CN113337583A - Micro-drop digital detection method for multiple target nucleic acids - Google Patents
Micro-drop digital detection method for multiple target nucleic acids Download PDFInfo
- Publication number
- CN113337583A CN113337583A CN202110641186.9A CN202110641186A CN113337583A CN 113337583 A CN113337583 A CN 113337583A CN 202110641186 A CN202110641186 A CN 202110641186A CN 113337583 A CN113337583 A CN 113337583A
- Authority
- CN
- China
- Prior art keywords
- primer
- target nucleic
- nucleic acid
- nucleic acids
- probe
- 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.)
- Pending
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 80
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 79
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 79
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 238000007397 LAMP assay Methods 0.000 claims abstract description 64
- 238000006243 chemical reaction Methods 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 48
- 230000003321 amplification Effects 0.000 claims abstract description 34
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 34
- 239000007788 liquid Substances 0.000 claims abstract description 31
- 239000011541 reaction mixture Substances 0.000 claims abstract description 18
- 239000000203 mixture Substances 0.000 claims abstract description 13
- 238000004458 analytical method Methods 0.000 claims abstract description 9
- 239000000523 sample Substances 0.000 claims description 59
- 241000725303 Human immunodeficiency virus Species 0.000 claims description 30
- 241000239226 Scorpiones Species 0.000 claims description 30
- 241000711549 Hepacivirus C Species 0.000 claims description 29
- 239000003921 oil Substances 0.000 claims description 21
- 230000000295 complement effect Effects 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 10
- 229940020947 fluorescein sodium Drugs 0.000 claims description 10
- 238000010791 quenching Methods 0.000 claims description 10
- 230000000171 quenching effect Effects 0.000 claims description 10
- 102100034343 Integrase Human genes 0.000 claims description 9
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 9
- 239000004094 surface-active agent Substances 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 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 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 241000711573 Coronaviridae Species 0.000 claims description 4
- 241000233866 Fungi Species 0.000 claims description 4
- 241000700721 Hepatitis B virus Species 0.000 claims description 4
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 4
- 241000315672 SARS coronavirus Species 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000004615 ingredient Substances 0.000 claims description 4
- 239000011777 magnesium Substances 0.000 claims description 4
- 229910052749 magnesium Inorganic materials 0.000 claims description 4
- 244000045947 parasite Species 0.000 claims description 4
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 4
- 241000712461 unidentified influenza virus Species 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 208000006400 Arbovirus Encephalitis Diseases 0.000 claims description 2
- 206010052369 Encephalitis lethargica Diseases 0.000 claims description 2
- 241000590002 Helicobacter pylori Species 0.000 claims description 2
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 claims description 2
- 241000318927 Shrimp white spot syndrome virus Species 0.000 claims description 2
- 241000282898 Sus scrofa Species 0.000 claims description 2
- 239000008157 edible vegetable oil Substances 0.000 claims description 2
- 229940037467 helicobacter pylori Drugs 0.000 claims description 2
- 239000002480 mineral oil Substances 0.000 claims description 2
- 235000010446 mineral oil Nutrition 0.000 claims description 2
- 230000001717 pathogenic effect Effects 0.000 claims description 2
- 229920002545 silicone oil Polymers 0.000 claims description 2
- 201000002498 viral encephalitis Diseases 0.000 claims description 2
- 238000010252 digital analysis Methods 0.000 abstract description 5
- 108020004414 DNA Proteins 0.000 description 35
- 239000000047 product Substances 0.000 description 16
- 238000001962 electrophoresis Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000006073 displacement reaction Methods 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000012295 chemical reaction liquid Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000012175 pyrosequencing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 241000023308 Acca Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 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 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007847 digital PCR Methods 0.000 description 1
- XZTWHWHGBBCSMX-UHFFFAOYSA-J dimagnesium;phosphonato phosphate Chemical compound [Mg+2].[Mg+2].[O-]P([O-])(=O)OP([O-])([O-])=O XZTWHWHGBBCSMX-UHFFFAOYSA-J 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- NJDNXYGOVLYJHP-UHFFFAOYSA-L disodium;2-(3-oxido-6-oxoxanthen-9-yl)benzoate Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=CC(=O)C=C2OC2=CC([O-])=CC=C21 NJDNXYGOVLYJHP-UHFFFAOYSA-L 0.000 description 1
- 229910000514 dolomite Inorganic materials 0.000 description 1
- 239000010459 dolomite Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000001921 nucleic acid quantification Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 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
- C12Q1/6844—Nucleic acid amplification reactions
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (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 provides a droplet-type digital detection method of multiple target nucleic acids, which comprises the following steps: (1) preparing a mixture required by the loop-mediated isothermal amplification reaction according to the multiple target nucleic acids; (2) taking the reaction mixture obtained in the step (1) as a dispersed phase and taking an oil phase as a continuous phase, and respectively injecting the dispersed phases into a microfluidic chip to generate monodisperse liquid drops; (3) placing the microfluidic chip in the step (2) at a constant temperature for amplification reaction; (4) and (4) carrying out fluorescence signal detection and analysis on the liquid drops in the microfluidic chip after the reaction in the step (3). The method can complete the digital analysis of the multiple target nucleic acids without complex chips, controllable temperature control equipment and other equipment with higher cost, simplifies the experimental steps, shortens the time by utilizing the advantages of the loop-mediated isothermal amplification and improves the efficiency.
Description
Technical Field
The invention belongs to the field of molecular biology, and relates to a droplet type digital detection method for multiple target nucleic acids.
