CN114717295B - Liquid phase chip method hybridization buffer solution, preparation method and liquid phase chip detection method - Google Patents
Liquid phase chip method hybridization buffer solution, preparation method and liquid phase chip detection method Download PDFInfo
- Publication number
- CN114717295B CN114717295B CN202210647571.9A CN202210647571A CN114717295B CN 114717295 B CN114717295 B CN 114717295B CN 202210647571 A CN202210647571 A CN 202210647571A CN 114717295 B CN114717295 B CN 114717295B
- Authority
- CN
- China
- Prior art keywords
- hybridization
- liquid phase
- phase chip
- hybridization buffer
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000009396 hybridization Methods 0.000 title claims abstract description 186
- 239000007791 liquid phase Substances 0.000 title claims abstract description 55
- 239000007853 buffer solution Substances 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 48
- 238000001514 detection method Methods 0.000 title claims description 24
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 14
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 13
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 13
- 239000000872 buffer Substances 0.000 claims description 79
- OKIZCWYLBDKLSU-UHFFFAOYSA-M N,N,N-Trimethylmethanaminium chloride Chemical compound [Cl-].C[N+](C)(C)C OKIZCWYLBDKLSU-UHFFFAOYSA-M 0.000 claims description 56
- 238000006243 chemical reaction Methods 0.000 claims description 56
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 34
- 239000004005 microsphere Substances 0.000 claims description 33
- 239000000523 sample Substances 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 19
- 235000019270 ammonium chloride Nutrition 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 239000008213 purified water Substances 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 238000003745 diagnosis Methods 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 239000002904 solvent Substances 0.000 claims 1
- 239000000047 product Substances 0.000 description 36
- 230000000052 comparative effect Effects 0.000 description 30
- 238000012360 testing method Methods 0.000 description 28
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 25
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 239000012496 blank sample Substances 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 7
- 238000000684 flow cytometry Methods 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 206010022000 influenza Diseases 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000342334 Human metapneumovirus Species 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- OHVLMTFVQDZYHP-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical class N1N=NC=2CN(CCC=21)C(CN1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O OHVLMTFVQDZYHP-UHFFFAOYSA-N 0.000 description 1
- VLEIUWBSEKKKFX-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O VLEIUWBSEKKKFX-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 101100452676 Arabidopsis thaliana INVB gene Proteins 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 244000309467 Human Coronavirus Species 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 101150114988 invA gene Proteins 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of liquid phase chips, and discloses a liquid phase chip method hybridization buffer solution, in particular to a hybridization buffer solution suitable for detecting multiple nucleic acids by a liquid phase chip method, and provides a preparation method and a method for detecting the liquid phase chip by using the hybridization buffer solution.
Description
Technical Field
The invention relates to a hybridization buffer solution by a liquid phase chip method, a preparation method and a liquid phase chip detection method, belonging to the field of liquid phase chips.
Background
The liquid phase chip is also called suspension array or flow type fluorescence technology, and the basic detection principle is that suspension microspheres are used as a liquid phase reaction carrier, flow cytometry is used as an analysis means, and the purpose of qualitative and quantitative detection is achieved by detecting microsphere coding fluorescence and reporting molecular fluorescence through two beams of red and green laser. The microspheres can be divided into more than 100 different colors by only doping two kinds of red and orange fluorescent dyes with different proportions to form a coding microsphere array, so that the one-time high-flux high-density analysis of multiple indexes is realized. Therefore, the liquid phase chip technology has great application potential in the large-scale detection of biomacromolecules such as nucleic acid, protein and the like.
The flow of nucleic acid detection using the liquid phase chip method includes: (1) extracting nucleic acid; (2) carrying out PCR amplification reaction; (3) carrying out hybridization reaction on the probe microspheres and the PCR product; (4) incubating a reporter fluorescent molecule; (5) and (4) performing machine detection on a flow cytometer or a liquid phase chip instrument.
In the detection of multi-target nucleic acid molecules, after multiple PCR amplification of various target genes, the efficiency of hybridization reaction between an amplification product and coding microspheres grafted with different oligonucleotide probes has important influence on the detection sensitivity. The efficiency of hybridization depends on the hybridization reaction environment, i.e., the hybridization buffer, in addition to the specificity of base complementary pairing between the probe and target sequences.
In most of the literature data, the hybridization buffer used in the liquid chip assay is prepared by the manufacturers of the liquid chip method, and the formulation includes 1.5 × hybridization buffer prepared from 4.5M tetramethylammonium chloride (TMAC), 0.15% SDS (sodium dodecyl sulfate), 75mM Tris (Tris-hydroxymethyl-aminomethane) and 6mM EDTA (ethylenediaminetetraacetic acid), and 50 μ l of hybridization reaction system includes 1.5 × hybridization buffer 33 μ l, Tris-EDTA (pH = 8) buffer 12 μ l, and 5 μ l of PCR product.
