WO2022206661A1 - 一种核酸等温扩增方法及其应用 - Google Patents

一种核酸等温扩增方法及其应用 Download PDF

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WO2022206661A1
WO2022206661A1 PCT/CN2022/083311 CN2022083311W WO2022206661A1 WO 2022206661 A1 WO2022206661 A1 WO 2022206661A1 CN 2022083311 W CN2022083311 W CN 2022083311W WO 2022206661 A1 WO2022206661 A1 WO 2022206661A1
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amplification
nucleic acid
reaction
reverse transcriptase
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韩序
段昆
高晓庆
王珺
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杭州杰毅生物技术有限公司
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  • the present disclosure relates to a new nucleic acid isothermal amplification detection technology, which optimizes the reaction system of TMA/NASBA by utilizing the combination of three enzymes of heat-resistant reverse transcriptase, RNA polymerase and RNase H, which can realize Amplification reactions of target RNA molecules.
  • the present disclosure also relates to the application of the nucleic acid isothermal amplification detection technology in pathogen detection.
  • Nucleic acid detection is currently the gold standard for most disease detection. For example, the diagnosis of influenza virus, coronavirus, etc. requires nucleic acid detection results as the gold standard for judgment.
  • concentration of nucleic acid molecules in samples is generally low. For example, patients with mild virus infection may only have a nucleic acid concentration of 1000-3000 copies/mL. Therefore, nucleic acid detection is basically composed of amplification methods and detection methods.
  • the most classic molecular detection method is the PCR method.
  • the traditional PCR detection method is PCR amplification, and then the PCR product is confirmed by electrophoresis or next-generation sequencing. If RNA needs to be detected, reverse transcriptase is added in the PCR step. This method is not commonly used at present because of its low efficiency and the need to open the tube to easily cause pollution.
  • the most commonly used PCR detection method is the fluorescence quantitative PCR method. Fluorescent probes or fluorescent dyes are added to the PCR reaction, so that the amplification process itself can generate a real-time fluorescent signal, and then by judging the fluorescent signal, the target can be detected. confirm.
  • the fluorescence quantitative PCR method is fast and simple. At the same time, the fluorescent quantitative PCR reaction does not need to open the tube to take out the amplification product, and the risk of contamination is also small.
  • the fluorescent quantitative PCR has become the gold standard method for molecular detection.
  • fluorescent quantitative PCR itself also has some inherent shortcomings. For example, fluorescent quantitative PCR needs to rely on a high-precision temperature cycler for detection, which has higher requirements for operators and stricter pollution control. Therefore, PCR-based RNA Testing is limited to a central laboratory, with significant limitations for rapid on-site testing.
  • LAMP Loop-mediated isothermal amplification
  • NASBA Nucleic acid sequence-dependent amplification
  • RPA technology mainly relies on three enzymes: recombinases that can bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand displacement DNA polymerases.
  • oligonucleotide primers oligonucleotide primers
  • SSBs single-stranded DNA binding proteins
  • strand displacement DNA polymerases The mixture of these three enzymes is also active at room temperature, and the amplification in an isothermal environment is realized by the joint action of these three enzymes.
  • the advantage is that the reaction system is simple, but the disadvantage of the existing NASBA is that in order to ensure the detection sensitivity, a step of pre-denaturation at a higher temperature (usually 60-65 °C) is required, and the amplification The increasing enzyme will be inactivated at this temperature, and the enzyme needs to be added when the temperature is lowered to 41-42 °C. This step leads to complicated operation, low degree of automation, and cannot be truly isothermal. However, if the pre-denaturation step is canceled, the amplification efficiency will be greatly reduced, which cannot meet the high sensitivity required for detection.
  • NASBA currently uses the probe method for detection. The probe method needs to read the fluorescence signal in real time, which occupies a large amount of resources of the fluorescence detector, which makes it difficult to improve the detection throughput.
  • a high temperature TMA method – Hi-TMA technology which utilizes the combination of high temperature of three enzymes, namely transcriptase/reverse transcriptase/RNase H, to achieve, for example, in 47-51 Isothermal amplification at °C temperature, and the amplification efficiency is greatly improved compared with the traditional NASBA.
  • this method can be combined with CRISPR/Cas13 detection technology, so that the method can also achieve accurate detection specificity by using the end-point method for detection, which improves the detection efficiency and detection throughput.
  • this paper provides an improved NASBA/TMA nucleic acid isothermal amplification method, wherein reverse transcriptase, RNA polymerase and RNase H are thermostable enzymes that are still active at least at 45°C, and the The amplification reaction is carried out at a constant temperature not lower than 45°C.
  • the reverse transcriptase, RNA polymerase, and RNase H are all thermostable enzymes that are active at least at 51°C, and the amplification reaction is isothermally performed at a temperature of 47°C-51°C .
  • the amplification reaction does not require a pre-denaturation step.
  • the nucleic acid includes DNA and/or RNA.
  • the reverse transcriptase is AMV reverse transcriptase or MMLV reverse transcriptase.
  • the RNA polymerase is a thermostable T7, T3, M13, or SP6 RNA polymerase.
  • the amplification reaction is performed in a metal bath, water bath, or air bath reaction vessel, and the amplification reaction time is 10-90 minutes.
  • the amplification reaction is performed in a buffer comprising: 10-100 mM Tris-HCl, 10-100 mM KCl, 10-50 mM MgCl2 , 0.1-10 mM NTPs, 0.1- 10 mM dNTPs, 5-40% glycerol, 0-25% DMSO (dimethyl sulfoxide), and 0.1-3 [mu]M amplification primer, pH 7.3-8.2.
  • the amplification reaction is performed in a buffer comprising: 20-100 mM Tris, 10-100 mM KCl, 10-50 mM MgCl2 , 0.1-10 mM NTP, 0.1-10 mM dNTP, 0.1-10 mM dNTP -10% glycerol, 0-10% DMSO, 0.1-3 ⁇ M amplification primers, 0-100 mM sorbitol, 0-10% betaine, 0-10 mM DTT (dithiothreitol) and 1-20 ⁇ g/ ⁇ L of BSA (Bovine Serum Albumin).
  • a buffer comprising: 20-100 mM Tris, 10-100 mM KCl, 10-50 mM MgCl2 , 0.1-10 mM NTP, 0.1-10 mM dNTP, 0.1-10 mM dNTP -10% glycerol, 0-10% DMSO, 0.1-3 ⁇ M amplification
  • the method includes detecting the amount of amplified product using a fluorescent probe method or CRISPR/Cas13 detection technology.
  • the crRNA required for the CRISPR/Cas13 detection technology is added to the amplification reaction in the form of crDNA.
  • nucleic acid isothermal amplification kit comprising reverse transcriptase, RNA polymerase and RNase H, wherein the reverse transcriptase, RNA polymerase and RNase H are all at least at 45°C Still active thermostable enzymes.
  • the reverse transcriptase, RNA polymerase, and RNase H are all thermostable enzymes that are active at least at 51°C.
  • the reverse transcriptase is AMV reverse transcriptase or MMLV reverse transcriptase.
  • the RNA polymerase is a thermostable T7, T3, M13, or SP6 RNA polymerase.
  • the kit further comprises a buffer for performing the amplification reaction, the buffer comprising the following components: 10-100 mM Tris-HCl, 10-100 mM KCl, 10-50 mM MgCl 2 , 0.1-10 mM NTPs, 0.1-10 mM dNTPs, 5-40% glycerol and 0-25% DMSO, pH 7.3-8.2.
  • the kit further comprises a buffer for performing the amplification reaction, the buffer comprising the following components: 20-100 mM Tris, 10-100 mM KCl, 10-50 mM MgCl 2 , 0.1-10 mM NTPs, 0.1-10 mM dNTPs, 0-10% glycerol, 0-10% DMSO, 0-100 mM sorbitol, 0-10% betaine, 0-10 mM DTT and 1-20 ⁇ g/ ⁇ L BSA.
