WO2022174377A1 - Nested recombinase-polymerase amplification method and use thereof - Google Patents

Nested recombinase-polymerase amplification method and use thereof Download PDF

Info

Publication number
WO2022174377A1
WO2022174377A1 PCT/CN2021/076773 CN2021076773W WO2022174377A1 WO 2022174377 A1 WO2022174377 A1 WO 2022174377A1 CN 2021076773 W CN2021076773 W CN 2021076773W WO 2022174377 A1 WO2022174377 A1 WO 2022174377A1
Authority
WO
WIPO (PCT)
Prior art keywords
rpa
primer pair
amplification
reaction
idsp
Prior art date
Application number
PCT/CN2021/076773
Other languages
French (fr)
Chinese (zh)
Inventor
白净卫
刘册
Original Assignee
清华大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 清华大学 filed Critical 清华大学
Priority to PCT/CN2021/076773 priority Critical patent/WO2022174377A1/en
Publication of WO2022174377A1 publication Critical patent/WO2022174377A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the invention relates to the field of biotechnology, in particular to a one-step, rapid and highly sensitive nested recombinase-polymerase amplification method, a method for rapid nucleic acid detection, and a nested recombinase-polymerase amplification method the kit.
  • Recombinase-polymerase Amplification technology (Recombinase Polymerase Amplification, RPA) is known as a nucleic acid detection technology that can replace PCR. Originally patented by ASM scientific, Inc. in 2003 (EP1499738B1). Such amplification reactions are also known as recombinase-aid amplification (RAA), Multienzyme Isothermal Rapid Amplification (MIRA) and body temperature amplification technology (STAMP).
  • RPA Recombinase-polymerase Amplification
  • the basic principle is that the recombinase and the primer form a combination (filament), which invades the template strand of the substrate nucleic acid, opens the double strand, and binds to the complementary position of the primer; then, under the action of DNA polymerase with stranded displacement ability, under the action of the primer The 3' end begins to synthesize complementary strands. Displaced single chains are bound by single-chain binding proteins to assemble the original double-stranded complex.
  • the above amplification reactions have been commercialized for the detection of target fragments and nucleic acids, and are usually also equipped with tag probes to improve specificity.
  • RPA technology mainly relies on three enzymes: recombinase that can bind single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase.
  • the basic principle is: the recombinase binds to the primer.
  • the formed protein-DNA complex (filament) invades the substrate nucleic acid template strand and can search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequence, a strand exchange reaction will occur to form and initiate DNA synthesis.
  • the complementary strand starts to be synthesized at the 3' end of the primer, and the target region on the template is exponentially amplified.
  • the displaced DNA strand binds to the SSB, preventing further displacement. In this system, a synthesis event is initiated by two opposing primers.
  • the key to RPA detection technology lies in the design of amplification primers and probes.
  • fluorescent RPA adopts one-step addition of a pair of primers and a probe, which is simple in operation, low in sensitivity and weak in fluorescence intensity.
  • Nest RPA uses two steps to add the outer primer pair and the inner primer pair (including the probe) respectively, which has high sensitivity and complicated operation.
  • the intermediate involves product transfer, and there is a risk of product leakage and contamination.
  • the One-pot Nest RPA of this application was obtained by simplifying the steps.
  • Four primers/probes can be added simultaneously in one step. The operation is simple, there is no risk of opening the lid, and the sensitivity is high.
  • the high fluorescence intensity enables visual detection with the naked eye.
  • the purpose of the present invention is to overcome the deficiencies of the prior art, to provide a one-step, fast, high-sensitivity nested recombinase-polymerase amplification method (named One-pot Nest RPA), and a nucleic acid rapid detection method Methods, kits for nested recombinase-polymerase amplification methods.
  • Nucleic acid can be detected quickly and with high sensitivity. There is no need to prepare two RPA reaction reagents during the reaction process. Moreover, two pairs of primers can be added to the reaction system at the same time in one step, avoiding the risk of opening the lid and leaking, and reducing the complexity of the operation.
  • a first aspect of the present invention provides a nested recombinase-polymerase amplification method, the method comprising:
  • the second primer pair is located in the template obtained by the first primer pair RPA or RT-RPA reaction amplification, and the second primer pair comprises the /idSp/ restriction site and the modification that prevents the 3' end amplification .
  • the 1) first primer pair, and, 2) the second primer pair and/or probe are added in one step.
  • the modification that prevents 3' end amplification can be any 3' end modification that is not suitable for DNA polymerase amplification.
  • Preference is given to unnatural deoxynucleotides whose 3'-terminal OH is chemically blocked and their modifications or DNA-OH complementary to the 3'-terminal. Among them, the unnatural deoxynucleotides such as hydroxymethyl azide.
  • the modification that prevents 3' end amplification is selected from hydroxymethyl azide, C18, C12, ddNTP, RNA, PNA or C3 spacer.
  • an endonuclease capable of recognizing /idSp/ is added to the RPA or RT-RPA reaction, and the endonuclease is preferably Exo (exonuclease III) or nfo (Endonuclease IV).
  • the second primer pair and/or the probe further comprise modification of a fluorescent reporter group and a fluorescence quenching group
  • the fluorescent reporter group is selected from FAM (6-carboxy-fluorescein, 6-carboxy-fluorescein, 6-carboxy-fluorescein Fluorescein), HEX (hexachlorofluorescein, hexachloro-6-methylfluorescein), TET (Tetrachlorofluorescein, tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethyl-4,5-dichloro- 6-carboxyfluorescein), Cy3 (Indodicarbocyanine) or TAMRA (Carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine), the fluorescence quenching group is selected from DABCYL (4-(4-oxaneaminophenylazo) ) benzoic acid), BHQ1 (Black Hole Qua fluorescent Qu
  • the fluorescent reporter group is modified at a position of 25-35 bp bases at the 5' end of the upstream primer sequence of the second primer pair. It is preferably at the position of 29-31 bp.
  • the fluorescence quenching group is modified at a position 10-20 bp away from the 3' end of the downstream primer sequence of the second primer pair. It is preferably at the position of 13-18 bp.
  • the fluorescent reporter group and the fluorescent quenching group are separated by 1-3 bases.
  • Preferred spacer bases comprise tetrahydrofuran (THF).
  • the /idSp/ modification is between the fluorescent reporter group and the fluorescent quencher group.
  • a 3'OH end that can be amplified is generated, and the amplification reaction is continued.
  • the second primer pair and the first primer pair do not overlap or overlap less than 10 bp.
  • the RPA or RT-RPA reaction can be used for DNA or RNA amplification, respectively.
  • the reaction time of the RPA or RT-RPA is 5-40 min, preferably 30 min.
  • the reaction temperature of the RPA or RT-RPA is 35°C-45°C, preferably 37°C-42°C.
  • the RPA or RT-RPA reaction is carried out in a reaction reagent, and the reaction reagent includes a first primer pair, a second primer pair, a buffer and/or an RPA reactant.
  • the RPA reactants can be any commercial reagents or kits required for the RPA reaction, or the reagents required for the RPA reaction obtained by the laboratory preparation, which can be liquid or dry powder.
  • Preferred include recombinases, polymerases, and single-chain binding proteins, among others.
  • the reaction reagent further comprises magnesium acetate, DEPC water, buffer, PEG and/or lysate.
  • the lysate can be any commercial lysate reagent or kit, etc., or the lysate can be prepared by oneself in the laboratory.
  • the method further includes the step of observing the detection result.
  • the described step of observing the detection result comprises using a 470nm LED lamp and/or a filter, and then judging the detection result by naked eyes, without opening the reaction tube in the whole process.
  • the LED lamp is a single-color LED lamp, which can be a fluorescent lamp of any color, preferably a red light, yellow light, blue light, green light or white light.
  • the outer pair of primers are conventional RPA amplification primers, and the inner pair of primers are similar in design to the commonly used probes for RPA (see Figure 5), both with Modifications such as /idSp/ restriction site and C3 spacer at the 3' end that prevent amplification of the 3 end. Therefore, the reaction is initiated by a pair of primers on the outside (one-pot 1 and one-pot4) to initiate amplification, and the amplified double-stranded product recombines with one-pot2 and one-pot3, causing /idSp/ to be endo-cut by Exo, etc.
  • Enzyme cleavage produces a 3'OH end that can be amplified, and the primer equivalent to the inner reaction is activated, triggering a nested reaction. Therefore, the reaction speed can be increased and the sensitivity can be increased.
  • FAM-BHQ is added to the probe of the inner reaction, it can also be used as a fluorescent probe, so it can be used as both a probe and an inner nested primer.
  • the inner nested primers are designed in this way, because the 3' ends of the two primers are blocked, so it can avoid the specific amplification caused by the overlapping of the primers in the absence of a template, and the resulting Primer consumption.
  • a second aspect of the present invention provides a method for rapid nucleic acid detection, the method comprising:
  • step B) using the above-mentioned nested recombinase-polymerase amplification method to amplify the nucleic acid of step A) to obtain a reaction product;
  • the reaction product can also be diluted and inserted into a colloidal gold test strip for colloidal gold color development.
  • the reaction product is added into DEPC water, mixed evenly for dilution, and the diluted product is drawn and placed in a colloidal gold spotting hole for color development.
  • the colloidal chromatography test paper can be set as: the surface of colloidal gold is streptavidin (or FAM/FITC antibody), the first line T line is FAM/FITC antibody (or streptavidin antibody), the second line is streptavidin (or FAM/FITC antibody).
  • Line C is the streptavidin antibody (or anti-IgG antibody).
  • the detection of the fluorescence of the reaction product includes a fluorescent probe detection step.
  • the method further includes the step of observing the detection result.
  • the described step of observing the detection result comprises using a 470nm LED lamp and/or a filter, and then judging the detection result by naked eyes, without opening the reaction tube in the whole process.
  • the LED lamp is a single-color LED lamp, which can be a fluorescent lamp of any color, preferably a red light, yellow light, blue light, green light or white light.
  • a third aspect of the present invention provides a kit comprising the first primer pair, the second primer pair, the probe, the reaction reagent, the colloidal gold test strip and/or any of the above-mentioned methods. or a combination thereof.
  • the fourth aspect of the present invention provides a nucleic acid detection or amplification kit, the kit comprises a first primer pair, a second primer pair, a buffer and/or an RPA reactant, and the The second primer pair is located in the template obtained by the reaction of the first primer pair RPA or RT-RPA, and the second primer pair includes an /idSp/ restriction site and a modification that prevents amplification of the 3' end.
  • a fifth aspect of the present invention provides a kit for a nested recombinase-polymerase amplification method, the kit comprising a first primer pair, a second primer pair, a buffer and/or RPA Reaction, the second primer pair is located in the template obtained by the first primer pair RPA or RT-RPA reaction amplification, the second primer pair comprises /idSp/ restriction site and prevents the 3' end Amplified modifications.
  • the RPA reactant comprises an endonuclease capable of recognizing /idSp/.
  • the kit further comprises magnesium acetate, DEPC water, buffer, PEG and/or lysate.
  • the sixth aspect of the present invention provides the application of any one of the first primer pair and/or the second primer pair described above in preparing a kit for nucleic acid amplification and/or detection.
  • the seventh aspect of the present invention provides the application of the above method and the above kit in viral nucleic acid amplification or detection.
  • the eighth aspect of the present invention provides the application of a first primer pair and/or a second primer pair in viral nucleic acid amplification or detection, where the second primer pair is located in the first primer pair RPA or RT-RPA reaction amplification Within the template obtained, the second primer pair contains an /idSp/ restriction site and a modification that prevents amplification of the 3' end.
  • the virus is a coronavirus, more preferably a SARS-Cov2 virus or a SARS-Cov2 pseudovirus.
  • the first primer pair is:
  • E-Primer R AGACCAGAAGATCAGGAACTCTAGAAGAA
  • the second primer pair is:
  • E-Primer F2 5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’
  • E-Primer R2 5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3 spacer-3’
  • the first primer pair is:
  • O-Primer F TGTAGTTGTGATCAACTCCGCGAACCCATGCT
  • the second primer pair is:
  • O-Primer F2 5’-AGCCCGTCTTACACCGTGCGGCACAGGCAC/i6FAMdT//idSp/g/iBHQ1dT/ACTGATGTCGTAT-C3 spacer-3’
  • O-Primer R2 5’-CTTGGAAGCGACAACAATTAGTTTTTAGGAA/i6FAMdT//idSp//iBHQ1dT/AGCAAAACCAGCTA-C3 spacer-3’
  • the "one-step" described in this application includes simultaneous but not limited to simultaneous, including a one-time or only one-time sample adding process before or during the reaction.
  • the RPA or RT-RPA reaction is added to the reaction system at an interval, sequentially or simultaneously before the start of the reaction.
  • the "/idSp/" described in this application is an internal D-spacer, which can also be called /thf/ or abasic.
  • a base modification it represents the addition of a tetrahydrofuran (THF) spacer inside the oligonucleotide.
  • THF tetrahydrofuran
  • nucleic acid described in this application is composed of nucleotide monomers, and the nucleotide monomers are composed of five-carbon sugars, phosphate groups and nitrogen-containing bases, wherein the nitrogen-containing bases are selected from A, T, C, G or U.
  • the nucleic acid can be natural or modified DNA or RNA.
  • the nucleic acid is RNA.
  • Described nucleic acid can come from any organism that contains nucleic acid, described organism includes eukaryotic organism and prokaryotic organism, and is specifically selected from human, non-human animal, plant, bacterium, cyanobacteria, mycoplasma, chlamydia, fungus, virus and many more.
  • the nucleic acid is from a virus, preferably a coronavirus, more preferably a SARS-Cov2 virus or a SARS-Cov2 pseudovirus.
  • Figure 1 One-pot Nest RPA fluorescence amplification curve, where NC is the negative control and RFU (relative fluorescence units) is the relative fluorescence intensity.
  • FIG. 1 Blue light excitation fluorescence image of One-pot Nest RPA.
  • Figure 3 One-pot Nest RPA probe concentration screening fluorescence amplification curve, where RFU is the relative fluorescence intensity.
  • Figure 4 One-pot Nest RPA probe concentration screening blue light excitation fluorescence map.
  • Figure 5 Schematic diagram of the comparison of primer design for One-pot Nest RPA reaction with that of other RPA reactions.
  • Figure 6 The detection sensitivity of One-pot Nest RPA (one pot) versus conventional RPA (single RPA) for nucleic acid detection, where RFU is the relative fluorescence intensity.
  • Figure 7 Blue light-excited fluorescence of One-pot Nest RPA (one pot) versus conventional RPA (single RPA).
  • Figure 8 Fluorescence graph of the sensitivity of One-Pot Nest RPA to detect SARS-Cov2 pseudovirus E gene.
  • Figure 9 Fluorescence amplification curve of SARS-Cov2 pseudovirus O gene detected by One-pot Nest RPA.
  • Figure 10 Blue light fluorescence image of SARS-Cov2 pseudovirus O gene detected by One-pot Nest RPA.
  • Embodiment 1 One-pot Nest RPA reaction detects SARS-Cov2 nucleic acid E gene
  • SARS-Cov2 virus RNA samples were purchased from the national standard material (standard material number: GBW(E)091099).
  • the RPA reaction kit was purchased from the British TwistDX company, the product model is exo RT kit.
  • qPCR Real-time quantitative PCR
  • E-Primer F ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
  • E-Primer R AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
  • E-Primer F2 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3 spacer-3'
  • E-Primer R2 5'(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
  • RNA added is different (set as x), and the total volume of DEPC water plus RNA is 12.1 ⁇ L.
  • RPA reaction dry powder derived from the RPA reaction kit
  • RNA template 1 ⁇ L, 0.4 ⁇ L, 0.2 ⁇ L and 0 ⁇ L (100 copies RNA) of RNA template to the reaction system respectively.
  • the temperature of the metal bath was set to 42°C, and after 40 minutes of reaction, the fluorescence of the sample was detected under a blue light, and the sample was photographed and recorded.
  • SARS-Cov2 virus RNA samples were purchased from the national standard material (standard material number: GBW(E)091099.
  • the RPA reaction kit was purchased from the British TwistDX company, the product model is exo RT kit.
  • qPCR Real-time quantitative PCR
  • the primer sequences are as follows:
  • E-Primer F ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
  • E-Primer R AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
  • E-Primer F2 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3spacer-3'
  • E-Primer R2 5'-(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
  • the volume of primers added is different (set to x).
  • the total volume of DEPC water plus E-Primer F/R2 is 13.1 ⁇ L.
  • RPA reaction dry powder derived from the RPA reaction kit
  • E-Primer F2 and E-Primer R2 at final concentrations of 0.1 ⁇ M, 0.2 ⁇ M, 0.4 ⁇ M and 0.8 ⁇ M to the reaction system, respectively.
  • the temperature of the metal bath was set to 42 degrees Celsius, and the fluorescence was detected by photographing under a blue light after 40 minutes of reaction.
  • Embodiment 3 One-pot Nest RPA compares conventional RPA detection sensitivity
  • This example attempts to verify the detection sensitivity of One-pot Nest RPA, wherein, from the primer design, the difference between One-pot Nest RPA and other RPAs is shown in Figure 5.
  • SARS-Cov2 virus RNA samples were purchased from the national standard material (standard material number: GBW(E)091099).
  • the RPA reaction kit was purchased from the British TwistDX company, the product model is exo RT kit.
  • qPCR Real-time quantitative PCR
  • the primer sequences are as follows:
  • E-Primer F ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
  • E-Primer R AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
  • E-Primer F2 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3spacer-3'
  • E-Primer R2 5'-(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
  • RPA reaction dry powder derived from the RPA reaction kit
  • the detection sensitivity of One-pot Nest RPA is 10copies/50 ⁇ L, which is higher than 50copies/50 ⁇ L of conventional RPA reaction. And under the same conditions, the fluorescence intensity of One-pot Nest RPA is much higher than that of conventional RPA.
  • One-pot Nest RPA can detect 10 copies/50 ⁇ L of amplification products, while the conventional RPA reaction cannot detect obvious fluorescence due to the low fluorescence intensity.
  • Embodiment 4 One-pot Nest RPA detects SARS-Cov2 pseudovirus E gene sensitivity test
  • SARS-Cov2 pseudovirus samples were purchased from Fubaiao (Suzhou) Biotechnology Co., Ltd. (product number: FNV2596).
  • the RPA reaction kit was purchased from the British TwistDX company, the product model is exo RT kit.
  • Triton X-100 was purchased from sigma company, product number: T9284-100ML.
  • NP-40 was purchased from sigma company, the product number is: NP40S-500ML.
  • the primer sequences are as follows:
  • E-Primer F ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
  • E-Primer R AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
  • E-Primer F2 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3spacer-3'
  • E-Primer R2 5'-(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
  • RPA reaction dry powder derived from the RPA reaction kit
  • Embodiment 5 One-pot Nest RPA detects SARS-Cov2 pseudovirus O gene sensitivity test
  • SARS-Cov2 pseudovirus samples were purchased from Fubaiao (Suzhou) Biotechnology Co., Ltd. (product number: FNV2596).
  • the RPA reaction kit was purchased from the British TwistDX company, the product model is exo RT kit.
  • Triton X-100 was purchased from sigma company, product number: T9284-100ML.
  • NP-40 was purchased from sigma company, the product number is: NP40S-500ML.
  • the primer sequences are as follows:
  • O-Primer F TGTAGTTGTGATCAACTCCGCGAACCCATGCT (SEQ ID NO: 7)
  • O-Primer R TCTTCATGTTGGTAGTTAGAGAAAGTGTGTCT (SEQ ID NO: 8)
  • O-Primer F2 5'(SEQ ID NO:9)/i6FAMdT//idSp/g/iBHQ1dT/(SEQ ID NO:10)-C3 spacer-3'
  • O-Primer R2 5'(SEQ ID NO:11)/i6FAMdT//idSp//iBHQ1dT/(SEQ ID NO:12)-C3spacer-3'
  • RPA reaction dry powder derived from the RPA reaction kit
  • reaction temperature of the metal bath was set to 42 degrees Celsius, and after 40 minutes of reaction, the fluorescent signal was detected under a blue light and photographed.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to a one-step, rapid and high-sensitivity nested recombinase-polymerase amplification method, a rapid nucleic acid detection method, and a kit for the nested recombinase-polymerase amplification method. Compared with the two-step reaction of a traditional nested RPA, in the present invention, two pairs of primers are simultaneously added into a reaction system to complete the rapid amplification and/or detection of nucleic acid in one step by means of introducing a special modification method. In addition, a detection result can be judged by means of using the naked eye in cooperation with a 470 nm monochromatic LED lamp and an optical filter, and there is no need to open a reaction tube in the whole process.

