接结合；
至少一第三试剂，该第三试剂内包含多个检测分子，该检测分子能够与靶分子特异性结合，或者能够与靶抗原免疫结合；
至少一第四试剂，该第四试剂内包含核酸检测试剂，该核酸检测试剂内包含信标检测的引物和探针、基因检测的引物和探针以及核酸聚合酶。
其中，该包覆有链霉亲合素琼脂的固相分离载体为链霉亲合素琼脂磁珠微粒、链霉亲合素磁珠微粒、链霉亲合素琼脂试纸或、链霉亲合素琼脂芯片、化学基团(羧基和氨基等)芯片。
其中，该连接捕捉分子为生物素化的单(多)克隆抗体、生物素化的适配体或、生物素化的核酸配基或带有化学基团(羧基和氨基等)的抗体和适配体。
其中，该检测分子为靶分子核酸信标配基、核酸信标配基检测分子或核酸信标配基免疫检测分子。
其中，该核酸检测试剂为包含信标检测的引物和探针、基因检测的引物和 探针以及核酸聚合酶的反转录多重实时定量-PCR反应液(滚环复制反应液)。
其中，该第一试剂的该固相分离载体为链霉亲合素琼脂磁珠微粒，并且该链霉亲和素琼脂磁珠微粒为悬浮于磷酸盐缓冲液中。
其中，该第一试剂内的该链霉亲和素琼脂磁珠微粒为溶于5-10mL的pH值为7.4的0.01-0.1M的磷酸盐缓冲液中。
其中，溶于磷酸盐缓冲液的该链霉亲和素琼脂磁珠微粒的体积比为50％。
其中，该链霉亲和素琼脂磁珠微粒粒径为5-50nm。
其中，该第一试剂内含重量比0.01％的叠氮钠防腐剂。
该第一试剂可以采用如下实施方式：每一该第一试剂包括5-10mL的pH值为7.4的0.01-0.1M的第一磷酸盐缓冲液，该第一磷酸盐缓冲液内溶有体积比50％粒径5-50nm的链霉亲和素琼脂磁珠微粒，并该第一磷酸盐缓冲液含重量比0.01％的叠氮钠防腐剂。
其中，该第二试剂的该连接捕捉分子(生物素化的单克隆抗体、生物素化的适配体或生物素化的核酸配基)为溶于磷酸盐缓冲液。
其中，该第二试剂内的该连接捕捉分子为溶于5-10mL的pH值为7.4的0.01-0.1M的磷酸盐缓冲液中。
其中，该第二试剂内的该连接捕捉分子的浓度为0.003-3μg/L。
其中，该第二试剂内含重量比0.01％的叠氮钠防腐剂。
该第二试剂可以采用如下实施方式：每一该第二试剂包括5-10mL的pH值为7.4的0.01-0.1M的第二磷酸盐缓冲液，该第二磷酸盐缓冲液内溶有生物素化的单克隆抗体0.003-3μg/L，并该第二磷酸盐缓冲液含重量比0.01％的叠氮钠防腐剂；
其中，该第三试剂内的该检测分子为靶分子核酸信标配基，该靶分子核酸信标配基为溶于结合缓冲溶液(binding buffer)中。
其中，该第三试剂内的该靶分子核酸信标配基为溶于5-10mL的pH值为7.4的磷酸根浓度为0.01-0.1M的结合缓冲溶液中。
其中，该结合缓冲溶液(binding buffer)内至少包括138mmol/L NaCl，2.7mmol/L KCl，8.1mmol/L Na2HPO4，1.1mmol/L KH2PO4及1mmol/L MgCl2，pH值为7.4。配制后以高压蒸汽灭菌20min，室温保存。
其中，该第三试剂内的该检测分子的浓度为0.001-4μg/L。
其中，该第三试剂内含重量比0.01％的叠氮钠防腐剂。
该第三试剂可以采用如下实施方式：每一该第三试剂包括5-10mL的pH值为7.4的磷酸根浓度为0.01-0.1M的结合缓冲溶液，该结合缓冲溶液内溶有靶分子核酸信标配基0.001-4μg/L，并该结合缓冲溶液含重量比0.01％的叠氮钠防腐剂；
其中，每一该第四试剂内包含1-3mL反转录多重实时定量-PCR反应液，每一该第四试剂内包含10-30单位核酸聚合酶，该反转录多重实时定量-PCR反应液内含多对引物和探针。此处核酸聚合酶的单位指生物学的活性单位，酶的催化能力的计量单位。
为实现上述目的，本发明还提供一种蛋白配体和基因同时检测方法，用于靶分子和基因同时实时定量-PCR检测，该方法包括下述步骤：
(1)将检测标本、生物素化的单克隆抗体和核酸信标配基一起孵育，形成抗原抗体核酸适配体夹心复合物；
(2)在该抗原抗体核酸适配体夹心复合物中添加包被链霉亲和素的琼脂磁珠微粒一起孵育，使该抗原抗体核酸适配体夹心复合物与该包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的作用结合，得一结合物；以及
(3)洗涤除去未结合的物质后，加入反转录多重实时定量-PCR反应液进行实时定量-PCR，记录结果。
其中，于步骤(1)中，检测标本为10-1000μL，生物素化的单克隆抗体为20-100μL，核酸信标配基为20-100μL。
其中，于步骤(1)中，所述孵育为在37℃下孵育30-60分钟。
即，步骤(1)可采用如下实施方式：将10-100μL检测标本、20-100μL生物素化的单克隆抗体和20-100μL核酸信标配基及500μL结合缓冲溶液一起在37℃下孵育30-60分钟，形成抗原抗体核酸适配体夹心复合物。该结合缓冲溶液(binding buffer)内至少包括138mmol/L NaCl，2.7mmol/L KCl，8.1mmol/L Na2HPO4，1.1mmol/L KH2PO4及1mmol/L MgCl2，pH值为7.4。配制后以高压蒸汽灭菌20min，室温保存。