  • the buffer comprising the following components: 20-100 mM Tris, 10-100 mM KCl, 10-50 mM MgCl 2 , 0.1-10 mM NTPs, 0.1-10 mM dNTPs, 0-10% glycerol, 0-10% DMSO, 0-100 mM sorbitol, 0-10% betaine, 0-10 mM DTT and 1-20 ⁇ g/ ⁇ L BSA.
  • the buffer further includes 0.1-3 ⁇ M amplification primers.
  • the kit further includes fluorescent probes and/or CRISPR/Cas13 detection reagents for detecting the amount of amplification reaction product.
  • the CRISPR/Cas13 detection reagent includes crRNA or crDNA used in conjunction with the Cas13 enzyme.
  • kits in pathogen nucleic acid detection.
  • the pathogen is an RNA virus.
  • the isothermal nucleic acid amplification method provided in this paper optimizes the TMA/NASBA reaction system, which can realize the amplification reaction of target nucleic acid molecules at a single temperature, and has better amplification efficiency and sensitivity.
  • Figure 1 shows a methodological comparison of TMA/NASBA and Hi-TMA.
  • Figure 2 is a flow chart of Hi-TMA binding to Cas13 for molecular detection.
  • Figure 3 shows the amplification efficiency results for Hi-TMA and common TMA methods.
  • Figure 4 shows the detection sensitivity results of Hi-TMA and common TMA methods.
  • Figure 5 shows the results of Hi-TMA and common TMA methods for amplification reactions at different temperatures
  • Figure 6 shows the results of the detection of influenza A virus samples with different concentrations using Hi-TMA.
  • Figure 7 shows the results of the detection of influenza A virus samples, influenza B virus samples and their mixed samples by Hi-TMA.
  • isothermal nucleic acid amplification method refers to that the amplification reaction can be carried out at a constant temperature relative to a variable temperature nucleic acid amplification method such as PCR. Since the amplification reaction can be carried out under constant conditions, the amplification method requires less amplification instrument and is more suitable for on-site rapid detection applications.
  • RNA polymerase or called transcriptase
  • RNase H which has the ability to hydrolyze RNA-DNA hybrid The activity of RNA strands on a molecule.
  • the amplification reaction also uses a pair of amplification primers, wherein the 3' end of the first primer is complementary to the 3' end sequence of the target amplification region of the nucleic acid to be tested, and the 5' end is a promoter sequence recognizable by RNA polymerase ;
  • the second primer is consistent with the sequence of the 5' end of the target amplification region of the nucleic acid to be detected.
  • Its amplification reaction includes an acyclic phase and a cyclic phase, where the acyclic phase includes a pre-denaturation step (eg, performed at 65°C or higher) for annealing primers to RNA molecules as templates to initiate the first cDNA strand Synthesis.
  • NASBA can be used to amplify RNA to obtain RNA products.
  • a slight modification of the process (adding a heat denaturation step) can be used to amplify DNA molecules, but the amplified product is still RNA. Except for the denaturation step, the NASBA amplification reaction was performed at around 42°C.
  • TMA Transcription Mediated Amplification
  • MMLV reverse transcriptase has both reverse transcriptase activity and RNase H activity, so there is no need to add RNase H in the amplification reaction.
  • the amplification reaction is usually carried out at around 41.5°C.
  • NASBA/TMA isothermal nucleic acid amplification method or the reverse transcriptase used in TMA itself can be regarded as two Enzymes: reverse transcriptase and RNase H.
  • NASBA/TMA isothermal nucleic acid amplification method or the reverse transcriptase used in TMA itself can be regarded as two Enzymes: reverse transcriptase and RNase H.
  • TMA T7 RNA polymerase is usually used, but it will be apparent to those skilled in the art that other RNA polymerases can be used to complete the amplification reaction.
  • thermoostable reverse transcriptase may include commonly used reverse transcriptases as long as they have reverse transcriptional activity at a temperature higher than 45°C or higher, such as AMV reverse transcriptase and MMLV reverse transcriptase.
  • Heat-stable RNA polymerase refers to a transcriptase that is transcriptionally active at temperatures above 45°C or higher, eg, T7 RNA transcriptase with multiple mutations, some of which are already commercially available, as implemented The manufacturer and catalog number of T7 transcriptase are given in the example.
  • Heat-stable RNase H refers to an RNase that has the activity of digesting RNA in RNA-DNA molecules at temperatures above 45°C or higher. It can be E. coli RNaseH with point mutations or RNaseH from thermotolerant bacteria. Likewise, some of them are also commercially available, such as RNaseH with the manufacturer and product number given in the Examples.
  • the "improved NASBA/TMA nucleic acid isothermal amplification method refers to a method obtained by improving the amplification principle of the above method.
  • the improved method involves the use of thermostable reverse transcriptase, RNA polymerase, and RNase H to perform amplification reactions at higher temperatures (eg, not lower than 45°C), increasing amplification efficiency.
  • the improved method performs the amplification reaction at a higher temperature (eg, not lower than 45°C) and does not employ a pre-denaturation step. When there is no pre-denaturation step, the "isothermal" of the entire amplification reaction is truly realized.
  • the inventors unexpectedly found that performing the amplification reaction at a higher temperature (eg, not lower than 45° C.) not only eliminates the need for a pre-denaturation step, but also achieves higher amplification efficiency and amplification sensitivity.
  • the improved process is carried out isothermally at a temperature of 47°C to 51°C.
  • the improved method is carried out at a constant temperature of 50°C.
  • detection of amplification products various methods known in the art can be used, including gel electrophoresis, use of fluorescent probes (for real-time monitoring or end-point detection), fluorescent dyes, and direct detection of light absorbance, and the like.
  • the inventors expect that under the reaction conditions of 47°C-51°C (for example, 50°C), the specificity of the reaction may decrease compared to the amplification reaction at about 41°C. detection, there will be a higher background signal.
  • detection of the amplified product is performed by CRISPR/Cas13 detection technology.
  • the CRISPR/Cas13 detection system is characterized by the use of a reaction combination of Cas13 and specific crRNA, which can be specifically activated by the crRNA-complementary template to generate a non-specific RNase signal, which can be achieved by an RNA-coupled fluorescent reporter probe.
  • the detection system can achieve high specificity detection reaction, but the detection sensitivity is insufficient, and can only achieve nM level detection.
  • the crRNA required for the CRISPR/Cas13 detection reaction can be placed in the form of crDNA and in the process of isothermal amplification reaction, the isothermal RNA polymerase (e.g. T7 RNA polymerase) transcription to generate crRNA required for CRISPR/Cas13 detection.
  • the isothermal RNA polymerase e.g. T7 RNA polymerase
  • the advantage is that the stability of DNA is much better than that of RNA, making the reagent more stable.
  • Some components used in this CRISPR/Cas13 detection technology may include:
  • Enzyme mixture eg Cas13 enzyme, RNase inhibitory protein
  • Cas13 enzyme includes but not limited to LbaCas13, LbuC13a, LwaCas13a, AspCas13b, BzoCas13b, CcaCas13b, PsmCas13b, PinCas13b, Pin2Cas13b, Pin3Cas13b, PbuCas13b, 3bguCas13b, PigCas13b RanCas13b, PspCas13b, EsCas13d, RspCas13d; if the detection reaction uses guide DNA, the enzyme mixture needs to include RNA polymerase, including T7 RNA polymerase, T3 RNA polymerase, Sp6 RNA polymerase, M13 RNA polymerase; if the detection reaction uses guide RNA, the enzyme The mixture does not require RNA polymerase;
  • the fluorescent reporter substrate is a fluorescently labeled single-stranded RNA molecule; the single-stranded RNA molecule is labeled with a fluorescent group at the 5' end and a fluorescent quenching group at the 3' end;
  • Detection buffer including 10-100 mM tris hydrochloride, 2-30 mM magnesium chloride, 10-100 mM potassium chloride, 1-10 mM dithiothreitol; the pH of the buffer is at 6.5 -8.0; if guide DNA is used, the buffer should also include NTPs;
  • the guide RNA sequence is an RNA sequence that binds to the Cas13 protein and is complementary to the target RNA molecule to be detected, and is used for the activation of Cas13; the guide DNA is used for transcription to generate a double-stranded guide RNA sequence.
