WO2022037623A1 - SARS-CoV-2病毒核酸等温快速检测试剂盒及检测方法 - Google Patents

SARS-CoV-2病毒核酸等温快速检测试剂盒及检测方法 Download PDF

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WO2022037623A1
WO2022037623A1 PCT/CN2021/113348 CN2021113348W WO2022037623A1 WO 2022037623 A1 WO2022037623 A1 WO 2022037623A1 CN 2021113348 W CN2021113348 W CN 2021113348W WO 2022037623 A1 WO2022037623 A1 WO 2022037623A1
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nucleic acid
acid molecule
target nucleic
guide
detection
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PCT/CN2021/113348
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French (fr)
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冯雁
李忠磊
叶星宇
郭翔
黄�俊
刘涛
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上海交通大学
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Priority to US18/042,265 priority Critical patent/US20230323486A1/en
Priority to EP21857715.3A priority patent/EP4202064A1/en
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and in particular, the invention relates to a kit and a detection method for isothermal rapid detection of SARS-CoV-2 virus nucleic acid.
  • new coronavirus 2019-nCoV, hereinafter referred to as "new coronavirus”
  • new coronavirus has spread to the whole country since the outbreak of infection.
  • the Chinese government took active action and launched an emergency prevention and control plan.
  • high-tech investment is required from the diagnosis to the treatment of infectious patients.
  • the accurate and quantitative detection of the new coronavirus, especially the low-abundance virus has a positive impact on the diagnosis, follow-up control and treatment of patients.
  • the release of viral genome sequencing results the development of detection products for specific nucleic acid sequences has become a focus. So far, the products of seven in vitro diagnostic companies have been put into the market, and more than 20 companies have also launched testing products.
  • Real-time PCR reverse transcription PCR
  • the principle of reverse transcription PCR is reverse transcription-polymerase chain reaction. PCR amplification is performed using cDNA as a template to obtain the target gene or detect gene expression.
  • nucleic acid diagnostic kits have cost and time advantages over conventional genome sequencing analysis. However, because they are mostly based on fluorescent PCR methods, there are still long detection times (about 2 hours) and high prices (200 yuan). /time), expensive equipment (Q-PCR instrument 300,000-600,000 yuan), and high professional requirements for operators. Therefore, the current diagnosis of confirmed and suspected patients is still the limiting factor for follow-up prevention and treatment.
  • reverse transcription LAMP is the combination of reverse transcription and Loop-Mediated Isothermal Amplification (loop-mediated isothermal amplification), which is specially used for rapid amplification of RNA.
  • LAMP is a novel isothermal nucleic acid amplification method that appeared in 2000. It uses 4 or 6 template-specific specific primers and DNA polymerase with strand displacement ability to complete the amplification in 15-60 minutes under isothermal conditions (about 63°C).
  • the technology is comparable to or even better than reverse transcription PCR technology in terms of sensitivity, specificity and detection range, and does not require template thermal denaturation, temperature cycling, electrophoresis and UV observation, and does not rely on any special equipment.
  • the purpose of the present invention is to provide a rapid, simple, efficient, highly sensitive and highly accurate method for detecting SARS-CoV-2.
  • a method for detecting a target nucleic acid molecule comprising the steps of:
  • cleavage reagent includes: two guide ssDNAs, a gene editing enzyme ( Ago), and a first reporter nucleic acid molecule, the first reporter nucleic acid molecule has a fluorescent group and a quenching group, and the two guide ssDNAs are adjacent to each other; and
  • the sample to be detected includes an unamplified sample and an amplified (or nucleic acid amplified) sample.
  • the sample to be detected is a sample obtained by amplification.
  • the amplification is selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling Loop amplification, HDA, SPIA, NEAR, TMA, SMAP2, EXPAR, or a combination thereof.
  • the first nucleotide at the 5' end of each guide ssDNA is T.
  • the lengths of the guide ssDNAs are each independently 10-60nt, preferably 10-40nt, more preferably 13-20nt.
  • the guide ssDNAs may be the same or different.
  • the lengths of the guide ssDNAs may be the same or different.
  • the guide ssDNA is a phosphorylated single-stranded DNA molecule.
  • the guide ssDNA is a 5'-phosphorylated single-stranded DNA molecule; and/or a 3'-phosphorylated single-stranded DNA molecule.
  • the gene editing enzyme Ago is selected from the following group: PfAgo (Pyrococcus furiosus Ago), MfAgo (Methanocaldococcus fervens Ago), TcAgo (Thermgladius calderae Ago), TfAgo (Thermus filiformis Ago), AaAgo (Aquifex aeolicus Ago), TpAgo (Thermus parvatiensis Ago), or a combination thereof.