The formulation has the following disadvantages: firstly, SDS added in the preparation process is extremely difficult to dissolve in a buffer solution. Secondly, the total ion concentration of the hybridization buffer solution is high, and crystallization is easy to generate under the environment with the temperature lower than 10 ℃, so that the use is difficult.
At present, no relevant report for screening and optimizing hybridization buffer solution by a liquid chip method exists, and the hybridization detection sensitivity needs to be further improved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a hybridization buffer solution for a liquid chip method, in particular to a hybridization buffer solution suitable for detecting multiple nucleic acids by the liquid chip method, and provides a preparation method and a method for detecting the liquid chip by using the hybridization buffer solution.
The technical scheme adopted by the invention for solving the technical problems is as follows:
in a first aspect, the present application provides a liquid phase chip hybridization buffer, the solute comprising only ammonium chloride and tetramethylammonium chloride.
The hybridization buffer solution of the liquid phase chip method provided by the application is easy to prepare, crystals are not easy to precipitate at low temperature, and the hybridization buffer solution also has the effect of improving the sensitivity of hybridization reaction.
Furthermore, the solution of the hybridization buffer solution of the liquid phase chip method is water.
Further, when the method is applied to liquid-phase chip hybridization reaction, the final concentration of the tetramethylammonium chloride in the system is 1-2 mol/L. The inventors also tried to use higher concentration of TMAC (> 2M, 1M =1 mol/L), but due to the higher viscosity of TMAC, magnetic separation after hybridization resulted in large microsphere loss, affecting microsphere fluorescence count statistical analysis.
Further, when the method is applied to liquid-phase chip hybridization reaction, the final concentration of the tetramethylammonium chloride in the system is 1mol/L-1.75 mol/L.
Further, when the method is applied to liquid-phase chip hybridization reaction, the final concentration of the tetramethylammonium chloride in the system is 1.5-1.75 mol/L. Most preferably 1.75M, to give the result the lowest background signal.
Further, when the method is applied to liquid phase chip hybridization reaction, the final concentration of the ammonium chloride in the system is 0.25mol/L-1 mol/L. The inventors have also tried to use 2M NH without TMAC 4 Cl was tested and the background signal was still acceptable, but the positive results for some indicators were poor, so TMAC and NH 4 The two Cl components are not indispensable.
Further, when the method is applied to liquid phase chip hybridization reaction, the final concentration of the ammonium chloride in the system is 0.25mol/L-0.5 mol/L. Most preferably 0.25M, to give the result the lowest background signal.
Furthermore, the hybridization buffer solution of the liquid phase chip method is a concentrated solution.
Further, the hybridization buffer solution of the liquid phase chip method was 1.5 × concentrated solution.
In a second aspect, the present application provides a method for preparing a hybridization buffer solution by a liquid phase chip method, comprising the steps of: dissolving ammonium chloride in purified water to obtain an ammonium chloride solution; and mixing the ammonium chloride solution with a tetramethylammonium chloride solution to obtain a liquid-phase chip hybridization buffer solution with a solute only containing ammonium chloride and tetramethylammonium chloride. The preparation process is simple, the obtained hybridization buffer solution of the liquid phase chip method is not easy to separate out crystals at low temperature, and in addition, the hybridization buffer solution also has the effect of improving the sensitivity of hybridization reaction.
In a third aspect, the present application provides a liquid-phase chip detection method, including: nucleic acid extraction-PCR amplification reaction-hybridization reaction of probe microspheres and PCR products-report fluorescent molecule incubation-detection by using a flow cytometer or a liquid phase chip instrument, wherein the step of hybridization reaction of the probe microspheres and the PCR products comprises the following steps:
mixing the hybridization buffer solution of the liquid phase chip method and the probe microsphere solution, adding a PCR product, uniformly mixing to obtain a hybridization reaction system, and placing the hybridization reaction system into molecular hybridization equipment for hybridization. The liquid-phase chip detection method applies the liquid-phase chip hybridization buffer solution with the solute only containing ammonium chloride and tetramethylammonium chloride, and can improve the reaction sensitivity. Wherein the molecular hybridization device is a PCR instrument or a molecular hybridization furnace.
The invention has the beneficial effects that: the hybridization buffer solution of the liquid chip method has simple components and easy preparation, and after the hybridization buffer solution is improved aiming at the requirement of not precipitating crystals at low temperature, the concentration of TMAC in the hybridization buffer solution of the traditional liquid chip method is adjusted, and unused ammonium chloride in the hybridization buffer solution of the liquid chip method is added, so that the background signal can be reduced in the detection of multiple PCR samples by the hybridization buffer solution of the liquid chip method, and the sensitivity of hybridization reaction can be improved.
Drawings
FIG. 1 is a histogram of background signals generated by testing a blank sample using a conventional hybridization buffer and a hybridization buffer of the present invention in comparative combination 1.
FIG. 2 is a bar graph generated by testing samples of low template concentration using conventional hybridization buffer and hybridization buffer of the present invention in comparative combination 1.
FIG. 3 is a bar graph generated by testing samples of high template concentration using conventional hybridization buffer and hybridization buffer of the invention in comparative combination 1.