  • RNA synthase for a 10 ⁇ l volume of Hi-TMA reaction system, it is preferable to include: reverse transcriptase: 5-20U; thermostable RNA synthase: 20-100U; thermostable NaseH: 0.1-0.5U ; Buffers are: 20-100 mM Tris, 10-100 mM KCl, 10-50 mM MgCl 2 , 0.1-10 mM NTP, 0.1-10 mM dNTP, 0-10% glycerol, 0-10% dimethyl sulfoxide, 0.1-3 ⁇ M Amplification primers, 0-100 mM sorbitol, 0-10% betaine, 0-10 mM DTT and 10-200 ⁇ g BSA); the amplification reaction temperature was 50 degrees, and the reaction time was 40 min.
  • the following components and conditions are used in a CRISPR detection reaction in a volume of 50 ⁇ L: Cas13 protein: 1-10 ⁇ g; RNase inhibitor: 20-60 U; buffer (Tris-HCl 8.0, 1-10 mM MgCl 2 , 1-10 mM DTT); RNase reporter (FAM-UUUUU-BHQ1): 0.1 ⁇ M-1 ⁇ M; the reaction temperature was 37 degrees, and the reaction time was 15 min.
  • the nucleic acid isothermal amplification detection method provided in this paper is more efficient than the traditional NASBA that needs to change temperature, and can realize the one-time addition of buffer and amplification enzyme, which simplifies the operation steps and improves the detection efficiency.
  • thermostable molecular enzymes including thermostable RNA transcriptase, reverse transcriptase AMV, thermostable RNase H, all of which are thermostable. It can withstand the reaction temperature of 47-52 degrees; because the reaction temperature of the system can be at the level of 50 degrees, the pre-denaturation step can be skipped and a good amplification effect can be achieved.
  • nucleic acid isothermal amplification detection methods include, but are not limited to:
  • Templates, primers, buffers and enzymes required for the reaction can be added to the reaction tube at one time, without the need for pre-denaturation to increase the step of opening the cap;
  • reaction efficiency is significantly better than the TMA reaction at 41°C, which can achieve higher amplification efficiency in the same time, or reduce the reaction time to achieve the same amplification efficiency;
  • iii) has a very high detection sensitivity (several nucleic acid copies per microliter).
  • Embodiment 1 Influenza A M gene detection—compare with existing common TMA method
  • Primer 1 AATTCTAATACGACTCACTATAGGGG ACCRATCCTGTCACCTCTGAC (SEQ ID NO: 1)
  • primer 2 GGGCATTYTGGACAAAKCGTCTACG (SEQ ID NO: 2)
  • R ⁇ Y ⁇ K are degenerate bases, and the promoter sequence is underlined in primer 1 .
  • the crDNA sequence used for amplification product detection is (SEQ ID NO: 3):
  • the reaction conditions are as follows:
  • the total reaction volume is 10 ⁇ L, including:
  • AMV reverse transcriptase (NEB, catalog number: M0277L): 15U;
  • T7 transcriptase (NEB, catalog number: M0251L): 100U;
  • RNase H (NEB, item number: M0297L): 2U;
  • crDNA 2ng
  • Amplification buffer 50 mM Tris-HCl, 80 mM KCl, 16 mM MgCl 2 , 4 mM NTP, 2 mM dNTP, 10% glycerol, 10% dimethyl sulfoxide, 2 ⁇ M amplification primers, 50 mM sorbitol, 10% betaine, 2 mM DTT, 100 ⁇ g BSA);
  • the reaction temperature is 65 degrees for pre-denaturation, and the amplification reaction is performed at 41 degrees (AMV, T7 and RNaseH are added after the system temperature drops to 41 degrees after pre-denaturation).
  • the default reaction time is 40 minutes.
  • the total reaction volume is 10 ⁇ L, including:
  • AMV reverse transcriptase (NEB, catalog number: M0277L): 15U;
  • Thermostable T7 Transcriptase (NEB, Cat. No.: M0251): 100U;
  • Heat-resistant RNaseH (NEB, item number: M0523S): 0.2U;
  • Amplification buffer 50 mM Tris; 100 mM KCl; 40 mM MgCl 2 ; 4 mM NTP; 2 mM dNTP; 10% glycerol; 10% dimethyl sulfoxide; 2 ⁇ M amplification primer; 100 mM sorbitol; 5% betaine; 2 mM DTT; 100 ⁇ g BSA);
  • the reaction temperature was 50 degrees, and the reaction time was 40 min by default.
  • the CRISPR detection reaction uses the following components and conditions:
  • the total reaction volume is 50 ⁇ L, including:
  • Cas13 protein (Case Bio, Cat. No.: KS001): 2 ⁇ g;
  • RNase enzyme inhibitor (Kess Biotech, product number: KS003): 20U;
  • Buffer Tris-HCl 8.0, 10 mM MgCl 2 , 2 mM DTT
  • RNase reporter (FAM-UUUUU-BHQ1): 0.2 ⁇ M;
  • the reaction temperature was 37 degrees, and the reaction time was 15 min.
  • Fluorescence detection Detected in a microplate reader (Tecan Infinite 200PRO), the fluorescence emission wavelength is 488mm, the detection wavelength is 520nm, and the fluorescence value is read (the fluorescence signal recording interval is 3 minutes).
  • Figure 3 shows the results of testing the same virus samples. It can be seen from the results that the amplification efficiency of Hi-TMA is much better than that of ordinary TMA reaction (reaction conditions are the same as above, and the amplification reaction time is compared from 0 to 40 min).
  • the inventors also studied the optimum reaction temperature of Hi-TMA.
  • the results are shown in Figure 5 (the temperature of the amplification reaction was set at 42-60°C, and the reaction conditions were the same as above). It can be seen from the figure that Hi-TMA can be carried out between 42°C and 60°C, but the optimal reaction temperature is between 47°C and 51°C. In contrast to this, ordinary TMA methods are not suitable to be carried out at 45°C or higher.
  • Embodiment 2 Detection of viral RNA
  • the following takes the detection of samples containing different concentrations of influenza A virus (Influenza A) as an example to illustrate the amplification and detection of viral RNA by the method of the present invention.
  • the specific method includes the following steps:
  • the crDNA sequence is (SEQ ID NO: 3):
  • the total reaction volume is 10 ⁇ L, including:
  • AMV reverse transcriptase (NEB, catalog number: M0277L): 15U;
  • Thermostable T7 Transcriptase (NEB, Cat. No.: M0251): 100U;
  • Heat-resistant RNaseH (NEB, item number: M0523S): 0.2U;
  • Buffer 50 mM Tris; 100 mM KCl; 40 mM MgCl 2 ; 4 mM NTP; 2 mM dNTP; 10% glycerol; 10% dimethyl sulfoxide; 2 ⁇ M amplification primer; 100 mM sorbitol; 5% betaine; );
  • the reaction temperature was 50 degrees, and the reaction time was 40 min by default.
  • the CRISPR detection reaction uses the following components and conditions:
  • the total reaction volume is 50 ⁇ L, including:
  • Cas13 protein (Case Bio, Cat. No.: KS001): 2 ⁇ g;
  • RNase enzyme inhibitor (Kess Biotech, product number: KS003): 20U;
  • Buffer Tris-HCl 8.0, 10 mM MgCl 2 , 2 mM DTT
  • RNase reporter (FAM-UUUUU-BHQ1): 0.2 ⁇ M;
  • the reaction temperature was 37 degrees, and the reaction time was 15 min.
