WO2023274330A1 - Procédé d'amplification isotherme de séquences cibles d'acides nucléiques - Google Patents

Procédé d'amplification isotherme de séquences cibles d'acides nucléiques Download PDF

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WO2023274330A1
WO2023274330A1 PCT/CN2022/102545 CN2022102545W WO2023274330A1 WO 2023274330 A1 WO2023274330 A1 WO 2023274330A1 CN 2022102545 W CN2022102545 W CN 2022102545W WO 2023274330 A1 WO2023274330 A1 WO 2023274330A1
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stranded
amplification
primer
target sequence
dna polymerase
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PCT/CN2022/102545
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Chinese (zh)
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李春燕
朱兆奎
昃白尘
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上海伯杰医疗科技股份有限公司北京分公司
上海伯杰医疗科技股份有限公司
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Priority to AU2022301095A priority Critical patent/AU2022301095A1/en
Priority to BR112023027256A priority patent/BR112023027256A2/pt
Publication of WO2023274330A1 publication Critical patent/WO2023274330A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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

Definitions

  • the invention belongs to the technical field of nucleic acid detection, in particular to a method for isothermally amplifying a nucleic acid target sequence.
  • Polymerase chain reaction (Polymerase chain reaction, PCR) is currently the most widely used nucleic acid amplification detection technology (Nucleic Acid Amplification Test, NAAT).
  • NAAT Nucleic Acid Amplification Test
  • the classic reaction of this technology includes three steps of denaturation, renaturation, and extension. It is a process that requires rapid temperature cycling. It requires a specific thermal cycler for high-precision temperature control and consumes a lot of power. At the same time, the reaction time is long, which cannot meet the requirements of point-of-care detection (POCT).
  • POCT point-of-care detection
  • RPA recombinase polymerase amplification
  • LAMP loop-mediated isothermal amplification
  • SDA strand displacement amplification
  • NEAR nick and extension amplification
  • TMA transcription amplification. mediation technology
  • RPA technology relies on three enzymes: recombinases that bind single-stranded nucleic acids, single-stranded DNA-binding proteins, and strand-displacing DNA polymerases.
  • the complementary sequence of single-stranded nucleic acid is recognized by recombinase, and they are combined, and the binding region is stabilized by single-strand binding protein, and the strand displaces DNA polymerase for extension.
  • the reaction is generally carried out at 37-42°C for 15-30 minutes, and special probes can be added to judge the result.
  • RPA involves many components, and the cost of reagents is too high.
  • LAMP uses strand displacement enzymes to complete the reaction.
  • SDA uses specially modified nucleotides, endonucleases, and strand-displacing DNA polymerases, and requires 4 primers.
  • the reaction time is generally 30-60min.
  • NEAR is similar to SDA in that it uses nickase and strand displacement and requires only 2 primers.
  • the distance between the two primers (3' ends) is 1-5 bases, through the effect of primer invasion, a product with nickase sites at both ends is formed, and the product is in the nickase and strand displacement DNA polymerase Under the effect of exponential amplification. Products can be analyzed by probes and dyes. Many products of this technology have been launched on the market. In the detection of new coronavirus nucleic acid, there have been reports of low sensitivity. Because the distance between the primers is too short, when the probe is used for real-time detection, false positives are prone to occur due to the homologous position between the primer and the probe. The reaction time is about 12 minutes.
  • TMA Transcription amplification-mediated technology
  • CN104726549A discloses a new method for detection of double-strand isothermal amplification based on nickase.
  • the method uses three primers, one of which can be designed as a beacon probe, and the product is obtained by dye method, fluorescence method, electrochemical method, etc. method, colorimetric method, and chemical reflection method for analysis, and the detection time is 30-60min.
  • Using methods other than the fluorescent method can easily lead to false positives, and the patent labels the primers so that the reaction cannot be carried out correctly. At the same time, the non-specific reaction of the labeled primers will bring false positive results. Due to factors such as too long reaction time and unreasonable product analysis, there is currently no product on the market.
  • the purpose of the invention is to solve the existing technical problems of long detection time and poor specificity.
