WO2014036972A1 - Procédé d'amplification d'acide nucléique - Google Patents

Procédé d'amplification d'acide nucléique Download PDF

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WO2014036972A1
WO2014036972A1 PCT/CN2013/083182 CN2013083182W WO2014036972A1 WO 2014036972 A1 WO2014036972 A1 WO 2014036972A1 CN 2013083182 W CN2013083182 W CN 2013083182W WO 2014036972 A1 WO2014036972 A1 WO 2014036972A1
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dna
restriction
enzyme
amplification
primer
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PCT/CN2013/083182
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Chinese (zh)
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周裕程
杨文秀
万强
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思洛生物技术股份有限公司
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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
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a nucleic acid amplification method.
  • PCR Polymerase Chain Reaction
  • PCR technology has long occupied a monopoly position in nucleic acid amplification technology
  • a series of isothermal nucleic acid amplification technologies developed in the future are gradually becoming an alternative to PCR technology and are widely used.
  • These amplification techniques employ different principles and methods to achieve amplification of nucleic acids (DNA or RNA) under a specific temperature condition (eg, 37 ° C).
  • NASBA Nucleic acid sequence based amplification
  • TMA transcription-mediated amplification
  • SDA Rolling Circle Amplification
  • HAD Helicase Dependant Amplification
  • SPIA Single Primer Isothermal Amplification
  • NEMA Incision Nicking endonuclease mediated amplification
  • NASBA nucleic acid sequence amplification technology
  • TMA transcription-mediated amplification technology
  • NEMA Incisionnicking endonuclease mediated amplification
  • RNA-dependent DNA polymerase activity DNA-dependent DNA polymerase activity
  • RNase H activity RNase H activity
  • RNA polymerase activity Some of these activities can be combined in one enzyme Medium, so usually only 2 or 3 enzymes are required.
  • the difference between the two methods is that the reverse transcriptase is different.
  • the NASBA method uses avian myoma virus (AMV) reverse transcriptase
  • the TMA method uses Moloney leukemia virus (M-MLV) reverse transcriptase.
  • AMV avian myoma virus
  • M-MLV Moloney leukemia virus
  • the target sequence is reverse transcribed under the action of reverse transcriptase, and the RNase H activity of the reverse transcriptase degrades the RNA on the hybrid chain to synthesize the double-stranded T7 promoter sequence.
  • the amplification product can be detected by some methods (such as adding a molecular beacon probe) to quantify or characterize the target nucleic acid. In fact, starting with the single-stranded RNA in the sample, once all the ingredients are put together and the mixture is placed at the appropriate temperature to allow the enzyme to be active, a whole series of events will occur without human intervention.
  • TMA or NASBA technology amplification methods are particularly effective for the amplification of single-stranded RNA.
  • a TMA or NASBA amplification method is applied to a target nucleic acid which is present only in the form of double-stranded DNA (cyclic or linear), it is necessary to convert the DNA into a single-stranded nucleic acid to synthesize RNA.
  • a method is described in EP397269, in which double-stranded DNA is pretreated with a restriction enzyme and then subjected to a heating separation step to form a single-stranded DNA.
  • the first primer P1 promoter primer
  • the 3 ⁇ portion contains a sequence which is exactly complementary to a 3, end of the DNA double strand, and the 5 ⁇ end contains a polymerase (eg, ⁇ 7 )
  • the identified promoter sequence eg, ⁇ 7
  • the 5 ⁇ promoter sequence of the first primer can serve as a template for the extension reaction from the 3 ⁇ end of the DNA strand.
  • a double-stranded promoter can be formed by a DNA-dependent DNA polymerase, and the resulting complex can be used as a template for a DNA-dependent RNA polymerase to synthesize multiple copies of RNA.
  • Subsequent reaction progress is identical to the normal NAS or NASBA method.
  • the reaction process is shown in Figure 1.
  • the restriction oligonucleotide (RP) or restriction primer contains a sequence complementary to the region including the restriction site of the target DNA, which is added together with the restriction enzyme, as shown in Fig. 2.
  • restriction oligonucleotide can be incorporated into an oligonucleotide containing a promoter sequence that can be recognized by a DNA-dependent RNA polymerase.
  • bioMerieux Using this method to solve the HBV isothermal amplification (NASBA) problem, the 95% detection rate was 242 WHO IU/mL, and the 50% detection rate was 35 WHO IU/mL.