FIG. 4 is a bar graph comparing the ratio of the signal of a sample at low template concentration to the background signal in combination 1 tested using conventional hybridization buffer and hybridization buffer of the invention.
FIG. 5 is a histogram of background signals generated by testing a blank sample with three hybridization buffers in comparative combination 2.
FIG. 6 is a bar graph generated by testing samples at low template concentrations using three hybridization buffers in comparative combination 2.
FIG. 7 is a bar graph generated by testing samples with high template concentrations using three hybridization buffers in comparative combination 2.
FIG. 8 is a bar graph comparing the ratio of sample signal to background signal for low template concentration tests using three hybridization buffers in combination 2.
FIG. 9 is a histogram of background signals generated by testing blank samples with C, D, E, F four sets of hybridization buffer in comparative combination 3.
FIG. 10 is a bar graph generated comparing the low template concentration samples tested in combination 3 using C, D, E, F four sets of hybridization buffers.
FIG. 11 is a bar graph comparing the ratio of sample signal to background signal for the low template concentration test using C, D, E, F four sets of hybridization buffers in combination 3.
FIG. 12 is a bar graph of background signals generated by test blanks in comparative combination 4 using hybridization buffers of the invention at pH adjusted to 5, 6, 7, and 8.
FIG. 13 is a bar graph generated by testing samples with low template concentrations at pH adjusted to 5, 6, 7, and 8 using hybridization buffer of the invention in comparative combination 4.
FIG. 14 is a bar graph generated by testing samples with high template concentrations at pH 5, 6, 7, and 8 using hybridization buffer of the invention in comparative combination 4.
FIG. 15 is a bar graph of background signals generated by testing blank samples with hybridization buffers containing different concentrations of SDS in comparative combination 5.
FIG. 16 is a bar graph comparing the ratio of the signal of low template concentration sample to the background signal in combination 5 using hybridization buffers containing different concentrations of SDS.
FIG. 17 is a bar graph comparing the ratio of sample signal to background signal for high template concentrations tested in combination 5 using hybridization buffers containing different concentrations of SDS.
FIG. 18 is a graph showing the results of the concentration of microspheres in the test group without SDS when the microsphere selection was performed in the FSC/SSC two-dimensional coordinate system in the flow cytometer test in comparative group 5.
FIG. 19 is a graph showing the results of the concentration of microspheres in the test group containing 0.05% SDS when the microsphere selection was performed in the two-dimensional FSC/SSC coordinate system in the flow cytometer test in comparative group 5.
FIG. 20 is a graph showing the results of the concentration of microspheres in the test group containing 0.1% SDS when the microsphere selection was performed in the two-dimensional FSC/SSC coordinate system in the flow cytometer test in comparative group 5.
FIG. 21 is a histogram of background signals generated by testing blank samples with J, K, L, M four sets of hybridization buffer in comparative combination 6.
FIG. 22 is a bar graph generated comparing the low template concentration samples tested in combination 6 using J, K, L, M four sets of hybridization buffers.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
Most of the buffers screened and optimized in the existing literature are PCR buffers, that is, in the above flow of nucleic acid detection by using the liquid-phase chip method, PCR buffers are used for PCR amplification reaction in the flow (2), and the present invention is directed to hybridization buffers used for hybridization reaction of probe microspheres and PCR products in the flow (3).
It should be noted that the buffer used in the hybridization reaction is different from the buffer used in the PCR reaction in terms of the composition, the purpose of use, and the effect. The PCR buffer solution mainly comprises Tris-HCl, KCl and MgCl 2 And the like, some merchants can additionally add other components according to use requirements, such as adding (NH) 4 ) 2 SO 4 Etc. for improving the specificity of the PCR reaction. The PCR buffer solution is used for providing an optimal enzyme catalysis reaction condition for DNA polymerase so as to ensure the smooth operation of PCR amplification. The hybridization reaction is a process of combining a PCR product with a single-stranded DNA probe on the surface of the microsphere, no enzyme participates in the process, and a reaction system only comprises three components of buffer solution, probe microspheres and the PCR product. The purpose of the hybridization reaction is to combine the DNA probes on the microspheres and the double-stranded product fragments carrying biotin after the target genes are amplified by PCR one-to-one so as to facilitate the subsequent connection with the avidin modified reporter molecules.
The inventor, in the face of the problem that the conventional 1.5 × hybridization buffer solution containing 4.5M TMAC, 0.15% SDS, 75mM Tris and 6mM EDTA is difficult to prepare and is easy to crystallize at low temperature (10 ℃), adjusts the concentration of TMAC, and adds some new substances (for the hybridization buffer solution of the liquid phase chip method) to change some components in the conventional formula.
Further experiments were conducted to find that the final concentration of TMAC and ammonium chloride in the hybridization reaction system was 1M-2M and 0.25M-0.75M, respectively, and the background signal was in the lowest range. And the optimal combination is selected to be that the final concentration of TMAC is 1.75M and the final concentration of ammonium chloride is 0.25M.