Description

一种巢式重组酶-聚合酶扩增的方法及其应用A nested recombinase-polymerase amplification method and application thereof 技术领域technical field
本发明涉及生物技术领域，具体涉及一种一步法、快速、高灵敏度的巢式重组酶-聚合酶扩增的方法、以及核酸快速检测的方法、用于巢式重组酶-聚合酶扩增方法的试剂盒。The invention relates to the field of biotechnology, in particular to a one-step, rapid and highly sensitive nested recombinase-polymerase amplification method, a method for rapid nucleic acid detection, and a nested recombinase-polymerase amplification method the kit.
背景技术Background technique
重组酶-聚合酶扩增技术(Recombinase Polymerase Amplification，RPA)，被称为是可以替代PCR的核酸检测技术。最初由ASM scientific,Inc.于2003年申请专利(EP1499738B1)。此类扩增反应又被称为recombinase-aid amplification(RAA)，Multienzyme Isothermal Rapid Amplification(MIRA)和体温扩增技术(STAMP)。其基本原理都为重组酶与引物形成组合体(filament)，入侵到底物核酸template strand进行，对双链进行打开，结合到引物互补的部位；接着具有stranded displacement能力DNA聚合酶作用下，在引物3’端开始合成互补链。Displaced的单链由单链结合蛋白所结合，组装原双链的复合。以上扩增反应已经被商业化，用来检测目的片段和核酸，通常也配有标签probe来提高特异性。Recombinase-polymerase amplification technology (Recombinase Polymerase Amplification, RPA) is known as a nucleic acid detection technology that can replace PCR. Originally patented by ASM scientific, Inc. in 2003 (EP1499738B1). Such amplification reactions are also known as recombinase-aid amplification (RAA), Multienzyme Isothermal Rapid Amplification (MIRA) and body temperature amplification technology (STAMP). The basic principle is that the recombinase and the primer form a combination (filament), which invades the template strand of the substrate nucleic acid, opens the double strand, and binds to the complementary position of the primer; then, under the action of DNA polymerase with stranded displacement ability, under the action of the primer The 3' end begins to synthesize complementary strands. Displaced single chains are bound by single-chain binding proteins to assemble the original double-stranded complex. The above amplification reactions have been commercialized for the detection of target fragments and nucleic acids, and are usually also equipped with tag probes to improve specificity.
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，其基本原理为：重组酶与引物结合形成的蛋白-DNA复合物(filament)，入侵到底物核酸template strand进行，能在双链DNA中寻找同源序列，一旦引物定位了同源序列，就会发生链交换反应形成并启动DNA合成，在DNA聚合酶作用下，在引物3’端开始合成互补链，对模板上的目标区域进行指数式扩增。被置换的DNA链与SSB结合，防止进一步替换。在这个体系中，由两个相对的引物起始一个合成事件。RPA technology mainly relies on three enzymes: recombinase that can bind single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase. The basic principle is: the recombinase binds to the primer. The formed protein-DNA complex (filament) invades the substrate nucleic acid template strand and can search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequence, a strand exchange reaction will occur to form and initiate DNA synthesis. Under the action of DNA polymerase, the complementary strand starts to be synthesized at the 3' end of the primer, and the target region on the template is exponentially amplified. The displaced DNA strand binds to the SSB, preventing further displacement. In this system, a synthesis event is initiated by two opposing primers.
RPA检测技术的关键在于扩增引物和探针的设计。目前，荧光法RPA采用一步加入一对引物和一条探针，操作简单，灵敏度低，荧光强度弱。Nest RPA采用分两步分别加入外引物对和内引物对(含探针)，灵敏度高，操作复杂，中间涉及到产物转移，有产物 泄露污染风险。然而，为保证灵敏度高，达到无泄漏风险的前提下，简化步骤获得了本申请的One-pot Nest RPA，可以一步同时加入四条引物/探针，操作简单，无开盖泄露风险，灵敏度高，荧光强度高，可实现肉眼可视化检测。The key to RPA detection technology lies in the design of amplification primers and probes. At present, fluorescent RPA adopts one-step addition of a pair of primers and a probe, which is simple in operation, low in sensitivity and weak in fluorescence intensity. Nest RPA uses two steps to add the outer primer pair and the inner primer pair (including the probe) respectively, which has high sensitivity and complicated operation. The intermediate involves product transfer, and there is a risk of product leakage and contamination. However, in order to ensure high sensitivity and no leakage risk, the One-pot Nest RPA of this application was obtained by simplifying the steps. Four primers/probes can be added simultaneously in one step. The operation is simple, there is no risk of opening the lid, and the sensitivity is high. The high fluorescence intensity enables visual detection with the naked eye.
发明内容SUMMARY OF THE INVENTION
本发明的目的是为了克服现有技术的不足，提供一种一步法、快速、高灵敏度的巢式重组酶-聚合酶扩增的方法(命名为One-pot Nest RPA)、以及核酸快速检测的方法、用于巢式重组酶-聚合酶扩增方法的试剂盒。可快速、高灵敏度的对核酸进行检测，反应过程无需配制两个RPA反应试剂，而且，两对引物可以一步同时加入反应体系中，避免开盖泄露风险，以及减少操作的复杂性。The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a one-step, fast, high-sensitivity nested recombinase-polymerase amplification method (named One-pot Nest RPA), and a nucleic acid rapid detection method Methods, kits for nested recombinase-polymerase amplification methods. Nucleic acid can be detected quickly and with high sensitivity. There is no need to prepare two RPA reaction reagents during the reaction process. Moreover, two pairs of primers can be added to the reaction system at the same time in one step, avoiding the risk of opening the lid and leaking, and reducing the complexity of the operation.
本发明的第一方面，提供了一种巢式重组酶-聚合酶扩增的方法，所述的方法包括：A first aspect of the present invention provides a nested recombinase-polymerase amplification method, the method comprising:
配制反应体系，加入1)第一引物对，和，2)第二引物对和/或探针；Prepare a reaction system, add 1) a first primer pair, and, 2) a second primer pair and/or probe;
进行RPA或RT-RPA反应扩增；Perform RPA or RT-RPA reaction amplification;
其中，第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。Wherein, the second primer pair is located in the template obtained by the first primer pair RPA or RT-RPA reaction amplification, and the second primer pair comprises the /idSp/ restriction site and the modification that prevents the 3' end amplification .
优选的，所述的1)第一引物对，和，2)第二引物对和/或探针，为一步加入。Preferably, the 1) first primer pair, and, 2) the second primer pair and/or probe are added in one step.
该方法中，两对引物同时加入，靠外面的第一引物对先进行反应，通过Exo(exonuclease III)或nfo(Endonuclease IV)等酶切第二引物对，产生可以扩增的3’OH末端，继续进行扩增反应。In this method, two pairs of primers are added at the same time, the first primer pair on the outside reacts first, and the second primer pair is cut by enzymes such as Exo (exonuclease III) or nfo (Endonuclease IV) to generate 3'OH ends that can be amplified. , continue the amplification reaction.
优选的，所述的阻止3’端扩增的修饰可以为任何不适合DNA聚合酶扩增的3’端修饰。优选3’端OH被化学阻碍的非自然脱氧核苷酸及其修饰或者与3’端互补的DNA-OH。其中，所述的非自然脱氧核苷酸例如羟甲基叠氮。Preferably, the modification that prevents 3' end amplification can be any 3' end modification that is not suitable for DNA polymerase amplification. Preference is given to unnatural deoxynucleotides whose 3'-terminal OH is chemically blocked and their modifications or DNA-OH complementary to the 3'-terminal. Among them, the unnatural deoxynucleotides such as hydroxymethyl azide.
在本发明的一个具体实施方式中，所述的阻止3’端扩增的修饰选自羟甲基叠氮、C18、C12、ddNTP、RNA、PNA或C3 spacer。In a specific embodiment of the present invention, the modification that prevents 3' end amplification is selected from hydroxymethyl azide, C18, C12, ddNTP, RNA, PNA or C3 spacer.
优选的，所述的RPA或RT-RPA反应中添加能够识别/idSp/的核酸内切酶，所述的核酸内切酶优选为Exo(exonuclease III)或nfo(Endonuclease IV)。Preferably, an endonuclease capable of recognizing /idSp/ is added to the RPA or RT-RPA reaction, and the endonuclease is preferably Exo (exonuclease III) or nfo (Endonuclease IV).
优选的，所述的第二引物对和/或探针还包含荧光报告基团和荧光淬灭基团的修饰，所述的荧光报告基团选自FAM(6-carboxy-fluorescein，6-羧基荧光素)、HEX (hexachlorofluorescein，六氯-6-甲基荧光素)、TET(Tetrachlorofluorescein，四氯-6-羧基荧光素)、JOE(2,7-二甲基-4,5-二氯-6-羧基荧光素)、Cy3(Indodicarbocyanine)或TAMRA(Carboxytetramethylrhodamine，6-羧基四甲基若丹明)，所述的荧光淬灭基团选自DABCYL(4-(4-恶烷氨基苯偶氮)苯甲酸)、BHQ1(Black Hole Quencher 1)、BHQ2(Black Hole Quencher 2)、BHQ3(Black Hole Quencher 3)Eclipse或MGB(Minor Groove Binder，小沟结合)。Preferably, the second primer pair and/or the probe further comprise modification of a fluorescent reporter group and a fluorescence quenching group, and the fluorescent reporter group is selected from FAM (6-carboxy-fluorescein, 6-carboxy-fluorescein, 6-carboxy-fluorescein Fluorescein), HEX (hexachlorofluorescein, hexachloro-6-methylfluorescein), TET (Tetrachlorofluorescein, tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethyl-4,5-dichloro- 6-carboxyfluorescein), Cy3 (Indodicarbocyanine) or TAMRA (Carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine), the fluorescence quenching group is selected from DABCYL (4-(4-oxaneaminophenylazo) ) benzoic acid), BHQ1 (Black Hole Quencher 1), BHQ2 (Black Hole Quencher 2), BHQ3 (Black Hole Quencher 3) Eclipse or MGB (Minor Groove Binder, minor groove binding).