Background
In recent years, a series of gene detection methods have been developed, and classical methods have been improved continuously, which greatly promotes the development of Loop-mediated isothermal amplification (LAMP) technologyDue to the development and application of detection research. The loop-mediated isothermal amplification technology is a novel nucleic acid specific amplification technology established by Notomi and the like, and has the advantages of strong specificity, high sensitivity, simple operation, easy detection of products and the like. This technique has been widely used in the field of molecular diagnostics. LAMP designs 4 core primers aiming at 6 specific parts of a target sequence, and Bst DNA polymerase with strand displacement activity is used for catalyzing new strand synthesis under a constant temperature condition, so that the target sequence is efficiently amplified. Of the 4 core primers, 2 are inner primers, i.e., FIP (ForWard inner primer, FIP) and BIP (Backward inner primer, BIP). FIP comprises Flc and F2 (the complement of the F2c region), i.e., 5' -Flc-F2; BIP comprises B1c (the complement of the B1 region) and B2, i.e., 5' -Blc-B2. The remaining two core primers were outer primers F3 and B3. Two additional Loop primers (Loop primers, LF and LB) were added to the reaction system to accelerate the LAMP reaction. The loop-mediated isothermal amplification reaction can be completed by only putting a gene template, a primer, a strand displacement DNA polymerase, dNTP and the like together at a certain temperature (60-65 ℃) through one step. The reaction amplification efficiency is extremely high and can be realized within 15-60min by 109-1010The amplification is multiplied, and the sensitivity and specificity are higher, so that the method is very suitable for being applied to various nucleic acid detections.
After LAMP amplification, the detection of the product can be observed by agarose electrophoresis followed by staining. The simple method is to add SYBR Green I into the product to dye, and the positive reaction is Green, and the negative reaction is orange red. The judgment can also be carried out by the turbidity of the amplified by-product magnesium pyrophosphate precipitate, and the positive reaction is carried out when the liquid is turbid, and the negative reaction is carried out when the liquid is centrifuged or white precipitate is generated. It is now a simpler method to add a visible dye to the reaction mixture, and the color of the positive reaction tubes changes from light grey to green, while the negative reaction tubes remain light grey. However, these methods can only detect the progress of the LAMP reaction, and cannot recognize specific amplification of a specific target sequence, and thus LAMP lacks specificity in determining the result when detecting a target sequence. Therefore, the detection of multiple target fragments is difficult to realize by the traditional LAMP detection, which greatly limits the wide application of LAMP.
In view of the above, some studies have been devoted to the development of multiplex LAMP detection techniques. The most common method for realizing multiple LAMP detection is to search restriction enzyme cutting sites in a target sequence, digest LAMP products by using restriction enzymes, and correspond the LAMP products after electrophoretic digestion to the corresponding target sequence according to different electrophoretic band sizes. However, the method needs two steps to be completed, when the restriction enzyme cuts the LAMP products with different sizes, the time consumption is long, the enzyme cutting is incomplete, so that one target sequence often corresponds to a plurality of electrophoresis strips, and the result of the multiple LAMP is difficult to judge. Another new technology for realizing multiplex LAMP detection is to combine LAMP amplification reaction and pyrosequencing. However, this method, like the restriction enzyme mediated multiplex LAMP detection technique, requires two steps, first LAMP amplification and then pyrosequencing to correspond to the corresponding target sequence. The method is complicated to operate, a specific kit is needed for purifying the LAMP product, special personnel are needed in the sequencing process, and a sequencer and sequencing reagents which cannot be borne by a common laboratory are needed. These disadvantages limit the widespread use of this approach.
An important application of microfluidics in nucleic acid research is droplet PCR technology, which produces picoliter-nanoliter-level droplets through microfluidic chips, which are small and independent reaction chambers, performs PCR in these uniform droplets, and performs quantitative analysis on target DNA using a fluorescent probe method. The technology can dilute the sample to a single molecule level, evenly distribute the sample into dozens to tens of thousands of units for reaction, and finally calculate the original concentration or content of the sample through direct counting or a Poisson distribution formula.
However, the amplification efficiency of the PCR reaction in the emulsion is not high, so that the signal amplification effect of the current droplet PCR technology after fluorescent labeling is limited, and usually, steps such as secondary amplification of the signal are required, and a more precise instrument and a more expensive temperature control device are required, which restricts the development of the digital nucleic acid quantification to a certain extent. In addition, the existing multiple LAMP detection technology can not realize rapid detection, and the time for completing the multiple LAMP detection is more than 2.5 hours. Because the sensitivity of LAMP reaction is extremely high, the uncovering operation of LAMP products causes great pollution to the subsequent LAMP experiment.
In summary, the LAMP in the prior art has the technical problem that the detection of single amplification or multiple amplification is difficult to overcome. Based on this, there is a need for a loop-mediated isothermal amplification method that can simultaneously detect multiple target nucleic acids.
Disclosure of Invention
Therefore, the present invention aims to provide a method for digital detection of multiple target nucleic acids in a droplet format, which overcomes the disadvantages of the prior art. The invention carries out loop-mediated isothermal amplification on target nucleic acid in a single liquid drop by a micro-drop digital detection method. Compared with the liquid drop digital PCR in the prior art, the method has higher nucleic acid amplification efficiency and signal amplification effect, and the nucleic acid sample does not need to be subjected to PCR amplification in advance, and can be directly subjected to counting analysis by a microscope after the amplification reaction. The method of the invention has no dependence on the target nucleic acid, and the digital analysis of the target nucleic acid can be carried out only by carrying out matched primer design. In addition, compared with the traditional single base mutation detection, the method of the invention has low cost and simple operation, and can realize the quantitative analysis of multiple nucleic acids.
The purpose of the invention is realized by the following technical scheme.
In one aspect, the present invention provides a method for digital detection of multiple target nucleic acids in a droplet format, the method comprising the steps of:
(1) preparing a mixture required by the loop-mediated isothermal amplification reaction according to the multiple target nucleic acids;
wherein the mixture comprises primer and probe sets designed for the multiplexed target nucleic acids, respectively, each of the primer and probe sets comprising 2 outer primers F3 and B3, 2 inner primers FIP and BIP, a loop primer, and a scorpion primer probe; a nucleic acid to be detected;
(2) taking the reaction mixture obtained in the step (1) as a dispersed phase and taking an oil phase as a continuous phase, and respectively injecting the dispersed phases into a microfluidic chip to generate monodisperse liquid drops;
(3) placing the microfluidic chip in the step (2) at a constant temperature for amplification reaction;
(4) and (4) carrying out fluorescence signal detection and analysis on the liquid drops in the microfluidic chip after the reaction in the step (3).