The following liquid phase chip method was used to detect 15 PCR products to show the differences between the conventional hybridization buffer and the hybridization buffer of the present invention.
Wherein the 15-fold PCR product comprises a virus combination 15-fold PCR product and a blank 15-fold PCR product. The 15-fold PCR product of the virus combination is a PCR product obtained by amplifying 15 plasmid samples designed aiming at 15 respiratory virus gene locus sequences in the same reaction tube through 45 PCR cycles, and the PCR product is used as a sample in the following experiments. The 15 respiratory viruses include influenza a subtypes H1N1, H3N2, H7N 9; influenza a major INVA; influenza b major INVB; rhinovirus HRV; human syncytial virus RSV; human metapneumovirus HMPV; an adenovirus HADV; four common human coronaviruses OC43, NL63, 229E, HKU 1; the novel coronavirus has two gene loci of ORF1ab and N gene. The blank 15-fold PCR product is obtained by adding enzyme-free water with the volume equal to that of the plasmid for 15-fold PCR amplification. The volume of the multiple PCR reaction system is 20 mul, and only 5 mul is taken as a sample in the subsequent liquid phase chip method hybridization reaction.
The samples include a blank sample, a low template concentration sample, and a high template concentration sample. Blank samples are samples taken from blank 15-fold PCR products. The low template concentration sample is 15 plasmid concentrations added into a PCR reaction tube, and the concentrations are all n x 10 1 Samples of copies levels. The concentration of 15 plasmid in the sample with high template concentration is n x 10 3 Samples of copies levels.
Comparative combination 1-comparison of the differences in the effect of using the hybridization buffer of the invention versus a conventional hybridization buffer in the detection of 15 PCR products:
a conventional 1.5 Xhybridization buffer contains 4.5M TMAC, 75mM Tris, 0.15% SDS and 6mM EDTA. The preparation method comprises the following steps: 75mg of SDS was weighed, added to 0.65mL of purified water, 3.75mL of Tris (1M), 0.6mL of EDTA (0.5M) and 45mL of TMAC (5M), and mixed by ultrasonic vortexing at room temperature to obtain a volume of 50mL of 1.5 Xconventional hybridization buffer.
The liquid phase chip hybridization buffer in this example contained 1.5M TMAC and 0.5M NH at the final concentration in the system (1 ×) 4 And (4) Cl. Preparing a hybridization buffer solution according to the preparation method of the invention: 5.35g of NH were weighed 4 Cl was dissolved in 20ml of purified water to give 5M NH 4 And (4) Cl solution. 22.5ml of TMAC (5M), 7.5Ml NH 4 The Cl solution (5M) was mixed with 20mL of purified water at room temperature to obtain 50mL of 1.5 XTMAC + NH 4 Cl hybridization buffer.
The hybridization reaction process is as follows:
preparing a hybridization reaction system: group a, conventional hybridization reaction system: dissolve 15 probe microspheres (2000 spheres/seed) in 12. mu.l TE buffer, add 33. mu.l 1.5 Xconventional hybridization buffer, add 5. mu.l 15-fold PCR product, vortex and mix well. Group B, the hybridization reaction system of the invention: 15 probe microspheres (2000 spheres/seed) were dissolved in 12. mu.l TE buffer, and 33. mu.l of 1.5 XTMAC + NH was added 4 Cl hybridization buffer, add 5. mu.l 15-fold PCR product, vortex and mix well.
A, B two groups of hybridization reaction tubes are put into a PCR instrument to react for 5min at 95 ℃ and then react for 10min at 60 ℃.
The hybridization reaction tube was removed from the PCR apparatus, placed on a magnetic rack for 1min, and the supernatant was aspirated.
At the same time, 50 μ l of PBS solution of SA-PE (R-phycoerythrin labeled streptavidin) with the concentration of 7mg/ml is added, mixed evenly by vortex, placed for 20min at room temperature and detected by a flow cytometer.
The PE-median signal values (fluorescence relative intensity, dimensionless) on each encoded microsphere in the two sets of hybridization products were measured using a beckmann CytoFlex flow cytometer under the same configuration parameters as shown in table 1, and the data in table 1 were statistically analyzed and processed to obtain histograms, which are compared with fig. 1 to 4. Wherein, FIGS. 1 to 3 show the results of measuring the difference in signal values by a flow cytometer after hybridization of 15 PCR amplification products of a blank sample, a sample with a low template concentration, and a sample with a high template concentration using a conventional hybridization buffer and a hybridization buffer according to the present invention, respectively; FIG. 4 shows the ratio of positive to background signal obtained by hybridization of samples at low template concentration using two hybridization buffers.
TABLE 1
Compared with the conventional hybridization buffer solution, the hybridization buffer solution has the advantages that the background signal (generated by a blank sample) obtained by testing is lower, the positive signal is higher, and the difference of positive and new signals of two groups of hybridization reaction systems is more obvious under the condition of low template concentration. The positive signal of each index of low template concentration is divided by the corresponding background signal to obtain a positive/negative ratio value, for example, as shown in FIG. 4, and the hybridization buffer solution of the present invention has a result obviously superior to that of the conventional hybridization buffer solution.