  • the present invention provides a method for isothermally amplifying a nucleic acid target sequence, comprising the steps of:
  • initial product formation comprises the following steps:
  • the amplification primer P1 and the displacement primer are complementary combined with the single-stranded target, and the amplification primer P1 is extended along the single-stranded target under the action of DNA polymerase replacing the amplification product of the amplification primer P1 with the replacement primer; using the product formed by the extension of the replaced amplification primer P1 as a single-stranded template;
  • the single-stranded template can be obtained by reacting in two ways:
  • the amplification primer P1, displacement primer and DNA polymerase are contacted with single-stranded RNA, and the single-stranded RNA is Reverse transcribe into cDNA under the action of enzymatic reverse transcription activity, and be replaced by a displacement primer to obtain a single-stranded template;
  • the DNA polymerase does not have the reverse transcription function, it is necessary to add a reverse transcriptase with RNase H activity, and contact the amplification primer P1 and the reverse transcriptase with the single-stranded RNA, and the single-stranded RNA Under the action of reverse transcriptase, it is reverse-transcribed into cDNA to form a cDNA-RNA composite double-stranded product, and the RNA strand in the composite double-stranded product is hydrolyzed by the RNase H activity of reverse transcriptase to obtain a single-stranded template.
  • the amplification primer P2 is complementary to the single-stranded template formed in step A, and the amplification primer P2 is extended along the single-stranded template under the action of DNA polymerase, and then the nicking enzyme acts on the extension product to extend at the nick and replacement to form a double-stranded initial product with one enzyme cleavage site at each end;
  • the exponential amplification signal collection comprises the following steps:
  • step D Complementary binding of amplification primer P1 or P2 to the single strand formed in step C, and extension under the action of DNA polymerase to form two double strand products each with one restriction site;
  • step E Contacting the nicking enzyme and the DNA polymerase with the two double-stranded products produced in step D, the two double-stranded products form nicks respectively under the action of the nicking enzyme, and the DNA polymerase amplifies from the nicking site and Replacement to obtain two single strands that can be complementary to the amplification primer P1 or P2 respectively; the single strand is then contacted with the amplification primer P1 or P2, and extended under the action of DNA polymerase to form a double-stranded product;
  • step E Repeat step E to obtain the amplification product exponentially;
  • Steps C to F also include complementary binding of the amplification system to molecular beacon probes to provide fluorescent signals;
  • the displacement primer is fully complementary to the target sequence
  • the molecular beacon probe is complementary to the target sequence or hybridizable to 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;
  • the single-stranded target when the single-stranded target is single-stranded DNA, the single-stranded target can be single-stranded DNA and the double-stranded DNA is contacted with the double-stranded DNA through a nickase and a DNA polymerase, and a nick is generated under the action of the nickase, and the DNA polymerizes
  • the enzyme amplifies and displaces the resulting single-stranded product from the nick;
  • the DNA polymerase has a strand displacement function
  • the methods are for non-disease diagnosis purposes.
  • the positions of the base regions complementary to the target sequence on the amplification primers P1 and P2 are modified, and the modification methods include 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.
  • Molecular beacons may contain conventional synthetic modifications similar to the primers described above.
  • the length of the molecular beacon is 13-80bp, and the binding position of the molecular beacon to the target sequence is not less than 12bp near the 5' end and 3'.
  • the length of the amplification primer is between 17-40bp
  • the replacement primer is between 10-30bp
  • the GC% content is between 20-80%
  • the probe length is between 20-40bp
  • the GC% content is 10% Between -80%.
  • the method for isothermally amplifying nucleic acid target sequences provided by the present invention is closed-tube real-time fluorescence detection. After the sample nucleic acid is loaded, the reaction is carried out on the machine, and there is no tube opening process in the middle, which avoids the possibility of product contamination caused by opening the cap.
  • the single-stranded target is 30-100 bases in length;
  • the amplification is implemented between 37°C-70°C;
  • the entire reaction time is 1-10 minutes.
  • the reaction time of the method is no more than 8 minutes. Positive and negative results can be obtained within 8 minutes. When there is a high concentration of positive target sequences in the sample, a positive result can be obtained within 1-2 minutes.
  • the nickase is selected from Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsrDI, Nb.BsmI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nb.BtsI, Nt.CviPII at least one.
  • the DNA polymerase is selected from one of Bst DNA polymerase, Bsu DNA polymerase, phi29 DNA polymerase.
  • the DNA polymerase is Bst 2.0 or Bst3.0.
  • one end of the molecular beacon probe is a fluorescent group
  • the other end is a fluorescent quenching group
  • the 5' end and 3' end of the probe are partially complementary in sequence, forming a stem-loop structure.