  • Fluorescence detection Detected in a microplate reader (Tecan Infinite 200PRO), the fluorescence emission wavelength is 488mm, the detection wavelength is 520nm, and the fluorescence value is read (the fluorescence signal recording interval is 3 minutes).
  • the Hi-TMA method is used to detect the A-flow sample, the B-flow sample and the A-B flow mixed sample.
  • a primer 1 containing the T7 promoter sequence at the 5' end was designed according to the amplified target sequence.
  • Primer 2 without promoter the sequence is as follows:
  • Primer 1 (SEQ ID NO: 6):
  • Primer 2 (SEQ ID NO: 7):
  • influenza A crDNA sequence (SEQ ID NO: 3):
  • the B-stream crDNA sequence is (SEQ ID NO: 8):
  • the total reaction volume is 10 ⁇ L, including:
  • AMV reverse transcriptase (NEB, catalog number: M0277L): 15U;
  • Thermostable T7 Transcriptase (NEB, Cat. No.: M0251): 100U;
  • Heat-resistant RNaseH (NEB, item number: M0523S): 0.2U;
  • Buffer 50 mM Tris; 100 mM KCl; 40 mM MgCl 2 ; 4 mM NTP; 2 mM dNTP; 10% glycerol; 10% dimethyl sulfoxide; 2 ⁇ M amplification primer; 100 mM sorbitol; 5% betaine; );
  • the reaction temperature was 50 degrees, and the reaction time was 40 min by default.
  • the components and conditions used in the CRISPR detection reaction are:
  • LwaCas13 protein 2 ⁇ g
  • PsmCas13 protein 4 ⁇ g
  • RNase enzyme inhibitor 40U;
  • Buffer Tris-HCl 8.0, 4mM MgCl2 , 2mM DTT
  • RNase reporter (FAM-UUUUU-BHQ1): 0.2 ⁇ M;
  • RNase reporter (HEX-AAAAA-BHQ1): 0.4 ⁇ M
  • the reaction temperature was 37 degrees, and the reaction time was 15 min.
  • Fluorescence detection Detected in a microplate reader (Tecan Infinite 200PRO), the fluorescence detection channels are 488/520nm and 530/560nm, and the fluorescence value is read.
  • the CRISPR detection reaction is simultaneously amplified and detected in a single tube.
  • the substrate specificity of the two Cas13 proteins LwaCas13 recognizes UUUUU after activation, and PsmCas13 recognizes AAAAA after activation
  • the specific target sequence of the amplified product can be determined.
  • reaction is within a temperature range that is easy to control, such as 47-51 °C;
  • the Cas13 detection reaction can be used to achieve high specificity of detection.

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Abstract

本发明公开了一种核酸的恒温扩增方法及其应用。该方法中所用的逆转录酶、RNA聚合酶以及RNA酶H均为至少在45℃仍具有活性的耐热酶，并且所述扩增反应在不低于45℃的温度下恒温进行。本发明的恒温核酸扩增方法具有快速、恒温、灵敏度和特异性高的特点，适合临床检验等领域的核酸检测。

Description

一种核酸等温扩增方法及其应用
本申请要求于2021年3月30日提交至中国专利局、申请号为202110338236.6的中国专利申请的优先权，在此通过引用将其全文并入本文。
技术领域
本公开涉及一种新的核酸等温扩增检测技术，利用耐热的逆转录酶、RNA聚合酶和RNA酶H三种酶的组合，优化了TMA/NASBA的反应体系，可以实现单一温度下对靶标RNA分子的扩增反应。本公开还涉及该核酸等温扩增检测技术在病原体检测方面的应用。
背景技术
核酸检测是目前大多数疾病检测的金标准，例如流感病毒、冠状病毒等的诊断都需要核酸检测检测结果作为金标准判定。样本中的核酸分子一般浓度都较低，例如病毒轻度感染的患者，其样本中的核酸浓度可能只有1000-3000copies/mL。所以核酸检测基本都是有扩增方法加检测方法组成。最为经典的分子检测方法就是PCR法，传统的PCR检测方法是PCR扩增，然后通过电泳或一代测序对PCR产物加以确认，若是需要对RNA进行检测，则在PCR的步骤中加入逆转录酶。这种方法因为效率低，且需要开管容易造成污染，所以目前已经不常用。
目前最为常用的PCR检测方法是荧光定量PCR法，在PCR反应中加入荧光探针或者荧光染料，使得扩增过程本身能产生实时的荧光信号，再通过对荧光信号的判定则可以实现对靶标的确认。荧光定量PCR法具有快速简单的特点，同时荧光定量PCR反应不需要开管取出扩增产物，污染风险也较小，荧光定量PCR已经成为分子检测的金标准方法。但荧光定量PCR本身也存在一些固有的短板，比如，荧光定量PCR需要依赖高精密的温度循环仪进行检测，对操作人员的要求也较高，且污染控制也较为严格，所以基于PCR的RNA检测局限于中心实验室，对于现场快速检测有很大的限制。
近年来发展出很多新的核酸等温扩增检测技术，例如1)环介导等温扩增技术(LAMP)可利用4对引物特异识别6个靶位点和具有链置换活性的DNA聚合酶，可在60-65℃条件下实现核酸快速扩增和检测(一般小于1小时)；2)依赖于核酸序列的扩增(NASBA)法，利用三种酶(逆转录酶、RNA酶和T7RNA聚合酶)和两个特异性引物的引导，在一恒定温度下温育45-90分钟，即可完成对RNA模板的快速扩增，同时利用荧光基团标记的发夹型核酸探针进行检测；3)滚环扩增技术(RCA)，利用DNA连接酶和DNA聚合酶，以滚环复制的模式，从一条或多条引物处进行环形模板的链置换合成，从而可实现部分病毒的环状基因组的扩增；4)重组酶聚合酶扩增技术(RPA)，是在由多 种酶和蛋白的参与下，在恒温条件下实现核酸指数扩增的技术。RPA技术主要依赖于三种酶：能结合单链核酸(寡核苷酸引物)的重组酶、单链DNA结合蛋白(SSB)和链置换DNA聚合酶。这三种酶的混合物在常温下也有活性，利用这三种酶的共同作用，实现在等温环境下的扩增。