  • the premise of the method is that it is necessary to first determine whether the sample to be tested is single-stranded or double-stranded DNA, and adopt different strategies (whether or not RP is added).
  • RP nucleotide sequence
  • restriction oligonucleotide Even if the function of the restriction oligonucleotide can be incorporated into an oligonucleotide containing a promoter sequence that can be recognized by a DNA-dependent RNA polymerase, it will still form two additional sequences after digestion. The two sequences are not necessary for the entire amplification reaction system, and the problem of increased non-specific products cannot be avoided.
  • the object of the present invention is to solve the problem of reduced amplification efficiency and increase of non-specific products caused by additionally adding RP in the prior art, and to provide a nicking enzyme-coupled transcription medium with high amplification efficiency and few non-specific products. Guided nucleic acid amplification method.
  • the present invention adopts the following technical solutions:
  • One method of nucleic acid amplification is to perform transcriptional amplification in a reaction mixture of restriction enzymes that cleave DNA at a selected restriction site.
  • the present invention utilizes restriction nicking enzymes for amplification of double-stranded DNA and/or single-stranded DNA.
  • No additional reagents such as the restriction oligonucleotide RP
  • RP restriction oligonucleotide
  • the method begins with DNA present in the sample, and specifically includes the following steps:
  • the incubation system comprising: one or more restriction nicking enzymes capable of cleaving DNA at a selected restriction site, the restriction nicking enzyme in the DNA Forming a specific 3 ⁇ end on one strand;
  • the 5 3 ⁇ 4 region of the promoter primer comprising a promoter sequence recognized by a DNA-dependent RNA polymerase and having a 3 ⁇ region that is inversely complementary to a specific 3 ⁇ terminus of the DNA strand;
  • a second or reverse primer having a polarity opposite to that of the promoter primer and comprising a 5 ⁇ terminus of the target sequence
  • reaction mixture thus formed is maintained under appropriate conditions for a sufficient period of time to effect digestion of the restriction nicking enzyme;
  • reaction mixture thus formed is maintained under appropriate conditions for a sufficient period of time for amplification.
  • the DNA in the sample is double-stranded and the DNA has one or more restriction nickase cleavage sites, the promoter primers being identifiable by DNA-dependent RNA polymerase
  • the promoter sequence and its 3 ⁇ region are inversely complementary to the DNase-specific 3 ⁇ terminus of the DNase. That is, for a double-stranded DNA template, the double-stranded DNA is first pretreated with a restriction nicking enzyme to form a nick at a specific position of one of the strands, and then the double-stranded DNA is separated from each other to form a single strand by a heating separation step, and is cleaved.
  • a chain that is engraved is broken at the nicking position to expose the 3 ⁇ end of the position.
  • a first primer P1 promoter primer
  • the 3 ⁇ portion of which contains a sequence which is exactly complementary to the exposed 3' end.
  • the 5 ⁇ terminus contains a promoter sequence that is recognized by an RNA polymerase (such as T7). Which should be the template.
  • RNA polymerase such as T7
  • a double-stranded promoter can be formed by a DNA-dependent DNA polymerase, and the resulting complex can be used as a template for a DNA-dependent RNA polymerase to synthesize multiple copies of RNA.
  • Subsequent reactions are common hydrazine or NASBA processes.
  • the reaction diagram is shown in Figure 3.
  • the DNA in the sample is single stranded and the DNA has one or more restriction nickase cleavage sites containing restriction sites comprising the target single stranded DNA A region-complementary sequence and a promoter sequence that is recognized by a DNA-dependent RNA polymerase. That is, for a single-stranded DNA template, the promoter primer first renatures with the single-stranded DNA to form a nickase cleavage site. Promoter primer used in this method, which portion contains the tangent point of the end 3 ⁇ exact complementary sequence, and the end 5 ⁇ promoter sequence may be recognized by an RNA polymerase (e.g. ⁇ 7).
  • an RNA polymerase e.g. ⁇ 7
  • the renatured product is pretreated with a restriction nicking enzyme to form a nick at a specific position of the DNA to be tested, and then the template DNA is cleaved at a cleavage site by a heating separation step to expose the 3 ⁇ end of the position.