Comparative combination 2-NaCl, KCl and NH were used in comparative hybridization buffer, respectively 4 Difference in Cl effect in detecting 15-fold PCR products:
TMAC is used to reduce the difference of the hybridization annealing temperature of a plurality of nucleic acid chains and improve the specificity of hybridization reaction, and the inventor reserves the component when optimizing the conventional hybridization buffer solution. The inventors speculate that NH 4 NH in Cl 4 + Can neutralize the negative charge of the phosphate group, reduce the repulsive force between DNA chains, increase the hybridization efficiency between the probe and the PCR product, and improve the sensitivity of the hybridization reaction.
The 15-fold PCR product, hybridization procedure, and detection procedure used in this example were the same as in comparative combination 1, except for the preparation of the hybridization buffer. The components of the three hybridization buffer solutions diluted to 1 × were 1.75M TMAC +0.25M NaCl, 1.75M TMAC +0.25M KCl, and 1.75M TMAC +0.25M NH 4 And (4) Cl. The preparation method comprises the following steps:
weighing 5.844g NaCl and dissolving in 20ml purified water to prepare 5M NaCl solution; 7.455g of KCl is weighed and dissolved in 20ml of purified water to prepare 5M KCl solution; 5.35g of NH are weighed 4 Cl was dissolved in 20ml purified water to obtain 5M NH 4 And (4) Cl solution.
43.75ml of TMAC (5M) and 6.25ml of NaCl (5M), KCl (5M), NH, respectively 4 Mixing with Cl (5M) to obtain three 2.5 × hybridization buffers with the concentrations of 4.375M TMAC and 0.625M NaCl, 4.375M TMAC and 0.625M KCl, and 4.375M TMAC and 0.625M NH 4 Cl。
The three hybridization buffers were hybridized with 5. mu.l of 15-fold PCR product, and the data of three sets of sample backgrounds, positive with low template concentration and positive with high template concentration obtained by flow cytometry are shown in Table 2. The data in Table 2 were summarized to give the columns of FIGS. 5 to 8Figure (a). FIGS. 5 to 7 show the use of NaCl or KCl or NH, respectively, in the same molar concentration 4 After the hybridization buffer solution of Cl is hybridized with 15 PCR amplification products of a blank sample, a sample with low template concentration and a sample with high template concentration, a flow cytometer tests the result of signal value difference; FIG. 8 shows the ratio of positive to background signal obtained by hybridization of samples at low template concentration using three hybridization buffers.
TABLE 2
As can be seen by combining the data with the histogram results, the NH content is increased under the condition that the molar concentrations of TMAC and the three ions are the same 4 The Cl hybridization buffer gave lower background signal and higher positive signal. Both potassium and sodium ions can neutralize the negative charge of the phosphate group, but the results indicate NH 4 Cl can achieve better effects, and the reason for the Cl needs to be further researched.
Comparative combination 3-comparative different TMAC and NH 4 The effect difference of hybridization buffer solution with Cl molar concentration ratio when detecting 15-fold PCR products is as follows:
the 15-fold PCR product and hybridization and detection procedures used in this example were the same as for comparative combination 1, except for the composition of the hybridization buffer. Preparing four kinds of hybridization buffer solutions, and the final molar concentrations in the liquid phase chip hybridization reaction system are different as follows: group C, 2M NH 4 Cl; group D, 1M TMAC +1M NH 4 Cl; group E, 1.5M TMAC +0.5M NH 4 Cl; group F, 2M TMAC. Four kinds of 2.5 Xhybridization buffers were prepared in 20ml each.
Group C: 5.35g of NH are weighed 4 Cl was dissolved in 20ml purified water.
Group D: 10ml TMAC (5M) and 10ml NH 4 Vortex Cl (5M) and mix well.
Group E: 15ml TMAC (5M) and 5ml NH 4 Vortex Cl (5M) and mix well.
And F group: 20ml of TMAC (5M).
Mixing the four hybridization buffers with 15 PCR productsIn addition, the flow cytometry test results in the positive signal ratio of background to low template concentration shown in fig. 9 and 10, and the positive signal ratio of low template concentration shown in fig. 11. As can be seen from the figure, four TMAC and NH 4 No NH is contained in the hybridization buffer solution under the Cl molar concentration ratio 4 The overall background was highest and the positive signal was lowest for the Cl 2M TMAC panel. 2M NH without TMAC 4 The Cl group background signal was still acceptable, but the positive results obtained for some indicators (e.g., H3N2, RSV, HMPV, etc.) were slightly inferior, thus TMAC and NH 4 The two Cl components are not indispensable.