以上几种技术都各有其内在缺陷，如LAMP法扩增一旦开盖容易形成气溶胶污染，加上目前国内大多数实验室不能严格分区，假阳性问题比较严重，并且其引物设计要求比较高，对于分型或点突变检测常常不能满足要求；RPA方法的酶组分较为复杂，对于点突变的检测能力有限；RCA的反应时间偏长(>4小时)，使其在应用方面特别是现场检测中的优势不甚明显。NASBA/TMA方法为一种RNA扩增方法，优点是反应体系简单，但现有NASBA的缺点是为了保证检测的灵敏度，需要进行一步温度较高的预变性(一般为60-65℃)，扩增酶在此温度下会失活，需要在降温到41-42℃时才能加入酶。这个步骤导致操作变得复杂，自动化程度低，不能做到真正的等温，但若取消预变性步骤，则扩增效率大幅降低，不能满足检测所需的高灵敏度。同时，NASBA目前常用探针法检测，探针法需要实时进行荧光信号的读取，对荧光检测仪的资源占用较大，导致检测通量难以提高。
发明内容
为了解决NASBA/TMA技术的痛点，我们开发了一种高温TMA方法–Hi-TMA技术，利用三种酶即转录酶/逆转录酶/RNA酶H的高温的组合，实现了例如在47-51℃温度下的等温扩增，且扩增效率相比传统的NASBA大幅提高。同时，该方法可以搭配CRISPR/Cas13检测技术，使得该方法采用终点法进行检测也可以实现精准的检测特异性，提升了检测效率和检测通量。
因此，在一方面，本文提供了一种改进的NASBA/TMA核酸恒温扩增方法，其中逆转录酶、RNA聚合酶以及RNA酶H均为至少在45℃仍具有活性的耐热酶，并且所述扩增反应在不低于45℃的温度下恒温进行。
在一些实施方案中，所述逆转录酶、RNA聚合酶以及RNA酶H均为至少在51℃仍具有活性的耐热酶，并且所述扩增反应在47℃-51℃的温度下恒温进行。
在一些实施方案中，所述扩增反应无需预变性步骤。
在一些实施方案中，所述核酸包括DNA和/或RNA。
在一些实施方案中，所述逆转录酶为AMV逆转录酶或MMLV逆转录酶。
在一些实施方案中，所述RNA聚合酶为耐热型T7、T3、M13、或SP6RNA聚合酶。
在一些实施方案中，所述扩增反应在金属浴、水浴或气浴反应容器中进行，扩增反应时间为10-90分钟。
在一些实施方案中，所述扩增反应在包括以下成分的缓冲液中进行：10-100mM的Tris-HCl、10-100mM的KCl、10-50mM的MgCl 2，0.1-10mM的NTPs，0.1-10mM的dNTPs、5-40％甘油、0-25％DMSO(二甲基亚砜)以及0.1-3μM扩增引物，pH为7.3-8.2。
在一些实施方案中，所述扩增反应在包括以下成分的缓冲液中进行：20-100mM的Tris、10-100mM的KCl、10-50mM MgCl 2、0.1-10mM NTP、0.1-10mM dNTP、0-10％甘油、0-10％的DMSO、0.1-3μM扩增引物、0-100mM山梨醇、0-10％甜菜碱、0-10mM DTT(二硫苏糖醇)以及1-20μg/μL的BSA(牛血清白蛋白)。
在一些实施方案中，所述方法包括采用荧光探针法或CRISPR/Cas13检测技术对扩增产物的量进行检测。
在一些实施方案中，所述CRISPR/Cas13检测技术所需的crRNA以crDNA的形式加入至所述扩增反应中。
在另一方面，本文提供了一种核酸恒温扩增试剂盒，其包括逆转录酶、RNA聚合酶以及RNA酶H，所述逆转录酶、RNA聚合酶以及RNA酶H均为至少在45℃仍具有活性的耐热酶。
在一些实施方案中，所述逆转录酶、RNA聚合酶以及RNA酶H均为至少在51℃仍具有活性的耐热酶。
在一些实施方案中，所述逆转录酶为AMV逆转录酶或MMLV逆转录酶。
在一些实施方案中，所述RNA聚合酶为耐热型T7、T3、M13、或SP6RNA聚合酶。
在一些实施方案中，所述试剂盒还包括用于进行扩增反应的缓冲液，所述缓冲液包括以下组分：10-100mM的Tris-HCl、10-100mM的KCl、10-50mM的MgCl 2、0.1-10mM的NTPs、0.1-10mM的dNTPs、5-40％甘油以及0-25％的DMSO，pH为7.3-8.2。
在一些实施方案中，所述试剂盒还包括用于进行扩增反应的缓冲液，所述缓冲液包括以下组分：20-100mM的Tris、10-100mM的KCl、10-50mM的MgCl 2、0.1-10mM的NTPs、0.1-10mM的dNTPs、0-10％甘油、0-10％的DMSO、0-100mM山梨醇、0-10％甜菜碱、0-10mM的DTT以及1-20μg/μL的BSA。
在一些实施方案中，所述缓冲液还包括0.1-3μM的扩增引物。
在一些实施方案中，所述试剂盒还包括用于检测扩增反应产物的量的荧光探针和/或CRISPR/Cas13检测试剂。
在一些实施方案中，所述CRISPR/Cas13检测试剂包括与Cas13酶配合使用的crRNA或crDNA。
在另一方面，本文提供了上述试剂盒在病原体核酸检测方面的应用。
在一些实施方案中，所述病原体为RNA病毒。
本文提供的恒温核酸扩增方法优化了TMA/NASBA反应体系，可以实现单一温度下对靶标核酸分子的扩增反应，并且具有更佳的扩增效率和灵敏度。
附图说明
图1显示了TMA/NASBA和Hi-TMA的方法学对比。
图2为Hi-TMA结合Cas13进行分子检测的流程图。
图3显示了Hi-TMA和普通TMA方法的扩增效率结果。
图4显示Hi-TMA和普通TMA方法的检测灵敏度结果。
图5显示了Hi-TMA和普通TMA方法在不同温度下进行扩增反应的结果
图6显示了用Hi-TMA对不同浓度甲流病毒样品进行检测的结果。
图7显示了用Hi-TMA对甲流病毒样品、乙流病毒样品以及它们的混合样品进行检测的结果。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
本文所用的“核酸恒温扩增方法”指，相对于变温核酸扩增方法如PCR而言，其扩增反应可以在恒定温度下进行。由于扩增反应可在恒定条件下进行，该扩增方法对扩增仪器的要求较低，更适合于现场快速检测应用。
“依赖核酸序列的扩增(Nuclear acid sequence-based amplification,NASBA)”为本领域已知的一种核酸恒温扩增方法，其扩增反应采用了三种酶：逆转录酶，其具有以RNA或DNA为模板合成DNA链的活性；RNA聚合酶(或称为转录酶)，其具有在启动子控制下以DNA为模板合成RNA链的活性；以及RNA酶H，其具有水解RNA-DNA杂交分子上RNA链的活性。该扩增反应还使用了一对扩增引物，其中第一引物的3’端与待测核酸目标扩增区的3’端序列互补配对，5’端为RNA聚合酶可识别的启动子序列；第二引物与待测核酸目标扩增区的5’端序列一致。其扩增反应包括非循环相和循环相，其中非循环相包括一个预变性步骤(如65℃或更高温度进行)，用于引物与作为模板的RNA分子退火，以便开始第一条cDNA链的合成。随后的扩增反应过程为本领域已知，在此不再赘述。通常情况下，NASBA可用于对RNA进行扩增，获得RNA产物。另外，也可以对该过程稍加改动(增加加热变性步骤)，用于对DNA分子进行扩增，但扩增产物仍然是RNA。除变性步骤外，NASBA扩增反应在42℃左右进行。
“转录介导的扩增(Transcription Mediated Amplification,TMA)”是本领域已知的另一种核酸恒温扩增方法，技术原理与NASBA基本一致，略有不同之处是TMA利用的逆转录酶(如MMLV逆转录酶)既有逆转录酶的活性又具有RNA酶H活性，所以扩增反应中不需要另外添加RNA酶H。其扩增反应通常在41.5℃左右进行。除另有说明外，为了描述简洁，在本文中对NASBA和TMA不加区分(统称为“NASBA/TMA核酸恒温扩增方法”)，或者也可将TMA中所用的逆转录酶本身视为两种酶：逆转录酶和RNA酶H。另外，还需要指出的是，本文提及NASBA或TMA时，只是为了说明本文提供的方法在扩增原理上与它们相似，并非必要地同时采用其中使用的具体酶。例如，对于NASBA，其通常采用T7RNA聚合酶，但是，本领域技术人员显然可以理解，也可以采用其他的RNA聚合酶来完成扩增反应。
“耐热逆转录酶”可包括常用的逆转录酶，只要其在高于45℃或更高温度下具有逆转录活性即可，例如AMV逆转录酶和MMLV逆转录酶。
“耐热RNA聚合酶”指在高于45℃或更高温度下具有转录活性的转录酶，例如，带有多个突变的T7RNA转录酶，它们有些是已经商品化可购得的，如实施例中给出了厂家和货号的T7转录酶。