  • the promoter primer is exactly complementary to the 3, terminus of the position of the tangent, and the 5 ⁇ promoter sequence of the primer can serve as a template for the extension reaction from the 3 ⁇ end of the DNA strand.
  • a double-stranded promoter can be formed by a DNA-dependent DNA polymerase, and the resulting complex can be used as a template for a DNA-dependent RNA polymerase to synthesize multiple copies of RNA.
  • Subsequent reactions are common hydrazine or NASBA processes.
  • the reaction diagram is shown in Figure 4.
  • the DNA in the sample is a mixture of single strands and double strands and the two or more DNAs have one or more restriction enzyme cleavage sites of the same or different, different Simultaneous detection of nucleic acid templates.
  • the restriction nicking enzyme recognizes the specific nucleic acid sequence, and cleaves one of the nucleic acid strands to form a slit;
  • the promoter primer (P1) binds to the single-stranded DNA to form a specific nucleic acid sequence recognized by a restriction nicking enzyme, and is cleaved on the template strand to form an incision;
  • the 3, part of the promoter primer (P1) is exactly complementary to the 3, end of the exposure, and the 5 ⁇ end contains a promoter sequence which is recognized by RNA polymerase, and the sequence can be used as a template for extending the reaction from the 3 ⁇ end of the DNA strand;
  • e an enzyme that relies on DNA polymerase activity to accurately recognize a double-stranded promoter sequence, and transcribes an RNA strand complementary to the DNA strand using double-stranded DNA as a template;
  • step (e) annealing the second primer P2 and the RNA transcript produced in step (e); g) extending the second primer in the reaction catalyzed by the RNA-dependent DNA polymerase activity to form the first RNA/cDNA hybrid nucleic acid molecule;
  • the activity of RNaseH selectively removes RNA of the first RNA/cDNA hybrid nucleic acid molecule to obtain a first single-stranded cDNA molecule
  • step (k) preparing a plurality of RNA transcripts identical in polarity to step e) using a first portion of the double-stranded DNA molecule of step (j) in a reaction catalyzed by a DNA-dependent RNA polymerase, said DNA-dependent RNA polymerase Having specificity for the promoter sequence in the promoter primer (P1);
  • the formation of by-products is reduced, and the detection sensitivity of the system can be improved, and the enzyme which depends on the RNA polymerase activity of the DNA at this time
  • the double-stranded promoter sequence is accurately recognized, and the RNA strand complementary to the DNA strand is transcribed from the single-stranded DNA as a template.
  • the sample may be any type of DNA, viral DNA to be detected, and genomic DNA.
  • the sample is a sample containing hepatitis B virus DNA or a sample of Treponema pallidum virus DNA.
  • the incubation temperature is between 35 and 45 °C. More preferably, it is 41 V. In some embodiments, the heating temperature is 92 to 98 °C. More preferably, it is 95 °C.
  • a restriction nicking enzyme is a specific type of endonuclease that cleaves a specific site of double-stranded DNA by discriminating a specific nucleotide sequence, which does not cleave the double strand of the DNA molecule, but only cleaves the double strand. a chain. Then, using the polymerase activity, the new strand is synthesized using the uncut single strand as a starting point and the uncut single strand as a starting point.
  • restriction nicking enzymes are all suitable for the nucleic acid amplification method of the present invention, for example, Nb.BtsI, Nt.CviPII, N.AlwI, Nb.BbvCK Nt.BbvCK N.BstNBL Nb.Bsm.
  • the restriction restriction nicking enzyme is Nb.BtsI or Nt.CviPIL.
  • the restriction region is considered in consideration of a conserved region.
  • the enzymatic enzyme is Nt.CviPII, and its restriction sites are as follows:
  • the present invention also provides a nucleic acid amplification kit comprising a promoter primer, a reverse primer, a molecular beacon probe, and a restriction nicking enzyme.
  • the kit may further include other components of nucleic acid amplification, such as a reaction buffer component, a TMA enzyme component, and the like.
  • the reaction buffer components include Tris-HCl, MgCl 2 , KC1, DMSO, DTT, various dNTPs and NTP (ie, dNTPs include dATP, dTTP, dGTP, and dCTP, and NTP includes ATP, UTP, GTP, and CTP)
  • the TMA enzyme component includes Trehalose, Bovine Serum Albumin (BSA), M-MLV And T 7 RNA polymerase.