其中，于步骤(2)中，添加的链霉亲和素的琼脂磁珠微粒为10-100μL。
其中，于步骤(2)中，所述孵育为在37℃下孵育30-60分钟。
即，步骤(2)可采用如下实施方式：在该抗原抗体核酸适配体夹心复合物中添加10-100μL包被链霉亲和素的琼脂磁珠微粒，在37度下孵育30-60分钟，使该抗原抗体核酸适配体夹心复合物与该包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的作用结合，得一结合物。
其中，于步骤(3)中，所述洗涤操作为利用电磁作用将该结合物吸附在磁极上，以TPBS缓冲液洗涤3-12次，每次3-10分钟，除去未结合的物质。
即，步骤(3)可采用如下实施方式：利用电磁作用将该结合物吸附在磁极上，以TPBS缓冲液洗涤3-12次，每次3-10分钟，除去未结合的物质，加入反转录多重实时定量-PCR反应液进行实时定量-PCR，实时定量-PCR仪器自动通过4点校正的定标曲线计算得到检测结果。
其中，该检测标本包括血清、尿液和体液，所述血清、尿液和体液中含有致病菌、致病粒子和配体分子(核酸、蛋白质、多肽、有机染料、ATP、金属离子类的任何分子)。
其中，该生物素化的单克隆抗体是将单克隆抗体化学耦联生物素。
其中，该核酸信标配基是单链配基和双链信标组成。该核酸信标配基可选用中国专利CN1521272《新型配体检测方法》中所公开的核酸信标配基。
其中，该反转录多重实时定量-PCR反应液是反转录和实时定量体系组成，能够将RNA反转录成DNA，再通过实时定量-PCR反应对基因和信标分子进行定量。
本发明所述蛋白配体和基因同时检测试剂盒，其有益效果在于：
本发明的检测试剂盒是通过核酸信标配基与靶分子(非核酸分子)结合将靶分子信号转换成核酸信号，完成蛋白信号与核酸信号的统一，再利用实时定量-PCR进行定量检测。该检测方法可使多种非核酸配体的信号通过核酸信标配基转换成核酸信号，具有快速、灵敏度高、特异性强、多配体微阵检测和可机械化完成等特点。
本发明是利用琼脂磁珠(等磁珠)作为载体对检测分子进行磁性分离，具有附着靶分子，可机械加样，分离，洗脱，孵育等，无须再将靶分子分离提纯。
本发明是利用连接在抗体或配基上的人为修饰DNA或RNA序列作为信号分子，通过对信号修饰序列的PCR扩增，实现对抗原或配体的检测。该修 饰DNA或RNA序列具有一对引物和中间的人为标记序列，针对被检测的靶分子和检测目的的要求这一对引物可以相同也可以不相同，中间的标记序列是靶分子的标记。如：在多种样品同时检测时一对相同引物可以使多种样品检测数据更切合实际，标记序列则是和检测靶分子一一对应。
本发明的检测仪器可通过对DNA或RNA修饰序列的变换来达到数据的效正和有效的消除污染。
本发明的检测仪器是通过用实时定量-PCR仪进行扩增产物检测或寡核苷酸芯片检测来数据采集和处理。便于单一样品或多种样品的检测。
本发明检测仪器如果通过用实时定量-PCR仪进行扩增产物检测来数据采集和处理，其整个过程都在PCR管内一次性完成，无须将产物转换地方，进行提纯、检测等步骤，并且PCR产物可在PCR管封闭销毁，避免逸出产物造成环境污染。
本发明检测仪器如果通过用寡核苷酸芯片检测来数据采集和处理，其过程是将PCR产物通过移液机转入寡核苷酸芯片检测体系内全自动化完成，其过程是全封闭的，无须进行人工提纯、分离、检测等步骤。
本发明检测仪器可机械化完成全部检测过程，包括：加样、孵育、洗管、实时定量-PCR仪(或微阵芯片)的数据检测和数据处理、发报告等。
为实现上述目的，本发明还提供一种蛋白配体和基因同时检测方法，用于靶分子和基因同时实时定量-PCR检测，该方法包括下述步骤：
(1)标本制备：按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固，然后在3000-6000转/min下离心，分离血清为标本；
(2)抗原抗体核酸适配体夹心复合物的形成：将10-100μL标本、25pmol生物素化的单克隆抗体和25pmol核酸信标配基一起孵育，形成抗原抗体核酸适配体夹心复合物；
(3)利用磁珠分离抗原抗体核酸适配体夹心复合物：在抗原抗体核酸适配体夹心复合物中添加20-100μL包被链霉亲和素的琼脂磁珠微粒进行孵育，使抗原抗体核酸适配体夹心复合物与包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的的作用结合，得一结合物；
(4)反转录-PCR扩增：利用电磁作用将结合物吸附在磁极上，洗涤除去未结合的物质，加入反转录多重实时定量-PCR反应液进行实时定量-PCR；
(5)数据采集和处理。
其中，换算方法：1μg/L/分子量＝1/分子量umol＝1/分子量×1000pmol。