“耐热RNA酶H”指在高于45℃或更高温度下具有消化RNA-DNA分子中RNA的活性的RNA酶。其可以是具有点突变的E.coli RNaseH或来自耐热菌的RNaseH。同样地，它们中的一些也是已经商品化可购得的，如实施例中给出了厂家和货号的RNaseH。
本文所用的“改进的NASBA/TMA核酸恒温扩增方法(本文中也简称为“Hi-TMA”)”指在上述方法扩增原理基础上进行了改进之后而获得的方法。在一些实施方案中，该改进后的方法涉及采用耐热逆转录酶、RNA聚合酶和RNA酶H，以便在较高的温度下(例如不低于45℃)进行扩增反应，提高扩增效率。在另一些实施方案中，该改进后的方法在较高的温度下(例如不低于45℃)进行扩增反应，并且不采用预变性步骤。没有预变性步骤时，才真正实现了整个扩增反应的“恒温”进行。发明人意外地发现，在更高的温度下(例如不低于45℃)进行扩增反应，不仅可以不采用预变性步骤，还可以在获得更高的扩增效率和扩增灵敏度。在优选的实施方案中，该改进后的方法在47℃-51℃的温度下恒温进行。在更优选的实施方案中，该改进后的方法在50℃恒温进行。本文的Hi-TMA方法和目前NASBA/TMA方法的扩增过程的比较可参加图1。
对于扩增产物的检测，可以采用本领域已知的多种方法，包括凝胶电泳、采用荧光探针(用于实时监测或终点检测)、荧光染料，以及直接检测光吸收度等。发明人预期，在47℃-51℃(例如50℃)的反应条件下，相比41℃左右的扩增反应，反应的特异性有可能会有所下降，若采用探针法或荧光法进行检测，会有更高的本底信号。因此，在一些优选的实施方案中，通过CRISPR/Cas13检测技术来对扩增产物进行检测。
CRISPR/Cas13检测系统的特点在于，利用一个Cas13和特异性crRNA的反应组合，可以特异性地被crRNA互补的模板所激活，产生非特异性的RNase信号，通过RNA偶连的荧光报告探针即可实现检测。该检测系统可以实现高特异性的检测反应，但检测灵敏度不足，仅仅能实现nM级别的检测。
在采用CRISPR/Cas13检测技术进行扩增产物的检测的实施方案中，可将CRISPR/Cas13检测反应所需的crRNA通过crDNA的方式放置与等温扩增反应过程中，利用等温反应的RNA聚合酶(例如T7RNA聚合酶)转录产生CRISPR/Cas13检测所需的crRNA。其优点在于，DNA的稳定性远优于RNA，使得试剂的稳定性变强。
该CRISPR/Cas13检测技术使用的一些组分可包括：
(1)酶混合物(例如Cas13酶，RNase抑制蛋白)，其中Cas13酶包括但不限于LbaCas13、LbuC13a、LwaCas13a、AspCas13b、BzoCas13b、CcaCas13b、PsmCas13b、PinCas13b、Pin2Cas13b、Pin3Cas13b、PbuCas13b、PguCas13b、PigCas13b、PsaCas13b、RanCas13b、PspCas13b、EsCas13d、RspCas13d；若检测反应使用向导DNA，则酶混合物需要包括RNA聚合酶，包括T7RNA聚合酶、T3RNA聚合酶、Sp6RNA聚合酶、M13RNA聚合酶；若检测反应使用向导RNA，则酶混合物不需要RNA聚合酶；
(2)荧光报告底物，为荧光标记的单链RNA分子；单链RNA分子为在5‘端标记荧光基团、3’端标记荧光淬灭基团；
(3)检测缓冲液，包括10-100mM三羟甲基氨基甲烷盐酸盐、2-30mM氯化镁、10-100mM氯化钾、1-10mM二硫苏糖醇；所述缓冲液pH值在6.5-8.0之间；若使用向导DNA，则所述缓冲液还应该包括NTPs；
(4)向导RNA或向导DNA，所述向导RNA序列为结合Cas13蛋白并与目标待检RNA分子互补的一段RNA序列，用于Cas13的激活；向导DNA是用于转录产生向导RNA序列的双链DNA分子，该向导DNA在5‘端具有T7或T3或Sp6或M13启动子序列。
采用CRISPR/Cas13检测来扩增产物时，本发明方法的总体过程可参加图2。
在一些具体实施方案中，对于例如一个10μl体积的Hi-TMA反应体系来说，优选包括：逆转录酶：5-20U；耐热RNA合成酶：20-100U；耐热NaseH：0.1-0.5U；缓冲液为：20-100mM Tris、10-100mM KCl、10-50mM MgCl 2、0.1-10mM NTP、0.1-10mM dNTP、0-10％甘油、0-10％二甲基亚砜、0.1-3μM扩增引物、0-100mM山梨醇、0-10％甜菜碱、0-10mM DTT以及10-200μg BSA)；扩增反应温度为50度，反应时间为40min。
在一些具体实施方案中，体积为50μL的CRISPR检测反应采用如下组分和条件：Cas13蛋白：1-10μg；RNase酶抑制剂：20-60U；缓冲液(Tris-HCl 8.0，1-10mM MgCl 2，1-10mM DTT)；RNase reporter(FAM-UUUUU-BHQ1)：0.1μM-1μM；反应温度为37度，反应时间为15min。
本文提供的核酸等温扩增检测方法，比传统的需要变温的NASBA效率更高，且可以实现缓冲液和扩增酶的一次性加入，简化了操作步骤，提高的检测效率。
本文提供了方法实现了一步法核酸等温扩增，其中参与等温扩增反应的全部酶都是耐热型分子酶，包括耐热型RNA转录酶、逆转录酶AMV、耐热RNA酶H，均能耐受47-52度反应温度；因为体系的反应温度可以在50度的级别，因此可以跳过预变性的步骤也能实现良好的扩增效果。
本文提供的核酸等温扩增检测方法的优点包括但不限于：
i)可一次性将反应所需的模板、引物、缓冲液和酶加入反应管，不需要预变性增加开盖的步骤；
ii)反应效率显著优于41℃的TMA反应，可以在同样时间内实现更高的扩增效率，或达到同样扩增效率减少反应时间；
iii)具有极高的检测灵敏度(毎微升数个核酸拷贝)。
以下通过具体实施例来进一步阐述本发明。实施例中的所用试剂和方法如无特别说明均为常规试剂和方法。
实施例1甲型流感M基因检测—与现有普通TMA方法进行比较
针对甲型流感M基因，发明人设计了如下引物对。引物1： AATTCTAATACGACTCACTATAGGGGACCRATCCTGTCACCTCTGAC(SEQ ID NO：1)，引物2：GGGCATTYTGGACAAAKCGTCTACG(SEQ ID NO：2)，(其中：R\Y\K为简并碱基，引物1中下划线标出了启动子序列)。
用于扩增产物检测的crDNA序列为(SEQ ID NO：3)：
Figure PCTCN2022083311-appb-000001
反应条件如下：
对于TMA反应体系：
总反应体系为10μL，包括：
AMV逆转录酶(NEB，货号：M0277L)：15U；
T7转录酶(NEB，货号：M0251L)：100U；
RNase H(NEB，货号：M0297L)：2U；
crDNA：2ng；
引物：1μM；
模板：1fM流感病毒RNA；
扩增缓冲液(50mM Tris-HCl、80mM KCl、16mM MgCl 2、4mM NTP、2mM dNTP、10％甘油、10％二甲基亚砜、2μM扩增引物、50mM山梨醇、10％甜菜碱、2mM DTT、100μg BSA)；
反应温度为65度预变性，41度进行扩增反应(AMV、T7和RNaseH在预变性后体系温度下降到41度后加入)，反应时间默认为40分钟。
对于Hi-TMA反应体系：
总反应体系为10μL，包括：
AMV逆转录酶(NEB，货号：M0277L):15U；
耐热T7转录酶(NEB，货号：M0251):100U；
耐热RNaseH(NEB，货号：M0523S):0.2U；
crDNA：2ng
扩增缓冲液(50mM Tris；100mM KCl；40mM MgCl 2；4mM NTP；2mM dNTP；10％甘油；10％二甲基亚砜；2μM扩增引物；100mM山梨醇；5％甜菜碱；2mM DTT；100μg BSA)；
反应温度为50度，反应时间默认为40min。
CRISPR检测反应采用如下组分和条件：
总反应体系为50μL，包括：
Cas13蛋白(恺思生物，货号：KS001)：2μg；
RNase酶抑制剂(恺思生物，货号：KS003)：20U；
缓冲液(Tris-HCl 8.0，10mM MgCl 2，2mM DTT)；
RNase reporter(FAM-UUUUU-BHQ1)：0.2μM；
反应温度为37度，反应时间为15min。
荧光检测：在酶标仪(Tecan Infinite 200PRO)中检测，荧光发射波长为488mm，检测波长为520nm，读取荧光值(荧光信号记录间隔为3分钟)。