优选的，所述的荧光报告基团修饰在第二引物对上游引物序列5′端碱基数25-35bp的位置上。优选为29-31bp的位置上。Preferably, the fluorescent reporter group is modified at a position of 25-35 bp bases at the 5' end of the upstream primer sequence of the second primer pair. It is preferably at the position of 29-31 bp.
优选的，所述的荧光淬灭基团修饰在第二引物对下游引物序列离3′端碱基数10-20bp的位置上。优选为13-18bp的位置上。Preferably, the fluorescence quenching group is modified at a position 10-20 bp away from the 3' end of the downstream primer sequence of the second primer pair. It is preferably at the position of 13-18 bp.
优选的，所述的荧光报告基团与荧光淬灭基团之间间隔1-3个碱基。优选间隔的碱基包含四氢呋喃(THF)。Preferably, the fluorescent reporter group and the fluorescent quenching group are separated by 1-3 bases. Preferred spacer bases comprise tetrahydrofuran (THF).
优选的，所述的/idSp/修饰在荧光报告基团与荧光淬灭基团之间。Preferably, the /idSp/ modification is between the fluorescent reporter group and the fluorescent quencher group.
优选的，所述的第二引物对和/或探针中的idSp被nfo或exo切割后，产生可以扩增的3’OH末端，继续进行扩增反应。Preferably, after the idSp in the second primer pair and/or probe is cleaved by nfo or exo, a 3'OH end that can be amplified is generated, and the amplification reaction is continued.
优选的，所述的第二引物对与所述第一引物对不重叠或小于10bp的重叠。Preferably, the second primer pair and the first primer pair do not overlap or overlap less than 10 bp.
优选的，所述的RPA或RT-RPA反应分别可用于DNA或RNA的扩增。Preferably, the RPA or RT-RPA reaction can be used for DNA or RNA amplification, respectively.
优选的，所述的RPA或RT-RPA反应时间为5-40min，优选为30min。Preferably, the reaction time of the RPA or RT-RPA is 5-40 min, preferably 30 min.
优选的，所述的RPA或RT-RPA反应温度为35℃-45℃，优选的是37℃-42℃。Preferably, the reaction temperature of the RPA or RT-RPA is 35°C-45°C, preferably 37°C-42°C.
优选的，所述的RPA或RT-RPA反应在反应试剂中进行，所述的反应试剂包括第一引物对、第二引物对、缓冲液和/或RPA反应物。Preferably, the RPA or RT-RPA reaction is carried out in a reaction reagent, and the reaction reagent includes a first primer pair, a second primer pair, a buffer and/or an RPA reactant.
所述的RPA反应物可以是商业化的任意的RPA反应所需的试剂或试剂盒等，也可自行实验室进行配制获得的RPA反应所需的试剂，其可以是液体，也可以是干粉。优选包括重组酶、聚合酶以及单链结合蛋白等等。The RPA reactants can be any commercial reagents or kits required for the RPA reaction, or the reagents required for the RPA reaction obtained by the laboratory preparation, which can be liquid or dry powder. Preferred include recombinases, polymerases, and single-chain binding proteins, among others.
优选的，所述的反应试剂还包含选自醋酸镁、DEPC水、缓冲液、PEG和/或裂解液。裂解液可以是商业化的任意的裂解液试剂或试剂盒等，也可自行实验室进行配制获得的裂解液。Preferably, the reaction reagent further comprises magnesium acetate, DEPC water, buffer, PEG and/or lysate. The lysate can be any commercial lysate reagent or kit, etc., or the lysate can be prepared by oneself in the laboratory.
优选的，所述的方法还包括观察检测结果的步骤。进一步优选的，所述的观察检测结果的步骤包括使用470nm LED灯和/或滤光片，然后通过裸眼判断检测结果，全 程无需打开反应管。在本发明的一个具体实施方式中，所述的LED灯为单色LED灯，其可以为任何颜色的荧光灯，优选为红光灯、黄光灯、蓝光灯、绿光灯或白光灯。Preferably, the method further includes the step of observing the detection result. Further preferably, the described step of observing the detection result comprises using a 470nm LED lamp and/or a filter, and then judging the detection result by naked eyes, without opening the reaction tube in the whole process. In a specific embodiment of the present invention, the LED lamp is a single-color LED lamp, which can be a fluorescent lamp of any color, preferably a red light, yellow light, blue light, green light or white light.
本发明所述的One-pot Nest RPA反应，外面的一对引物是常规的RPA扩增引物，靠里侧的一对引物，设计上与RPA常用的probe类似(参见图5)，都带有/idSp/酶切位点和3’端的C3 spacer等阻止3端扩增的修饰。因此，反应起始由外面的一对引物(one-pot 1和one-pot4)引发扩增，扩增的双链产物与one-pot2和one-pot3重组，引起/idSp/被Exo等内切酶酶切，产生可以扩增的3’OH末端，相当于内侧反应的引物被激活，引发巢式反应。因此可以提高反应速度，增加灵敏度。内侧反应的probe如果加入了FAM-BHQ，还可以作为荧光的探针，因此既可以作为探针，又可以作为内部巢式引物。此外，内部巢式引物的如此设计，由于两条引物的3’端都被block住了，因此可以避免在没有模板的情况下，引物互搭造成的特异性扩增，以及由此带来的引物消耗。In the One-pot Nest RPA reaction of the present invention, the outer pair of primers are conventional RPA amplification primers, and the inner pair of primers are similar in design to the commonly used probes for RPA (see Figure 5), both with Modifications such as /idSp/ restriction site and C3 spacer at the 3' end that prevent amplification of the 3 end. Therefore, the reaction is initiated by a pair of primers on the outside (one-pot 1 and one-pot4) to initiate amplification, and the amplified double-stranded product recombines with one-pot2 and one-pot3, causing /idSp/ to be endo-cut by Exo, etc. Enzyme cleavage produces a 3'OH end that can be amplified, and the primer equivalent to the inner reaction is activated, triggering a nested reaction. Therefore, the reaction speed can be increased and the sensitivity can be increased. If FAM-BHQ is added to the probe of the inner reaction, it can also be used as a fluorescent probe, so it can be used as both a probe and an inner nested primer. In addition, the inner nested primers are designed in this way, because the 3' ends of the two primers are blocked, so it can avoid the specific amplification caused by the overlapping of the primers in the absence of a template, and the resulting Primer consumption.
本发明的第二方面，提供了一种核酸快速检测的方法，所述的方法包括：A second aspect of the present invention provides a method for rapid nucleic acid detection, the method comprising:
A)获得待检测核酸；部分样品可进行稀释，也可不进行稀释直接扩增。A) Obtain the nucleic acid to be detected; some samples can be diluted or directly amplified without dilution.
B)使用上述的巢式重组酶-聚合酶扩增的方法扩增步骤A)的核酸，获得反应产物；B) using the above-mentioned nested recombinase-polymerase amplification method to amplify the nucleic acid of step A) to obtain a reaction product;
C)检测反应产物的荧光。C) Detect the fluorescence of the reaction product.
优选的，所述的方法还可以将反应产物稀释后插入胶体金试纸条进行胶体金显色。具体为：将反应产物加入到DEPC水中混合均匀进行稀释，吸取稀释产物置于胶体金点样孔中显色。Preferably, in the method, the reaction product can also be diluted and inserted into a colloidal gold test strip for colloidal gold color development. Specifically, the reaction product is added into DEPC water, mixed evenly for dilution, and the diluted product is drawn and placed in a colloidal gold spotting hole for color development.
第二引物对被切割后的带有FAM(或FITC)的产物与反应带有biotin的引物组成引物对，参与扩增并形成一端带有FAM(或FITC)，一端带有biotin的双链DNA，此时在胶体金层析中通过夹心法被显色。此时胶体层析试纸可设置为：胶体金表面为链霉亲和素(或者FAM/FITC抗体)，第一条线T线为FAM/FITC抗体(或者链霉亲和素抗体)，第二条线C线为链霉亲和素抗体(或者anti-IgG抗体)。The product with FAM (or FITC) after the second primer pair is cleaved and the primer with biotin in the reaction form a primer pair, which participates in amplification and forms a double-stranded DNA with FAM (or FITC) at one end and biotin at one end , and was developed by the sandwich method in colloidal gold chromatography. At this time, the colloidal chromatography test paper can be set as: the surface of colloidal gold is streptavidin (or FAM/FITC antibody), the first line T line is FAM/FITC antibody (or streptavidin antibody), the second line is streptavidin (or FAM/FITC antibody). Line C is the streptavidin antibody (or anti-IgG antibody).
优选的，所述的检测反应产物的荧光包括荧光探针检测步骤。Preferably, the detection of the fluorescence of the reaction product includes a fluorescent probe detection step.
优选的，所述的方法还包括观察检测结果的步骤。进一步优选的，所述的观察检测结果的步骤包括使用470nm LED灯和/或滤光片，然后通过裸眼判断检测结果，全 程无需打开反应管。在本发明的一个具体实施方式中，所述的LED灯为单色LED灯，其可以为任何颜色的荧光灯，优选为红光灯、黄光灯、蓝光灯、绿光灯或白光灯。Preferably, the method further includes the step of observing the detection result. Further preferably, the described step of observing the detection result comprises using a 470nm LED lamp and/or a filter, and then judging the detection result by naked eyes, without opening the reaction tube in the whole process. In a specific embodiment of the present invention, the LED lamp is a single-color LED lamp, which can be a fluorescent lamp of any color, preferably a red light, yellow light, blue light, green light or white light.
本发明的第三方面，提供了一种试剂盒，所述的试剂盒包含上述任一所述方法的第一引物对、第二引物对、探针、反应试剂、胶体金试纸条和/或其组合。A third aspect of the present invention provides a kit comprising the first primer pair, the second primer pair, the probe, the reaction reagent, the colloidal gold test strip and/or any of the above-mentioned methods. or a combination thereof.
本发明的第四方面，提供了一种核酸的检测或扩增的试剂盒，所述的试剂盒中包含第一引物对、第二引物对、缓冲液和/或RPA反应物，所述的第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。The fourth aspect of the present invention provides a nucleic acid detection or amplification kit, the kit comprises a first primer pair, a second primer pair, a buffer and/or an RPA reactant, and the The second primer pair is located in the template obtained by the reaction of the first primer pair RPA or RT-RPA, and the second primer pair includes an /idSp/ restriction site and a modification that prevents amplification of the 3' end.