本发明所述蛋白配体和基因同时检测方法，其有益效果在于：机械化成度高；利用本发明仪器可使加样、加试剂、洗涤、PCR、检测及数据处理等，可全机械化完成；人为修饰序列储存信息量大。本发明的修饰核酸序列为特异寡核苷酸配基末端人为加上的一些不影响其结合活性的核酸序列(可以是ssDNA、dsDNA和RNA)，修饰序列的长短，碱基序列可根据检测目的设计，不同的修饰序列可通过寡核苷酸配基与配体的特异结合反映配体的性质和数量；灵敏度高、特异性强；本发明仪器的靶分子检测是采用新型免疫-PCR和修饰配基(核酸(类)抗体)的检测方法，是利用抗原抗体的特异结合和配体配基的特异结合，使检测的特异性增强；在配基和配体直接结合形成稳定的复合物时，由于在配体特异结合的寡核苷酸配基上有人为修饰序列，可直接通过PCR指数级富集，对配体信息进行传递和放大，因此其灵敏度有明显的提高；检测样品灵活。可用实时荧光定量PCR检测单一或少量样品，也可以用微阵芯片检测多种样品；准确的信息采集和处理系统。由于本发明仪器的信息采集和处理系统是通过实时定量-PCR检测和微阵芯片检测，故有校准的数据报告；使用器皿单一，操作过程简单、安全。检测时使用器皿单一，即一次性PCR管，操作过程简单，数据采集及处理可在全封闭的情况下完成，扩增产物可封闭销毁，有效保证环境的洁净；本发明的操作简单，易于普及应用。由于在反应时只需将配体和配基室温孵育20-45分钟就能完成配体和配基的结合反应，因此操作简便，便于一般实验室或临床检验科室的普及应用；利用本发明组装的检测试剂盒或构建的生物芯片可以广泛应用于基础研究与临床检测，可带来一定的经济效益与社会效益。
综上所述，本发明提出一种利用核酸信标配基将蛋白配体等非核酸分子转换成核酸信号，通过(多重)实时定量-PCR(或滚环复制)扩增核酸信标和基因核酸分子，实施对配体和基因同时检测的技术方法。同时检测蛋白(等非核酸靶分子)和基因的试剂盒是利用核酸信标配基分子(该分子已申请《新型 配体检测方法》CN1521272专利)的配基与蛋白配体结合，形成配体-核酸信标配基分子复合物，使靶分子配体信息转换成核酸信标信息，使靶分子的信息与核酸基因信息统一，再利用实时定量-PCR对靶分子和核酸基因进行同时检测。该试剂盒不仅提高配体靶分子的灵敏度，而且使配体和基因在同一水平进行分子检测，检测技术具有快速、灵敏度高、特异性强、多配体微阵检测和可机械化完成等特点。对病原体，基因和蛋白质等组学的研究具有重要意义。
以下结合附图和具体实施例对本发明进行详细描述，但不作为对本发明的限定。
附图简要说明
图1为本发明一实施例提供的固定分离靶分子后混合基因检测方法流程示意图；
图2为本发明一实施例提供的同时固定分离靶分子和基因检测方法流程示意图；
图3为本发明一实施例提供的免疫核酸信标配基靶分子对靶分子基因一体检测方法流程示意图；
图4a为本发明一实施例中免疫PCR检测HBsAg的标准实时定量-PCR曲线图；
图4b为本发明一实施例中免疫PCR检测HBsAg的(图4a)标准曲线图；
图4c为本发明一实施例中免疫PCR检测HBsAg的重复检测16个结果的实时定量-PCR曲线图；
图4d为本发明一实施例中免疫PCR对HBsAg的重复检测16个结果的统计分析图。
其中，附图标记：
1：P24抗体
2：磁珠
3：P24抗原
4：引物
5：HIV病毒
6：探针
7：P24核酸信标配基
8：HIV-RNA
9：gp120抗体
10：gp120核酸信标配基
11：HBV表面抗体
12：Fc核酸信标配基
13：小鼠抗人HBs-IgG抗体
14：HBV病毒
15：HBV-DNA
16：信标扩增序列
17：基因扩增序列
实现本发明的最佳方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的其中一个实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
为可以利用核酸信标配基将蛋白配体等非核酸分子转换成核酸信号，通过(多重)实时定量-PCR(或滚环复制)扩增核酸信标和基因核酸分子，实施对配体和基因同时检测，本发明提供一种蛋白配体和基因同时检测试剂盒，用于靶分子和基因同时实时定量-PCR检测，该试剂盒包括：
至少一第一试剂，该第一试剂内包含多个包覆有链霉亲合素琼脂的固相分离载体，该固相分离载体能够分离复合分子；
至少一第二试剂，该第二试剂内包含多个连接捕捉分子，该连接捕捉分子能够捕捉靶分子，该固相分离载体与该连接捕捉分子之间能够连接结合；
至少一第三试剂，该第三试剂内包含多个检测分子，该检测分子能够与靶分子特异性结合，或者能够与靶抗原免疫结合；
至少一第四试剂，该第四试剂内包含核酸检测试剂，该核酸检测试剂内包含信标检测的引物和探针、基因检测的引物和探针以及核酸聚合酶。
其中，该包覆有链霉亲合素琼脂的固相分离载体为链霉亲合素琼脂磁珠微粒、链霉亲合素磁珠微粒、链霉亲合素试纸、链霉亲合素芯片、化学基团(羧基和氨基等)芯片。
其中，该连接捕捉分子为生物素化的单(多)克隆抗体、生物素化的适配体、生物素化的核酸配基或带有化学基团(羧基和氨基等)的抗体和适配体。
其中，该检测分子为靶分子核酸信标配基、核酸信标配基检测分子或核酸信标配基免疫检测分子。
其中，该核酸检测试剂为包含信标检测的引物和探针、基因检测的引物和探针以及核酸聚合酶的反转录多重实时定量-PCR反应液(滚环复制反应液)。
其中，该第一试剂的该固相分离载体为链霉亲合素琼脂磁珠微粒，并且该链霉亲和素琼脂磁珠微粒为悬浮于磷酸盐缓冲液中。