The method according to the present invention, wherein, in step (1), the upstream region of the scorpion primer probe comprises a hairpin structure, and the downstream region comprises a fragment specifically complementary to the loop primer, preferably the complementary fragment has the same length as the loop primer; and the 5' end base of the scorpion primer probe is marked with a fluorescent group, and the corresponding base which is complementary and matched with the fluorescent group is marked with a quenching group.
Preferably, the fluorophores labeled by the scorpion primer probes of each of the primer and probe sets are different;
preferably, the radial region of the scorpion primer probe comprises 7-8 bases, and the loop region comprises 7-8 bases;
preferably, the fluorescent group is selected from FAM, TAMRA and/or Cy5 and the quenching group is selected from BHQ1 and/or BHQ 2.
According to different target sequences, different loop primers and different scorpion-shaped primer probes are designed, different fluorescent groups are marked, and the detection of multiple targets can be realized. Wherein, the fluorescent group and the quenching group of the scorpion primer probe aiming at different targets are different.
The method according to the present invention, wherein, in step (1), the reaction mixture further comprises dNTPs and a polymerase, preferably Bst 2.0WarmStartTMA polymerase;
when the nucleic acid to be detected comprises RNA, reverse transcriptase is also included; preferably an AMV reverse transcriptase;
preferably, the reaction mixture further comprises one or more of fluorescein sodium, fluorescein isothiocyanate, or tetraethylrhodamine, more preferably comprises fluorescein sodium;
preferably, the reaction mixture further comprises one or more ingredients selected from the group consisting of: mg (magnesium)2+、K+、NH4 +、H+、Cl-、SO4 2-Tris-HCl and fineA cell surfactant;
more preferably, the reaction mixture comprises Tris-HCl, KCl, (NH)4)2SO4、MgSO4And
X-100。
in a preferred embodiment, the reagents for detecting two target nucleic acids include:
the two target nucleic acids correspond to outer primers F3 and B3, inner primers FIP and BIP, loop primer LF, scorpion primer probe SP, and 10 × Thermopol buffer solution (200mM Tris-HCl, 100mM (NH)4)2SO4，100mM KCl，20mM MgSO4，1％
X-100, pH 8.8), two target nucleic acids, MgSO4dNTP, AMV reverse transcriptase, Bst 2.0WarmStartTMA polymerase; fluorescein sodium.
The method according to the present invention, wherein, in the step (2), the dispersed phase is dispersed in the continuous phase in the form of micro volume units; preferably, the micro volume unit is 10-12～10-15L；
Preferably, the oil phase is one or a mixture of fluorinated oil, mineral oil, silicone oil and/or edible oil;
preferably, the oil comprises a surfactant;
more preferably, the oil phase is Pico-Surf-containingTM1 fluorinated oils of surfactants;
preferably, the microfluidic chip is a flow focusing structure, including:
an oil phase inlet for injecting a continuous phase;
a water phase inlet for injecting a dispersed phase; and
an outlet for droplet collection.
The process according to the invention, wherein, in step (3), the temperature of the reaction is 60-65 ℃, preferably 63-64 ℃.
The method according to the present invention, wherein, in the step (4), the detection is performed using a fluorescence microscope.
According to the method of the present invention, the multiple target nucleic acids and/or the nucleic acids to be detected are RNA and/or DNA; preferably, the multiplex target nucleic acid and/or the nucleic acid to be detected is selected from the group consisting of RNA and/or DNA of two or more viruses, bacteria, fungi, pathogenic parasites;
preferably, the multiple target nucleic acids and/or the nucleic acids to be detected are selected from RNA and/or DNA of helicobacter pylori, shrimp white spot syndrome virus and/or swine epidemic encephalitis B virus;
preferably, the multiple target nucleic acids and/or the nucleic acids to be detected are RNA and/or DNA of two, three, four, five or six of Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), avian influenza virus, SARS virus and novel coronaviruses;
more preferably, the multiplex target nucleic acid and/or the nucleic acid to be detected is RNA and/or DNA of Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV);
more preferably, the multiplex target nucleic acid and/or the nucleic acid to be detected is an RNA of avian influenza virus, SARS virus and novel coronavirus.
In a specific embodiment, the primer and probe sets comprise one or more of the sequences set forth as SEQ ID NOS 1-12;
preferably, the primer and probe set comprises the primer and probe set shown in SEQ ID NO. 1-6 and/or the primer and probe set shown in SEQ ID NO. 6-12;
more preferably, the primer and probe sets are set forth in SEQ ID NOS: 1-12.
In another aspect, the present invention also provides a digital detection kit of the droplet type for detecting multiple target nucleic acids, comprising a reaction mixture comprising primers and probe sets designed for multiple target nucleic acids, each of the primers and probe sets comprising 2 outer primers F3 and B3, 2 inner primers FIP and BIP, a loop primer, and a scorpion primer probe;
preferably, the kit further comprises: dNTPs and polymerase, preferably Bst 2.0WarmStartTMA polymerase;
when the nucleic acid to be detected comprises RNA, the mixture further comprises a reverse transcriptase, preferably AMV reverse transcriptase;
preferably, the kit further comprises one or more of fluorescein sodium, fluorescein isothiocyanate or tetraethylrhodamine, more preferably fluorescein sodium;
preferably, the reaction mixture further comprises one or more ingredients selected from the group consisting of: mg (magnesium)2+、K+、NH4 +、H+、Cl-、SO4 2-Tris-HCl and a cell surfactant; more preferably, the reaction mixture comprises Tris-HCl, KCl, (NH)4)2SO4、MgSO4、
X-100；
Preferably, the upstream region of the scorpion primer probe comprises a hairpin structure and the downstream region comprises a segment that is specifically complementary to the loop primer, preferably the complementary segment is the same length as the loop primer; in addition, a fluorescent group is marked on a base at the 5' end of the scorpion primer probe, and a quenching group is marked on a corresponding base which is complementarily paired with the fluorescent group;
preferably, the fluorescent groups labeled by the scorpion primer probes of each of the primer and probe sets are different;
preferably, the radial region of the scorpion primer probe comprises 7-8 bases, and the loop region comprises 7-8 bases;
preferably, the fluorescent group is selected from FAM, TAMRA and/or Cy5 and the quenching group is selected from BHQ1 and/or BHQ 2.
Preferably, the kit further comprises an oil phase as the continuous phase.
The invention also provides a method for detecting a microorganism, which comprises detecting nucleic acid from a microorganism to be detected by using the method or the kit;
wherein the microorganism is two or more fungi, bacteria, parasites and/or viruses.
The reaction mechanism of the method for the digital detection of multiple target nucleic acids in droplet form according to the present invention is briefly described as follows, with reference to FIG. 1 of the specification:
in the loop-mediated isothermal amplification reaction, a scorpion primer probe is added in addition to 2 outer primers, 2 inner primers and 1 loop primer required by the reaction. The upstream region of the scorpion primer probe is a common hairpin structure, the diameter region has 7 basic groups, the loop region has 8 basic groups, the 5' end basic group is marked with a fluorescent group, the complementary matched corresponding basic group is marked with a quenching group, the downstream region is specifically complementary with the target loop primer, and the sequence length is the same as that of the loop primer.
The first stage of the reaction is the formation of dumbbell chains. Under the condition of isothermal 64 ℃, the double-stranded DNA template firstly specifically recognizes the F2c region of the template from the F2 region of the inner primer FIP, and forms a new double-stranded DNA under the action of DNA polymerase, and at the moment, the outer primer F3 replaces a new strand synthesized by FIP to form a self DNA double strand by complementary pairing with the F3c region of the newly synthesized double-stranded DNA. The replaced FIP new strand 5' end has the complementary regions of F1c and F1, and can form a loop structure by itself. Similarly, the other inner primer BIP can hybridize with the above circular single strand at this time, and the circular structure is opened to synthesize a complementary strand. Under the action of the outer primer B3, a new double-stranded DNA is formed. The second phase of the reaction is the cyclic amplification phase. The dumbbell-shaped structure takes self as a template and self-guides to synthesize a DNA chain under the action of an inner primer. FIP primer F2 hybridizes with F2c to form a new DNA strand, and B1 and B1c can complementarily bind to form a loop structure again to replace the previous complementary strand. Similarly, B2 of BIP primer is combined with B2c complementarily to form a new DNA chain, and the previously synthesized DNA chain is replaced, F1 in the new chain is combined with F1c complementarily to form a circular structure, and the final product is a mixture of some DNA with stem-loop structure of different stem length and some DNA with multiple loops and similar Cabrous structure.
The loop primers in the reaction also hybridize through the stem-loop structure, initiating strand displacement DNA synthesis, with binding regions located between F1 and F2 and between B1 and B2, respectively. The addition of the loop primer has no influence on the combination of the original inner primer, and can combine other loop structures which cannot be combined by the inner primer and initiate the synthesis of strand displacement DNA, so that all the formed loop single-chain structures in the amplification reaction are ensured to be combined by the corresponding primers to initiate the synthesis of DNA, and the LAMP reaction speed is greatly improved.
On the basis of the reaction mechanism, the technical scheme of the invention takes the microfluidic chip as a platform to generate a single water-in-oil droplet, collects the generated droplet in a liquid storage tank of the glass chip and takes each droplet as a reaction chamber. When each liquid drop has a target chain, a loop-mediated isothermal amplification reaction is carried out, a large number of dumbbell-type single-chain structures are generated in the reaction process, scorpion-shaped primer probes are hybridized with the dumbbell-shaped single-chain structures to carry out polymerization extension, the hairpin structures are opened, fluorescence energy resonance transfer is carried out, the fluorescence of the droplets is recovered, the fluorescence intensity of each liquid drop is observed under a microscope, the number of bright liquid drops is counted, and the purpose of digital nucleic acid analysis on target DNA is achieved. In the present invention, the LAMP reaction solution contains sodium fluorescein, and excitation is performed by 488nm laser to generate a strong green fluorescence signal, so that droplets are prepared by using the reaction solution, and each droplet has a green fluorescence signal when observed under a microscope. The invention simultaneously detects two target nucleic acid molecules of HCV and HIV, the reaction solution simultaneously contains amplification primers of HCV and HIV, a scorpion primer probe label TAMRA for detecting HCV and a scorpion primer probe label Cy5 for detecting HIV, when HCV molecules exist in the liquid drop, a yellow fluorescent signal is generated, and when HIV molecules exist in the liquid drop, a red fluorescent signal is generated. Because the number of the generated droplets is far larger than that of the target molecules, each droplet at most contains one target molecule according to Poisson distribution, and single-molecule amplification and detection are realized. Under a microscope platform, the total number of the reaction liquid drops can be known by counting the number of the green liquid drops, the number of the yellow liquid drops is counted, the number of HCV molecules is counted, the number of the red liquid drops is counted, and the number of HIV molecules is counted. By the method and the platform, the aim of simultaneously detecting multiple target nucleic acid molecules by LAMP reaction is fulfilled. The establishment of the method provides a simple, rapid and high-sensitivity nucleic acid digital analysis means for diagnosing various diseases. .
Compared with the prior art, the invention has the following advantages:
1. the method provided by the invention can complete quantitative analysis of nucleic acid without complex controllable temperature control equipment, has simple experimental steps, shortens reaction time by utilizing the advantages of loop-mediated isothermal amplification, improves reaction efficiency, has high repeatability and good universality, is favorable for high-throughput application, and provides a nucleic acid analysis method with high sensitivity, simplicity and convenience in detection and time saving for researches such as gene diagnosis and treatment.
2. The method provided by the invention has no dependence on target nucleic acid sequences, and can be used for detecting and analyzing the target nucleic acids by selecting a proper amplification region and carrying out matched primer design aiming at each target nucleic acid.
3. The method can complete the digital analysis of the multiple target nucleic acid without complex chips, controllable temperature control equipment and other equipment with higher cost, simplifies the experimental steps, shortens the time and improves the efficiency by utilizing the advantages of loop-mediated isothermal amplification, and provides a digital analysis means of the multiple target nucleic acid with simple operation, wide applicability and high sensitivity for the diagnosis and research of infectious diseases.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a reaction scheme of a digital detection method of a droplet type according to an embodiment of the present invention; multiple LAMP single-molecule amplification occurs in the microfluidic droplets, the green droplets of the chip liquid storage tank are droplets generated by the microfluidic chip, the yellow droplets are fluorescent signals generated by LAMP amplification of HCV target molecules, and the red droplets are fluorescent signals generated by LAMP amplification of HIV target molecules;
FIG. 2 is a microscope image of droplets generated by a chip of the digital droplet inspection method according to an embodiment of the present invention, and as can be seen from FIG. 2, the droplets generated by the method have a diameter of about 50 μm;
FIG. 3 is a microscopic image of a droplet after LAMP reaction in the digital detection method of droplet type according to one embodiment of the present invention, as shown in FIG. 3, in which the green droplet is a negative droplet, which indicates that the droplet does not undergo the loop-mediated isothermal amplification reaction, and does not contain the target nucleic acid strand, and there is no negative droplet in which the scorpion primer probe hybridizes to the single-stranded dumbbell structure for polymerization extension, the yellow droplet is a positive droplet containing the HCV target molecule, and the red droplet is a positive droplet containing the HIV target molecule, which indicates that the droplet undergoes the loop-mediated isothermal amplification reaction, and there is the target nucleic acid strand, and there is a positive droplet in which the scorpion primer probe hybridizes to the single-stranded dumbbell structure for polymerization extension;
FIG. 4 is a real-time fluorescence curve of the LAMP reaction according to the embodiment of the present invention;
FIG. 5 is a diagram of gel electrophoresis according to an embodiment of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example 1 detection of HCV and HIV according to the methods of the present invention
(1) Designing a primer: loop-mediated isothermal amplification (LAMP) primers were designed for HCV and HIV genes. Primer sequences are shown in table 1:
table 1: primer sequence Listing
The cDNA sequences of CV and HIV are as follows:
HCV target gene base sequence SEQ ID NO 13
GTTTAGGATTCGTGCTCATGGTGCACGGTCTACGAGACCTCCCGGGGCACTCGCAAGCACCCTATCAGGCAGTACCACAAGGCCTTTCGCGACCCAACACTACTCGGCTAGCAGTCTCGCGGGGGCACGCCCAAATCTCCAGGCATTGAGCGGGTTGATCCAAGAAAGGACCCGGTCGTCCTGGCAATTCCGGTGTACTCACCGGTTCCGCAGACCACTATGGCTCTCCCGGGAGGGGGGGTCCTGGAGGCTGCACGACACTCATACTAACGCCATGGCTAGACGCTTTCTGCGTGAAGACAGTAGTTCCTCACAGGGGAGTGATTCATGGTGGAGTGTCGCCCCCATCAGGGGGCTGGC
HIV target gene base sequence SEQ ID NO 14
ATTTTATTTAATCCCAGGATTATCCATCTTTTATAAATTTCTCCTACTGGGATAGGTGGATTATTTGTCATCCATCCTATTTGTTCCTGAAGGGTACTAGTAGTTCCTGCTATGTCACTTCCCCTTGGTTCTCTCATCTGGCCTGGTGCAATAGGCCCTGCATGCACTGGATGCACTCTATCCCATTCTGCAGCTTCCTCATTGATGGTCTCTTTTAACATTTGCATGGCTGCTTGATGTCCCCCCACTGTGTTTAGCATGGTGTTTAAATCTTGTGGGGTGGCTCCTTCTGATAATGCTGAAAACATGGGTATCACTTCTGGGCTGAAAGCCTTCTCTTCTACTACTTTTACCCATGCATTTAAAGTTCTAGGTGATATGGCCTGATGTACCA
(2) Preparing LAMP reaction liquid: outer primers HCV-F3 (5. mu.M), HIV-F3 (5. mu.M), HCV-B3 (5. mu.M), HIV-B3 (5. mu.M) each 8. mu.L, inner primers HCV-FIP (40. mu.M), HCV-BIP (40. mu.M), HIV-FIP (40. mu.M), HIV-BIP (40. mu.M) each 8. mu.L, loop primers HCV-LF (10. mu.M), HIV-LF (10. mu.M) each 8. mu.L, scorpion primers HCV-SP (10. mu.M), HIV-SP (10. mu.M) each 8. mu.L, 10 XThermopol buffer solution (200 mM-Tris, 100mM (NH-M)4)2SO4，100mM KCl，20mM MgSO4，1％
X-100，pH 8.8)20μL，MgSO4(10mM)8μL，dNTP(10mM)32μL，Bst 2.0WarmStartTMMixing 8 μ L of polymerase and 20 μ M of fluorescein sodium 5 μ L, adding sterilized ultrapure water to 100 μ L;
(3) preparing an oil phase: 10mL of the solution containing 1% of Pico-S.mu.rf TM1 fluorinated oil Novec-7500 of surfactant for use;
(4) droplet preparation: preparing liquid drops by using a 2Reagent Droplet Chip purchased from Dolomite, wherein a water phase 1 is 100 mu L of LAMP reaction liquid, a water phase 2 is 100 mu L of target nucleic acid, 2 oil phases are 200 mu L, the flow rates of the water phase and the oil phase are respectively 3 mu L/min and 10 mu L/min, an outlet is connected with a glass Chip liquid storage tank, collecting the generated liquid drops, and collecting for 3 min;
(5) LAMP reaction: putting the glass chip liquid storage tank into a constant-temperature water bath kettle, and reacting for 1 hour at 64 ℃;
(6) and (3) microscopic detection: adjusting fluorescence microscope parameters, simultaneously opening 488, 560 and 640 laser channels, wherein the number of green liquid drops is the total number of liquid drops, the number of yellow liquid drops is HCV positive liquid drops, the number of red liquid drops is HIV positive liquid drops, and the number of the obtained positive liquid drops is in direct proportion to the addition amount of the target nucleic acid;
(7) real-time fluorescence quantitative analysis
Preparing a reaction solution: outer primers HCV-F3 (5. mu.M), HIV-F3 (5. mu.M), HCV-B3 (5. mu.M), HIV-B3 (5. mu.M) each in 1. mu.L, inner primers HCV-FIP (40. mu.M), HCV-BIP (40. mu.M), HIV-FIP (40. mu.M), HIV-BIP (40. mu.M) each in 1. mu.L, loop primers HCV-LF (10. mu.M), HIV-LF (10. mu.M) each in 1. mu.L, scorpion primers HCV-SP (10. mu.M), HIV-SP (10. mu.M) each in 1. mu.L, 10 XThermopol buffer solution (200 mM-Tris, 100mM (NH-Tris, 10. mu.M)4)2SO4，100mM KCl，20mM MgSO4，1％
X-100, pH 8.8) 2.5. mu.L, target nucleic acid, MgSO4(10mM)1μL，dNTP(10mM)4μL，Bst 2.0WarmStartTMPolymerase 1. mu.L, sterile ultrapure water was added to 25. mu.L. The reaction temperature was 64 ℃ and the apparatus used was a C1000Thermal Cycler (Bio-Rad, Herc. mu. les, CA, M SA) including a CFX96 in situ detection system. Fluorescence was monitored in real time for the selected FAM, ROX, Cy5 channels, and fluorescence values were read every 30s, with the results shown in FIG. 4: curve a represents the non-target added, curve b represents the HCV target added, and curve c represents the HIV target added.
(8) Gel electrophoresis analysis
And (3) carrying out agarose gel electrophoresis analysis on the LAMP reaction product. The gel formation and electrophoresis were carried out at room temperature, the electrophoresis analysis was carried out using 1.5% agarose and 0.5 XTBE buffer (45mM Tris, 45mM Boric Acid, 10mM EDTA, pH 8.0), staining with 0.5. mu.g/mL GoldView and 0.5. mu.g/mL ethidium bromide, adding 10. mu.L of the sample mixture to the above sample, subjecting the sample mixture to electrophoresis at 100V for 90 minutes, and after the electrophoresis was completed, observing and photographing the bands with a Tanon 4200SF gel imaging system (Shanghai Nature science Co., Ltd., China), and the results were shown in FIG. 5: from left to right in sequence: DNA marker band; a LAMP amplification product band with an HCV primer and an HIV template; a LAMP amplification product band with HCV primers and HCV templates; a LAMP amplification product band with HIV primers and HCV template; a LAMP amplification product band with an HIV primer and an HIV template; a LAMP amplification product band with HCV primers and HIV primers; a LAMP amplification product band with an HCV primer, an HIV primer and an HCV template; a LAMP amplification product band with an HCV primer, an HIV primer and an HIV template; there are bands of LAMP amplification products of HCV primer, HIV primer, HCV template and HIV template. The electrophoresis result chart shows that the LAMP reaction can only occur if corresponding target primers and target templates exist, and the existence of another target can not interfere with the multiplex LAMP, thereby proving the specificity of the reaction.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> university of Hunan
<120> droplet type digital detection method for multiple target nucleic acids
<130> DIC20110097
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tggtctgcgg aaccgg 16
<210> 2
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggggcactcg caagca 16
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acgcccaaat ctccaggcat tgcattgcca ggacgaccgg 40
<210> 4
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccgcgagact gctagccgac cctatcaggc agta 34
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agcgggttga tccaagaaag gac 23
<210> 6
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_binding
<222> (1)..(1)
<223> labeling of fluorescent group TAMRA
<220>
<221> misc_binding
<222> (22)..(22)
<223> labeled quencher group BHQ2
<400> 6
agcgcggata tctcaccgcg cttgttgggt cgcgaaaggc c 41
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
attatcagaa ggagccacc 19
<210> 9
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gagtgcatcc agtgcatgca ctgctatgtc acttcccct 39
<210> 10
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ccattctgca gcttcctcat tgaacaccat gctaaacaca gt 42
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
<210> 12
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_binding
<222> (1)..(1)
<223> fluorescent group labeling Cy5
<220>
<221> misc_binding
<222> (22)..(22)
<223> labeled quencher group BHQ2
<400> 12
agcgcggata tctcaccgcg ctatggctgc ttgatgtccc c 41
<210> 13
<211> 360
<212> DNA
<213> Hepatitis C virus
<400> 13
gtttaggatt cgtgctcatg gtgcacggtc tacgagacct cccggggcac tcgcaagcac 60
cctatcaggc agtaccacaa ggcctttcgc gacccaacac tactcggcta gcagtctcgc 120
gggggcacgc ccaaatctcc aggcattgag cgggttgatc caagaaagga cccggtcgtc 180
ctggcaattc cggtgtactc accggttccg cagaccacta tggctctccc gggagggggg 240
gtcctggagg ctgcacgaca ctcatactaa cgccatggct agacgctttc tgcgtgaaga 300
cagtagttcc tcacagggga gtgattcatg gtggagtgtc gcccccatca gggggctggc 360
<210> 14
<211> 394
<212> DNA
<213> Human immunodeficiency virus
<400> 14
attttattta atcccaggat tatccatctt ttataaattt ctcctactgg gataggtgga 60
ttatttgtca tccatcctat ttgttcctga agggtactag tagttcctgc tatgtcactt 120
ccccttggtt ctctcatctg gcctggtgca ataggccctg catgcactgg atgcactcta 180
tcccattctg cagcttcctc attgatggtc tcttttaaca tttgcatggc tgcttgatgt 240
ccccccactg tgtttagcat ggtgtttaaa tcttgtgggg tggctccttc tgataatgct 300
gaaaacatgg gtatcacttc tgggctgaaa gccttctctt ctactacttt tacccatgca 360
tttaaagttc taggtgatat ggcctgatgt acca 394
Claims (11)
1. A method for digital detection of multiple target nucleic acids in microdroplet format, the method comprising the steps of:
(1) preparing a mixture required by the loop-mediated isothermal amplification reaction according to the multiple target nucleic acids;
wherein the mixture comprises primer and probe sets designed for the multiplexed target nucleic acids, respectively, each of the primer and probe sets comprising 2 outer primers F3 and B3, 2 inner primers FIP and BIP, a loop primer, and a scorpion primer probe; a nucleic acid to be detected;
(2) taking the reaction mixture obtained in the step (1) as a dispersed phase and taking an oil phase as a continuous phase, and respectively injecting the dispersed phases into a microfluidic chip to generate monodisperse liquid drops;
(3) placing the microfluidic chip in the step (2) at a constant temperature for amplification reaction;
(4) and (4) carrying out fluorescence signal detection and analysis on the liquid drops in the microfluidic chip after the reaction in the step (3).