  • the amplification reaction system includes Tris HCl buffer, BSA, NaCl, KCl, dNTP, Mg2+, (NH4)2SO4 and additives.
  • the additive includes at least one of trehalose, betaine, dimethyl sulfoxide, gelatin, Tween 20, Triton-x100, and NP-40.
  • the present invention provides a kit for realizing the method, which at least includes the amplification primers P1 and P2, displacement primers, molecular beacon probes and amplification reaction system in the above-mentioned method.
  • the method for isothermally amplifying a nucleic acid target sequence provided by the invention has the following beneficial effects:
  • the novel method for rapid isothermal amplification and nucleic acid detection provided by the present invention.
  • This method is applicable to double-stranded DNA, single-stranded DNA, and single-stranded RNA, including the joint reaction of nickase and strand displacement enzyme.
  • three primers and one probe are used, while When performing single-stranded RNA detection, it can be 3 primers and 1 probe, or 2 primers and 1 probe.
  • the probe is a molecular beacon, which does not degrade during the amplification process and is only used to specifically bind the target fragment to provide a fluorescent signal and ensure the specificity of the reaction.
  • the entire reaction process of the present invention is carried out under isothermal conditions, and there is no need to denature the target sequence before amplification, which is easier to operate than variable temperature nucleic acid amplification detection technology.
  • Both the upstream and downstream amplification primers of the present invention introduce the nucleic acid sequence of the nicking enzyme recognition site, and the 5' and 3' ends of the double-stranded initial product that can be obtained have a nicking enzyme cleavage recognition site, which can effectively improve
  • the reaction efficiency of the subsequent exponential amplification stage can complete the reaction in a shorter time; and the present invention uses locked nucleic acid to modify the primer, so that the efficiency and stability of primer and template binding are better than conventional primers; at the same time, the reaction system increases the reaction efficiency
  • the additives and the upgraded version of DNA polymerase with strand displacement activity further improve the reaction efficiency of the reaction system, making the reaction time shorter, and the reaction is completed within 8 minutes, while the general isothermal amplification reaction takes more than 30-60 minutes.
  • the present invention is more in line with the POCT detection requirement.
  • the present invention uses a beacon probe that does not overlap with the primer binding region on the target sequence to judge the result in real time.
  • the beacon probe has strong specificity in binding to the target sequence, and avoids the use of dye method or electrochemical method False positives caused by other protocols; at the same time, the tube is not opened after the reaction to further avoid false positives caused by product contamination.
  • Fig. 1 is a schematic diagram of detecting double-stranded DNA in a preferred embodiment of the present invention.
  • Fig. 2 is a schematic diagram of initial template formation when detecting single-stranded DNA and single-stranded RNA in a preferred embodiment of the present invention.
  • Fig. 3 is a schematic diagram of initial template formation when detecting single-stranded RNA in a preferred embodiment of the present invention.
  • Fig. 4 is a diagram showing the amplification effect of a plasmid carrying human gene PSMB2 in a preferred embodiment of the present invention.
  • Fig. 5 is a diagram showing the effect of amplification of samples for detecting Mycoplasma pneumoniae in a preferred embodiment of the present invention.
  • Fig. 6 is a diagram showing the amplification effect of detecting influenza B samples in a preferred embodiment of the present invention.
  • Fig. 7 is a diagram showing the amplification effect of detecting parvovirus in a preferred embodiment of the present invention.
  • Fig. 8 is a schematic diagram of the strand displacement amplification of the sample itself in a preferred embodiment of the present invention.
  • Fig. 9 is a graph showing the amplification effect of the sample self-strand displacement amplification reaction in a preferred embodiment of the present invention.
  • the invention provided by the invention provides a new method for rapid isothermal amplification and detection of double-stranded DNA, single-stranded DNA and single-stranded RNA. Including the following steps:
  • the reaction includes an initial product generation stage (double-stranded initial product formation stage) and an exponential amplification signal acquisition stage.
  • the nickase acts on the nickase cut site on the double-stranded DNA template to form a nick
  • the strand displacement enzyme DNA polymerase with strand displacement function
  • the primer with a single-strand enzyme cleavage site F/R, that is, the amplification primer P1
  • the displacement primer B
  • an enzyme-containing The single-stranded product of the cleavage site, another primer (R/F, amplification primer P2) with a single-stranded restriction site binds to and extends the single-stranded product, and then undergoes restriction digestion and strand displacement to form an initial
  • the product is a double-stranded initial product with two restriction sites at both ends ( Figure 1).