  • the kit when the sample is containing hepatitis B virus DNA, the kit includes a promoter primer, a reverse primer, a molecular beacon probe, and a restriction nicking enzyme, wherein the promoter primer Having the nucleotide sequence shown in SEQ ID No: 1, the reverse primer has the nucleotide sequence shown in SEQ ID No: 2, and the molecular beacon probe has the nucleotide sequence shown in SEQ ID No: 3. , the restriction nicking enzyme is Nt. CviPIL specific sequence is shown in Table 1.
  • a kit for amplifying hepatitis B virus DNA comprises 50 mM various kinds of pH 8.5 Tris-HCK 8.66 mM MgCl 2 , 70 mM KC1, 11 v/v % DMSO, 3.3 mM DTT, 1.25 mM. dNTP and NTP, ⁇ . ⁇ promoter primer F201111-1 having the nucleotide sequence shown in SEQ ID No: 1, ⁇ . ⁇ reverse primer R708 having a nucleotide sequence of SEQ ID No: 2, 0.05 ⁇ Molecular beacon probe MB713 having a nucleotide sequence of SEQ ID No: 3, 1U restriction nicking enzyme Nt. CviPIL, 0.07M Trehalose, 22.5 g BSA, 400 UM-MLV and 100 U T7 RNA polymerase.
  • the kit when the sample is containing Treponema pallidum DNA, the kit includes a promoter primer, a reverse primer, a molecular beacon probe, and a restriction nicking enzyme, wherein the promoter primer has SEQ a nucleotide sequence represented by ID No: 4, wherein the reverse primer has the nucleotide sequence shown in SEQ ID No: 5, and the molecular beacon probe has the SEQ ID No: 6 nucleotide sequence, the restriction nicking enzyme is Nb. BtsL specific sequence is shown in the table
  • a kit for amplifying Treponema pallidum DNA comprises
  • the promoter primer ends are modified with an amino group.
  • the 13th position of the 5 ⁇ end of the reverse primer is a locked nucleobase.
  • the nicking enzyme ⁇ chain transcription-mediated nucleic acid amplification of the present invention does not require the addition of any additional reagents (such as a restriction oligonucleotide) in addition to the addition of a restriction nicking enzyme.
  • RP restriction oligonucleotide
  • the methods and kits of the present invention can be used with any type of DNA, viral DNA to be detected in a sample, and genomic DNA, particularly for amplification and detection of DNA from hepatitis B virus (HBV).
  • the methods and kits of the present invention can be used not only for multiplex detection of DNA mixed samples, ie, containing multiple DNA templates, single strands and/or double strands; but also for multiplex detection of mixed DNA and RNA samples, such as HBV. - HCV-HIV triple screening. Combined with other related technologies, it can be widely used in molecular diagnostics, scientific research, epidemic prevention testing, forensic identification and other fields.
  • Figure 1 is a schematic diagram showing the mechanism of double-stranded DNA-NASBA reaction
  • Figure 2 is a schematic diagram showing the mechanism of single-strand DNA-NASBA reaction
  • Figure 3 is a schematic diagram showing the mechanism of double-strand DNA-NETAS reaction of the nicking enzyme ⁇ chain transcription-mediated nucleic acid amplification method of the present invention
  • Figure 4 is a schematic diagram showing the single-strand DNA-NETAS reaction mechanism of the nicking enzyme ⁇ chain transcription-mediated nucleic acid amplification method of the present invention
  • Figure 5 shows an amplification curve of amplifying (control) of different concentrations of nucleic acid using restriction endonucleases
  • Figure 6 shows an amplification curve of amplifying the different concentrations of nucleic acid using a restriction nicking enzyme (this protocol group);
  • Figure 7 is a graph showing the results of polyacrylamide gel electrophoresis of the amplified product of Example 1, wherein the absolute input of nucleic acids 1-8 (control group) is 50000 IU, 5000 IU, 500 IU, 50 IU, 5 IU, 2.55 IU, 1.25, respectively.