Comparative combination 4-comparative use of 1.5M TMAC +0.5M NH 4 Effect of hybridization reaction pH on background and positive signal when Cl hybridization buffer:
the 15 PCR products and hybridization and detection procedures used were the same as in comparative combination 1, and the hybridization buffer components were the same as in comparative combination 3, except that 1M HCl or 1M NaOH was used to adjust the pH of the system to 5, 6, 7, 8 after the hybridization system was complete. Before the adjustment, the pH value of the hybridization reaction was measured by a pH meter to be 7.24. Hybridization reactions with 15-fold PCR products were performed at four pH values, and the results of the tests are shown in fig. 12 to 14. As can be seen from the comparison of the histograms, the background signal obtained from the hybridization assay is almost indistinguishable from the positive signal in the pH range from 5 to 8, so that the hybridization buffer of the invention can be used normally in the pH range from 5 to 8.
Comparative combination 5-different concentrations of SDS were added to the hybridization buffer to explore its effect on background versus positive signal:
the 15 PCR products and hybridization and detection processes used are the same as those of the comparative combination 1, and the components of one group of the hybridization buffer solution without SDS are the same as those of the group E in the comparative combination 3, i.e., the molar concentration ratio of the final hybridization buffer solution in the reaction system is 1.5M TMAC +0.5M NH 4 And (4) Cl. To the hybridization buffer (same as group E in comparative group 3), SDS was added at concentrations of 0.05% and 0.1% in the other two groups, respectively, wherein the final concentration of SDS in the hybridization reaction system was 0.1% which was the same as that used in the conventional hybridization buffer formulation.
The three hybridization buffers were: group G, 1.5MTMAC+0.5M NH 4 Cl; group H, 1.5M TMAC +0.5M NH 4 Cl +0.05% SDS; group I, 1.5M TMAC +0.5M NH 4 The data of the background, positive low-template concentration and positive high-template concentration signals of the three groups of samples obtained by the flow cytometry test are shown in table 3.
TABLE 3
As can be seen from the data in Table 3, the background signal of each index increased significantly after SDS was added, and the positive signal also increased slightly. However, after calculating the ratio of the positive signal to the background signal and plotting the histogram 15 to 17, it can be seen that the positive-negative ratio (i.e., the signal-to-noise ratio) of each index is significantly reduced after adding SDS. FIG. 15 shows the difference in background signal values of hybridization buffers containing different concentrations of SDS applied to hybridization assays of 15-fold PCR products; FIGS. 16 and 17 show the ratio of positive to background signals obtained by hybridization of samples at low and high template concentrations using hybridization buffers containing different concentrations of SDS.
Meanwhile, in the flow cytometry test, the addition of SDS can obviously influence the state of the microspheres. As shown in FIGS. 18 to 20, when the two-dimensional FSC/SSC coordinate system is used for the frame selection of the microspheres, the ratio of the area occupied by the microspheres in the test group without SDS is as high as 98.04%, and after SDS is added, the same size of the frame can only count 76.28% and 71.26% of the microspheres, which indicates that the aggregation of the microspheres occurs, which will seriously affect the hybridization test result and the counting statistical analysis of the microspheres, so the SDS component should not be added into the hybridization buffer of the present invention.
Comparative combination 6-feasibility of adding DMSO in hybridization buffer:
since dimethyl sulfoxide (DMSO) can reduce the secondary structure of DNA, 1.5M TMAC +0.5M NH was used in this example 4 2% -10% DMSO was added to Cl hybridization buffer. The 15-fold PCR product and hybridization and detection procedures used in this example were the same as for comparative combination 1, and the hybridization buffer components were: group J, 1.5M TMAC +0.5M NH 4 Cl；KGroup 1.5M TMAC +0.5M NH 4 Cl +2% DMSO; l group 1.5M TMAC +0.5M NH 4 Cl +6% DMSO; group M, 1.5M TMAC +0.5M NH 4 Cl +10% DMSO. The background and low template concentration positive signal data for the three groups of samples obtained by flow cytometry are shown in table 4.
TABLE 4
The data were collated to obtain histograms as shown in fig. 21 and 22. As can be seen from the data and the histogram, the addition of DMSO had no significant effect on the background signal for most indicators, except for OC43, but the positive signal showed a significantly decreasing trend with increasing DMSO concentration. It is assumed that DMSO is not suitable for addition to the hybridization buffer of the present invention because DMSO reduces the stability of double-stranded binding between the probe and the product, although DMSO reduces the formation of DNA secondary structures.
The hybridization buffer solution for detecting multiple nucleic acids by the liquid-phase chip method has simple components, convenient preparation and low cost, and compared with the conventional hybridization buffer solution which is used in the widest application range at present, the hybridization buffer solution for detecting multiple nucleic acids by the liquid-phase chip method has the advantages of lower background signal and higher positive signal, namely, the efficiency of the hybridization reaction is improved, the nonspecific combination among nucleic acid molecules is reduced, and the improvement of the sensitivity of detecting multiple target nucleic acids based on the liquid-phase chip method is facilitated.
In the description of the present specification, reference to the terms "one embodiment," "certain embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (7)
1. The liquid phase chip method hybridization buffer solution is characterized by being used in the range of pH 5-8, the solutes are ammonium chloride and tetramethylammonium chloride, the solvent is water, the working concentration of the tetramethylammonium chloride in the system is 1-2 mol/L, and the working concentration of the ammonium chloride in the system is 0.25-1 mol/L.