本发明的第五方面，提供了一种用于巢式重组酶-聚合酶扩增方法的试剂盒，所述的试剂盒中包含第一引物对、第二引物对、缓冲液和/或RPA反应物，所述的第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。A fifth aspect of the present invention provides a kit for a nested recombinase-polymerase amplification method, the kit comprising a first primer pair, a second primer pair, a buffer and/or RPA Reaction, the second primer pair is located in the template obtained by the first primer pair RPA or RT-RPA reaction amplification, the second primer pair comprises /idSp/ restriction site and prevents the 3' end Amplified modifications.
优选的，所述的RPA反应物包含能够识别/idSp/的核酸内切酶。Preferably, the RPA reactant comprises an endonuclease capable of recognizing /idSp/.
优选的，所述的试剂盒中还包含选自醋酸镁、DEPC水、缓冲液、PEG和/或裂解液。Preferably, the kit further comprises magnesium acetate, DEPC water, buffer, PEG and/or lysate.
本发明的第六方面，提供了上述任一所述的第一引物对和/或第二引物对在制备核酸的扩增和/或检测的试剂盒中的应用。The sixth aspect of the present invention provides the application of any one of the first primer pair and/or the second primer pair described above in preparing a kit for nucleic acid amplification and/or detection.
本发明的第七方面，提供了上述的方法、上述的试剂盒在病毒核酸扩增或检测中的应用。The seventh aspect of the present invention provides the application of the above method and the above kit in viral nucleic acid amplification or detection.
本发明的第八方面，提供了第一引物对和/或第二引物对在病毒核酸扩增或检测中的应用，第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。The eighth aspect of the present invention provides the application of a first primer pair and/or a second primer pair in viral nucleic acid amplification or detection, where the second primer pair is located in the first primer pair RPA or RT-RPA reaction amplification Within the template obtained, the second primer pair contains an /idSp/ restriction site and a modification that prevents amplification of the 3' end.
优选的，所述的病毒为冠状病毒，进一步优选为SARS-Cov2病毒或SARS-Cov2假病毒。Preferably, the virus is a coronavirus, more preferably a SARS-Cov2 virus or a SARS-Cov2 pseudovirus.
当检测的病毒核酸为SARS-Cov2病毒E基因RNA时，所述第一引物对为：When the viral nucleic acid detected is SARS-Cov2 virus E gene RNA, the first primer pair is:
E-Primer F：ATGTACTCATTCGTTTCGGAAGAGACAGGE-Primer F: ATGTACTCATTCGTTTCGGAAGAGACAGG
E-Primer R：AGACCAGAAGATCAGGAACTCTAGAAGAAE-Primer R: AGACCAGAAGATCAGGAACTCTAGAAGAA
第二引物对为：The second primer pair is:
E-Primer F2：5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’E-Primer F2: 5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’
E-Primer R2：5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3 spacer-3’E-Primer R2: 5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3 spacer-3’
当检测的病毒核酸为SARS-Cov2病毒O基因RNA时，所述第一引物对为：When the detected viral nucleic acid is SARS-Cov2 virus O gene RNA, the first primer pair is:
O-Primer F：TGTAGTTGTGATCAACTCCGCGAACCCATGCTO-Primer F: TGTAGTTGTGATCAACTCCGCGAACCCATGCT
O-Primer R：TCTTCATGTTGGTAGTTAGAGAAAGTGTGTCTO-Primer R: TCTTCATGTTGGTAGTTAGAGAAAGTGTGTCT
第二引物对为：The second primer pair is:
O-Primer F2：5’-AGCCCGTCTTACACCGTGCGGCACAGGCAC/i6FAMdT//idSp/g/iBHQ1dT/ACTGATGTCGTAT-C3 spacer-3’O-Primer F2: 5’-AGCCCGTCTTACACCGTGCGGCACAGGCAC/i6FAMdT//idSp/g/iBHQ1dT/ACTGATGTCGTAT-C3 spacer-3’
O-Primer R2：5’-CTTGGAAGCGACAACAATTAGTTTTTAGGAA/i6FAMdT//idSp//iBHQ1dT/AGCAAAACCAGCTA-C3 spacer-3’O-Primer R2: 5’-CTTGGAAGCGACAACAATTAGTTTTTAGGAA/i6FAMdT//idSp//iBHQ1dT/AGCAAAACCAGCTA-C3 spacer-3’
本申请所述的“一步”包括同时但不限于同时，包括反应前或反应过程中的一次性或者只开盖一次的加样过程。优选为RPA或RT-RPA反应开始前间隔一段时间、按顺序或者同时加入反应体系。The "one-step" described in this application includes simultaneous but not limited to simultaneous, including a one-time or only one-time sample adding process before or during the reaction. Preferably, the RPA or RT-RPA reaction is added to the reaction system at an interval, sequentially or simultaneously before the start of the reaction.
本申请所述的“/idSp/”为internal D-spacer，也可以叫做/thf/或abasic，其作为一种碱基修饰，代表在寡核苷酸内部添加一个四氢呋喃(THF)间隔基。The "/idSp/" described in this application is an internal D-spacer, which can also be called /thf/ or abasic. As a base modification, it represents the addition of a tetrahydrofuran (THF) spacer inside the oligonucleotide.
本申请所述的“核酸”由核苷酸单体组成，而核苷酸单体由五碳糖、磷酸基和含氮碱基组成，其中，含氮碱基选自A、T、C、G或U。优选的，所述的核酸可以为天然的或经过修饰的DNA或RNA。在本发明的一个具体实施方式中，所述的核酸为RNA。所述的核酸可以来自任何包含核酸的生物体中，所述的生物体包括真核生物和原核生物，具体的选自人、非人动物、植物、细菌、蓝藻、支原体、衣原体、真菌、病毒等等。在本发明的一个具体实施方式中，所述的核酸来自病毒，优选为冠状病毒，进一步优选为SARS-Cov2病毒或SARS-Cov2假病毒。The "nucleic acid" described in this application is composed of nucleotide monomers, and the nucleotide monomers are composed of five-carbon sugars, phosphate groups and nitrogen-containing bases, wherein the nitrogen-containing bases are selected from A, T, C, G or U. Preferably, the nucleic acid can be natural or modified DNA or RNA. In a specific embodiment of the present invention, the nucleic acid is RNA. Described nucleic acid can come from any organism that contains nucleic acid, described organism includes eukaryotic organism and prokaryotic organism, and is specifically selected from human, non-human animal, plant, bacterium, cyanobacteria, mycoplasma, chlamydia, fungus, virus and many more. In a specific embodiment of the present invention, the nucleic acid is from a virus, preferably a coronavirus, more preferably a SARS-Cov2 virus or a SARS-Cov2 pseudovirus.
附图说明Description of drawings
以下，结合附图来详细说明本发明的实施例，其中：Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:
图1：One-pot Nest RPA荧光扩增曲线，其中，NC为阴性对照，RFU(relative fluorescence units)为相对荧光强度。Figure 1: One-pot Nest RPA fluorescence amplification curve, where NC is the negative control and RFU (relative fluorescence units) is the relative fluorescence intensity.
图2：One-pot Nest RPA蓝光激发荧光图。Figure 2: Blue light excitation fluorescence image of One-pot Nest RPA.
图3：One-pot Nest RPA探针浓度筛选荧光扩增曲线，其中，RFU为相对荧光强度。Figure 3: One-pot Nest RPA probe concentration screening fluorescence amplification curve, where RFU is the relative fluorescence intensity.
图4：One-pot Nest RPA探针浓度筛选蓝光激发荧光图。Figure 4: One-pot Nest RPA probe concentration screening blue light excitation fluorescence map.
图5：One-pot Nest RPA反应引物设计与其它RPA反应引物设计的对比示意图。Figure 5: Schematic diagram of the comparison of primer design for One-pot Nest RPA reaction with that of other RPA reactions.
图6：One-pot Nest RPA(one pot)对比常规RPA(single RPA)对核酸的检测灵敏度，其中，RFU为相对荧光强度。Figure 6: The detection sensitivity of One-pot Nest RPA (one pot) versus conventional RPA (single RPA) for nucleic acid detection, where RFU is the relative fluorescence intensity.
图7：One-pot Nest RPA(one pot)对比常规RPA(single RPA)的蓝光激发荧光。Figure 7: Blue light-excited fluorescence of One-pot Nest RPA (one pot) versus conventional RPA (single RPA).
图8：One-Pot Nest RPA检测SARS-Cov2假病毒E基因灵敏度荧光图。Figure 8: Fluorescence graph of the sensitivity of One-Pot Nest RPA to detect SARS-Cov2 pseudovirus E gene.
图9：One-pot Nest RPA检测SARS-Cov2假病毒O基因荧光扩增曲线。Figure 9: Fluorescence amplification curve of SARS-Cov2 pseudovirus O gene detected by One-pot Nest RPA.
图10：One-pot Nest RPA检测SARS-Cov2假病毒O基因蓝光灯荧光图。Figure 10: Blue light fluorescence image of SARS-Cov2 pseudovirus O gene detected by One-pot Nest RPA.
具体实施方式Detailed ways
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.
实施例1 One-pot Nest RPA反应检测SARS-Cov2核酸E基因 Embodiment 1 One-pot Nest RPA reaction detects SARS-Cov2 nucleic acid E gene
1.试剂仪器准备1. Reagent instrument preparation
SARS-Cov2病毒RNA样品购自国家标准物质(标准物质编号：GBW(E)091099)。SARS-Cov2 virus RNA samples were purchased from the national standard material (standard material number: GBW(E)091099).
RPA反应试剂盒购自英国TwistDX公司，产品型号为
Figure PCTCN2021076773-appb-000001
exo RT kit。 The RPA reaction kit was purchased from the British TwistDX company, the product model is
Figure PCTCN2021076773-appb-000001
exo RT kit.
实时定量荧光PCR(qPCR)仪购自美国伯乐公司，仪器型号为Bio-Rad CFX96。Real-time quantitative PCR (qPCR) instrument was purchased from Bio-Rad Company, USA, and the instrument model was Bio-Rad CFX96.
2.实验步骤2. Experimental steps
1)将SARS-Cov2病毒RNA按照E基因浓度，稀释至100copies/μL(每μL含100拷贝的RNA)。1) Dilute the SARS-Cov2 virus RNA to 100 copies/μL according to the E gene concentration (each μL contains 100 copies of RNA).
2)将SARS-Cov2病毒E基因引物用DEPC水稀释至10μM。引物序列如下：2) Dilute the SARS-Cov2 virus E gene primer to 10 μM with DEPC water. The primer sequences are as follows:
E-Primer F：ATGTACTCATTCGTTTCGGAAGAGACAGG(SEQ ID NO：1)E-Primer F: ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
E-Primer R：AGACCAGAAGATCAGGAACTCTAGAAGAA(SEQ ID NO：2)E-Primer R: AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