  • the primer (F/R) and the displacement primer (B) with a single-stranded restriction site bind to the single-stranded product, and the initial product is formed through the same process as above ( Figure 2) .
  • the template is single-stranded RNA
  • the primer (B) when using a reverse transcriptase with RNase H activity, the primer (B) does not need to be replaced, as shown in Figure 3, through reverse transcription and RNase H, a single strand with an enzyme cleavage site is formed, and the subsequent The reaction is the same as above; the second one uses reverse transcriptase (such as Bst 3.0) to generate a single strand with a restriction site through reverse transcription and strand displacement, and the process is similar to a single-stranded DNA template.
  • reverse transcriptase such as Bst 3.0
  • the nicking enzyme creates nicks on the initial product to form two double-stranded DNAs with enzyme cleavage sites on one side, as shown in the "exponential amplification" area of Figure 4, the first product Under the action of nickase and amplification enzyme, a single-stranded product can be generated, and the product can be further combined with the amplification primer and extended to form the second product; conversely, the second product can also generate the first product, and the two form exponential expansion.
  • Molecular beacon probes can be combined with one of the single-stranded products, and a suitable fluorescent detection system can collect the amplification signal.
  • This method uses 2 amplification primers, 1 displacement primer and 1 molecular beacon probe when detecting double-stranded DNA, single-stranded DNA and single-stranded RNA; it can also be 2 amplification primers when detecting single-stranded RNA Primers and 1 molecular beacon probe.
  • the length of the molecular beacon is 13-80bp, and the binding position of the molecular beacon and the target sequence is not less than 12bp near the 5' end and 3'.
  • the amplification enzyme used in the present invention has the function of synthesizing DNA with DNA as a template, and at the same time has the function of strand displacement, and some types of amplification enzymes also have the function of reverse transcription into DNA with RNA as a template.
  • the length of the specific region of the initial product (not counting sequences such as restriction sites introduced by primer amplification) is between 30-100 bp.
  • the molecular signal probe When the molecular signal probe binds to the single-stranded product, it does not overlap with 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.
  • the temperature is constant during the reaction, and the reaction can be completed within 8 minutes.
  • the present invention uses 3 primers and 1 beacon probe (when detecting single-stranded RNA, it can be 2 primers and 1 probe), nickase and strand displacement DNA polymerase, and can complete nucleic acid amplification within 8 minutes and real-time fluorescence detection of the product.
  • the method is isothermal amplification, the temperature in the reaction is constant, and the reaction temperature is between 37-70°C.
  • the reaction time of the method does not exceed 8 minutes, positive and negative results are obtained within 8 minutes, and positive results can be obtained within 1-2 minutes when there is a high concentration of positive target sequences in the sample.
  • the method is closed-tube real-time fluorescence detection. After adding the sample nucleic acid, the reaction is carried out on the machine, and there is no process of opening the tube in the middle.
  • the primer is a single-stranded nucleotide polymer, and if necessary, the primer may contain conventional synthetic modifications such as locked nucleic acid (LNA) and methylation.
  • LNA locked nucleic acid
  • 1 is a strand displacement primer
  • 2 are amplification primers.
  • the strand displacement primer is completely complementary to the template, and the amplification primer contains 3 regions, which are the specific binding region, the enzyme cleavage site region and the stable region. .
  • the beacon probe refers to a single-stranded nucleotide polymer modified with a fluorescent group and a quencher group, and artificial sequences at the 5' and 3' ends are complementary to form a stem-loop structure. If necessary, routine synthetic modifications similar to those described above for the primers may be included, as well as spacer modifications at the 5' and 3' ends to increase their length. Beacon probes and primers have no overlapping parts on the target sequence, ensuring their specificity.
  • the nickase is a kind of special enzyme that recognizes the specific sequence of double-stranded DNA and forms a nick thereon, 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 having the same function.
  • the strand-displacing DNA polymerase is a kind of polymerase that has the activity of polymerizing at the 3' terminal of nucleic acid and also has the function of displacing nucleic acid in the direction of polymerization.