2. The liquid phase chip hybridization buffer according to claim 1, wherein the working concentration of tetramethylammonium chloride in the system is 1mol/L to 1.75mol/L when applied to the liquid phase chip hybridization reaction.
3. The liquid phase chip hybridization buffer according to claim 2, wherein the working concentration of tetramethylammonium chloride in the system is 1.5mol/L to 1.75mol/L when applied to the liquid phase chip hybridization reaction.
4. The liquid phase chip hybridization buffer according to claim 1, wherein the working concentration of ammonium chloride in the system is 0.25mol/L to 0.5mol/L when the buffer is applied to the liquid phase chip hybridization reaction.
5. The liquid phase chip hybridization buffer according to claim 1, wherein said liquid phase chip hybridization buffer is a concentrated solution.
6. A method for preparing a hybridization buffer according to the liquid phase chip method of any of claims 1 to 5, comprising the steps of: dissolving ammonium chloride in purified water to obtain an ammonium chloride solution; mixing the ammonium chloride solution with a tetramethylammonium chloride solution to obtain the liquid chip hybridization buffer according to any one of claims 1 to 5.
7. A liquid chip detection method for non-disease diagnosis purposes comprises the following steps: nucleic acid extraction-PCR amplification reaction-hybridization reaction of probe microspheres and PCR products-report fluorescent molecule incubation-detection by using a flow cytometer or a liquid phase chip instrument, and the hybridization reaction of the probe microspheres and the PCR products comprises the following steps:
mixing the hybridization buffer solution of the liquid phase chip method according to any one of claims 1 to 5 with the probe microsphere solution, then adding the PCR product, uniformly mixing to obtain a hybridization reaction system, and putting the hybridization reaction system into molecular hybridization equipment for hybridization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210647571.9A CN114717295B (en) | 2022-06-09 | 2022-06-09 | Liquid phase chip method hybridization buffer solution, preparation method and liquid phase chip detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210647571.9A CN114717295B (en) | 2022-06-09 | 2022-06-09 | Liquid phase chip method hybridization buffer solution, preparation method and liquid phase chip detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114717295A CN114717295A (en) | 2022-07-08 |
| CN114717295B true CN114717295B (en) | 2022-09-30 |
Family
ID=82232476
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210647571.9A Active CN114717295B (en) | 2022-06-09 | 2022-06-09 | Liquid phase chip method hybridization buffer solution, preparation method and liquid phase chip detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114717295B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5633134A (en) * | 1992-10-06 | 1997-05-27 | Ig Laboratories, Inc. | Method for simultaneously detecting multiple mutations in a DNA sample |
| CN1580283A (en) * | 2003-08-13 | 2005-02-16 | 清华大学 | Method for detecting nucleic acid molecule |
| CN101550457A (en) * | 2009-05-14 | 2009-10-07 | 江苏省疾病预防控制中心 | Liquid phase chip for detecting pathogeny of hand foot and mouth disease, preparation method and applications thereof |
| EP2224018A1 (en) * | 2009-02-25 | 2010-09-01 | QIAGEN GmbH | Hybridization and detection buffer |
| CN102628009A (en) * | 2011-02-03 | 2012-08-08 | 斯泰拉化工公司 | Cleaning liquid and cleaning method |
| CN104293937A (en) * | 2014-09-28 | 2015-01-21 | 广东省妇幼保健院 | Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization |
| CN105131932A (en) * | 2015-08-19 | 2015-12-09 | 昆山京昆油田化学科技开发公司 | Fracturing fluid prepared by utilizing fracturing flow-back fluid and preparation method thereof |
| CN107988317A (en) * | 2017-12-14 | 2018-05-04 | 中源协和基因科技有限公司 | A kind of buffer solution that hybrid capture is carried out to target set nucleic acid region |
| CN110373456A (en) * | 2018-04-12 | 2019-10-25 | 合度精密生物科技有限公司 | Capture includes the method for nucleic acid and application thereof of target oligonucleotide sequences |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030170706A1 (en) * | 2002-02-01 | 2003-09-11 | Roland Green | Use of a volatile hybridization wash buffer |
| TW201512405A (en) * | 2013-09-23 | 2015-04-01 | Zhao-De Chen | Method of nucleic acid hybridization through using microfluidic channel contained chip |
| US10450601B2 (en) * | 2013-09-26 | 2019-10-22 | Toyo Kohan Co., Ltd. | Buffer composition for hybridization use, and hybridization method |
| WO2017041005A1 (en) * | 2015-09-03 | 2017-03-09 | Abbott Molecular Inc. | Hybridization buffers comprising an alkyl diester |
| JP2019502386A (en) * | 2016-01-08 | 2019-01-31 | アボツト・モレキユラー・インコーポレイテツド | Hybridization buffer containing guanidinium thiocyanate |