E-Primer F2：5’(SEQ ID NO：3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO：4)-C3 spacer-3’E-Primer F2: 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3 spacer-3'
5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’
E-Primer R2：5’(SEQ ID NO：5)/idSp/(SEQ ID NO：6)-C3 spacer-3’E-Primer R2: 5'(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3 spacer-3’5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3 spacer-3’
3)RPA反应体系配置3) RPA reaction system configuration
Figure PCTCN2021076773-appb-000002
Figure PCTCN2021076773-appb-000002
注：根据实验分组不同，RNA加入的体积不同(设置为x)，DEPC水加RNA的总体积是12.1μL。Note: According to different experimental groups, the volume of RNA added is different (set as x), and the total volume of DEPC water plus RNA is 12.1 μL.
上述成分配置好后，直接加入到RPA反应干粉(来源于RPA反应试剂盒)中形成RPA反应体系。After the above components are prepared, they are directly added to the RPA reaction dry powder (derived from the RPA reaction kit) to form an RPA reaction system.
4)分别在反应体系中加入1μL、0.4μL、0.2μL、0μL(100copies RNA)RNA模板。设置金属浴温度为42℃，反应40分钟后，在蓝光灯下检测样品荧光，拍照记录。4) Add 1 μL, 0.4 μL, 0.2 μL and 0 μL (100 copies RNA) of RNA template to the reaction system respectively. The temperature of the metal bath was set to 42°C, and after 40 minutes of reaction, the fluorescence of the sample was detected under a blue light, and the sample was photographed and recorded.
5)分别在反应体系中加入1μL、0.4μL、0.2μL、0μL(100copies RNA)RNA模板。 设置qPCR仪器温度为42℃，荧光通道为FAM，每分钟记录一次荧光强度值，共记录30min。5) Add 1 μL, 0.4 μL, 0.2 μL, and 0 μL (100 copies RNA) of RNA template to the reaction system, respectively. Set the temperature of the qPCR instrument to 42 °C, the fluorescence channel to FAM, and record the fluorescence intensity value every minute for a total of 30 min.
3.实验结论3. Experimental conclusion
1)实验结果如图1，图2所示。1) The experimental results are shown in Figure 1 and Figure 2.
2)qPCR仪结果显示，One-pot Nest RPA可以检测20copies E基因核酸，且荧光强度随拷贝数呈梯度变化。2) The results of the qPCR instrument showed that the One-pot Nest RPA could detect the nucleic acid of the 20copies E gene, and the fluorescence intensity changed in a gradient with the number of copies.
3)荧光拍照结果显示，One-pot Nest RPA可以在40min内检测出20copies E基因核酸扩增产物。3) The results of fluorescence photography show that One-pot Nest RPA can detect 20copies E gene nucleic acid amplification products within 40 minutes.
实施例2 One-pot Nest RPA探针浓度筛选Example 2 One-pot Nest RPA Probe Concentration Screening
1、试剂仪器准备1. Preparation of reagents and instruments
SARS-Cov2病毒RNA样品购自国家标准物质(标准物质编号：GBW(E)091099。SARS-Cov2 virus RNA samples were purchased from the national standard material (standard material number: GBW(E)091099.
RPA反应试剂盒购自英国TwistDX公司，产品型号为
Figure PCTCN2021076773-appb-000003
exo RT kit。 The RPA reaction kit was purchased from the British TwistDX company, the product model is
Figure PCTCN2021076773-appb-000003
exo RT kit.
实时定量荧光PCR(qPCR)仪购自美国伯乐公司，仪器型号为Bio-Rad CFX96。Real-time quantitative PCR (qPCR) instrument was purchased from Bio-Rad Company, USA, and the instrument model was Bio-Rad CFX96.
2、实验步骤：2. Experimental steps:
1)将SARS-Cov2病毒RNA按照E基因浓度，稀释至20copies/μL。1) Dilute the SARS-Cov2 virus RNA to 20 copies/μL according to the E gene concentration.
2)将SARS-Cov2病毒E基因引物用DEPC水稀释至10μM。2) Dilute the SARS-Cov2 virus E gene primer to 10 μM with DEPC water.
引物序列如下：The primer sequences are as follows:
E-Primer F：ATGTACTCATTCGTTTCGGAAGAGACAGG(SEQ ID NO：1)E-Primer F: ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
E-Primer R：AGACCAGAAGATCAGGAACTCTAGAAGAA(SEQ ID NO：2)E-Primer R: AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
E-Primer F2：5’(SEQ ID NO：3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO：4)-C3spacer-3’E-Primer F2: 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3spacer-3'
5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’
E-Primer R2：5’-(SEQ ID NO：5)/idSp/(SEQ ID NO：6)-C3 spacer-3’E-Primer R2: 5'-(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3spacer-3’5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3spacer-3’
3)RPA反应体系配置3) RPA reaction system configuration
Figure PCTCN2021076773-appb-000004
Figure PCTCN2021076773-appb-000004
注：根据实验分组不同，引物加入的体积不同(设置为x)，DEPC水加E-Primer F/R2的总体积是13.1μL。Note: According to different experimental groups, the volume of primers added is different (set to x). The total volume of DEPC water plus E-Primer F/R2 is 13.1 μL.
上述成分配置好后，直接加入到RPA反应干粉(来源于RPA反应试剂盒)中形成RPA反应体系。After the above components are prepared, they are directly added to the RPA reaction dry powder (derived from the RPA reaction kit) to form an RPA reaction system.
4)在反应体系中分别加入终浓度为0.1μM，0.2μM，0.4μM，0.8μM的E-Primer F2和E-Primer R2。设置金属浴的温度为42摄氏度，反应40分钟后在蓝光灯下拍照检测荧光。4) Add E-Primer F2 and E-Primer R2 at final concentrations of 0.1 μM, 0.2 μM, 0.4 μM and 0.8 μM to the reaction system, respectively. The temperature of the metal bath was set to 42 degrees Celsius, and the fluorescence was detected by photographing under a blue light after 40 minutes of reaction.
5)在反应体系中分别加入终浓度为0.1μM，0.2μM，0.4μM，0.8μM的E-Primer F2和E-Primer R2。设置qPCR仪的反应温度为42摄氏度，荧光通道为FAM，每2分钟记录一次荧光值，共记录60分钟。5) Add E-Primer F2 and E-Primer R2 at final concentrations of 0.1 μM, 0.2 μM, 0.4 μM and 0.8 μM to the reaction system, respectively. The reaction temperature of the qPCR instrument was set to 42 degrees Celsius, the fluorescence channel was FAM, and the fluorescence value was recorded every 2 minutes for a total of 60 minutes.
3、实验结论3. Experimental conclusion
1)实验结果如图3，图4所示。1) The experimental results are shown in Figure 3 and Figure 4.
2)0.4μM的探针浓度具有最佳反应强度。2) The probe concentration of 0.4 μM has the best response intensity.
实施例3 One-pot Nest RPA对比常规RPA检测灵敏度 Embodiment 3 One-pot Nest RPA compares conventional RPA detection sensitivity
本实施例试图验证One-pot Nest RPA的检测灵敏度，其中，从引物设计上，One-pot Nest RPA与其他RPA的区别参见图5。This example attempts to verify the detection sensitivity of One-pot Nest RPA, wherein, from the primer design, the difference between One-pot Nest RPA and other RPAs is shown in Figure 5.
1、试剂仪器准备1. Preparation of reagents and instruments
SARS-Cov2病毒RNA样品购自国家标准物质(标准物质编号：GBW(E)091099)。SARS-Cov2 virus RNA samples were purchased from the national standard material (standard material number: GBW(E)091099).
RPA反应试剂盒购自英国TwistDX公司，产品型号为
Figure PCTCN2021076773-appb-000005
exo RT kit。 The RPA reaction kit was purchased from the British TwistDX company, the product model is
Figure PCTCN2021076773-appb-000005
exo RT kit.
实时定量荧光PCR(qPCR)仪购自美国伯乐公司，仪器型号为Bio-Rad CFX96。Real-time quantitative PCR (qPCR) instrument was purchased from Bio-Rad Company, USA, and the instrument model was Bio-Rad CFX96.
2、实验步骤：2. Experimental steps:
1)将SARS-Cov2病毒RNA按照E基因浓度，稀释至20copies/μL。1) Dilute the SARS-Cov2 virus RNA to 20 copies/μL according to the E gene concentration.
2)将SARS-Cov2病毒E基因引物用DEPC水稀释至10μM。2) Dilute the SARS-Cov2 virus E gene primer to 10 μM with DEPC water.
引物序列如下：The primer sequences are as follows:
E-Primer F：ATGTACTCATTCGTTTCGGAAGAGACAGG(SEQ ID NO：1)E-Primer F: ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
E-Primer R：AGACCAGAAGATCAGGAACTCTAGAAGAA(SEQ ID NO：2)E-Primer R: AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
E-Primer F2：5’(SEQ ID NO：3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO：4)-C3spacer-3’E-Primer F2: 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3spacer-3'
5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/GTGCGTACTGCTG-C3 spacer-3’
E-Primer R2：5’-(SEQ ID NO：5)/idSp/(SEQ ID NO：6)-C3 spacer-3’E-Primer R2: 5'-(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3spacer-3’5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3spacer-3’
3)One-pot Nest RPA反应体系配制3) Preparation of One-pot Nest RPA reaction system
Figure PCTCN2021076773-appb-000006
Figure PCTCN2021076773-appb-000006
Figure PCTCN2021076773-appb-000007
Figure PCTCN2021076773-appb-000007
上述成分配置好后，直接加入到RPA反应干粉(来源于RPA反应试剂盒)中形成RPA反应体系。After the above components are prepared, they are directly added to the RPA reaction dry powder (derived from the RPA reaction kit) to form an RPA reaction system.
4)常规RPA反应体系配制4) Preparation of conventional RPA reaction system
Figure PCTCN2021076773-appb-000008
Figure PCTCN2021076773-appb-000008
5)在RPA反应体系中分别加入100copies，50copies，10copies，5copies，0copy模板，震荡混匀。设置金属浴温度为42℃，加热40min。在蓝光灯下观察反应产物荧光强度，并拍照。5) Add 100copies, 50copies, 10copies, 5copies, and 0copy template to the RPA reaction system, respectively, and shake and mix. Set the temperature of the metal bath to 42 °C and heat for 40 min. The fluorescence intensity of the reaction product was observed under blue light and photographed.