  • Bst DNA polymerase including Bst 2.0, Bst3.0 and other upgraded products
  • Bst DNA polymerase large fragment Bsu DNA polymerase
  • Bsu DNA polymerase large fragment phi29 DNA polymerase, etc.
  • the method also includes various substances used in common nucleic acid amplification reactions, such as Tris HCl buffer, BSA, NaCl, KCl, dNTP, Mg2+, (NH4)2SO4 and other reactions Commonly used buffers and ionic components, as well as additives such as trehalose, betaine, dimethyl sulfoxide, gelatin, Tween 20, Triton-x100, NP-40, etc.
  • Example 1 The detection comparison of the plasmid carrying the human gene PSMB2
  • Experimental group using upgraded DNA polymerase, primers to introduce locked nucleic acid markers, reaction system adding reaction enhancer and control group: using low-level version of DNA polymerase, primers not locked nucleic acid markers, reaction system without adding reaction Enhancers were compared for reaction times.
  • composition of the amplification reaction system of the present invention and the control group, additives and the modified situation of the primers used are as follows in Table 1:
  • the primer probe sequence (5'-3') is as follows:
  • PSMB2-B (primer): CCCAGCACTTT
  • PSMB2-F (primer): TTCAGACTATTGAGTCTATTCTGACCA A CAT
  • PSMB2-R (primer): GTCAGACTATTGAGTCTTCTCCCAGCT A AT
  • PSMB2-P (probe): ATGGTAGTAGAGACGGGGTTTTTACCAT
  • the primer probe sequence (5'-3') is as follows:
  • PSMB2-B (primer): CCCAGCACTTT
  • PSMB2-F (primer): TTCAGACTATTGAGTCTATTCTGACCAACAT
  • PSMB2-R (primer): GTCAGACTATTGAGTCTTCTCCCAGCTAAT
  • PSMB2-P (probe): ATGGTAGTAGAGACGGGGTTTTTACCAT
  • Control group 1 Control group 2
  • Control group 3 Control group 4 detection time 2.5-4min 5-9min 5.5-9min 7-10min 9-12min
  • the detection time of the present invention is significantly earlier than that of the control groups, indicating that the present invention has a better time advantage in the application of double-stranded DNA nucleic acid detection.
  • Experimental group using upgraded DNA polymerase, primers for nucleic acid-locking labeling, reaction system adding reaction enhancer and control group: using low-level version of DNA polymerase, primers for nucleic acid-locking labeling, no reaction added to the reaction system Enhancers were compared for reaction times.
  • composition of the amplification reaction system of the present invention and the control group, the additives and the modified conditions of the primers used are as follows in Table 3:
  • the primer probe sequence (5'-3') is as follows:
  • Mp-P (probe): CGCAGCTGGTTACGGGAATACTGCG
  • the primer probe sequence (5'-3') is as follows:
  • Mp-P (probe): CGCAGCTGGTTACGGGAATACTGCG
  • the instrument was LightCycler480II. Detected 8 lung branch samples, and 8 other respiratory pathogens: influenza A virus, influenza B virus, Chlamydia pneumoniae, respiratory syncytial virus, human parvovirus B19, Staphylococcus aureus, human respiratory adenovirus, rhinovirus, The results of each group are shown in Table 4, and the graph of the detection results of the present invention is shown in Figure 5. The following table shows the comparison results of the detection of Mycoplasma pneumoniae.
  • Control group 1 Control group 2
  • Control group 3 Control group 4 detection time 3-7min 10-15min 14-19min 15-19min 18-22min Detection of non-specific 0/8 2/8 0/8 1/8 2/8
  • the present invention has obvious advantages in the detection time and detection specificity of double-stranded DNA nucleic acid.
  • Example 3 Influenza B virus (single-stranded RNA virus) clinical sample detection comparison
  • Experimental group using upgraded DNA polymerase, primers to introduce locked nucleic acid markers, adding reaction enhancer to the reaction system and control group: using a low-level version of DNA polymerase, primers for locked nucleic acid markers, no reaction added to the reaction system Enhancers were compared for reaction times.
  • composition of the amplification reaction system of the present invention and the control group, the additives and the modification of the primers used are as follows in Table 5:
  • the primer probe sequence (5'-3') is as follows:
  • the primer probe sequence (5'-3') is as follows:
  • the instrument was LightCycler 480II. Detected 8 clinical samples of influenza B virus, and 8 other respiratory pathogens: influenza A virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, human parvovirus B19, Staphylococcus aureus, human respiratory adenovirus, rhinovirus To verify the specificity of the reaction system, the results of each group are shown in Table 3, and the graph of the detection results of the present invention is shown in Figure 6.