其中，该第一试剂内的该链霉亲和素琼脂磁珠微粒为溶于5-10mL的pH值为7.4的0.01-0.1M的磷酸盐缓冲液中。
其中，溶于磷酸盐缓冲液的该链霉亲和素琼脂磁珠微粒的体积比为50％。
其中，该链霉亲和素琼脂磁珠微粒粒径为5-50nm。
其中，该第一试剂内含重量比0.01％的叠氮钠防腐剂。
该第一试剂可以采用如下实施方式：每一该第一试剂包括5-10mL的pH值为7.4的0.01-0.1M的第一磷酸盐缓冲液，该第一磷酸盐缓冲液内溶有体积比50％粒径5-50nm的链霉亲和素琼脂磁珠微粒，并该第一磷酸盐缓冲液含重量比0.01％的叠氮钠防腐剂。
其中，该第二试剂的该连接捕捉分子(生物素化的单(多)克隆抗体、生物素化的适配体或生物素化的核酸配基)为溶于磷酸盐缓冲液。
其中，该第二试剂内的该连接捕捉分子为溶于5-10mL的pH值为7.4的0.01-0.1M的磷酸盐缓冲液中。
其中，该第二试剂内的该连接捕捉分子的浓度为0.003-3μg/L。
其中，该第二试剂内含重量比0.01％的叠氮钠防腐剂。
该第二试剂可以采用如下实施方式：每一该第二试剂包括5-10mL的pH值为7.4的0.01-0.1M的第二磷酸盐缓冲液，该第二磷酸盐缓冲液内溶有生物素化的单克隆抗体0.003-3μg/L，并该第二磷酸盐缓冲液含重量比0.01％的叠氮钠防腐剂；
其中，该第三试剂内的该检测分子为靶分子核酸信标配基，该靶分子核酸信标配基为溶于结合缓冲溶液(binding buffer)中。
其中，该第三试剂内的该靶分子核酸信标配基为溶于5-10mL的pH值为7.4的磷酸根浓度为0.01-0.1M的结合缓冲溶液中。
其中，该结合缓冲溶液(binding buffer)内至少包括138mmol/L NaCl，2.7mmol/L KCl，8.1mmol/L Na2HPO4，1.1mmol/L KH2PO4及1mmol/L MgCl2，pH值为7.4。配制后以高压蒸汽灭菌20min，室温保存。
其中，该第三试剂内的该检测分子的浓度为0.001-4μg/L。
其中，该第三试剂内含重量比0.01％的叠氮钠防腐剂。
该第三试剂可以采用如下实施方式：每一该第三试剂包括5-10mL的pH值为7.4的磷酸根浓度为0.01-0.1M的结合缓冲溶液，该结合缓冲溶液内溶有靶分子核酸信标配基0.001-4μg/L，并该结合缓冲溶液含重量比0.01％的叠氮钠防腐剂；
其冲，每一该第四试剂内包含1-3mL反转录多重实时定量-PCR反应液，10-30单位核酸聚合酶，该反转录多重实时定量-PCR反应液内含多对引物和探针。此处核酸聚合酶的单位指生物学的活性单位，酶的催化能力的计量单位。
本发明还提供一种蛋白配体和基因同时检测方法，用于靶分子和基因同时实时定量-PCR检测，该方法包括下述步骤：
(1)将检测标本、生物素化的单克隆抗体和核酸信标配基一起孵育，形成抗原抗体核酸适配体夹心复合物；
(2)在该抗原抗体核酸适配体夹心复合物中添加包被链霉亲和素的琼脂磁珠微粒一起孵育，使该抗原抗体核酸适配体夹心复合物与该包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的作用结合，得一结合物；以及
(3)洗涤除去未结合的物质后，加入反转录多重实时定量-PCR反应液进行实时定量-PCR，记录结果。
其中，于步骤(1)中，检测标本为10-1000μL，生物素化的单克隆抗体为20-100μL，核酸信标配基为20-100μL。
其中，于步骤(1)中，所述孵育为在37℃下孵育30-60分钟。
即，步骤(1)可采用如下实施方式：将10-100μL检测标本、20-100μL生物素化的单克隆抗体和20-100μL核酸信标配基及500μL结合缓冲溶液一 起在37℃下孵育30-60分钟，形成抗原抗体核酸适配体夹心复合物。该结合缓冲溶液(binding buffer)内至少包括138mmol/L NaCl，2.7mmol/L KCl，8.1mmol/L Na2HPO4，1.1mmol/L KH2PO4及1mmol/L MgCl2，pH值为7.4。配制后以高压蒸汽灭菌20min，室温保存。
其中，于步骤(2)中，添加的链霉亲和素的琼脂磁珠微粒为10-100μL。
其中，于步骤(2)中，所述孵育为在37℃下孵育30-60分钟。
即，步骤(2)可采用如下实施方式：在该抗原抗体核酸适配体夹心复合物中添加10-100μL包被链霉亲和素的琼脂磁珠微粒，在37度下孵育30-60分钟，使该抗原抗体核酸适配体夹心复合物与该包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的作用结合，得一结合物。
其中，于步骤(3)中，所述洗涤操作为利用电磁作用将该结合物吸附在磁极上，以TPBS缓冲液洗涤3-12次，每次3-10分钟，除去未结合的物质。
即，步骤(3)可采用如下实施方式：利用电磁作用将该结合物吸附在磁极上，以TPBS缓冲液洗涤3-12次，每次3-10分钟，除去未结合的物质，加入反转录多重实时定量-PCR反应液进行实时定量-PCR，实时定量-PCR仪器自动通过4点校正的定标曲线计算得到检测结果。
其中，该检测标本包括血清、尿液和体液，所述血清、尿液和体液中含有致病菌、致病粒子和配体分子(核酸、蛋白质、多肽、有机染料、ATP、金属离子类的任何分子)。