  • lanes 9-16 are absolute input of nucleic acid of 50000 IU, 5000 IU, 500 IU, 50 IU, 5 IU, 2.55 IU, 1.25 IU, negative control;
  • lane M is RNA marker, top to bottom 1000 nt, 800 nt, 600 nt, 400 nt, 300 nt, 200 nt, lOOnt;
  • the target band A is the control product of the control group;
  • the target band B is the target product of the amplification of the program group;
  • Figure 8 is a graph showing the results of 10% polyacrylamide gel electrophoresis of Nt.CviPII digesting activity of HBV DNA amplified in Example 2, wherein lane 1 is an incubation product without addition of Nt.CviPII; lane 2 is added. Incubation product of Nt.CviPII; lane D is single-stranded DNA marker;
  • Figure 9 is a graph showing the amplification detection of the natural HBV virus nucleic acid by the method of Nt. CviPII without the addition of a restriction nicking enzyme;
  • Fig. 10 is a graph showing the amplification detection of the natural HBV virus nucleic acid by the restriction nicking enzyme Nt.CviPII;
  • Figure 11 is a graph showing the results of 10% polyacrylamide gel electrophoresis of the amplified Nb.BtsI digestion activity of TP DNA of Example 3, wherein the D lane is a single-stranded DNA marker; the lane 1 is a non-added Nb.BtsI The product was incubated; lane 2 was the incubation product with the addition of Nb.BtsI;
  • Figure 12 is a graph showing the detection and amplification of the treponema virulence nucleic acid of Example 3 without the addition of a restriction nicking enzyme Nb.BtsI;
  • Figure 13 is a graph showing the detection and amplification of the detection of Treponema pallidum virus nucleic acid by the restriction nicking enzyme Nb.BtsI in Example 3;
  • Figure 14 is a graph showing the results of polyacrylamide gel electrophoresis detection of the amplification product of Treponema pallidum virus in Example 3, wherein lanes 1-8 are not added with Nb.BtsI group, and 3L, 4L, 5L from left to right. , 6L, 7L, 8L, 9L, negative control; lanes 9-16 are added Nb.BtsI group, from left to right are 3L, 4L, 5L, 6L, 7L, 8L, 9L, negative control; R lane is RNA marker. detailed description
  • the present invention discloses a method for nucleic acid amplification mediated by cleavage. Those skilled in the art can learn from the contents of this paper and appropriately improve the process parameters. It is specifically to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method of the present invention has been described by the preferred embodiments, and it is obvious that those skilled in the art can change and adapt and combine the methods described herein to implement and apply the techniques of the present invention without departing from the scope of the present invention. .
  • Example 1 Different concentrations of nucleic acids were amplified by restriction endonucleases and restriction nicking enzymes, respectively, to compare detection sensitivity and specificity of amplification products.
  • each assay uses 5 ⁇ extract (absolute input is 50000IU, 5000IU, 500IU, 50IU, 5IU, 2.5 IU, 1.25 IU)
  • 5 ⁇ extract absolute input is 50000IU, 5000IU, 500IU, 50IU, 5IU, 2.5 IU, 1.25 IU
  • 5 ⁇ extract absolute input is 50000IU, 5000IU, 500IU, 50IU, 5IU, 2.5 IU, 1.25 IU
  • 5 ⁇ extract absolute input is 50000IU, 5000IU, 500IU, 50IU, 5IU, 2.5 IU, 1.25 IU
  • 5 mM DTT 1 mM of various dNTPs
  • 2mMATP 2mM CTP, 2mM UTP, 1.5mM GTP
  • 0.5mM ITP 0.2 ⁇ forward primer S-p3.8 in Table 3
  • 0.2 ⁇ reverse primer in Table 3 S-p4.5
  • the enzyme mixture (22.5 g BSA, 400 U M-MLV and 100 U T7) was added.
  • RNA polymerase RNA polymerase
  • the reaction mixture is mixed by gently repeated pipetting and short-time centrifugation, and then amplification and experimental detection are started.
  • Mx3000P STRATAGENE 41 ° C incubation 90min, and detecting fluorescence per minute. The reaction was excited at 485nm, emission at 520nm measured signal.
  • a conserved restriction nicking enzyme, Nt.CviPII was encoded in the conserved region of S-gen of HBV DNA (nt257-262, -TGG ⁇ TGG ⁇ A- according to the EcoRI site).
  • a promoter primer is added, and a promoter primer is used in the method, the portion of which contains a sequence which is exactly complementary to the 3 ⁇ end of the tangent point position, and 5 ⁇ The end contains a promoter sequence that is recognized by an RNA polymerase such as ⁇ 7. Thereby a double-strand restriction site is formed for all genomic DNA present.