  • Table 6 is the detection and comparison results of influenza B virus.
  • Control group 1 Control group 2
  • Control group 3 Control group 4 detection time 3-5min 7-10min 9-13min 11-15min 20-24min Detection of non-specific 0/8 1/8 0/8 1/8 2/8
  • Experimental group using upgraded DNA polymerase, primers to introduce locked nucleic acid markers, adding reaction enhancer to the reaction system and control group: using a low-level version of DNA polymerase, primers for locked nucleic acid markers, no reaction added to the reaction system Enhancers were compared for reaction times.
  • composition of the amplification reaction system of the present invention and the control group, the additives and the modified conditions of the primers used are as follows in Table 7:
  • CVP-F (primer): GAACTTTTGAGTCTTTTACTATAC A CATC
  • CVP-R (primer): GAACTTTTGAGTCTTTTTCCCAGTTTTC A T
  • CVP-P (probe): CGCCAGGAAAAGTACCAGAATGGCG
  • CVP-F (primer): GAACTTTTGAGTCTTTTACTATAC A CATC
  • CVP-R (primer): GAACTTTTGAGTCTTTTTCCCAGTTTTC A T
  • CVP-P (probe): CGCCAGGAAAAGTACCAGAATGGCG
  • the present invention has obvious advantages in detection time and detection specificity of single-stranded DNA nucleic acid.
  • the following table 8 is the detection and comparison results of canine parvovirus.
  • Control group 1 Control group 2
  • Control group 3 Control group 4 detection time 3.5-5min 8-12min 9-15min 13-17min 15-22min Detection of non-specific 0/8 2/8 0/8 2/8 3/8
  • the reaction was carried out at 55°C, and the signal was collected every 1 min for a total of 60 cycles.
  • the instrument was LightCycler 480II.
  • the samples were nucleic acid stock solution extracted from throat swabs, 10 times and 100 dilutions of the stock solution, and each was repeated twice.
  • the results are shown in Figure 9, the nucleic acid sample extracted from the throat swab showed an amplification signal in about 12 minutes.
  • strand displacement enzyme and nickase are used for amplification, self-amplification of the sample is inevitable.
  • CN104726549A uses the dye method to judge the result, when the reaction is carried out for 30-60 minutes, the occurrence of this false positive phenomenon cannot be avoided.

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Abstract

La présente invention concerne un procédé d'amplification isotherme de séquences cibles d'acides nucléiques. Le procédé convient pour l'ADN double brin, l'ADN simple brin et l'ARN simple brin, et comprend une réaction combinée de nickase et d'enzyme de déplacement de brin. Dans la détection d'ADN double brin et d'ADN simple brin, trois amorces et une sonde sont utilisées, et dans la détection d'ARN simple brin, trois amorces et une sonde peuvent être utilisées, ou deux amorces et une sonde peuvent être utilisées. La sonde est une balise moléculaire ne se dégradant pas au cours d'un processus d'amplification et uniquement utilisée pour se lier particulièrement à un fragment cible afin de fournir un signal fluorescent pour assurer la spécificité de la réaction. Dans la présente invention, la sonde balise ne chevauchant pas les amorces dans une région de liaison sur une séquence cible est utilisée pour déterminer le résultat en temps réel. La sonde balise présente une haute spécificité de liaison à la séquence cible et les tubes ne doivent pas être ouverts après la réaction pour éviter la génération de faux positifs. La réaction s'effectue à une température constante et requiert peu de temps, et la détection peut être achevée en 8 minutes, ce qui correspond davantage aux exigences de la détection POCT.
PCT/CN2022/102545 2021-06-30 2022-06-29 Procédé d'amplification isotherme de séquences cibles d'acides nucléiques WO2023274330A1 (fr)

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AU2022301095A AU2022301095A1 (en) 2021-06-30 2022-06-29 Method for isothermal amplification of nucleic acid target sequences
BR112023027256A BR112023027256A2 (pt) 2021-06-30 2022-06-29 Método para amplificar isotermicamente uma sequência alvo de ácidos nucleicos

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