其中，该生物素化的单克隆抗体是将单克隆抗体化学耦联生物素。
其中，该核酸信标配基是单链配基和双链信标组成。该核酸信标配基可选用中国专利CN1521272《新型配体检测方法》中所公开的核酸信标配基。
其中，该反转录多重实时定量-PCR反应液是反转录和实时定量体系组成，能够将RNA反转录成DNA，再通过实时定量-PCR反应对基因和信标分子进行定量。
本发明所述蛋白配体和基因同时检测试剂盒，其有益效果在于：
本发明的检测试剂盒是通过核酸信标配基与靶分子(非核酸分子)结合将靶分子信号转换成核酸信号，完成蛋白信号与核酸信号的统一，再利用实时定量-PCR进行定量检测。该检测方法可使多种非核酸配体的信号通过核酸信标配基转换成核酸信号，具有快速、灵敏度高、特异性强、多配体微阵检测和可 机械化完成等特点。
本发明是利用琼脂磁珠(等磁珠)作为载体对检测分子进行磁性分离，具有附着靶分子，可机械加样，分离，洗脱，孵育等，无须再将靶分子分离提纯。
本发明是利用连接在抗体或配基上的人为修饰DNA或RNA序列作为信号分子，通过对信号修饰序列的PCR扩增，实现对抗原或配体的检测。该修饰DNA或RNA序列具有一对引物和中间的人为标记序列，针对被检测的靶分子和检测目的的要求这一对引物可以相同也可以不相同，中间的标记序列是靶分子的标记。如：在多种样品同时检测时一对相同引物可以使多种样品检测数据更切合实际，标记序列则是和检测靶分子一一对应。
本发明的检测仪器可通过对DNA或RNA修饰序列的变换来达到数据的效正和有效的消除污染。
本发明的检测仪器是通过用实时定量-PCR仪进行扩增产物检测或寡核苷酸芯片检测来数据采集和处理。便于单一样品或多种样品的检测。
本发明检测仪器如果通过用实时定量-PCR仪进行扩增产物检测来数据采集和处理，其整个过程都在PCR管内一次性完成，无须将产物转换地方，进行提纯、检测等步骤，并且PCR产物可在PCR管封闭销毁，避免逸出产物造成环境污染。
本发明检测仪器如果通过用寡核苷酸芯片检测来数据采集和处理，其过程是将PCR产物通过移液机转入寡核苷酸芯片检测体系内全自动化完成，其过程是全封闭的，无须进行人工提纯、分离、检测等步骤。
本发明检测仪器可机械化完成全部检测过程，包括：加样、孵育、洗管、实时定量-PCR仪(或微阵芯片)的数据检测和数据处理、发报告等。
本发明还提供一种蛋白配体和基因同时检测方法，用于靶分子和基因同时实时定量-PCR检测，该方法包括下述步骤：
(1)标本制备：按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固，然后在3000-6000转/min下离心，分离血清为标本；
(2)抗原抗体核酸适配体夹心复合物的形成：将10-100μL标本、25pmol生物素化的单克隆抗体和25pmol核酸信标配基一起孵育，形成抗原抗体核酸适配体夹心复合物；
(3)利用磁珠分离抗原抗体核酸适配体夹心复合物：在抗原抗体核酸适配体夹心复合物中添加20-100μL包被链霉亲和素的琼脂磁珠微粒进行孵育，使抗原抗体核酸适配体夹心复合物与包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的的作用结合，得一结合物；
(4)反转录-PCR扩增：利用电磁作用将结合物吸附在磁极上，洗涤除去未结合的物质，加入反转录多重实时定量-PCR反应液进行实时定量-PCR；
(5)数据采集和处理。
其中，换算方法：1μg/L/分子量＝1/分子量umol＝1/分子量×1000pmol。
本发明所述蛋白配体和基因同时检测方法，其有益效果在于：机械化成度高；利用本发明仪器可使加样、加试剂、洗涤、PCR、检测及数据处理等，可全机械化完成；人为修饰序列储存信息量大。本发明的修饰核酸序列为特异寡核苷酸配基末端人为加上的一些不影响其结合活性的核酸序列(可以是ssDNA、dsDNA和RNA)，修饰序列的长短，碱基序列可根据检测目的设计，不同的修饰序列可通过寡核苷酸配基与配体的特异结合反映配体的性质和数量；灵敏度高、特异性强；本发明仪器的靶分子检测是采用新型免疫-PCR和修饰配基(核酸(类)抗体)的检测方法，是利用抗原抗体的特异结合和配体配基的特异结合，使检测的特异性增强；在配基和配体直接结合形成稳定的复合物时，由于在配体特异结合的寡核苷酸配基上有人为修饰序列，可直接通过PCR指数级富集，对配体信息进行传递和放大，因此其灵敏度有明显的提高；检测样品灵活。可用实时荧光定量PCR检测单一或少量样品，也可以用微阵芯片检测多种样品；准确的信息采集和处理系统。由于本发明仪器的信息采集和处理系统是通过实时定量-PCR检测和微阵芯片检测，故有校准的数据报告；使用器皿单一，操作过程简单、安全。检测时使用器皿单一，即一次性PCR管，操作过程简单，数据采集及处理可在全封闭的情况下完成，扩增产物可封闭销毁，有效保证环境的洁净；本发明的操作简单，易于普及应用。由于在反应时只需将配体和配基室温孵育20-45分钟就能完成配体和配基的结合反应，因此操作简便，便于一般实验室或临床检验科室的普及应用；利用本发明组装的检测试剂盒或构建的生物芯片可以广泛应用于基础研究与临床检测，可带来一定的经济效益与社会效益。