6)在RPA反应体系中分别加入100copies，50copies，10copies，5copies，0copy模板， 震荡混匀。设置qPCR仪反应温度为42℃，检测通道为FAM。每2分钟记录一次荧光值，共记录60分钟。6) Add 100copies, 50copies, 10copies, 5copies, and 0copy template to the RPA reaction system, respectively, and shake to mix. Set the reaction temperature of the qPCR instrument to 42 °C and the detection channel to FAM. Fluorescence values were recorded every 2 minutes for a total of 60 minutes.
3、实验结论3. Experimental conclusion
1)实验结果见图6，图7。1) The experimental results are shown in Figure 6 and Figure 7.
2)对于qPCR仪上的反应，One-pot Nest RPA检测灵敏度为10copies/50μL，高于常规RPA反应的50copies/50μL。且同等条件下One-pot Nest RPA的荧光强度远高于常规RPA。2) For the reaction on the qPCR instrument, the detection sensitivity of One-pot Nest RPA is 10copies/50μL, which is higher than 50copies/50μL of conventional RPA reaction. And under the same conditions, the fluorescence intensity of One-pot Nest RPA is much higher than that of conventional RPA.
3)对于蓝光灯下的拍照结果，One-pot Nest RPA可以检测到10copies/50μL的扩增产物，而常规RPA反应由于荧光强度较低，无法检测到明显荧光。3) For the photographing results under blue light, One-pot Nest RPA can detect 10 copies/50 μL of amplification products, while the conventional RPA reaction cannot detect obvious fluorescence due to the low fluorescence intensity.
实施例4 One-pot Nest RPA检测SARS-Cov2假病毒E基因灵敏度测试 Embodiment 4 One-pot Nest RPA detects SARS-Cov2 pseudovirus E gene sensitivity test
1、试剂仪器准备1. Preparation of reagents and instruments
SARS-Cov2假病毒样品购自复百澳(苏州)生物科技有限公司(产品货号：FNV2596)。SARS-Cov2 pseudovirus samples were purchased from Fubaiao (Suzhou) Biotechnology Co., Ltd. (product number: FNV2596).
RPA反应试剂盒购自英国TwistDX公司，产品型号为
Figure PCTCN2021076773-appb-000009
exo RT kit。 The RPA reaction kit was purchased from the British TwistDX company, the product model is
Figure PCTCN2021076773-appb-000009
exo RT kit.
Triton X-100购自sigma公司，产品货号为：T9284-100ML。Triton X-100 was purchased from sigma company, product number: T9284-100ML.
NP-40购自sigma公司，产品货号为：NP40S-500ML。NP-40 was purchased from sigma company, the product number is: NP40S-500ML.
2、实验步骤2. Experimental steps
1)将SARS-Cov2假病毒用DEPC水稀释至合适浓度。1) Dilute the SARS-Cov2 pseudovirus with DEPC water to an appropriate concentration.
2)配制SARS-Cov2假病毒核酸释放剂，成分为5％Triton X-100，5％NP-40。2) Prepare a SARS-Cov2 pseudovirus nucleic acid release agent, the components are 5% Triton X-100, 5% NP-40.
3)将SARS-Cov2假病毒E基因引物用DEPC水稀释至10μM。3) Dilute the SARS-Cov2 pseudovirus E gene primer with DEPC water to 10 μM.
引物序列如下：The primer sequences are as follows:
E-Primer F：ATGTACTCATTCGTTTCGGAAGAGACAGG(SEQ ID NO：1)E-Primer F: ATGTACTCATTCGTTTCGGAAGAGACAGG (SEQ ID NO: 1)
E-Primer R：AGACCAGAAGATCAGGAACTCTAGAAGAA(SEQ ID NO：2)E-Primer R: AGACCAGAAGATCAGGAACTCTAGAAGAA (SEQ ID NO: 2)
E-Primer F2：5’(SEQ ID NO：3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO：4)-C3spacer-3’E-Primer F2: 5'(SEQ ID NO:3)/i6FAMdT//idSp/G/iBHQ1dT/(SEQ ID NO:4)-C3spacer-3'
5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/ GTGCGTACTGCTG-C3 spacer-3’5’-TTACACTAGCCATCCTTACTGCGCTTCGA/i6FAMdT//idSp/G/iBHQ1dT/ GTGCGTACTGCTG-C3 spacer-3’
E-Primer R2：5’-(SEQ ID NO：5)/idSp/(SEQ ID NO：6)-C3 spacer-3’E-Primer R2: 5'-(SEQ ID NO:5)/idSp/(SEQ ID NO:6)-C3 spacer-3'
5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3spacer-3’5’-AGAATTCAGATTTTTAACACGAGAGTAAACG/idSp/AAAAAGAAGGTTTTa-C3spacer-3’
4)One-pot Nest RPA反应体系配制4) Preparation of One-pot Nest RPA reaction system
Figure PCTCN2021076773-appb-000010
Figure PCTCN2021076773-appb-000010
上述成分配置好后，直接加入到RPA反应干粉(来源于RPA反应试剂盒)中形成RPA反应体系。After the above components are prepared, they are directly added to the RPA reaction dry powder (derived from the RPA reaction kit) to form an RPA reaction system.
5)在RPA反应体系中分别加入100copies，50copies，20copies，0copies假病毒模板。设置金属浴温度为42摄氏度，反应40分钟后在蓝光灯下检测荧光，并拍照。5) 100copies, 50copies, 20copies and 0copies pseudovirus templates were added to the RPA reaction system respectively. The temperature of the metal bath was set to 42 degrees Celsius, and after 40 minutes of reaction, the fluorescence was detected under a blue light and photographed.
3、实验结论3. Experimental conclusion
1)实验结果见图8。1) The experimental results are shown in Figure 8.
2)One-pot Nest RPA检测SARS-Cov2假病毒E基因灵敏度为50copies/50μL2) The sensitivity of One-pot Nest RPA to detect SARS-Cov2 pseudovirus E gene is 50copies/50μL
实施例5 One-pot Nest RPA检测SARS-Cov2假病毒O基因灵敏度测试Embodiment 5 One-pot Nest RPA detects SARS-Cov2 pseudovirus O gene sensitivity test
1、试剂仪器准备1. Preparation of reagents and instruments
SARS-Cov2假病毒样品购自复百澳(苏州)生物科技有限公司(产品货号：FNV2596)。SARS-Cov2 pseudovirus samples were purchased from Fubaiao (Suzhou) Biotechnology Co., Ltd. (product number: FNV2596).
RPA反应试剂盒购自英国TwistDX公司，产品型号为
Figure PCTCN2021076773-appb-000011
exo RT kit。 The RPA reaction kit was purchased from the British TwistDX company, the product model is
Figure PCTCN2021076773-appb-000011
exo RT kit.
Triton X-100购自sigma公司，产品货号为：T9284-100ML。Triton X-100 was purchased from sigma company, product number: T9284-100ML.
NP-40购自sigma公司，产品货号为：NP40S-500ML。NP-40 was purchased from sigma company, the product number is: NP40S-500ML.
2、实验步骤2. Experimental steps
1)将SARS-Cov2假病毒用DEPC水稀释至合适浓度。1) Dilute the SARS-Cov2 pseudovirus with DEPC water to an appropriate concentration.
2)配制SARS-Cov2假病毒核酸释放剂，成分为5％Triton X-100，5％NP-40。2) Prepare a SARS-Cov2 pseudovirus nucleic acid release agent, the components are 5% Triton X-100, 5% NP-40.
3)将SARS-Cov2假病毒O基因引物用DEPC水稀释至10μM。3) Dilute the SARS-Cov2 pseudovirus O gene primer with DEPC water to 10 μM.
引物序列如下：The primer sequences are as follows:
O-Primer F：TGTAGTTGTGATCAACTCCGCGAACCCATGCT(SEQ ID NO：7)O-Primer F: TGTAGTTGTGATCAACTCCGCGAACCCATGCT (SEQ ID NO: 7)
O-Primer R：TCTTCATGTTGGTAGTTAGAGAAAGTGTGTCT(SEQ ID NO：8)O-Primer R: TCTTCATGTTGGTAGTTAGAGAAAGTGTGTCT (SEQ ID NO: 8)
O-Primer F2：5’(SEQ ID NO：9)/i6FAMdT//idSp/g/iBHQ1dT/(SEQ ID NO：10)-C3 spacer-3’O-Primer F2: 5'(SEQ ID NO:9)/i6FAMdT//idSp/g/iBHQ1dT/(SEQ ID NO:10)-C3 spacer-3'
5’-AGCCCGTCTTACACCGTGCGGCACAGGCAC/i6FAMdT//idSp/g/iBHQ1dT/ACTG ATGTCGTAT-C3 spacer-3’5’-AGCCCGTCTTACACCGTGCGGCACAGGCAC/i6FAMdT//idSp/g/iBHQ1dT/ACTG ATGTCGTAT-C3 spacer-3’
O-Primer R2：5’(SEQ ID NO：11)/i6FAMdT//idSp//iBHQ1dT/(SEQ ID NO：12)-C3spacer-3’O-Primer R2: 5'(SEQ ID NO:11)/i6FAMdT//idSp//iBHQ1dT/(SEQ ID NO:12)-C3spacer-3'
5’-CTTGGAAGCGACAACAATTAGTTTTTAGGAA/i6FAMdT//idSp//iBHQ1dT/AGCA AAACCAGCTA-C3 spacer-3’5’-CTTGGAAGCGACAACAATTAGTTTTTAGGAA/i6FAMdT//idSp//iBHQ1dT/AGCAAAACCAGCTA-C3 spacer-3’
4)One-pot Nest RPA反应体系配制4) Preparation of One-pot Nest RPA reaction system
Figure PCTCN2021076773-appb-000012
Figure PCTCN2021076773-appb-000012
Figure PCTCN2021076773-appb-000013
Figure PCTCN2021076773-appb-000013
上述成分配置好后，直接加入到RPA反应干粉(来源于RPA反应试剂盒)中形成RPA反应体系。After the above components are prepared, they are directly added to the RPA reaction dry powder (derived from the RPA reaction kit) to form an RPA reaction system.
5)在反应体系中分别加入100copies，50copies，20copies，0copy SARS-Cov2假病毒模板。设置金属浴反应温度为42摄氏度，反应40分钟后蓝光灯下检测荧光信号，并拍照。5) 100copies, 50copies, 20copies, 0copy SARS-Cov2 pseudovirus template were added to the reaction system respectively. The reaction temperature of the metal bath was set to 42 degrees Celsius, and after 40 minutes of reaction, the fluorescent signal was detected under a blue light and photographed.
6)在反应体系中分别加入100copies，50copies，20copies，0copy SARS-Cov2假病毒模板。设置qPCR仪反应温度为42摄氏度，检测通道为FAM。每2分钟记录一次荧光值，共记录60分钟。6) 100copies, 50copies, 20copies, 0copy SARS-Cov2 pseudovirus template were added to the reaction system respectively. Set the reaction temperature of the qPCR instrument to 42 degrees Celsius and the detection channel to FAM. Fluorescence values were recorded every 2 minutes for a total of 60 minutes.
3、实验结论3. Experimental conclusion
1)实验结果见图9，图10所示。1) The experimental results are shown in Figure 9 and Figure 10.
2)One-pot Nest RPA检测SARS-Cov2假病毒O基因的灵敏度为20copies/50μL(qPCR仪)，50copies/50μL(蓝光灯)。2) The sensitivity of One-pot Nest RPA to detect SARS-Cov2 pseudovirus O gene is 20copies/50μL (qPCR instrument), 50copies/50μL (blue light).
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention are described in detail above, but the present invention is not limited to the specific details of the above-mentioned embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。In addition, it should be noted that the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner unless they are inconsistent. In order to avoid unnecessary repetition, the present invention provides The combination method will not be specified otherwise.