  • each assay uses 5 ⁇ extract (absolute input is 50000IU, 5000IU, 500IU, 50IU, 5IU, 2.5 IU, 1.25 IU).
  • Restriction nicking digestion was carried out under the following conditions: buffer 40 mM Tris-HCl pH 8.5, 12 mM MgCl 2 , 70 mM KCl, 15% v/v DMSO, 5 mM DTT, 1 mM of various dNTPs, 2 mM ATP, 2 mM CTP , 2mM UTP, 1.5mM GTP, 0.5mM ITP, ⁇ . ⁇ Promoter primer F201111-1 in Table 4, ⁇ . ⁇ Reverse primer R708 in Table 4, 0.067 ⁇ Molecular beacon probe MB713 in Table 4 And 1U restriction nicking enzyme Nt.CviPII (experimental results show that there is no significant difference in the sensitivity of the amplification assay between NU.CviPII and 1U-3U, 1U is slightly better).
  • the restriction nicking enzyme was inactivated by heating, and the DNA template was denatured at 95 ° C for 5 min, and the reaction mixture was cooled to 41 ° C for 5 min, during which hybridization of the primer occurred.
  • Enzymes (22.5 ug BSA, 400 U of M-MLV and 100 U of T7 RNA polymerase;) were then added, and the reaction mixture was mixed by gentle repeated pipetting and short time centrifugation, and then amplification and experimental detection were started.
  • the reaction mixture was incubated in Mx3000P (STRATAGENE) at 41 °C for 90 min and fluorescence detection was performed every minute. The reaction was excited at 485 nm and the emission signal was measured at 520 nm.
  • the amplification curves are shown in Figure 5 and Figure 6.
  • the amplified products were detected by polyacrylamide gel electrophoresis.
  • the results are shown in Figure 7. It can be seen from Fig. 5 and Fig. 6 that the time-to-point (TTP) of the method using the restriction nicking enzyme amplification method of the present invention is later than the restriction within the same nucleic acid input amount.
  • the method of dicer amplification; however, the method of restriction endonuclease amplification can only detect nucleic acid samples with absolute input quantities of 50000 IU and 5000 IU, and the method of restriction nicking amplification can detect absolute input.
  • Nucleic acid samples of 50,000 IU and 5000 IU, 500 IU, 50 IU, 5 IU, 2.5 IU were more sensitive than the control group by three orders of magnitude.
  • each lane of the method using restriction endonuclease amplification has a diffuse band with a large number of non-specific bands at the bottom; and the method of limiting nicking enzyme amplification according to the present invention is expanded.
  • the product is relatively clean, and the nicking enzyme-coupled transcription-mediated amplification method for non-specific amplification product nicking enzyme amplification is superior to the endo-type transcription-mediated amplification using restriction endonuclease amplification.
  • Amplification method is the nicking enzyme-coupled transcription-mediated amplification method for non-specific amplification product nicking enzyme amplification.
  • a conserved restriction enzyme Nt.CviPII is encoded in the conserved region of S-gen of HBV DNA (nt257-262, -TGG ⁇ TGG ⁇ A- according to EcoRI site) flesh when this part of S-region can
  • a promoter primer is added, and a promoter primer is used in the method, and a part thereof contains a sequence which is exactly complementary to the 3' end of the tangent point position, and the 5 ⁇ end contains an RNA polymerase ( The promoter sequence recognized by ⁇ 7) thus forms a double-strand restriction site for all genomic DNA present. 6 L of extract is used for each assay.
  • Restriction nick digestion is performed under the following conditions: TMA buffer 50 mM Tris -HCl pH 8.5, 8.66 mM MgCl 2 , 70 mM KC1, 11% v/v DMSO, 3.3 mM DTT, 1.25 mM of various dNTPs and NTP, ⁇ . ⁇ in Table 5 Promoter primer F201111-1, ⁇ . ⁇ Reverse primer R708 in Table 5, 0.067 ⁇ Molecular beacon probe MB713 in Table 5 and 1U restriction nicking enzyme Nt.CviPII (Experimental results show that Nt. There was no significant difference in the sensitivity of CviPII between 1U and 3U, and 1U was slightly better.
  • the restriction nicking enzyme was inactivated by heating, and the DNA template was denatured at 95 °C for 5 min, and the reaction mixture was cooled to 41 V for 5 min, during which hybridization of the primer occurred.