-
2022
- 2022-06-09 CN CN202210647571.9A patent/CN114717295B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5633134A (en) * | 1992-10-06 | 1997-05-27 | Ig Laboratories, Inc. | Method for simultaneously detecting multiple mutations in a DNA sample |
| CN1580283A (en) * | 2003-08-13 | 2005-02-16 | 清华大学 | Method for detecting nucleic acid molecule |
| EP2224018A1 (en) * | 2009-02-25 | 2010-09-01 | QIAGEN GmbH | Hybridization and detection buffer |
| CN101550457A (en) * | 2009-05-14 | 2009-10-07 | 江苏省疾病预防控制中心 | Liquid phase chip for detecting pathogeny of hand foot and mouth disease, preparation method and applications thereof |
| CN102628009A (en) * | 2011-02-03 | 2012-08-08 | 斯泰拉化工公司 | Cleaning liquid and cleaning method |
| CN104293937A (en) * | 2014-09-28 | 2015-01-21 | 广东省妇幼保健院 | Group of probes, detection kit and detection method for detecting thalassemia gene point mutation based on liquid chip of locked nucleic acid sensibilization |
| CN105131932A (en) * | 2015-08-19 | 2015-12-09 | 昆山京昆油田化学科技开发公司 | Fracturing fluid prepared by utilizing fracturing flow-back fluid and preparation method thereof |
| CN107988317A (en) * | 2017-12-14 | 2018-05-04 | 中源协和基因科技有限公司 | A kind of buffer solution that hybrid capture is carried out to target set nucleic acid region |
| CN110373456A (en) * | 2018-04-12 | 2019-10-25 | 合度精密生物科技有限公司 | Capture includes the method for nucleic acid and application thereof of target oligonucleotide sequences |
Non-Patent Citations (3)
| Title |
|---|
| Development and application of a novel Bio–Plex suspension array system for high–throughput multiplexed nucleic acid detection of seven respiratory and reproductive pathogens in swine;Lu Xiao 等;《Journal of Virological Methods》;20180824;第261卷;第104-111页 * |
| 减阻剂对表面活性剂性能的影响研究;丁飞 等;《长江大学学报(自科版)》;20160910;第13卷(第25期);第42页最后1段 * |
| 同系物季铵盐离子液体阳离子的离子交换色谱法分析;刘永强 等;《分析测试学报》;20141030;第33卷(第10期);第1155页第1段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114717295A (en) | 2022-07-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101985665B (en) | Method for detecting various respiratory viruses and primers and probes thereof | |
| US9920380B2 (en) | Methods for detection and quantification of analytes in complex mixtures | |
| ES2320604T3 (en) | IDENTIFICATION OF DNA POLYMORPHISMS THROUGH THE USE OF FLOW CYTOMETRY. | |
| US6391544B1 (en) | Method for using unequal primer concentrations for generating nucleic acid amplification products | |
| CN103074434B (en) | CYP2C19 gene polymorphyism detection kit and detection method thereof | |
| AU2002327202A1 (en) | Methods for detection and quantification of analytes in complex mixtures | |
| EP2971139A2 (en) | Systems and methods for detection of genomic copy number changes | |
| BRPI0707155A2 (en) | probe, probe set, spacer, kit, and method for detecting any interaction between a probe and a target nucleic acid | |
| Wedemeyer et al. | Flow cytometry: an ‘old’tool for novel applications in medical genetics | |
| Li et al. | High-throughput and ultra-sensitive single-cell profiling of multiple microRNAs and identification of human cancer | |
| CN101570784B (en) | Signal combination coding-based DNA ligation sequencing method | |
| CN100338227C (en) | Method of real time detecting No.21 human chromosome number by quantitative PCR technology | |
| CN114717295B (en) | Liquid phase chip method hybridization buffer solution, preparation method and liquid phase chip detection method | |
| CN113564278A (en) | Method for detecting novel coronavirus and other respiratory viruses by liquid-phase chip | |
| CN108949929A (en) | For detecting the product and its methods and applications of MTHFR and MTRR gene pleiomorphism simultaneously | |
| CN103045716B (en) | Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology | |
| CN110499361B (en) | Preparation method and application of terminal base flow type fluorescence sequencing microspheres | |
| CN112359143A (en) | Isothermal index amplification method based on Y-type probe set and application thereof | |
| WO2009144497A1 (en) | Method for detecting respiratory viral agents in a test sample | |
| CN110331202A (en) | It is a kind of for detecting the kit and method of BRAC1 gene SNP | |
| CN112553379B (en) | Method and kit for detecting respiratory infectious disease virus based on liquid chip | |
| CN102191328B (en) | Nucleotide sequence analysis method based on magnetic separation and high-fidelity polymerase correction function | |
| CN115521979A (en) | Single-molecule nucleic acid detection method, product and related application | |
| CN108130383A (en) | A kind of fluorescence quantitative detection kit for fowl metapneumovirus Classification Identification | |
| RU2697096C1 (en) | Method for identification of genetic polymorphisms affecting metabolism of anticancer drugs using biological microchips |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |