AU2022301095A1 - Method for isothermal amplification of nucleic acid target sequences - Google Patents
Method for isothermal amplification of nucleic acid target sequences Download PDFInfo
- Publication number
- AU2022301095A1 AU2022301095A1 AU2022301095A AU2022301095A AU2022301095A1 AU 2022301095 A1 AU2022301095 A1 AU 2022301095A1 AU 2022301095 A AU2022301095 A AU 2022301095A AU 2022301095 A AU2022301095 A AU 2022301095A AU 2022301095 A1 AU2022301095 A1 AU 2022301095A1
- Authority
- AU
- Australia
- Prior art keywords
- stranded
- amplification
- primer
- dna polymerase
- double
- 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
- 238000000034 method Methods 0.000 title claims abstract description 54
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 34
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 29
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 29
- 238000011901 isothermal amplification Methods 0.000 title abstract description 8
- 230000003321 amplification Effects 0.000 claims abstract description 109
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 109
- 239000000523 sample Substances 0.000 claims abstract description 82
- 238000006243 chemical reaction Methods 0.000 claims abstract description 75
- 108020004414 DNA Proteins 0.000 claims abstract description 40
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims abstract description 38
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims abstract description 38
- 102000053602 DNA Human genes 0.000 claims abstract description 35
- 238000006073 displacement reaction Methods 0.000 claims abstract description 34
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 16
- 230000027455 binding Effects 0.000 claims abstract description 16
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 64
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 64
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 48
- 239000011780 sodium chloride Substances 0.000 claims description 24
- 230000029087 digestion Effects 0.000 claims description 20
- 230000004048 modification Effects 0.000 claims description 20
- 238000012986 modification Methods 0.000 claims description 20
- 102100034343 Integrase Human genes 0.000 claims description 17
- 230000000295 complement effect Effects 0.000 claims description 17
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 15
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 15
- 229960003237 betaine Drugs 0.000 claims description 15
- 239000000654 additive Substances 0.000 claims description 14
- 230000035484 reaction time Effects 0.000 claims description 14
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 11
- 230000000694 effects Effects 0.000 claims description 11
- 230000000996 additive effect Effects 0.000 claims description 9
- 238000010839 reverse transcription Methods 0.000 claims description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 101710203526 Integrase Proteins 0.000 claims description 6
- 239000002299 complementary DNA Substances 0.000 claims description 6
- 230000000087 stabilizing effect Effects 0.000 claims description 5
- 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 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- -1 dNTP Substances 0.000 claims description 4
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 3
- 229920000159 gelatin Polymers 0.000 claims description 3
- 239000008273 gelatin Substances 0.000 claims description 3
- 235000019322 gelatine Nutrition 0.000 claims description 3
- 235000011852 gelatine desserts Nutrition 0.000 claims description 3
- 230000011987 methylation Effects 0.000 claims description 3
- 238000007069 methylation reaction Methods 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 230000005758 transcription activity Effects 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 31
- 102000004190 Enzymes Human genes 0.000 abstract description 18
- 108090000790 Enzymes Proteins 0.000 abstract description 18
- 239000012634 fragment Substances 0.000 abstract description 4
- 238000012123 point-of-care testing Methods 0.000 abstract description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 42
- 229910001629 magnesium chloride Inorganic materials 0.000 description 21
- 239000003623 enhancer Substances 0.000 description 8
- 241000282414 Homo sapiens Species 0.000 description 6
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 6
- 230000000241 respiratory effect Effects 0.000 description 6
- 241000713196 Influenza B virus Species 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241000701931 Canine parvovirus Species 0.000 description 4
- ZTKGXSXZEFBBJI-UHFFFAOYSA-N Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl Chemical compound Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl.Cl ZTKGXSXZEFBBJI-UHFFFAOYSA-N 0.000 description 4
- 238000007397 LAMP assay Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000001917 fluorescence detection Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 241001647372 Chlamydia pneumoniae Species 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- 241000709661 Enterovirus Species 0.000 description 3
- 101000611053 Homo sapiens Proteasome subunit beta type-2 Proteins 0.000 description 3
- 241000702617 Human parvovirus B19 Species 0.000 description 3
- 241000712431 Influenza A virus Species 0.000 description 3
- 102000018120 Recombinases Human genes 0.000 description 3
- 108010091086 Recombinases Proteins 0.000 description 3
- 241000725643 Respiratory syncytial virus Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 2
- 208000034809 Product contamination Diseases 0.000 description 2
- 108091027568 Single-stranded nucleotide Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000005518 electrochemistry Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 241001678559 COVID-19 virus Species 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 101710116602 DNA-Binding protein G5P Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010014594 Heterogeneous Nuclear Ribonucleoprotein A1 Proteins 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 101710162453 Replication factor A Proteins 0.000 description 1
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 description 1
- 101710176276 SSB protein Proteins 0.000 description 1
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000037048 polymerization activity Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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
Abstract
The present invention provides a method for isothermal amplification of nucleic acid target sequences. The method is suitable for double-stranded DNA, single-stranded DNA, and single-stranded RNA, and comprises a combined reaction of nickase and strand displacement enzyme. In double-stranded DNA and single-stranded DNA detection, three primers and one probe are used, and in single-stranded RNA detection, three primers and one probe may be used, or two primers and one probe may be used. The probe is a molecular beacon, which does not degrade during an amplification process and is only used for specifically binding to a target fragment to provide a fluorescent signal to ensure the specificity of the reaction. In the present invention, the beacon probe that does not overlap with the primers in a binding region on a target sequence is used to determine the result in real time; the beacon probe has high specificity in binding to the target sequence; and it is unnecessary to open a tube after the reaction to further prevent generation of false positives. The reaction is carried out at a constant temperature and consumes short time, and the detection can be completed within 8 minutes, which is more in line with POCT detection requirements.
Description
The invention relates to the technical field of nucleic acid detection, and particularly, to a method for isothermally amplifying a target nucleic acid sequence.
Polymerase chain reaction (PCR) is the most commonly used nucleic acid amplification test (NAAT) technique. The classical reactions of the technology include three procedures: denaturation, renaturation and extension. PCR is a process requiring rapid temperature circulation, and requires a specific thermal cycler for high-precision temperature control, which consumes massive electric power. Meanwhile, PCR features a prolonged reaction time, which does not meet the requirement of point-of-care testing (POCT). Although products that can complete the reaction in 15-30 minutes have been known in recent years, such products are cost-inefficient due to complicated industrial designs.
In order to solve the numerous problems of PCR, a series of isothermal amplification techniques have been developed. Commonly used techniques are recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nicking enzyme amplification reaction (NEAR), transcription-mediated amplification (TMA), and the like.
RPA depends on three enzymes: a recombinase binding to a single-stranded nucleic acid, a single-stranded DNA binding protein, and a strand-displacing DNA polymerase. The recombinase recognizes a complementary sequence of the single-stranded nucleic acid and binds them, the single strand binding protein stabilizes the binding region, and the strand-displacing DNA polymerase extends the strand. The reaction is generally conducted at 37-42 °C within 15-30 min, and a special probe can be added to judge the result. RPA involves a large number of components and is thus cost-inefficient.
LAMP utilizes a strand-displacing enzyme to complete the reaction, and by designing 4 or 6 primers and the formation of stem-loop products, the reaction is continuously initiated at the stem-loop under the action of the strand-displacing enzyme. The process needs 30-45 min, and generally adopts dyes for judging the endpoint. LAMP reagents are cost-efficient, but the use of dyes may lead to false positive results and thus difficulties in designing the primer.
SDA uses specially modified nucleotides, endonucleases and strand-displacing DNA polymerases, and requires 4 primers. The product with digestion sites at both ends is generated by reacting a melting primer and an amplification primer with the template. Since the end with the modified nucleotide cannot be cleaved by the endonuclease, nicks are generated in the product under the action of the endonuclease, and displacement and extension are conducted under the action of a strand-displacing DNA polymerase, thus achieving exponential amplification. The double-stranded DNA template requires melting and primer annealing before the enzyme is added for reaction. The reaction time is generally 30-60 min.
NEAR is similar to SDA, requiring nickases, strand-displacing enzymes, and only 2 primers. The distance between the 2 primers (3' ends) is 1-5 bases, and by the action of primer invasion, a product with nickase sites at both ends is formed. The product can be exponentially amplified under the action of the nickase and strand-displacing DNA polymerase. The product can be analyzed by using probes and dyes. Some products of this technique have been approved, but were reported with low sensitivity in SARS-CoV-2 nucleic acid detection. Because of the short distance between the primers, when the probes are used for real-time detection, false positive results may occur due to the homologous positions of the primers and the probes. The reaction time is about 12 min.
Transcription-mediated amplification (TMA) is conducted by reactions of reverse transcriptase and RNA polymerase, and the major product is RNA. The reaction time is 15-60 min.
CN104726549A discloses a novel detection method for isothermally amplifying double strands !0 based on nickase, which adopts 3 primers. One of the amplification primers can be designed as a beacon probe, and the product is analyzed by staining, fluorescence, electrochemistry, colorimetry, and chemiluminescence. The detection time is 30-60 min. Methods other than fluorescence may easily lead to false positive results, and the labeled primers according to the patent may result in incorrect reactions. In addition, non-specific reactions in the labeled primers also bring false positive results. Due to factors of overlong reaction time, improper product analysis and the like, no products are on the market at present.
The present invention is intended to solve the technical problems of prolonged detection time and poor specificity in the prior art.
In order to achieve the above intention, in a first aspect, the present invention provides a method for isothermally amplifying a target nucleic acid sequence, comprising:
I, preparing an initial product, comprising:
Al, for a single-stranded DNA target, complementing an amplification primer P1 and a displacement primer with the single-stranded target, and displacing an amplification product of the amplification primer P1 with the displacement primer while extending the amplification primer P1 along the single-stranded target under the action of a DNA polymerase, wherein the displaced product formed by extending the amplification primer P1 is used as a single-stranded template; or
A2, for a single-stranded RNA target, acquiring a single-stranded template through two ways of reaction:
(1) for a DNA polymerase having polymerase functionality, strand displacement functionality, and reverse transcription functionality, contacting an amplification primer P1, a displacement primer and the DNA polymerase with the single-stranded RNA, wherein the single-stranded RNA is reversely transcribed into a cDNA under the reverse transcription activity of the DNA polymerase, and the cDNA is displaced by the displacement primer to give a single-stranded template; or
(2) for a DNA polymerase with no reverse transcription functionality, adding a reverse transcriptase with RNase H activity, and contacting an amplification primer P1 and the reverse transcriptase with the single-stranded RNA, wherein the single-stranded RNA is reversely transcribed into a cDNA under the action of the reverse transcriptase to give a cDNA-RNA double-stranded complex product, and the RNA strand in the double-stranded complex product is hydrolyzed under the action of the RNase H activity of the reverse transcriptase to give a single-stranded template; and
B, complementing an amplification primer P2 with the single-stranded template formed in step A, extending the amplification primer P2 along the single-stranded template under the action of a DNA polymerase, digesting the extension product with a nickase, and extending and displacing at the nick to give a double-stranded initial product with 1 digestion site at each of the two ends; and
II, acquiring an exponential amplification signal, comprising:
C, contacting a nickase and a DNA polymerase with a double-stranded template, wherein a double-stranded nick is generated on the double-stranded template under the action of the '0 nickase, and the DNA polymerase amplifies the strand from the nick and displaces the strand to give a single strand complementary to the amplification primer P1 or P2;
D, complementing the amplification primer P1 or P2 with the single strand formed in step C, and extending the amplification primer under the action of a DNA polymerase to give two double-stranded products each having 1 digestion site;
E, contacting a nickase and a DNA polymerase with the two double-stranded products generated in step D, wherein nicks are generated on the double-stranded products under the action of the nickase, and the DNA polymerase amplifies the strands from the nicks and displaces the strands to give two single strand respectively complementary to the amplification primer P1 or P2; the single strands then contact with the amplification primer P1 or P2, and are extended under the action of the DNA polymerase to give double-stranded products; and
F, repeating step E to exponentially produce an amplification product;
wherein the above steps are conducted under an isothermal condition without denaturing the target sequence prior to the amplification;
steps C to F also comprise complementing the amplification system and a molecular beacon probe to give a fluorescent signal;
the amplification primers P1 and P2 sequentially comprise a stabilizing region, a nickase recognition site region and a base region complementary to the target sequence in the 5'-3' direction; wherein the stabilizing region has a length of 6-20 bp;
the displacement primer is completely complementary to the target sequence;
the molecular beacon probe is complementary to the target sequence or can be hybridized with the target sequence, and the molecular beacon probe does not overlap with the binding regions !0 of the amplification primers P1 and P2 on the target sequence;
when for a single-stranded DNA target, the single-stranded target may be a single-stranded DNA and a single-stranded product obtained from a double-stranded DNA by contacting a nickase and a DNA polymerase with the double-stranded DNA, wherein a double-stranded nick is generated under the action of the nickase, and the DNA polymerase amplifies the strand from the nick and displaces the strand;
the DNA polymerase has strand displacement functionality;
the method is for non-diagnostic purposes.
Preferably, the base positions complementary to the target sequence on the amplification primers P1 and P2 are modified, and the mode of modification includes locked nucleic acid modification and methylation modification; the distance between the 3' terminal bases of the amplification primers P1 and P2 on the target sequence is not less than 10 bp.
The molecular beacon may comprise conventional synthetic modifications similar to the primers described above.
Preferably, the molecular beacon has a length of 13-80 bp, and the binding position of the molecular beacon and the target sequence is a position close to the 5' end and not less than 12 bp to the 3' end.
Preferably, the amplification primer has a length of 17-40 bp; the displacement primer has a length of 10-30 bp and a GC% content of 20-80%; the probe has a length of 20-40 bp and a GC% content of 10-80%.
The method provided herein is a closed-tube real-time fluorescence detection. After the nucleic acid sample is loaded, the tube will not be open during the process, thus eliminating the possibility of product contamination due to sample exposure.
Preferably, the single-stranded target has a length of 30-100 bases;
the amplification is conducted at 37 °C to 70 °C;
the overall reaction time is 1-10 min; preferably, the reaction time of the method is not greater than 8 min. Positive and negative results are obtained within 8 min, and the positive result in a sample with a high concentration of the positive target sequence can be obtained within 1-2 min.
Preferably, the nickase is selected from at least one of Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsrDI, Nb.BsmI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nb.BtsI, and Nt.CviPII.
Preferably, the DNA polymerase is selected from one of Bst DNA polymerase, Bsu DNA polymerase, and phi29 DNA polymerase.
Preferably, the DNA polymerase is Bst 2.0 or Bst 3.0.
Preferably, one end of the molecular beacon probe is a fluorescent group, and the other end of the molecular beacon probe is a fluorescence-quenching group; the 5' end and the 3' end of the probe are partially complementary in sequence, and can form a stem-loop structure.
Preferably, the amplification reaction system comprises a Tris HCl buffer, BSA, NaCl, KCl, dNTP, Mg 2 , (NH4)2SO4, and an additive.
Preferably, the additive comprises at least one of trehalose, betaine, dimethyl sulfoxide, gelatin,
Tween 20, Triton-x100, and NP-40.
In a second aspect, the present invention provides a kit for implementing the method, comprising at least the amplification primers P1 and P2, the displacement primer, the molecular beacon probe, and the amplification reaction system in the method.
Compared with the prior art, the method for isothermally amplifying a target nucleic acid sequence has the following beneficial effects:
1. The present invention provides a novel method for rapid isothermal amplification and detection of nucleic acid. The method is suitable for double-stranded DNA, single-stranded DNA, and single-stranded RNA, comprising a combined reaction of a nickase and a strand-displacing enzyme. The method adopts 3 primers and 1 probe for double-stranded DNA or single-stranded DNA assay, or 3 primers and 1 probe or 2 primers and 1 probe for single-stranded RNA assay. The probe is a molecular beacon, and will not be degraded in the amplification process. It is only used for specifically binding to a target fragment and provides a fluorescent signal, thereby ensuring the specificity of the reaction.
2. The whole reaction process is implemented under an isothermal condition, and does not require a denaturation of the target sequence before amplification, thus featuring simpler operation compared with the temperature-variable nucleic acid amplification and detection techniques.
3. According to the present invention, recognition site nucleic acid sequences for nickase !0 digestion are introduced into both upstream and downstream amplification primers, such that both 5' and 3' ends of the generated double-stranded initial product are provided with one nickase digestion recognition site, which can effectively improve the reaction efficiency in the subsequent exponential amplification stage and allows a faster completion of the reaction. In addition, the present invention adopts locked nucleic acid-modified primers, such that the efficiency and stability of the binding of the primer and the template are better than those of conventional primers. Also, an additive for promoting the reaction efficiency and an upgraded DNA polymerase with strand displacement activity are added in the reaction system to further improve the reaction efficiency of the reaction system, such that the reaction time is shortened and the reaction can be completed within 8 min. Compared with conventional isothermal amplification reactions that require more than 30-60 min, the method provided herein is more compliant with the POCT detection requirement.
The present invention adopts a beacon probe that does not overlap with the binding region between primers in the target sequence and has strong specificity for binding the target sequence to determine the result in real time, which avoids false positive results due to the use of staining or electrochemistry. The tube will not be opened after the reaction, thus further avoiding false positive results due to product contamination.
FIG. 1 is a schematic of the detection of a double-stranded DNA according to a preferred embodiment of the present invention.
FIG. 2 is a schematic of the initial template formation in detecting a single-stranded DNA and single-stranded RNA according to a preferred embodiment of the present invention.
FIG. 3 is a schematic of the initial template formation in detecting a single-stranded RNA according to a preferred embodiment of the present invention.
FIG. 4 is a diagram illustrating the amplification effect in detecting a plasmid carrying human gene PSMB2 according to a preferred embodiment of the present invention.
FIG. 5 is a diagram illustrating the amplification effect in detecting a Mycoplasma pneumoniae sample according to a preferred embodiment of the present invention.
FIG. 6 is a diagram illustrating the amplification effect in detecting influenza B virus according to a preferred embodiment of the present invention.
FIG. 7 is a diagram illustrating the amplification effect in detecting canine parvovirus according to a preferred embodiment of the present invention.
FIG. 8 is a schematic of the self strand displacement amplification in a sample according to a preferred embodiment of the present invention.
FIG. 9 is a diagram illustrating the amplification effect of the self strand displacement amplification reaction in a sample according to a preferred embodiment of the present invention.
To help understand the present invention, a more detailed description is provided with reference to the accompanying drawings, which illustrate several examples of the present invention. However, the present invention can be implemented in various forms, and is not limited to the examples described herein. On the contrary, the examples are provided to completely illustrate the disclosure of the present invention.
The present invention provides a novel method for the rapid isothermal amplification and detection of double-stranded DNA, single-stranded DNA, and single-stranded RNA. The method comprises: an initial product generation stage (double-stranded initial product formation stage) and an exponential amplification signal acquisition stage.
1. In the initial product generation stage, there are slight differences depending on the template situation and the enzyme system used:
a) When the template is a double-stranded DNA, the nickase acts on the nickase digestion sites on the double-stranded DNA template to form nicks. The strand-displacing enzyme (DNA polymerase with strand displacement functionality) initiates the extension and strand displacement from the nicks to form single-stranded products. A primer with a single-stranded digestion site (F/R, or the amplification primer P1) and the displacement primer (B) bind to the single-stranded products to form single-stranded products with the digestion sites through extension and strand displacement. The other primer with a single-stranded digestion site (R/F, or the amplification primer P2) binds to the single-stranded products and is subjected to extension, digestion and strand displacement to form an initial product, i.e., a double-stranded initial product with 2 digestion sites at the two ends (FIG. 1).
b) When the template is a single-stranded DNA, the primer with a single-stranded digestion site (F/R) and the displacement primer (B) bind to the single-stranded product, and the initial product is formed by the same procedure as described above (FIG. 2).
!0 c) When the template is a single-stranded RNA, there are 2 different ways to give the initial template. In the first method, when a reverse transcriptase with RNase H activity is used, a single strand with a digestion site is formed by reverse transcription and the action of RNase H without the use of displacement primer (B) as shown in FIG. 3, and the subsequent reaction is the same as that described above; in the second method, a reverse transcriptase (e.g., Bst 3.0) is used to generate a single strand with a digestion site by reverse transcription and the strand displacement functionality, and the process is similar to that of the single-stranded DNA template.
2. In the exponential amplification signal acquisition stage, the nickase cleaves the initial product to form two double-stranded DNAs with a digestion site on one side, as shown in the "exponential amplification" region of FIG. 4. The first product can generate a single-stranded product under the action of the nickase and the amplification enzyme, and the product further binds to the amplification primer and extends to form the second product; vice versa, the second product may also produce the first product. The two together lead to the exponential amplification. The molecular beacon probe can bind to one of the single-stranded products, and the amplification signal can be acquired by a suitable fluorescence detection system.
When detecting a double-stranded DNA, a single-stranded DNA or single-stranded RNA, the method adopts 2 amplification primers, 1 displacement primer and 1 molecular beacon probe; when a single-stranded RNA is detected, the method may also adopt 2 amplification primers and 1 molecular beacon probe.
The molecular beacon has a length of 13-80 bp, and the binding position of the molecular beacon and the target sequence is a position close to the 5' end and not less than 12 bp to the 3' end.
The amplification enzyme used in the present invention has the functionality of synthesizing DNA using a DNA template and strand displacement functionality, and some amplification enzymes also have the functionality of reversely transcribing an RNA template into DNA.
The length of the specific region (excluding the sequences such as digestion sites introduced by primer amplification) in the initial product is 30-100 bp.
The molecular signaling probe binds to the single-stranded product without overlapping the binding region of the amplification primer on the single-stranded product. The distance between the 3' terminal bases of the amplification primers P1 and P2 on the target sequence is not less than 10 bp. In designing the primers and probes, sufficient specific sites should be ensured for binding of the probe to the target sequence without base overlap between the probe and the amplification primer on the target sequence.
The temperature is constant in the reaction process, and the reaction can be completed within 8 min.
The present invention adopts 3 primers and 1 beacon probe (2 primers and 1 probe can be used for detecting a single-stranded RNA), nickase, and strand-displacing DNA polymerase, and the nucleic acid amplification and the real-time fluorescence detection of the product can be completed within 8 min.
The method features isothermal amplification. The temperature is kept constant in the reaction at 37-70 °C.
The reaction time of the method is not greater than 8 min. Positive and negative results are obtained within 8 min, and the positive result in a sample with a high concentration of the positive target sequence can be obtained within 1-2 min. The method is a closed-tube real-time fluorescence detection. After the nucleic acid sample is loaded, the tube will not be open during the process.
The primer is a single-stranded nucleotide polymer, and if necessary, the primer may comprise conventional synthetic modifications such as locked nucleic acid (LNA), methylation and the like. Of the 3 primers, 1 is a strand-displacing primer, and 2 are amplification primers. The strand-displacing primer is completely complementary to the template, and the amplification primer comprises 3 regions: a specific binding region, a digestion site region, and a stabilizing region.
The beacon probe is a single-stranded nucleotide polymer modified by a fluorescent group and a quenching group, and the 5' end and the 3' end of the artificial sequence are complementary to form a stem-loop structure. If necessary, the molecular beacon may comprise conventional synthetic modifications similar to the primers described above, and possibly, spacer modifications at the 5' and 3' ends to increase its length. The beacon probe and the primer have no overlaps on the target sequence, thus ensuring the specificity.
The nickase is a special enzyme for recognizing a specific sequence of a double-stranded DNA and forming a nick on the double-stranded DNA, such as Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsrDI, Nb.BsmI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nb.BtsI, Nt.CviPII, or other enzymes with the same functionality.
The strand-displacing DNA polymerase is a polymerase having nucleic acid 3' terminal !0 polymerization activity and functionality of displacing nucleic acid in the polymerization direction. Such as Bst DNA polymerase (including Bst 2.0, Bst 3.0, and other upgraded products), large Bst DNA polymerase fragments, Bsu DNA polymerase, large Bsu DNA polymerase fragments, phi29 DNA polymerase, etc.
In addition to the above primers, probes and enzymes, the method also comprises various substances used in common nucleic acid amplification reactions, such as Tris HCl buffer, BSA, NaCl, KCl, dNTP, Mg2 , (NH4)2SO4and other buffers and ionic components commonly used in reactions, and also comprises additives, such as trehalose, betaine, dimethyl sulfoxide, gelatin, Tween 20, Triton-x100, NP-40 and the like.
The following examples are intended to illustrate the present invention, rather than limiting the scope of the present invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001, or follow the conditions recommended by the manufacturer's instructions.
Example 1. Comparison of detections of plasmids carrying human gene PSMB2
The experimental group (the present invention) using upgraded DNA polymerases, primers introduced with locked nucleic acid labels and a reaction system added with a reaction enhancer, and the control groups using a low-grade version of DNA polymerase, primers without locked nucleic acid labels and a reaction system without a reaction enhancer were compared for the reaction time.
The amplification reaction system composition, additives and the modification of the primer used in the experimental group and the control groups are shown in Table 1:
Reaction The present Control Control Control Control system invention group 1 group 2 group 3 group 4 Tris-HCl Tris-HCl Tris-HCl Tris-HCl Tris-HCl pH8.0, pH8.0, pH8.0, pH8.0, pH8.0, 50mM 50mM 50mM 50mM 50mM
(NH4)2 (NH4)2 (NH4)2 (NH4)2 (NH4)2 Amplificatio S04,20mM S04,20mM S04,20mM S04,20mM S04,20mM reaction MgCl2, MgCl2, MgCl2, MgCl2, MgCl2, system 10mM 10mM 10mM 10mM 10mM NaCl, 30mM NaCl, 30mM NaCl, 30mM NaCl, 30mM NaCl, 30mM KCl, 10mM KCl, 10mM KCl, 10mM KCl, 10mM KCl, 10mM dNTP, 1mM dNTP, 1mM dNTP, 1mM dNTP, 1mM dNTP, 1mM Betaine, 0.5 Betaine, 0.5 Betaine, 0.5 Additive None None M M M PSMB2-B, PSMB2-B, PSMB2-B, PSMB2-B, PSMB2-B, 200mM 200mM 200mM 200mM 200mM PSMB2-F, PSMB2-F, PSMB2-F, PSMB2-F, PSMB2-F, Primer and 300mM 300mM 300mM 300mM 300mM probe PSMB2-R, PSMB2-R, PSMB2-R, PSMB2-R, PSMB2-R, 300mM 300mM 300mM 300mM 300mM PSMB2-P, PSMB2-P, PSMB2-P, PSMB2-P, PSMB2-P, 300mM 300mM 300mM 300mM 300mM
Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nickase 3U 3U 3U 3U 3U Bst 2.0 Bst 2.0 Bst 1.0 Bst 1.0 Bst 1.0 DNA polymerase warmstart, warmstart, warmstart, warmstart, warmstart, 4.8U 4.8U 4.8U 4.8U 4.8U Whether the Yes No Yes No No primer has a modification
Table 1
(1) Primer and probe sequences of the present invention:
The primer and probe sequences (5'-3') are as follows:
PSMB2-B (primer): CCCAGCACTTT
PSMB2-F (primer): TTCAGACTATTGAGTCTATTCTGACCAACAT
PSMB2-R (primer): GTCAGACTATTGAGTCTTCTCCCAGCTAAT
PSMB2-P (probe): ATGGTAGTAGAGACGGGGTTTTACCAT
Note: "A" is modified with LNA.
(2) Unmodified primers and probe are as follows:
The primer and probe sequences (5'-3') are as follows:
PSMB2-B (primer): CCCAGCACTTT
PSMB2-F (primer): TTCAGACTATTGAGTCTATTCTGACCAACAT
PSMB2-R (primer): GTCAGACTATTGAGTCTTCTCCCAGCTAAT
PSMB2-P (probe): ATGGTAGTAGAGACGGGGTTTTACCAT
The reactions in all groups were conducted at 55 °C and signals were acquired every 10 s with the instrument LightCycler 48011. Plasmid samples 1E5, 1E4, 1E3, 1E2, and 1E1 were tested. The results are shown in Table 1, and the test result profile of the present invention is shown in FIG. 4. Table 2. Comparison results of detections of plasmids carrying human gene PSMB2
Group The Control Control Control Control present group 1 group 2 group 3 group 4 invention Detection time 2.5-4min 5-9min 5.5-9min 7-10min 9-12min Table 2
Therefore, the detection time of the present invention is significantly shorter than those of the control groups, suggesting that the present invention has a time advantage in the application of double-stranded DNA nucleic acid detection.
Example 2. Comparison of Mycoplasma pneumoniae detection in clinical samples
The experimental group (the present invention) using upgraded DNA polymerases, primers introduced with locked nucleic acid labels and a reaction system added with a reaction enhancer, and the control groups using a low-grade version of DNA polymerase, primers without locked nucleic acid labels and a reaction system without a reaction enhancer were compared for the reaction time.
The amplification reaction system composition, additives and the modification of the primer used in the experimental group and the control groups are shown in Table 3:
Reaction The present Control Control Control Control system invention group 1 group 2 group 3 group 4 Tris-HCl Tris-HCl Tris-HCl Tris-HCl Tris-HCl pH8.0, pH8.0, pH8.0, pH8.0, pH8.0, 50mM 50mM 50mM 50mM 50mM
(NH4)2 (NH4)2 (NH4)2 (NH4)2 (NH4)2 Amplification S04,20mM S04,20mM S04,20mM S04,20mM S04,20mM reaction MgCl2, MgCl2, MgCl2, MgCl2, MgCl2, system 8mM 8mM 8mM 8mM 8mM NaCl, 30mM NaCl, 30mM NaCl, 30mM NaCl, 30mM NaCl, 30mM KCl, 10mM KCl, 10mM KCl, 10mM KCl, 10mM KCl, 10mM dNTP, 1mM dNTP, 1mM dNTP, 1mM dNTP, 1mM dNTP, 1mM Betaine, 0.5 Betaine, 0.5 Betaine, 0.5 Additive None None M M M Mp-B, Mp-B, Mp-B, Mp-B, Mp-B, 200mM 200mM 200mM 200mM 200mM Mp-F, Mp-F, Mp-F, Mp-F, Mp-F, Primer and 400mM 400mM 400mM 400mM 400mM probe Mp-R, Mp-R, Mp-R, Mp-R, Mp-R, 400mM 400mM 400mM 400mM 400mM Mp-P, Mp-P, Mp-P, Mp-P, Mp-P, 300mM 300mM 300mM 300mM 300mM
Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nickase 3U 3U 3U 3U 3U Bst 2.0 Bst 2.0 Bst 1.0 Bst 1.0 Bst 1.0 DNA polymerase warmstart, warmstart, warmstart, warmstart, warmstart, 4.8U 4.8U 4.8U 4.8U 4.8U Whether the primer has a Yes No Yes No No modification?
Table 3
(1) Primer and probe sequences of the present invention:
The primer and probe sequences (5'-3') are as follows:
Mp-B (primer): CTCTCCACTAA
Mp-F (primer): CATAGACTTATGAGTCTTCTATTCGCTTC
Mp-R (primer): GTTAGACTTTTGAGTCTTCTTGCTCTGGT
Mp-P (probe): CGCAGCTGGTTACGGGAATACTGCG
Note: "A" is modified with LNA.
(2) Unlabeled primer and probe sequences are as follows:
The primer and probe sequences (5'-3') are as follows:
Mp-B (primer): CTCTCCACTAA
Mp-F (primer): CATAGACTTATGAGTCTTCTATTCGCTTC
Mp-R (primer): GTTAGACTTTTGAGTCTTCTTGCTCTGGT
Mp-P (probe): CGCAGCTGGTTACGGGAATACTGCG
The reactions in all groups were conducted at 55 °C and signals were acquired every 10 s with the instrument LightCycler 48011. 8 Mycoplasma pneumoniae samples were tested, as well as 8 other respiratory pathogens: influenza A virus, influenza B virus, Chlamydia pneumoniae, respiratory syncytial virus, human parvovirus B19, Staphylococcus aureus, human respiratory adenovirus, and rhinovirus. The results are shown in Table 4, and the test result profile of the present invention is shown in FIG. 5. The following table shows the comparison results of detection with Mycoplasma pneumoniae.
Group The Control Control Control Control present group 1 group 2 group 3 group 4 invention Detection time 3-7min 10-15min 14-19min 15-19min 18-22min Non-specific 0/8 2/8 0/8 1/8 2/8 results Table 4
Therefore, the present invention has significant advantages in time and specificity for double-stranded DNA nucleic acid detection.
Example 3. Comparison of influenza B virus (single-stranded RNA virus) detection in clinical samples
The experimental group (the present invention) using upgraded DNA polymerases, primers introduced with locked nucleic acid labels and a reaction system added with a reaction enhancer, and the control groups using a low-grade version of DNA polymerase, primers without locked nucleic acid labels and a reaction system without a reaction enhancer were compared for the reaction time.
The amplification reaction system composition, additives and the modification of the primer used in the experimental group and the control groups are shown in Table 5:
Reaction The present Control Control Control Control system invention group 1 group 2 group 3 group 4 Tris-HCl Tris-HCl Tris-HCl Tris-HCl Tris-HCl pH8.0, pH8.0, pH8.0, pH8.0, pH8.0, 50mM 50mM 50mM 50mM 50mM
(NH4)2 (NH4)2 (NH4)2 (NH4)2 (NH4)2 Amplification S04,20mM S04,20mM S04,20mM S04,20mM S04,20mM reaction MgCl2, MgCl2, MgCl2, MgCl2, MgCl2, system 8mM 8mM 8mM 8mM 8mM NaCl, 30mM NaCl, 30mM NaCl, 30mM NaCl, 30mM NaCl, 30mM KCl, 10mM KCl, 10mM KCl, 10mM KCl, 10mM KCl, 10mM dNTP, 1mM dNTP, 1mM dNTP, 1mM dNTP, 1mM dNTP, 1mM Betaine, 0.5 Betaine, 0.5 Betaine, 0.5 Additive None None M M M FluB-B, FluB-B, FluB-B, FluB-B, FluB-B, 200mM 200mM 200mM 200mM 200mM FluB -F, FluB -F, FluB -F, FluB -F, FluB -F, Primer and 400mM 400mM 400mM 400mM 400mM probe FluB -R, FluB -R, FluB -R, FluB -R, FluB -R, 400mM 400mM 400mM 400mM 400mM FluB -P, FluB -P, FluB -P, FluB -P, FluB -P, 300mM 300mM 300mM 300mM 300mM
Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nickase 3U 3U 3U 3U 3U DNA Bst 3.0, 6U Bst 3.0, 6U Bst 1.0, 6U Bst 1.0, 6U Bst 1.0, 6U polymerase
Whether the primer has a Yes No Yes No No modification? Table 5
(1) Primer and probe sequences of the present invention:
The primer and probe sequences (5'-3') are as follows:
FluB-B (primer): TGTTGCTAAACT
FluB-F (primer): CTACTGATGAGTCTTTTAGTGGAGGAT
FluB-R (primer): CCTTCATTGAGTCTTTTGAAGAGTGA
FluB-P (probe): ACGGCCATCGGATCCTCAAGCCGT
Note: "A" is modified with LNA.
(1) Unlabeled primer and probe sequences are as follows:
The primer and probe sequences (5'-3') are as follows:
FluB-B (primer): TGTTGCTAAACT
FluB-F (primer): CTACTGATGAGTCTTTTAGTGGAGGAT
FluB-R (primer): CCTTCATTGAGTCTTTTGAAGAGTGA
FluB-P (probe): ACGGCCATCGGATCCTCAAGCCGT
The reactions in all groups were conducted at 55 °C and signals were acquired every 10 s with the instrument LightCycler 48011. 8 influenza B virus samples were tested to verify the specificity of the reaction system, as well as 8 other respiratory pathogens: influenza A virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, human parvovirus B19, Staphylococcus aureus, human respiratory adenovirus, and rhinovirus. The results are shown in Table 6, and the test result profile of the present invention is shown in FIG. 6.
Therefore, the present invention has significant advantages in time and specificity for single-stranded RNA nucleic acid detection. Table 6 shows the comparison results of influenza B virus detection.
Group The Control Control Control Control present group 1 group 2 group 3 group 4 invention Detection time 3-5min 7-10min 9-13min 11-15min 20-24min Non-specific 0/8 1/8 0/8 1/8 2/8 results Table 6
Example 4. Comparison of canine parvovirus detection
The experimental group (the present invention) using upgraded DNA polymerases, primers introduced with locked nucleic acid labels and a reaction system added with a reaction enhancer, and the control groups using a low-grade version of DNA polymerase, primers without locked nucleic acid labels and a reaction system without a reaction enhancer were compared for the reaction time.
The amplification reaction system composition, additives and the modification of the primer used in the experimental group and the control groups are shown in Table 7:
Reaction The present Control Control Control Control system invention group 1 group 2 group 3 group 4 Tris-HCl Tris-HCl Tris-HCl Tris-HCl Tris-HCl pH8.0, pH8.0, pH8.0, pH8.0, pH8.0, 50mM 50mM 50mM 50mM 50mM (NH4)2 (NH4)2 (NH4)2 (NH4)2 (NH4)2 Amplification S04,20mM S04,20mM S04,20mM S04,20mM S04,20mM reaction MgCl2, MgCl2, MgCl2, MgCl2, MgCl2, system 8mM 8mM 8mM 8mM 8mM NaCl, NaCl, NaCl, NaCl, NaCl, 30mM 30mM 30mM 30mM 30mM KCl,10mM KCl,10mM KCl,10mM KCl,10mM KCl,10mM dNTP,1mM dNTP,1mM dNTP,1mM dNTP,1mM dNTP,1mM Betaine, 0.5 Betaine, 0.5 Betaine, 0.5 Additive None None M M M CVP-B, CVP-B, CVP-B, CVP-B, CVP-B, 200mM 200mM 200mM 200mM 200mM
CVP-F, CVP-F, CVP-F, CVP-F, CVP-F, Primer and 500mM 500mM 500mM 500mM 500mM probe CVP-R, CVP-R, CVP-R, CVP-R, CVP-R, 500mM 500mM 500mM 500mM 500mM CVP-P, CVP-P, CVP-P, CVP-P, CVP-P,
300mM 300mM 300mM 300mM 300mM
Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nt.BstNBI, Nickase 3U 3U 3U 3U 3U DNA Bst 3.0, 6U Bst 3.0, 6U Bst 1.0, 6U Bst 1.0, 6U Bst 1.0, 6U polymerase Whether the primer has a Yes No Yes No No modification?
Table 7
(1) Primer and probe sequences of the present invention:
CVP-F (primer): GAACTTTTGAGTCTTTTACTATACACATC
CVP-R (primer): GAACTTTTGAGTCTTTTCCCAGTTTTCAT
CVP-B (primer): AGTCTTTGCAACCT
CVP-P (probe): CGCCAGGAAAAGTACCAGAATGGCG
Note: "A" is modified with LNA.
(2) Unlabeled primer and probe sequences are as follows:
CVP-F (primer): GAACTTTTGAGTCTTTTACTATACACATC
CVP-R (primer): GAACTTTTGAGTCTTTTCCCAGTTTTCAT
CVP-B (primer): AGTCTTTGCAACCT
CVP-P (probe): CGCCAGGAAAAGTACCAGAATGGCG
The reactions in all groups were conducted at 55 °C and signals were acquired every 10 s with the instrument LightCycler 48011. 5 canine parvovirus samples were tested, as well as 8 other respiratory pathogens: influenza A virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, human parvovirus B19, Staphylococcus aureus, human respiratory adenovirus, and rhinovirus. The results are shown in Table 4, and the test result profile of the present invention is shown in FIG. 7.
Therefore, the present invention has significant advantages in time and specificity for single-stranded DNA nucleic acid detection. The following Table 8 shows the comparison results of canine parvovirus detection.
Group The present Control Control Control Control invention group 1 group 2 group 3 group 4 Detection time 3.5-5min 8-12min 9-15min 13-17min 15-22min Non-specific 0/8 2/8 0/8 2/8 3/8 results Table 8
Example 5. Self strand-displacing amplification of samples
When the sample is amplified using a strand-displacing enzyme and a nickase, the sample itself may undergo strand-displacing amplification due to the massive digestion sites present in the sample. The process is similar to the multiple displacement amplification, and the principle is shown in FIG. 8, with the exception that the use of primers and probes is not required. An exemplary reaction is as follows:
The following reaction system was prepared:
Tris-HCl pH 8.0, 50 mM
o (NH4)2SO4,20 mM
MgCl2, 8 mM
NaCl, 30 mM
KCl, 10 mM
dNTP, 1 mM
Evagreen 1x
Nt.BstNBI, 3 U
Bst 3.0, 6 U
The reaction was conducted at 55 °C and signals were acquired every 1 min with the instrument LightCycler 48011 in a total of 60 cycles. The samples were stock nucleic acid sample solutions extracted via throat swabs, and 10- and 100-fold diluted samples of the stock solutions in duplicate. The results are shown in FIG. 9. The amplification signals in the nucleic acid sample extracted via throat swabs were observed at about 12 min. When the strand-displacing enzyme and nickase are used in amplification, self amplification is inevitable. According to the staining method disclosed in CN104726549A, the false positive results cannot be avoided either after 30-60 min of reaction. The above examples are only specific and detailed descriptions of certain embodiments of the present invention, and should not be construed as limitations to the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the present invention, and these changes and modifications are all within the protection scope of the present invention. Therefore, the protection scope of the present patent should be determined with reference to the appended claims.
Claims (11)
1. A method for isothermally amplifying a target nucleic acid sequence, comprising:
I, preparing an initial product, comprising:
Al, for a single-stranded DNA target, complementing an amplification primer P1 and a displacement primer with the single-stranded target, and displacing an amplification product of the amplification primer P1 with the displacement primer while extending the amplification primer P1 along the single-stranded target under the action of a DNA polymerase, wherein the displaced product formed by extending the amplification primer P1 is used as a single-stranded template; or
A2, for a single-stranded RNA target, acquiring a single-stranded template through two ways of reaction:
(1) for a DNA polymerase having polymerase functionality, strand displacement functionality, and reverse transcription functionality, contacting an amplification primer P1, a displacement primer and the DNA polymerase with the single-stranded RNA, wherein the single-stranded RNA is reversely transcribed into a cDNA under the reverse transcription activity of the DNA polymerase, and the cDNA is displaced by the displacement primer to give a single-stranded template; or
(2) for a DNA polymerase with no reverse transcription functionality, adding a reverse transcriptase with RNase H activity, and contacting an amplification primer P1 and the reverse transcriptase with the single-stranded RNA, wherein the single-stranded RNA is reversely transcribed into a cDNA under the action of the reverse transcriptase to give a cDNA-RNA double-stranded complex product, and the RNA strand in the double-stranded complex product is hydrolyzed under the action of the RNase H activity of the reverse transcriptase to give a single-stranded template; and
B, complementing an amplification primer P2 with the single-stranded template formed in step A, extending the amplification primer P2 along the single-stranded template under the action of a DNA polymerase, digesting the extension product with a nickase, and extending and displacing at the nick to give a double-stranded initial product with 1 digestion site at each of the two ends; and
II, acquiring an exponential amplification signal, comprising:
C, contacting a nickase and a DNA polymerase with a double-stranded template, wherein a double-stranded nick is generated on the double-stranded template under the action of the nickase, and the DNA polymerase amplifies the strand from the nick and displaces the strand to give a single strand complementary to the amplification primer P1 or P2;
D, complementing the amplification primer P1 or P2 with the single strand formed in step C, and extending the amplification primer under the action of a DNA polymerase to give two double-stranded products each having 1 digestion site;
E, contacting a nickase and a DNA polymerase with the two double-stranded products generated in step D, wherein nicks are generated on the double-stranded products under the action of the nickase, and the DNA polymerase amplifies the strands from the nicks and displaces the strands to give two single strand respectively complementary to the amplification primer P1 or P2; the single strands then contact with the amplification primer P1 or P2, and are extended under the action of the DNA polymerase to give double-stranded products; and
F, repeating step E to exponentially produce an amplification product;
wherein the above steps are conducted under an isothermal condition without denaturing the target sequence prior to the amplification;
steps C to F also comprise complementing the amplification system and a molecular beacon probe to give a fluorescent signal;
the amplification primers P1 and P2 sequentially comprise a stabilizing region, a nickase recognition site region and a base region complementary to the target sequence in the 5'-3' direction; wherein the stabilizing region has a length of 6-20 bp;
the displacement primer is completely complementary to the target sequence;
the molecular beacon probe is complementary to the target sequence or can be hybridized with the target sequence, and the molecular beacon probe does not overlap with the binding regions of the amplification primers P1 and P2 on the target sequence;
when for a single-stranded DNA target, the single-stranded target may be a single-stranded DNA and a single-stranded product obtained from a double-stranded DNA by contacting a nickase and a DNA polymerase with the double-stranded DNA, wherein a double-stranded nick is generated under the action of the nickase, and the DNA polymerase amplifies the strand from the nick and displaces the strand;
the DNA polymerase has strand displacement functionality;
the method is for non-diagnostic purposes.
2. The method according to claim 1, wherein:
the base positions complementary to the target sequence on the amplification primers P1 and P2 are modified, and the mode of modification includes locked nucleic acid modification and methylation modification; the distance between the 3' terminal bases of the amplification primers P1 and P2 on the target sequence is not less than 10 bp.
3. The method according to claim 1, wherein: the molecular beacon has a length of 13-80 bp, and the binding position of the molecular beacon and the target sequence is a position close to the 5' end and not less than 12 bp to the 3' end.
4. The method according to claim 1, wherein:
the single-stranded target has a length of 30-100 bases;
the amplification is conducted at 37 °C to 70 °C;
the overall reaction time is 1-10 min.
5. The method according to claim 1, wherein: the nickase is selected from at least one of Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsrDI, Nb.BsmI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nb.BtsI, and Nt.CviPII.
6. The method according to claim 1, wherein: the DNA polymerase is selected from one of Bst DNA polymerase, Bsu DNA polymerase, and phi29 DNA polymerase.
7. The method according to claim 1, wherein: the DNA polymerase is Bst 2.0 or Bst 3.0.
8. The method according to claim 1, wherein: one end of the molecular beacon probe is a fluorescent group, and the other end of the molecular beacon probe is a fluorescence-quenching group; the 5' end and the 3' end of the probe are partially complementary in sequence, and can form a stem-loop structure.
9. The method according to claim 1, wherein: the amplification reaction system comprises a Tris HCl buffer, BSA, NaCl, KCl, dNTP, Mg 2+, (NH4)2SO4, and an additive.
10. The method according to claim 1, wherein: the additive comprises at least one of trehalose, betaine, dimethyl sulfoxide, gelatin, Tween 20, Triton-x100, and NP-40.
11. The method according to claim 1, wherein: the kit comprises the amplification primers P1 and P2, the displacement primer, the molecular beacon probe, and the amplification reaction system in the method according to any one of claims 1-10.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110733555.7A CN113481283A (en) | 2021-06-30 | 2021-06-30 | Method for isothermal amplification of nucleic acid target sequences |
| CN202110733555.7 | 2021-06-30 | ||
| PCT/CN2022/102545 WO2023274330A1 (en) | 2021-06-30 | 2022-06-29 | Method for isothermal amplification of nucleic acid target sequences |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2022301095A1 true AU2022301095A1 (en) | 2024-01-18 |
Family
ID=77937009
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2022301095A Pending AU2022301095A1 (en) | 2021-06-30 | 2022-06-29 | Method for isothermal amplification of nucleic acid target sequences |
Country Status (4)
| Country | Link |
|---|---|
| CN (2) | CN113481283A (en) |
| AU (1) | AU2022301095A1 (en) |
| BR (1) | BR112023027256A2 (en) |
| WO (1) | WO2023274330A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113481283A (en) * | 2021-06-30 | 2021-10-08 | 上海伯杰医疗科技有限公司北京分公司 | Method for isothermal amplification of nucleic acid target sequences |
| CN113981047B (en) * | 2021-11-08 | 2023-11-07 | 中国科学院合肥物质科学研究院 | Reverse transcription-strand displacement amplification method for miRNA detection and application thereof |
| CN114350756A (en) * | 2021-11-22 | 2022-04-15 | 西安交通大学 | Whole genome self-priming amplification method and kit based on DNA nicking/polymeric strand displacement cycle reaction |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10590474B2 (en) * | 2013-03-11 | 2020-03-17 | Elitechgroup B.V. | Methods for true isothermal strand displacement amplification |
| CN104726549B (en) * | 2014-10-10 | 2020-01-21 | 青岛耐德生物技术有限公司 | Novel nicking enzyme-based double-stranded nucleic acid isothermal amplification detection method |
| CN108642144B (en) * | 2018-05-18 | 2020-06-09 | 贠红岩 | Constant temperature strand displacement amplification technology and kit |
| CN111850100B (en) * | 2020-07-21 | 2022-12-13 | 北京艾克伦医疗科技有限公司 | Nucleic acid amplification method and application thereof |
| CN113481283A (en) * | 2021-06-30 | 2021-10-08 | 上海伯杰医疗科技有限公司北京分公司 | Method for isothermal amplification of nucleic acid target sequences |
-
2021
- 2021-06-30 CN CN202110733555.7A patent/CN113481283A/en active Pending
-
2022
- 2022-06-29 CN CN202210751117.8A patent/CN115074419A/en active Pending
- 2022-06-29 AU AU2022301095A patent/AU2022301095A1/en active Pending
- 2022-06-29 WO PCT/CN2022/102545 patent/WO2023274330A1/en active Application Filing
- 2022-06-29 BR BR112023027256A patent/BR112023027256A2/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN113481283A (en) | 2021-10-08 |
| BR112023027256A2 (en) | 2024-03-12 |
| WO2023274330A1 (en) | 2023-01-05 |
| CN115074419A (en) | 2022-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2018201671B2 (en) | Compositions and methods for quantifying a nucleic acid sequence in a sample | |
| US9487807B2 (en) | Compositions and methods for producing single-stranded circular DNA | |
| JP6542771B2 (en) | Nucleic acid probe and genomic fragment detection method | |
| AU2022301095A1 (en) | Method for isothermal amplification of nucleic acid target sequences | |
| Chen et al. | Recombinase assisted loop-mediated isothermal DNA amplification | |
| JP6126381B2 (en) | Target nucleic acid detection method and kit | |
| CA2810856C (en) | Compositions and methods for quantifying a nucleic acid sequence in a sample | |
| Wang et al. | A ligation/recombinase polymerase amplification assay for rapid detection of SARS-CoV− 2 | |
| CN113308519B (en) | Primer and probe for detecting single base mutation site and detection method | |
| US10920285B2 (en) | Highly specific and sensitive methods for detecting enterohemorrhagic Escherichia coli serotypes O157:H7 and/or O145:H28 | |
| Li et al. | One-pot, ultrasensitive, and multiplex detection of SARS-CoV-2 genes utilizing self-priming hairpin-mediated isothermal amplification | |
| JP2023518217A (en) | Loop primer and loop de loop method for detecting target nucleic acid | |
| JP2020178681A (en) | Nucleic acid detection method, and reagent kit | |
| CN110577985A (en) | Method for detecting DNA methylation | |
| CN110577986A (en) | DNA methylation detection probe | |
| US20220081712A1 (en) | Isothermal amplification | |
| CN110603328A (en) | Quantitative PCR amplification primer pair and application thereof | |
| Nair et al. | Mitigating the non-specific rolling circle amplification: a comprehensive assessment of the role of ligation and digestion methods | |
| WO2024017763A1 (en) | Method for nucleic acid detection using signal-mediated amplification of rna technology and rna aptamers | |
| WO2023092178A1 (en) | Improved isothermal amplification | |
| CN114921529A (en) | Method for detecting single base mutation of gene by utilizing Tth Ago enzyme shearing and application thereof |