综上所述，本发明提出一种利用核酸信标配基将蛋白配体等非核酸分子转 换成核酸信号，通过(多重)实时定量-PCR(或滚环复制)扩增核酸信标和基因核酸分子，实施对配体和基因同时检测的技术方法。同时检测蛋白(等非核酸靶分子)和基因的试剂盒是利用核酸信标配基分子(该分子已申请《新型配体检测方法》CN1521272专利)的配基与蛋白配体结合，形成配体-核酸信标配基分子复合物，使靶分子配体信息转换成核酸信标信息，使靶分子的信息与核酸基因信息统一，再利用实时定量-PCR对靶分子和核酸基因进行同时检测。该试剂盒不仅提高配体靶分子的灵敏度，而且使配体和基因在同一水平进行分子检测，检测技术具有快速、灵敏度高、特异性强、多配体微阵检测和可机械化完成等特点。对病原体，基因和蛋白质等组学的研究具有重要意义。
实施例1
请参考图1，图1为本发明一实施例提供的固定分离靶分子后混合基因检测方法流程示意图，该方法包括如下步骤：
1.1(血清)标本制备
首先备齐用物，贴好标签，核对无误后按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固。离心3000-6000转/min分离血清为标本，4℃存放，血清中含P24抗原3。
1.2P24抗原抗体核酸信标配基磁珠夹心复合物的形成
10-100μL包被P24抗体1的琼脂磁珠2微粒加入血清100-1000μL标本，结合形成磁珠-抗体-P24抗原复合物，吸出血清，用洗涤液0.01-0.1M PBS，含吐温-20 0.01-0.1M 400μL，洗涤3-12次，3-6min/次后，加入P24核酸信标配基7溶液孵育形成P24抗体-抗原-核酸信标配基磁珠夹心复合物。再利用电磁极吸附磁珠，分离除去未结合的核酸信标配基，加入洗涤液0.01-0.1M PBS，含吐温-20 0.01-0.1M 400μL，洗涤3-12次，3-6min/次。然后再将吸出的血清加入磁珠中。
1.3提取核酸
加入裂解液，氯仿酚抽提，乙醇沉淀，提取HIV病毒5的HIV-RNA8。
1.4反转录-PCR扩增
加入反转录多重实时定量-PCR反应液，含P24核酸信标配基探针6，以 及两对引物4进行实时定量-PCR。
1.5数据采集和处理
1.6打印检测报告
实施例2
请参考图2，图2为本发明一实施例提供的同时固定分离靶分子和基因检测方法流程示意图，该方法包括如下步骤：
2.1(血清)标本制备
首先备齐用物，贴好标签，核对无误后按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固。离心3000转/min分离血清为标本，4℃存放，血清中含P24抗原3和HIV病毒5。
2.2P24抗原抗体核酸信标配基和HIV gp120抗体核酸信标配基夹心复合物的形成
取10-1000μL标本、20-100μL生物素化的P24单克隆抗体1、20-100μL生物素化的gp120单克隆抗体9、20-100μLP24核酸信标配基7和gp120核酸信标配基10一起孵育，形成P24抗原-抗体-核酸信标配基夹心复合物和gp120抗体-HIV-病毒表面核酸信标配基夹心复合物。
2.3利用磁珠分离夹心复合物
添加10-100μL包被链霉亲和素的琼脂磁珠微粒2进行孵育，复合物与磁珠通过生物素和链霉亲和素的作用结合。
2.4洗涤
利用电磁极吸附磁珠，分离除去未结合的生物素化的单克隆抗体和核酸信标配基，加入洗涤液0.01-0.1M PBS，含吐温-20 0.01-0.1M 400-1000μL，洗涤3-12次，3-6min/次。
2.5提取核酸
弃去洗涤液，提取HIV病毒5的HIV-RNA8，加入P24核酸信标配基探针6、gp120基因探针6和病毒表面核酸信标配基探针6，以及三对引物4，PCR体系。
2.6反转录-PCR扩增
进行反转录多重实时定量-PCR实时定量。
2.7数据采集和处理
2.8打印检测报告
实施例3
请参考图3，图3为本发明一实施例提供的免疫核酸信标配基靶分子对靶分子基因一体检测方法流程示意图，该方法包括如下步骤：
3.1(血清)标本制备
首先备齐用物，贴好标签，核对无误后按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固。离心6000转/min分离血清为标本，4度存放，血清中含HBV病毒14。
3.2抗原抗体核酸信标配基夹心复合物的形成
首先将20-100μL小鼠抗人HBs-IgG单克隆抗体13和小鼠IgG的Fc片段特异性核酸信标配基12一起孵育，使其形成IgG-核酸信标配基复合物；取10-1000μL标本、20-100μL生物素化羊抗人-McAnti-HBs抗体11和20-100μL小鼠抗人HBs-IgG抗体-核酸信标配基复合物一起孵育，形成McAnti-HBs抗体-HBV-小鼠抗人HBs-IgG抗体-核酸信标配基复合物。
3.3利用磁珠分离夹心复合物
添加10-100μL包被链霉亲和素的琼脂磁珠2微粒进行孵育，复合物与磁珠通过生物素和链霉亲和素的作用结合。
3.4洗涤