Claims (17)

  1. 一种巢式重组酶-聚合酶扩增的方法，其特征在于，所述的方法包括：A method for nested recombinase-polymerase amplification, characterized in that the method comprises:
    配制反应体系，加入1)第一引物对，和，2)第二引物对和/或探针；Prepare a reaction system, add 1) a first primer pair, and, 2) a second primer pair and/or probe;
    进行RPA或RT-RPA反应扩增；Perform RPA or RT-RPA reaction amplification;
    其中，第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。Wherein, the second primer pair is located in the template obtained by the first primer pair RPA or RT-RPA reaction amplification, and the second primer pair comprises the /idSp/ restriction site and the modification that prevents the 3' end amplification .
  2. 根据权利要求1所述的方法，其特征在于，所述的阻止3’端扩增的修饰选自羟甲基叠氮、C18、C12、ddNTP、RNA、PNA或C3 spacer。The method according to claim 1, wherein the modification that prevents 3' end amplification is selected from the group consisting of hydroxymethyl azide, C18, C12, ddNTP, RNA, PNA or C3 spacer.
  3. 根据权利要求1或2所述的方法，其特征在于，所述的1)第一引物对，和，2)第二引物对和/或探针，为一步加入。The method according to claim 1 or 2, wherein the 1) first primer pair, and, 2) the second primer pair and/or probe are added in one step.
  4. 根据权利要求1-3任一所述的方法，其特征在于，所述的RPA或RT-RPA反应中添加能够识别/idSp/的核酸内切酶，所述的核酸内切酶优选为Exo或nfo。The method according to any one of claims 1-3, wherein an endonuclease capable of recognizing /idSp/ is added in the RPA or RT-RPA reaction, and the endonuclease is preferably Exo or nfo.
  5. 根据权利要求1-4任一所述的方法，其特征在于，所述的第二引物对和/或探针还包含荧光报告基团和荧光淬灭基团的修饰，所述的荧光报告基团选自FAM、HEX、TET、JOE、Cy3或TAMRA，所述的荧光淬灭基团选自DABCYL、BHQ1、BHQ2或BHQ3、Eclipse、MGB。The method according to any one of claims 1-4, wherein the second primer pair and/or the probe further comprises modification of a fluorescent reporter group and a fluorescence quenching group, and the fluorescent reporter group The group is selected from FAM, HEX, TET, JOE, Cy3 or TAMRA, and the fluorescence quenching group is selected from DABCYL, BHQ1, BHQ2 or BHQ3, Eclipse, MGB.
  6. 根据权利要求1-5任一所述的方法，其特征在于，所述的第二引物对和/或探针中的/idSp/被nfo或exo切割后，产生可以扩增的3’OH末端，继续进行扩增反应。The method according to any one of claims 1-5, wherein after /idSp/ in the second primer pair and/or probe is cleaved by nfo or exo, a 3'OH end that can be amplified is generated , continue the amplification reaction.
  7. 根据权利要求1-6任一所述的方法，其特征在于，所述的第二引物对与所述第一引物对不重叠或小于10bp的重叠。The method according to any one of claims 1-6, wherein the second primer pair and the first primer pair do not overlap or overlap less than 10 bp.
  8. 根据权利要求1-7任一所述的方法，其特征在于，所述的RPA或RT-RPA反应分别可用于DNA或RNA的扩增。The method according to any one of claims 1-7, wherein the RPA or RT-RPA reaction can be used for DNA or RNA amplification, respectively.
  9. 根据权利要求1-8任一所述的方法，其特征在于，所述的RPA或RT-RPA反应时间为5-40min，优选为30min。The method according to any one of claims 1-8, wherein the RPA or RT-RPA reaction time is 5-40min, preferably 30min.
  10. 根据权利要求1-9任一所述的方法，其特征在于，所述的RPA或RT-RPA反应温度为35℃-45℃，优选的是37℃-42℃。The method according to any one of claims 1-9, wherein the reaction temperature of the RPA or RT-RPA is 35°C-45°C, preferably 37°C-42°C.
  11. 根据权利要求1-10任一所述的方法，其特征在于，所述的RPA或RT-RPA反应在反应试剂中进行，所述的反应试剂包括第一引物对、第二引物对、缓冲液和/或RPA反应物。The method according to any one of claims 1-10, wherein the RPA or RT-RPA reaction is carried out in a reaction reagent, and the reaction reagent comprises a first primer pair, a second primer pair, a buffer and/or RPA reactants.
  12. 一种核酸快速检测的方法，其特征在于，所述的方法包括：A method for rapid nucleic acid detection, characterized in that the method comprises:
    A)获得待检测核酸；A) obtain nucleic acid to be detected;
    B)使用权利要求1-11任一所述的巢式重组酶-聚合酶扩增的方法扩增步骤A)的核酸，获得反应产物；B) using the nested recombinase-polymerase amplification method according to any one of claims 1-11 to amplify the nucleic acid of step A) to obtain a reaction product;
    C)检测反应产物的荧光。C) Detect the fluorescence of the reaction product.
  13. 一种核酸的检测或扩增的试剂盒，其特征在于，所述的试剂盒中包含第一引物对、第二引物对、缓冲液和/或RPA反应物，所述的第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。A test kit for the detection or amplification of nucleic acid, characterized in that the test kit comprises a first primer pair, a second primer pair, a buffer and/or an RPA reactant, and the second primer pair is located at In the template obtained by the first primer pair RPA or RT-RPA reaction amplification, the second primer pair includes an /idSp/ restriction site and a modification to prevent the amplification of the 3' end.
  14. 一种用于巢式重组酶-聚合酶扩增方法的试剂盒，其特征在于，所述的试剂盒中包含第一引物对、第二引物对、缓冲液和/或RPA反应物，所述的第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。A test kit for nested recombinase-polymerase amplification method, characterized in that the test kit comprises a first primer pair, a second primer pair, a buffer and/or an RPA reactant, and the The second primer pair is located in the template obtained by the amplification of the first primer pair RPA or RT-RPA reaction, and the second primer pair comprises an /idSp/ restriction site and a modification to prevent amplification of the 3' end.
  15. 根据权利要求13或14所述的试剂盒，其特征在于，所述的RPA反应物包含能够识别/idSp/的核酸内切酶。The kit according to claim 13 or 14, wherein the RPA reactant comprises an endonuclease capable of recognizing /idSp/.
  16. 一种权利要求1-12任一所述的方法、权利要求13-15任一所述的试剂盒在病毒核酸扩增或检测中的应用。An application of the method according to any one of claims 1-12 and the kit according to any one of claims 13-15 in viral nucleic acid amplification or detection.
  17. 第一引物对和/或第二引物对在病毒核酸扩增或检测中的应用，其特征在于，第二引物对位于所述第一引物对RPA或RT-RPA反应扩增获得的模板内，所述的第二引物对包含/idSp/酶切位点和阻止3’端扩增的修饰。The application of the first primer pair and/or the second primer pair in viral nucleic acid amplification or detection, wherein the second primer pair is located in the template obtained by the first primer pair RPA or RT-RPA reaction amplification, The second primer pair contains an /idSp/ restriction site and a modification that prevents amplification of the 3' end.
PCT/CN2021/076773 2021-02-19 2021-02-19 Nested recombinase-polymerase amplification method and use thereof WO2022174377A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/076773 WO2022174377A1 (en) 2021-02-19 2021-02-19 Nested recombinase-polymerase amplification method and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/076773 WO2022174377A1 (en) 2021-02-19 2021-02-19 Nested recombinase-polymerase amplification method and use thereof