  • a TMA enzyme mixture (0.07M Trehalose, 22.5ug BSA, 400U M-MLV and 100U T7 RNA polymerase) was added, and the reaction mixture was mixed by gentle repeated suction and short time centrifugation, and then amplification and experimentation were started. Detection.
  • the reaction mixture was incubated in Mx3000P (STRATAGENE) at 41 °C for 90 min and fluorescence detection was performed every minute. The reaction was excited at 485 nm and the emission signal was measured at 520 nm.
  • Reaction buffer 50 mM Tris-HCl pH 8.5, 8.66 mM MgC12, 70 mM KCl, 11% v/v DMSO, 3.3 mM DTT, 1.25 mM of various dNTPs and NTP, restriction nicking of primers F201111-l, 2U
  • the enzyme Nt.CviPII a partial template sequence ⁇ ij containing a cleavage site (see Table 6 for specific sequences). After incubation at 41 °C for 15 min, the restriction enzyme was inactivated by heating. The results of 10% polyacrylamide gel electrophoresis are shown in Fig. 8.
  • F201111-1 5 -TTCTAATACGACTCACTATAGGGAGACGTGGTGGACTTCTCT
  • Nt.CviPII restriction sites There are two Nt.CviPII restriction sites in CBN1013, so the CBN1013 oligonucleotide is cleaved to obtain four bands of 16mer+34mer and 19mer+31mer, and the 34mer and 19mer sequences are obtained, and there is also a restriction enzyme digestion of Nt.CviPII.
  • the site is cut off by 3 bases, resulting in two bands of 31mer and 16mer. Therefore, in theory, the 34mer, 31mer and 16mer bands are clearly visible on the electropherogram. It can be seen from Fig. 8 that the incubation product without Nt.CviPII has only two template bands; the incubation product with Nt.CviPII is clearly visible on the electropherogram.
  • the 34mer, 3 lmer and 16mer bands and a template band are visible. Consistent, indicating that Nt.CviPII is active in the HBV TMA system.
  • the amplification template is a natural HBV virus nucleic acid.
  • Multiple HBV clinically positive samples were mixed and the genome was extracted by High Pure Viral Nucleic Acid Kit (Roche, lot 11858874001).
  • the obtained nucleic acid was determined by HBV second generation international standard (WHO, 97/750), which was 2.4583e+ 008 IU/mL.
  • the nucleic acid was diluted 10 times in 10 gradients with absolute nucleic acid input of 150,000 IU/reaction, 15,000 IU/reaction, 1500 IU/reaction, 150 IU/reaction and 15 IU/reaction, respectively.
  • the amplification effects of the restriction enzymes were compared using the five concentrations of nucleic acid as templates, and the amplification curve morphology and TTP were used for evaluation. The results are shown in Fig. 9 and Fig. 10.
  • the amplification sensitivity is improved by three orders of magnitude after the addition of Nt.CviPII.
  • the TTP added to the Nt.CviPII group was about 10 min less than the Nt.CviPII group.
  • the results indicate that the nicking-mediated transcription-mediated amplification method of the present invention can be used for detection of HBV natural virus nucleic acid with a minimum detection sensitivity of 15 IU/reaction or lower.
  • a conserved restriction nicking enzyme, the Nb.BtsI site, is encoded in the conserved region of the DNA polymerase I gene (polA) of TP DNA.
  • polyA DNA polymerase I gene
  • the cleavage site is 3, -CGTCAC v ⁇ -5 ⁇ .
  • the TP DNA in the natural state is double-stranded, and 6 L of TP DNA solution is used for each measurement.
  • Restriction nicking digestion was carried out under the following conditions: TMA buffer (50 mM Tris-HCl pH 8.5, 8.66 mM MgCl 2 , 70 mM KC1, 11% v/v DMSO, 3.3 mM DTT, 1.25 mM of various dNTPs and NTP, ⁇ . ⁇ Promoter primer F400806 in Table 7, ⁇ . ⁇ Reverse primer R400806-3 in Table 7, 0.067 ⁇ Molecular beacon probes in Table 7 ⁇ 3 and 2U restriction nicking enzyme Nb.