利用电磁极吸附磁珠，分离除去未结合的生物素化的单克隆抗体和核酸信标配基，加入洗涤液0.01-0.1M PBS，含吐温-20 0.01-0.1M 400μL，洗涤3-12次，3-6min/次。
3.5实时定量-PCR扩增
弃去洗涤液，提取HBV病毒14的HBV-DNA15，加入实时定量-PCR反应液，含HBV基因和信标探针6和引物4，进行实时定量-PCR，得到信标扩增序列16及基因扩增序列17。
3.6数据采集和处理
3.7打印检测报告
以下实施例结合具体检测实验数据对本发明的技术效果进行说明：
请参考图4a至图4d。本实施例为羧基琼脂磁珠核酸信标配基免疫-PCR对HBsAg(乙型肝炎病毒表面抗原)的检测实验。
以羧基琼脂糖磁珠为载体，包被100倍稀释浓度为0.02mg/ml的乙肝山羊抗人多克隆抗体，检测4个105-108倍梯度稀释浓度为1000pg，100pg，10pg，1pg的乙肝表面抗原，1个阴性对照和1个体系空白，用小鼠抗人乙肝表面抗原IgG和小鼠IgG的Fc核酸信标配基复合物作为检测分子，以核酸信标配基作为DNA模板进行检测，每管加入模板6ul。图4a为免疫PCR检测HBsAg的定量曲线：横坐标为循环数(Cycle)，纵坐标为荧光值(Norm Fluoro)，另有阈值(Threshold)，检测抗原浓度分别为(a)：1000pg(b)：100pg(c)：10pg(d)：1pg，结果显示：实时定量-PCR检测的Ct值随着抗原浓度的降低而增加；图4b为检测HBsAg的标准曲线：横坐标是抗原浓度，纵坐标是实时定量-PCR的Cts回归系数。如图4a、如图4b可以看到在105-108的定量域内HBsAg具有线性关系，可以检测出待测蛋白量。
进一步的，以羧基琼脂糖磁珠为载体，包被100倍稀释浓度为0.02mg/ml的乙肝山羊抗人多克隆抗体，在同样的反应条件下设置16孔重复检测107倍稀释的HBsAg样品5mg/ml和乙肝表面抗原的待检液，再加入1000稀释得小鼠抗人乙肝表面抗原IgG单克隆抗体和小鼠IgG的Fc核酸信标配基，经孵育、洗涤后，实时定量-PCR检测，已验证样品检测的稳定性。图4c为磁珠免疫PCR对HBsAg的重复检测的定量曲线，横坐标为循环数(Cycle)，纵坐标为荧光值(Norm Fluoro)，另有阈值(Threshold)，图4d为免疫PCR对HBsAg的重复检测16个结果的统计分析，横坐标为标准样品107倍稀释浓度为10pg和待检测的乙肝表面抗原，纵坐标为Ct值。图4c可以看到HBsAg检测结果具有重复性。图4d统计结果表明，16个检测值待检液Ct值SD值不足0.05，优势显著，重复性高。
本实施例以羧基琼脂糖磁珠为分离载体，基于核酸信标配基的抗原抗体特异性反应构建了磁珠-多抗-抗原-单抗-配基信标复合物夹心法模型结合Real Time-PCR检测技术，成功检测到了微量乙肝表面抗原蛋白(HBsAg)，由 此可证本发明的方法确实可以达到预期的技术效果。与ELISA方法相比，本发明的优点在于用核酸信标配基代替二抗，结合Real time-PCR检测技术具有操作简单、直观、重复率强、灵敏度高的特点，其灵敏度达到103-104倍能够方便地拓展到基于其他适配子的微量蛋白的检测。
当然，本发明还可有其它多种实施例，在不背离本发明精神及其实质的情况下，熟悉本领域的技术人员当可根据本发明作出各种相应的改变和变形，但这些相应的改变和变形都应属于本发明所附的权利要求的保护范围。
工业应用性
本发明提出了蛋白配体和基因同时检测方法及试剂盒，属于分子生物学领域。它包括蛋白配体和核酸配基的分子识别，通过核酸信标配基将蛋白信号转化成核酸信息，实现与基因检测信息的统一，利用实时定量PCR完成对蛋白和基因的同时检测。本发明的优点是利用核酸信标配基将靶蛋白信号转换成统一核酸信号；利用实时定量PCR对信标扩增检测提高靶分子蛋白的检测灵敏的；靶分子蛋白核酸信息的转换实现蛋白和基因在检测信息的统一；靶分子蛋白核酸信息的转换实现蛋白和基因在检测灵敏度的统一。因此，该项发明对蛋白质组学和基因组学研究与临床分子检测具有重要意义。

Claims (16)

  1. 一种蛋白配体和基因同时检测试剂盒，用于靶分子和基因同时实时定量-PCR检测，其特征在于，该试剂盒包括：
    至少一第一试剂，该第一试剂内包含多个包覆有链霉亲合素琼脂的固相分离载体，该固相分离载体能够分离复合分子；
    至少一第二试剂，该第二试剂内包含多个连接捕捉分子，该连接捕捉分子能够捕捉靶分子，该固相分离载体与该连接捕捉分子之间能够连接结合；
    至少一第三试剂，该第三试剂内包含多个检测分子，该检测分子能够与靶分子特异性结合，或者能够与靶抗原免疫结合；
    至少一第四试剂，该第四试剂内包含核酸检测试剂，该核酸检测试剂内包含信标检测的引物和探针、基因检测的引物和探针以及核酸聚合酶。
  2. 根据权利要求1所述的蛋白配体和基因同时检测试剂盒，其特征在于，该包覆有链霉亲合素琼脂的固相分离载体为链霉亲合素琼脂磁珠微粒、链霉亲合素琼脂试纸或链霉亲合素琼脂芯片；该连接捕捉分子为生物素化的单克隆抗体、生物素化的适配体或生物素化的核酸配基；该检测分子为靶分子核酸信标配基、核酸信标配基检测分子或核酸信标配基免疫检测分子；该核酸检测试剂为包含信标检测的引物和探针、基因检测的引物和探针以及核酸聚合酶的反转录多重实时定量-PCR反应液。