Publications (1)

Publication Number Publication Date
WO2022174377A1 true WO2022174377A1 (en) 2022-08-25

Family

ID=82931875

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/076773 WO2022174377A1 (en) 2021-02-19 2021-02-19 Nested recombinase-polymerase amplification method and use thereof

Country Status (1)

Country Link
WO (1) WO2022174377A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109072289A (en) * 2016-03-04 2018-12-21 爱乐圣地亚哥公司 Automate nido recombinase polymeric enzymatic amplification
CN109971886A (en) * 2018-11-22 2019-07-05 上海出入境检验检疫局动植物与食品检验检疫技术中心 A kind of kit for detecting CPV nucleic acid, RPA primer pair, probe and method
CN110066885A (en) * 2019-04-30 2019-07-30 南京林业大学 A kind of primer and probe combination and its application based on RPA- Sidestream chromatography technology detection chestnut Heisui River phytophthora

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109072289A (en) * 2016-03-04 2018-12-21 爱乐圣地亚哥公司 Automate nido recombinase polymeric enzymatic amplification
CN109971886A (en) * 2018-11-22 2019-07-05 上海出入境检验检疫局动植物与食品检验检疫技术中心 A kind of kit for detecting CPV nucleic acid, RPA primer pair, probe and method
CN110066885A (en) * 2019-04-30 2019-07-30 南京林业大学 A kind of primer and probe combination and its application based on RPA- Sidestream chromatography technology detection chestnut Heisui River phytophthora

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LOBATO IVAN MAGRIñá; O'SULLIVAN CIARA K.: "Recombinase polymerase amplification: Basics, applications and recent advances", TRAC TRENDS IN ANALYTICAL CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 98, 1 January 1900 (1900-01-01), AMSTERDAM, NL , pages 19 - 35, XP085315007, ISSN: 0165-9936, DOI: 10.1016/j.trac.2017.10.015 *
ZACHARY CRANNELL, ALEJANDRO CASTELLANOS-GONZALEZ, GAYATRI NAIR, ROJELIO MEJIA, A. CLINTON WHITE, REBECCA RICHARDS-KORTUM: "Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 88, no. 3, 2 February 2016 (2016-02-02), US , pages 1610 - 1616, XP055754697, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.5b03267 *

Similar Documents

Publication Publication Date Title
Zhou et al. Point-of-care COVID-19 diagnostics powered by lateral flow assay
Park et al. Optimization of primer sets and detection protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real-time PCR
CN111560472B (en) Probe, primer, kit, detection method and application for detecting novel coronavirus SARS-CoV-2
US10851406B2 (en) Nicking and extension amplification reaction for the exponential amplification of nucleic acids
WO2022257659A1 (en) Crispr/cas system-based sars-cov-2 double-target rapid detection method and kit
US8685649B2 (en) RT-LAMP assay for the detection of pan-serotype dengue virus
JP2020503030A (en) 2-part mediator probe
CN109913565B (en) Kit, primer pair, probe and method for detecting vibrio parahaemolyticus
WO2023284514A1 (en) Ultrasensitive antibody detection method
CN107619885A (en) A kind of fluorescence RT RAA primers, probe and detection method for being used to detect dengue virus
Ruang-Areerate et al. Distance-based paper device using combined SYBR safe and gold nanoparticle probe LAMP assay to detect Leishmania among patients with HIV
CA3067868A1 (en) Molecular fingerprinting methods to detect and genotype dna targets through polymerase chain reaction
WO2022099487A1 (en) Nested recombinase-polymerase amplification method and application thereof
WO2022174377A1 (en) Nested recombinase-polymerase amplification method and use thereof
JP2020533974A (en) Respiratory syncytial virus species nicking and elongation amplification reaction (NEAR)
WO2022257663A1 (en) Method and kit for detecting and screening n501y mutation in covid-19
Gulinaizhaer et al. Isothermal nucleic acid amplification technology in HIV detection
CN115820939A (en) CrRNA for detecting monkeypox virus, nucleic acid molecule composition, detection system and application
Jiang et al. New design of probe and central-homo primer pairs to improve TaqMan™ PCR accuracy for HBV detection
WO2021216573A1 (en) Scalable, easy-to-deploy system and reagents thereof for crispr-associated nuclease-based detection of pathogen genetic material
CN113151589A (en) Nested recombinase-polymerase amplification method and application thereof
CN106191314B (en) LAMP detection kit, detection method and application of DNA virus
CN114410793B (en) Method for detecting FEN1 activity by label-free fluorescence
KR102226356B1 (en) Primers for detecting Dengue virus by LAMP
KR20120020066A (en) Kit for detecting hiv-1 and method for detecting hiv-1 using the same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21926099

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21926099

Country of ref document: EP

Kind code of ref document: A1