  • the portion of the promoter primer (F400806) contains a sequence that is exactly complementary to the end of the tangent point position, and the 5 ⁇ end contains a promoter that can be recognized by an RNA polymerase (such as ⁇ 7).
  • RNA polymerase such as ⁇ 7
  • Subsequence Subsequent addition of chymase mixture (0.07 ⁇ Trehalose, 22.5ug BSA, 400U M-MLV and 100U) T 7 RNA polymerase ;), and mix the reaction mixture by gentle repeated suction and short time centrifugation, then start amplification and real-time detection.
  • RNA polymerase transcribed to form a large amount of RNA.
  • the reaction mixture was incubated in Mx3000P (STRATAGENE) for 90 min at 41 ° C, and fluorescence detection was performed every minute. The reaction was excited at 485 nm, and the emission signal was measured at 520 nm.
  • Reaction buffer 50 mM Tris-HCl pH 8.5, 8.66 mM MgCl 2 , 70 mM KCl, 11% v/v DMSO, 3.3 mM DTT, 1.25 mM of various dNTPs and NTP, 2U restriction nicking enzyme Nb.BtsI, TP1 oligoacid sequence containing Nb.BtsI cleavage site and its reverse complementation Sequence cTPl (see Table 8 for specific sequences). After incubation at 41 °C for 15 min, the inactive restriction nicking enzyme was inactivated by heating. The results of 10% polyacrylamide gel electrophoresis are shown in Figure 11. Table 8 Quantification of TP DNA by Nb.BtsI Digestion Activity Verification of Oligonucleotides
  • Nb.BtsI restriction site there is a Nb.BtsI restriction site in cTP1, so the cTP1 oligonucleotide should be cleaved to obtain two bands of 28mer and 32mer.
  • the incubation product without Nb.BtsI was two bands larger than 60 mer; the incubation product with Nb.BtsI added only one band larger than 60 mer, and two strips of 32 mer and 28 mer were added at the same time. Band, consistent with expectations, indicates that Nb.BtsI is active in the TP TMA system.
  • the reagent material name and amount and specific operations are as described above.
  • the double-stranded DNA including the position of the primer and the probe sequence was constructed by PCR. After agarose electrophoresis, the gel is recovered and used.
  • the obtained template nucleic acid (6.03 X 10 n copies/ul ) was diluted 10-fold into 9 gradients (1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, respectively).
  • 3L, 4L, 5L, 6L, 7L, 8L, and 9L nucleic acids as templates, compare the amplification effects with and without restriction nicking enzymes, and use amplification curve morphology and TTP and polyacrylamide gel electrophoresis (PAGE).

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Abstract

La présente invention concerne un procédé d'amplification d'acide nucléique et un kit de réactif d'essai. Le procédé utilise une enzyme de coupure simple brin de restriction pour amplifier un ADN bicaténaire et/ou un ADN monocaténaire et est applicable dans la détection multiple d'un échantillon d'ADN mixte et la détection multiple d'un échantillon mixte d'ADN et d'ARN.
PCT/CN2013/083182 2012-09-10 2013-09-10 Procédé d'amplification d'acide nucléique WO2014036972A1 (fr)

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EP4253560A1 (fr) * 2022-03-31 2023-10-04 Consejo Superior de Investigaciones Cientificas Détection améliorée d'acides nucléiques simple brin à l'aide d'une digestion assistée par fokl

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CN108913736B (zh) * 2018-07-10 2021-08-24 中国海洋大学 单链寡核苷酸的制备方法
CN109852670A (zh) * 2019-03-04 2019-06-07 中国医科大学 一种高特异性核酸检测试剂及其使用方法
CN114350756A (zh) * 2021-11-22 2022-04-15 西安交通大学 基于dna切刻/聚合链置换循环反应的全基因组自引发扩增方法及试剂盒

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CN1582339A (zh) * 2001-03-07 2005-02-16 拜奥默里克斯有限公司 用基于转录的扩增来扩增和检测dna的方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4253560A1 (fr) * 2022-03-31 2023-10-04 Consejo Superior de Investigaciones Cientificas Détection améliorée d'acides nucléiques simple brin à l'aide d'une digestion assistée par fokl
WO2023187193A1 (fr) * 2022-03-31 2023-10-05 Consejo Superior De Investigaciones Cientificas Détection améliorée d'acides nucléiques monocaténaires à l'aide d'une digestion assistée par foki

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