  3. 根据权利要求1或2所述的蛋白配体和基因同时检测试剂盒，其特征在于，该第一试剂的该固相分离载体为链霉亲合素琼脂磁珠微粒，并且该链霉亲和素琼脂磁珠微粒为悬浮于磷酸盐缓冲液中，溶于磷酸盐缓冲液的该链霉亲和素琼脂磁珠微粒的体积比为50％，磁珠微粒粒径为5-50nm。
  4. 根据权利要求1或2所述的蛋白配体和基因同时检测试剂盒，其特征在于，该第二试剂内的该连接捕捉分子的浓度为0.003-3μg/L。
  5. 根据权利要求1或2所述的蛋白配体和基因同时检测试剂盒，其特征在于，该第三试剂内的该检测分子的浓度为0.001-4μg/L。
  6. 根据权利要求1或2所述的蛋白配体和基因同时检测试剂盒，其特征在于，每一该第四试剂内包含1-3mL反转录多重实时定量-PCR反应液，10-30单位核酸聚合酶，该反转录多重实时定量-PCR反应液内含多对引物和探针。
  7. 一种蛋白配体和基因同时检测方法，用于靶分子和基因同时实时定量 -PCR检测，其特征在于，该方法包括下述步骤：
    (1)将检测标本、生物素化的单克隆抗体和核酸信标配基一起孵育，形成抗原抗体核酸适配体夹心复合物；
    (2)在该抗原抗体核酸适配体夹心复合物中添加包被链霉亲和素的琼脂磁珠微粒一起孵育，使该抗原抗体核酸适配体夹心复合物与该包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的作用结合，得一结合物；以及
    (3)洗涤除去未结合的物质后，加入反转录多重实时定量-PCR反应液进行实时定量-PCR，记录结果。
  8. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，于步骤(1)中，检测标本为10-1000μL，生物素化的单克隆抗体为20-100μL，核酸信标配基为20-100μL。
  9. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，于步骤(1)及/或步骤(2)中，所述孵育为在37℃下孵育30-60分钟。
  10. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，于步骤(2)中，添加的链霉亲和素的琼脂磁珠微粒为10-100μL。
  11. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，于步骤(3)中，所述洗涤操作为利用电磁作用将该结合物吸附在磁极上，以TPBS缓冲液洗涤3-12次，每次3-10分钟，除去未结合的物质。
  12. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，该检测标本包括血清、尿液和体液，所述血清、尿液和体液中含有致病菌、致病粒子和配体分子。
  13. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，该生物素化的单克隆抗体是将单克隆抗体化学耦联生物素。
  14. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，该核酸信标配基是单链配基和双链信标组成。
  15. 根据权利要求7所述的蛋白配体和基因同时检测方法，其特征在于，该反转录多重实时定量-PCR反应液是反转录和实时定量体系组成，能够将RNA反转录成DNA，再通过实时定量-PCR反应对基因和信标分子进行定量。
  16. 一种蛋白配体和基因同时检测方法，用于靶分子和基因同时实时定量-PCR检测，其特征在于，该方法包括下述步骤：
    (1)标本制备：按静脉穿刺法采取所需血量，立即卸下针头，将血液注入盛有抗凝剂的试管内，立即轻轻摇动，使血液和抗凝剂混匀，以防血液凝固，然后在3000-6000转/min下离心，分离血清为标本；
    (2)抗原抗体核酸适配体夹心复合物的形成：将10-100μL标本、25pmol生物素化的单克隆抗体和25pmol核酸信标配基一起孵育，形成抗原抗体核酸适配体夹心复合物；
    (3)利用磁珠分离抗原抗体核酸适配体夹心复合物：在抗原抗体核酸适配体夹心复合物中添加20-100μL包被链霉亲和素的琼脂磁珠微粒进行孵育，使抗原抗体核酸适配体夹心复合物与包被链霉亲和素的琼脂磁珠微粒通过生物素和链霉亲和素的的作用结合，得一结合物；
    (4)反转录-PCR扩增：利用电磁作用将结合物吸附在磁极上，洗涤除去未结合的物质，加入反转录多重实时定量-PCR反应液进行实时定量-PCR；以及
    (5)数据采集和处理。
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CN110498858B (zh) * 2019-07-26 2024-01-23 深圳市达科为生物工程有限公司 一种动态检测单细胞外泌蛋白分泌情况的方法
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