2. The method of claim 1, wherein in step (1), the upstream region of the scorpion primer probe comprises a hairpin structure and the downstream region comprises a segment that is specifically complementary to the loop primer, preferably the complementary segment is the same length as the loop primer; and the 5' end base of the scorpion primer probe is marked with a fluorescent group, and the corresponding base which is complementary and matched with the fluorescent group is marked with a quenching group.
3. The method of claim 1 or 2, wherein in step (1), the fluorophores labeled by the scorpion primer probes of each of the primer and probe sets are different;
preferably, the radial region of the scorpion primer probe comprises 7-8 bases, and the loop region comprises 7-8 bases;
preferably, the fluorescent group is selected from FAM, TAMRA and/or Cy5 and the quenching group is selected from BHQ1 and/or BHQ 2.
4. The method of any one of claims 1 to 3, wherein in step (1), the reaction mixture further comprises dNTPs and a polymerase, preferably Bst 2.0WarmStartTMA polymerase;
when the nucleic acid to be detected comprises RNA, the mixture further comprises a reverse transcriptase; preferably an AMV reverse transcriptase;
preferably, the reaction mixture further comprises one or more of fluorescein sodium, fluorescein isothiocyanate, or tetraethylrhodamine, more preferably comprises fluorescein sodium;
preferably, the reaction mixture further comprises one or more ingredients selected from the group consisting of: mg (magnesium)2+、K+、NH4 +、H+、Cl-、SO4 2-Tris-HCl and a cell surfactant;
more preferably, the reaction mixture comprises Tris-HCl, KCl, (NH)4)2SO4、MgSO4、
X-100。
5. The method according to any one of claims 1 to 4, wherein, in step (2), the dispersed phase is dispersed in the continuous phase in the form of micro volume units; preferably, the micro volume unit is 10-12～10-15L；
Preferably, the oil phase is one or a mixture of fluorinated oil, mineral oil, silicone oil and/or edible oil;
preferably, the oil comprises a surfactant;
more preferably, the oil phase is Pico-Surf-containingTM1 fluorinated oils of surfactants;
preferably, the microfluidic chip is a flow focusing structure, including:
an oil phase inlet for injecting a continuous phase;
a water phase inlet for injecting a dispersed phase; and
an outlet for droplet collection.
6. The process according to any one of claims 1 to 5, wherein in step (3) the temperature of the reaction is 60-65 ℃, preferably 63-64 ℃.
7. The method of any one of claims 1 to 6, wherein in step (4), the detection is performed using a fluorescence microscope.
8. The method according to any one of claims 1 to 7, wherein the multiplex target nucleic acids and/or the nucleic acids to be detected are RNA and/or DNA; preferably, the multiplex target nucleic acid and/or the nucleic acid to be detected is selected from the group consisting of RNA and/or DNA of two or more viruses, bacteria, fungi, pathogenic parasites;
preferably, the multiple target nucleic acids and/or the nucleic acids to be detected are selected from RNA and/or DNA of helicobacter pylori, shrimp white spot syndrome virus and/or swine epidemic encephalitis B virus;
preferably, the multiple target nucleic acids and/or the nucleic acids to be detected are RNA and/or DNA of two, three, four, five or six of hepatitis b virus, hepatitis c virus, human immunodeficiency virus, avian influenza virus, SARS virus and novel coronavirus;
more preferably, the multiplex target nucleic acid and/or the nucleic acid to be detected is RNA and/or DNA of hepatitis b virus, hepatitis c virus and human immunodeficiency virus;
more preferably, the multiplex target nucleic acid and/or the nucleic acid to be detected is an RNA of avian influenza virus, SARS virus and novel coronavirus.
9. The method of claim 8, wherein the primer and probe set comprises one or more of the sequences set forth as SEQ ID NOs 1-12;
preferably, the primer and probe set comprises the primer and probe set shown in SEQ ID NO. 1-6 and/or the primer and probe set shown in SEQ ID NO. 6-12;
preferably, the primer and probe sets are set forth in SEQ ID NOS: 1-12.
10. A digital detection kit of the droplet type for detecting a multiple target nucleic acid, comprising a reaction mixture comprising primers and probe sets designed for a multiple target nucleic acid, each of the primer and probe sets comprising 2 outer primers F3 and B3, 2 inner primers FIP and BIP, a loop primer, and a scorpion primer probe;
preferably, the kit further comprises: dNTPs and polymerase, preferably Bst 2.0WarmStartTMA polymerase;
when the nucleic acid to be detected comprises RNA, the mixture further comprises a reverse transcriptase, preferably AMV reverse transcriptase;
preferably, the kit further comprises one or more of fluorescein sodium, fluorescein isothiocyanate or tetraethylrhodamine, more preferably fluorescein sodium;
preferably, the reaction mixture further comprises one or more ingredients selected from the group consisting of: mg (magnesium)2+、K+、NH4 +、H+、Cl-、SO4 2-Tris-HCl and a cell surfactant; more preferably, the reaction mixture comprises Tris-HCl, KCl, (NH)4)2SO4、MgSO4、
X-100；
Preferably, the upstream region of the scorpion primer probe comprises a hairpin structure and the downstream region comprises a segment that is specifically complementary to the loop primer, preferably the complementary segment is the same length as the loop primer; in addition, a fluorescent group is marked on a base at the 5' end of the scorpion primer probe, and a quenching group is marked on a corresponding base which is complementarily paired with the fluorescent group;
preferably, the fluorescent groups labeled by the scorpion primer probes of each of the primer and probe sets are different;
preferably, the radial region of the scorpion primer probe comprises 7-8 bases, and the loop region comprises 7-8 bases;
preferably, the fluorescent group is selected from FAM, TAMRA and/or Cy5, and the quenching group is selected from BHQ1 and/or BHQ 2;
preferably, the kit further comprises an oil phase as the continuous phase.
11. A method for detecting a microorganism, which comprises detecting a nucleic acid from a microorganism to be detected by the method according to any one of claims 1 to 9 or the kit according to claim 10;
wherein the microorganism is two or more fungi, bacteria, parasites and/or viruses.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110641186.9A CN113337583A (en) | 2021-06-09 | 2021-06-09 | Micro-drop digital detection method for multiple target nucleic acids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110641186.9A CN113337583A (en) | 2021-06-09 | 2021-06-09 | Micro-drop digital detection method for multiple target nucleic acids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113337583A true CN113337583A (en) | 2021-09-03 |
Family
ID=77475664
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110641186.9A Pending CN113337583A (en) | 2021-06-09 | 2021-06-09 | Micro-drop digital detection method for multiple target nucleic acids |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113337583A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116497095A (en) * | 2023-01-09 | 2023-07-28 | 深圳大学 | Multiple nucleic acid quantification method, device and medium based on sediment bright field image processing |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102712944A (en) * | 2009-11-05 | 2012-10-03 | 贝克顿·迪金森公司 | Sequence-specific methods for homogenous, real-time detection of lamp products |
| CN106916898A (en) * | 2017-05-04 | 2017-07-04 | 湖南大学 | The digital miRNA analysis methods of ring mediated isothermal amplification are carried out in a kind of emulsion |
| CN107022644A (en) * | 2017-06-14 | 2017-08-08 | 山东省农业科学院农业质量标准与检测技术研究所 | Six kinds of multiple LAMP detection primers of food-borne pathogens, detection kit and detection method in fruits and vegetables |
-
2021
- 2021-06-09 CN CN202110641186.9A patent/CN113337583A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102712944A (en) * | 2009-11-05 | 2012-10-03 | 贝克顿·迪金森公司 | Sequence-specific methods for homogenous, real-time detection of lamp products |
| CN106916898A (en) * | 2017-05-04 | 2017-07-04 | 湖南大学 | The digital miRNA analysis methods of ring mediated isothermal amplification are carried out in a kind of emulsion |
| CN107022644A (en) * | 2017-06-14 | 2017-08-08 | 山东省农业科学院农业质量标准与检测技术研究所 | Six kinds of multiple LAMP detection primers of food-borne pathogens, detection kit and detection method in fruits and vegetables |
Non-Patent Citations (2)
| Title |
|---|
| YA-LING TAN等: "Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs", 《CHEMICAL SCIENCE》 * |
| 彭承琳等: "《生物医学传感器-原理与应用》", 31 August 1992 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116497095A (en) * | 2023-01-09 | 2023-07-28 | 深圳大学 | Multiple nucleic acid quantification method, device and medium based on sediment bright field image processing |
| CN116497095B (en) * | 2023-01-09 | 2024-07-12 | 深圳大学 | Dual nucleic acid quantification method, device and medium based on sediment bright field image processing |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108774639B (en) | Directional polymerization fluorescent probe PCR | |
| CN101638685B (en) | Method for amplifying target nucleic acid sequence by using cross primer and kit for amplifying target nucleic acid sequence and application thereof | |
| CN113201583B (en) | Method for synthesizing nucleic acid under constant temperature condition, kit and application | |
| CN103255227B (en) | Primer-mediated cyclized constant-temperature nucleic acid rolling circle amplification method and kit | |
| CN105803074B (en) | primer type nucleic acid fluorescent probe displaced by bidirectional strand | |
| CN105154556B (en) | Real-time fluorescence constant temperature exponential amplification methods | |
| CN105555971B (en) | Probe for improving molten chain resolution and multiplicity in nucleic acid determination | |
| CN102762745A (en) | Real-time multiplexing detection of target nucleic acid sequences with elimination of false signals | |
| CN113832258A (en) | Ribonucleic acid amplification and detection using attenuated probes | |
| US20240229123A1 (en) | Multiplex nucleic acid detection method, composition and kit | |
| CN105018593A (en) | Methods for detecting pathogen in coldwater fish | |
| CN113337583A (en) | Micro-drop digital detection method for multiple target nucleic acids | |
| CN108085372B (en) | Primer end enzymolysis dimer method PCR | |
| CN117070669B (en) | Novel Coronavirus Whole Genome Capture Kit | |
| CN106868198B (en) | Multiplex PCR primer group for simultaneously detecting four pathogenic bacteria of catfishes and monitoring method | |
| CN113201584B (en) | Detection method, kit and application of multiple target nucleic acids | |
| CN107904284A (en) | The nucleic acid constant-temperature amplification method of programmatic method and its kit application | |
| CN112029829B (en) | Nucleic acid isothermal amplification method based on hairpin structure and kit application thereof | |
| KR20190041314A (en) | Oligonucleotide set for detection of chikungunya virus and uses thereof | |
| CN113151416A (en) | Micro-drop digital detection method for detecting mutant nucleic acid | |
| EP3269820A1 (en) | Kit for the amplification of a sequence from a ribonucleic acid | |
| CN106498095A (en) | A kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus | |
| WO2021156882A1 (en) | Primer sets, biomarkers, kit and applications thereof | |
| CN115595382B (en) | Primer group for detecting porcine enterovirus and application thereof | |
| CN114008217B (en) | Combination, method and kit for detecting nucleic acid |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220316 Address after: 410036 No. 506, building a-5 to building A-8, Huigu science and Technology Industrial Park, No. 336, bachelor street, Yuelu District, Changsha City, Hunan Province Applicant after: Hunan Rongjian Biotechnology Co.,Ltd. Address before: 410082 Yuelu District Lushan South Road Lushan Gate, Changsha City, Hunan Province Applicant before: HUNAN University |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210903 |
|
| RJ01 | Rejection of invention patent application after publication |