图3显示了对相同病毒样品进行检测的结果。从结果可看出，Hi-TMA的扩增效率大幅优于普通TMA反应(反应条件同上所述，扩增反应时间从0-40min做比较)。
随后，本发明人对不同浓度的病毒样品进行了检测(图4)。从结果可看出，Hi-TMA的检测灵敏度也优于普通TMA反应(反应条件同上所述，扩增反应时间为40min)。
另外，采用上述反应条件，发明人还对Hi-TMA的最佳反应温度进行了研究。结果显示在图5中(扩增反应的温度设定为42-60℃，反应条件同上)。从图中可看出，Hi-TMA可以在42℃至60℃之间进行，但最佳反应温度在47℃-51℃。与此相反，普通TMA方法不适合于在45℃或更高温度下进行。
实施例2病毒RNA的检测
以下以含不同浓度甲流病毒(Influenza A)的样品的检测为例，说明用本发明的方法对病毒RNA进行的扩增检测。具体方法包括以下步骤：
对甲型流感病毒的M1基因序列(atgagtcttctaaccgaggtcgaaacgtacgttctctctattgtcccgtcaggccccctcaaagccgagatcgcacagagacttgaagatgtctttgctgggaagaacaccgatcttgaggctctcatggaatggctaaagacaagaccaatcctgtcacctctgactagggggattttgggatttgtattcacgctcaccgt gcccagtgagcgaggactgcagcgtagacgctttgtccaaaatgccctcaatgggaatggggatccaaataacatggacagagcagttaaactgtatagaaagcttaagggggagataacattccatggggccaaagaaatagcgctcagttattctgctggtgcacttgccagttgtatgggcctcatatacaacaggatgggggctgtgaccactgaagtggcctttggcctggtatgtgcaacctgtgaacagattgctgactcccagcataggtctcataggcaaatggtgacaacaaccaatccactaataagacatgagaacaggatggttctggccagcactacagctaaggctatggagcaaatggctggatcgagtgagcaagcagcagaggccatggaggttgctagtcaggccaggcaaatggtgcaggcaatgagagccattgggactcatcctagctccagtgctggtctgaaagatgatcttcttgaaaatttgcaggcctatcagaaacgaatgggggtgcagatgcaacgattcaagtgaccctcttgttgttgccgcgagtatcattgggatcttgcacttgatattgtggattcttgatcgtctttttttcaaatgcatttatcgcttctttaaacacggtctgaaaagagggccttctacggaaggagtaccagagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatagtcattttgtcagcatagagctggagta，(SEQ ID NO：4)，下划线为crRNA靶向序列)，根据扩增靶序列设计了一个5’端包含T7启动子序列的引物1和不带启动子的引物2，序列如下：
引物1(SEQ ID NO：1)：
Figure PCTCN2022083311-appb-000002
引物2(SEQ ID NO：2)：
Figure PCTCN2022083311-appb-000003
crDNA序列为(SEQ ID NO：3):
Figure PCTCN2022083311-appb-000004
Hi-TMA反应体系：
总反应体系为10μL，包括：
AMV逆转录酶(NEB，货号：M0277L):15U；
耐热T7转录酶(NEB，货号：M0251):100U；
耐热RNaseH(NEB，货号：M0523S):0.2U；
crDNA：2ng
缓冲液(50mM Tris；100mM KCl；40mM MgCl 2；4mM NTP；2mM dNTP；10％甘油；10％二甲基亚砜；2μM扩增引物；100mM山梨醇；5％甜菜碱；2mM DTT；100μg BSA)；
反应温度为50度，反应时间默认为40min。
CRISPR检测反应采用如下组分和条件：
总反应体系为50μL，包括：
Cas13蛋白(恺思生物，货号：KS001)：2μg；
RNase酶抑制剂(恺思生物，货号：KS003)：20U；
缓冲液(Tris-HCl 8.0，10mM MgCl 2，2mM DTT)；
RNase reporter(FAM-UUUUU-BHQ1)：0.2μM；
反应温度为37度，反应时间为15min。
荧光检测：在酶标仪(Tecan Infinite 200PRO)中检测，荧光发射波长为488mm，检测波长为520nm，读取荧光值(荧光信号记录间隔为3分钟)。
结果显示在图6中。从图6可看出，本发明Hi-TMA方法的检测灵敏度极高，低至毎微升样品甚至仅含数个拷贝的病毒。
实施例3利用Hi-TMA结合Cas13对甲型流感病毒和乙型流感病毒进行双重扩增检测
本实施例采用Hi-TMA方法对甲流样本、乙流样本以及甲乙流混合样品进行检测。
对甲型流感病毒的M1基因序列(atgagtcttctaaccgaggtcgaaacgtacgttctctctattgtcccgtcaggccccctcaaagccgagatcgcacagagacttgaagatgtctttgctgggaagaacaccgatcttgaggctctcatggaatggctaaagacaagaccaatcctgtcacctctgactagggggattttgggatttgtattcacgctcaccgt gcccagtgagcgaggactgcagcgtagacgctttgtccaaaatgccctcaatgggaatggggatccaaataacatggacagagcagttaaactgtatagaaagcttaagggggagataacattccatggggccaaagaaatagcgctcagttattctgctggtgcacttgccagttgtatgggcctcatatacaacaggatgggggctgtgaccactgaagtggcctttggcctggtatgtgcaacctgtgaacagattgctgactcccagcataggtctcataggcaaatggtgacaacaaccaatccactaataagacatgagaacaggatggttctggccagcactacagctaaggctatggagcaaatggctggatcgagtgagcaagcagcagaggccatggaggttgctagtcaggccaggcaaatggtgcaggcaatgagagccattgggactcatcctagctccagtgctggtctgaaagatgatcttcttgaaaatttgcaggcctatcagaaacgaatgggggtgcagatgcaacgattcaagtgaccctcttgttgttgccgcgagtatcattgggatcttgcacttgatattgtggattcttgatcgtctttttttcaaatgcatttatcgcttctttaaacacggtctgaaaagagggccttctacggaaggagtaccagagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatagtcattttgtcagcatagagctggagta，(SEQ ID NO：4)，下划线为crRNA靶向序列))，根据扩增靶序列设计了一个5’端包含T7启动子序列的引物1和不带启动子的引物2，序列如下：
引物1(SEQ ID NO：1)：
Figure PCTCN2022083311-appb-000005
引物2(SEQ ID NO：2)：
GGGCATTYTGGACAAAKCGTCTACG
对乙型流感病毒的M1基因序列(atgtcgctgtttggagacacaattgcctacctgctttcattgacagaagatggagaaggcaaagcagaactagcagaaaaattacactgttggttcggtggg aaagaatttgacctagactctgccttggaatggataaaaaacaaaagatgcttaactgatatacagaaagcactaattggtgcctctatctgctttttaaaacccaaagaccaggaaagaaaaagaagattcatcacagagcccttatcgggaatgggaacaacagcaacaaaaaagaagggcctgactctggctgagagaaaaatgagaaaatgtgtaagctttcatgaagcatttgaaatagcagaaggccatgaaagctcagcgctactatattgtctcatggtcatgtacctgaatcctggaaattattcaatgcaagtaaaactaggaacgctctgtgctttgtgcgaaaaacaagcatcacattcacacagagctcatagcagagcagcgagatcctcagtgccaggagtgagacgggaaatgcagatggtctcagctatgaacacagcaaaaacaatgaatggaatgggaaaaggagaagacgtccaaaaactggcagaagaactgcaaagcaacattggagtattgagatcccttggggcaagtcagaagaatggggaaggaattgcaaaggatgtaatggaagtgctaaagcagagctctatgggaaattcagctcttgtgaagaaatacctataa，(SEQ ID NO：5)，下划线为crRNA靶向序列))，根据扩增靶序列设计了一个5’端包含T7启动子序列的引物1和不带启动子的引物2，序列如下：
引物1(SEQ ID NO：6)：
Figure PCTCN2022083311-appb-000006
引物2(SEQ ID NO：7)：
Figure PCTCN2022083311-appb-000007
甲流crDNA序列为(SEQ ID NO：3)：
Figure PCTCN2022083311-appb-000008
乙流crDNA序列为(SEQ ID NO：8)：
Figure PCTCN2022083311-appb-000009
Hi-TMA反应体系：
总反应体系为10μL，包括：
AMV逆转录酶(NEB，货号：M0277L):15U；
耐热T7转录酶(NEB，货号：M0251):100U；
耐热RNaseH(NEB，货号：M0523S):0.2U；
crDNA：2ng
缓冲液(50mM Tris；100mM KCl；40mM MgCl 2；4mM NTP；2mM dNTP；10％甘油；10％二甲基亚砜；2μM扩增引物；100mM山梨醇；5％甜菜碱；2mM DTT；100μg BSA)；
反应温度为50度，反应时间默认为40min。
CRISPR检测反应所用的组分和条件为：
LwaCas13蛋白：2μg；
PsmCas13蛋白：4μg；
RNase酶抑制剂：40U；
缓冲液(Tris-HCl 8.0，4mM MgCl 2，2mM DTT)；
RNase reporter(FAM-UUUUU-BHQ1)：0.2μM；
RNase reporter(HEX-AAAAA-BHQ1)：0.4μM；
反应温度为37度，反应时间为15min。
荧光检测：在酶标仪(Tecan Infinite 200PRO)中检测，荧光检测通道为488/520nm以及530/560nm，读取荧光值。
对于混合样品，CRISPR检测反应是在单管中进行同时的扩增和检测的。利用两种Cas13蛋白的底物特异性(LwaCas13激活后识别UUUUU，PsmCas13激活后识别AAAAA)，可以判断出扩增产物具体是那种靶序列。
结果显示在图7中，说明本文提供的Hi-TMA方法可特异性地检测目标病原体，不受其他同时存在的类似病原体的干扰。
综上，本发明方法的的主要优点包括但不限于：
(1)快速，例如扩增反应时间大概在40分钟左右；
(2)无变温；
(3)反应在易于控制的温度范围内，例如47-51℃；
(4)利用耐热TMA反应实现高效率扩增，保证了检测的高灵敏度；
(5)可利用Cas13检测反应实现检测的高特异性。

Claims (22)

  1. 改进的NASBA/TMA核酸恒温扩增方法，其中逆转录酶、RNA聚合酶以及RNA酶H均为至少在45℃仍具有活性的耐热酶，并且所述扩增反应在不低于45℃的温度下恒温进行。
  2. 如权利要求1所述的NASBA/TMA核酸恒温扩增方法，其中所述逆转录酶、RNA聚合酶以及RNA酶H均为至少在51℃仍具有活性的耐热酶，并且所述扩增反应在47℃-51℃的温度下恒温进行。
  3. 如权利要求1或2所述的方法，其中所述扩增反应无需预变性步骤。
  4. 如权利要求1-3任一项所述的方法，其中所述核酸包括DNA和/或RNA。
  5. 如权利要求1-4任一项所述的方法，其中所述逆转录酶为AMV逆转录酶或MMLV逆转录酶。
  6. 如权利要求1-5任一项所述的方法，其中所述RNA聚合酶为耐热型T7、T3、M13、或SP6 RNA聚合酶。
  7. 如权利要求1-6任一项所述的方法，其中所述扩增反应在金属浴、水浴或气浴反应容器中进行，扩增反应时间为10-90分钟。
  8. 如权利要求1-7任一项所述的方法，其中所述扩增反应在包括以下成分的缓冲液中进行：10-100mM的Tris-HCl、10-100mM KCl、10-50mM MgCl 2、0.1-10mM NTPs、0.1-10mM dNTPs、5-40％甘油、0-25％的DMSO以及0.1-3μM扩增引物，pH为7.3-8.2。
  9. 如权利要求1-8任一项所述的方法，其中所述扩增反应在包括以下成分的缓冲液中进行：20-100mM Tris、10-100mM的KCl、10-50mM的MgCl 2、0.1-10mM的NTPs、0.1-10mM的dNTPs、0-10％甘油、0-10％的DMSO、0.1-3μM扩增引物、0-100mM山梨醇、0-10％甜菜碱、0-10mM的DTT以及1-20μg/μL的BSA。
  10. 如权利要求1-9任一项所述的方法，包括采用荧光探针法或CRISPR/Cas13检测技术对扩增产物的量进行检测。
  11. 如权利要求1-10任一项所述的方法，其中所述CRISPR/Cas13检测技术所需的crRNA以crDNA的形式加入至所述扩增反应中。
  12. 核酸恒温扩增试剂盒，包括逆转录酶、RNA聚合酶以及RNA酶H，它们均为至少在45℃仍具有活性的耐热酶。
  13. 如权利要求12所述的核酸恒温扩增试剂盒，其中所述逆转录酶、RNA聚合酶以及RNA酶H均为至少在51℃仍具有活性的耐热酶。
  14. 如权利要求12或13所述的核酸恒温扩增试剂盒，其中所述逆转录酶为AMV逆转录酶或MMLV逆转录酶。
  15. 如权利要求12-14任一项所述的核酸恒温扩增试剂盒，其中所述RNA聚合酶为耐热型T7、T3、M13、或SP6 RNA聚合酶。
  16. 如权利要求12-15任一项所述的核酸恒温扩增试剂盒，还包括用于进行扩增反应的缓冲液，所述缓冲液包括以下组分：10-100mM的Tris-HCl、10-100mM KCl、10-50mM MgCl 2、0.1-10mM NTPs、0.1-10mM dNTPs、5-40％甘油以及0-25％的DMSO，pH为7.3-8.2。
  17. 如权利要求12-16任一项所述的核酸恒温扩增试剂盒，还包括用于进行扩增反应的缓冲液，所述缓冲液包括以下组分：20-100mM的Tris-HCl、10-100mM的KCl、10-50mM的MgCl 2、0.1-10mM的NTPs、0.1-10mM的dNTPs、0-10％甘油、0-10％的DMSO、0-100mM山梨醇、0-10％甜菜碱、0-10mM的DTT以及1-20μg/μL的BSA。
  18. 如权利要求12-17任一项所述的核酸恒温扩增试剂盒，其中所述缓冲液还包括0.1-3μM的扩增引物。
  19. 如权利要求12-18任一项所述的核酸恒温扩增试剂盒，还包括用于检测扩增反应产物的量的荧光探针和/或CRISPR/Cas13检测试剂。
  20. 如权利要求12-19任一项所述的核酸恒温扩增试剂盒，其中所述CRISPR/Cas13检测试剂包括与Cas13酶配合使用的crRNA或crDNA。
  21. 权利要求12-20任一项的试剂盒在病原体核酸检测方面的应用。
  22. 如权利要求21所述的应用，其中所述病原体为RNA病毒。
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