  • the gene editing enzyme Ago includes PfAgo (Pyrococcus furiosus Ago).
  • the Ago includes wild-type and mutant Ago.
  • the working temperature of the gene editing enzyme is 87-99°C.
  • a secondary guide ssDNA is generated after the target nucleic acid molecule (or its amplified product) is cleaved by the gene editing enzyme Ago.
  • the length of the secondary guide ssDNA is 10-60nt, preferably 10-40nt, more preferably 15-17nt.
  • the secondary guide ssDNA is complementary to the sequence of the fluorescent reporter nucleic acid (first reporter nucleic acid molecule).
  • the gene editing enzyme Ago is guided to the fluorescent reporter nucleic acid (the first reporter nucleic acid).
  • reporter nucleic acid molecules are cleaved, resulting in a detectable signal (eg, fluorescence).
  • the fluorescent group and the quenching group are independently located at the 5' end and the 3' end of the fluorescent reporter nucleic acid.
  • the fluorescent group is selected from the group consisting of FAM, HEX, CY3, CY5, ROX, VIC, JOE, TET, Texas Red, NED, TAMRA, or a combination thereof.
  • the quenching group is selected from the group consisting of TAMRA, BHQ, DABSYL, or a combination thereof.
  • the length of the fluorescent reporter nucleic acid (the first reporter nucleic acid molecule) is 9-100 nt, preferably 10-60 nt, more preferably 15-40 nt.
  • the target nucleic acid molecule is selected from the group consisting of nucleic acid molecules of pathogenic microorganisms, nucleic acid molecules with genetic mutations, and specific target nucleic acid molecules.
  • the pathogenic microorganisms include viruses, bacteria, chlamydia, and mycoplasmas.
  • the virus includes plant virus or animal virus.
  • the virus includes: coronavirus, influenza virus, HIV, hepatitis virus, parainfluenza virus.
  • the virus is a coronavirus.
  • the virus is selected from the following group: SARS, SARS-CoV-2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV, or its combination.
  • the target nucleic acid molecule includes wild-type or mutant DNA.
  • the target nucleic acid molecule includes single-stranded cDNA.
  • the target nucleic acid molecule includes DNA obtained by reverse transcription or amplification of RNA, such as cDNA.
  • the target nucleic acid molecule includes a single base mutation in the SARS-CoV-2 virus.
  • the target nucleic acid molecule includes single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) mutants of SARS-CoV-2 virus ORF1ab, N, E, S genes and the like.
  • single nucleotide polymorphism Single Nucleotide Polymorphism, SNP
  • the target nucleic acid molecule includes a mutation at position 614 of the spike protein sequence expressed by the S gene of SARS-CoV-2 virus, and the amino acid mutation type is D614G, that is, the amino acid at position 614 is composed of Aspartic acid (D) becomes glycine (G).
  • the target nucleic acid molecule further includes a mutant in which the 23,403 nucleotide of the SARS-CoV-2 virus is mutated from A to G at the nucleotide level.
  • the target nucleic acid molecule further includes, at the nucleotide level, mutants of T8782C, G11083T, G26144T, and C28144T of SARS-CoV-2 virus.
  • the target nucleic acid molecule further includes mutants of N501Y, E484Q and L452R of SARS-CoV-2 virus at the amino acid level.
  • the method is used to detect whether the nucleic acid at the target site is in SNP, point mutation, deletion, and/or insertion.
  • the method is used for virus typing detection, that is, to determine whether the virus is a wild type or a mutant type.
  • the cleavage reagent when used for virus typing and detection, further includes two additional reporter nucleic acid molecules (a second reporter nucleic acid molecule and a third reporter nucleic acid molecule).
  • the lengths of the second reporter nucleic acid molecule and the third reporter nucleic acid molecule are each independently 9-200nt, preferably 10-60nt, more preferably 15-40nt.
  • the gene editing enzyme Ago is guided to cut the second reporter nucleic acid molecule, thereby producing a detectable signal (such as fluorescence).
  • the third reporter nucleic acid molecule cannot be complementary to the sequence of the secondary guide ssDNA produced by the wild-type virus, so the gene editing enzyme Ago does not cleave the third reporter nucleic acid molecule, thereby failing to produce A detectable signal (eg, fluorescence).
  • the second reporter nucleic acid molecule cannot be complementary to the sequence of the secondary guide ssDNA produced by the mutant virus, so the gene editing enzyme Ago does not cleave the second reporter nucleic acid molecule, so that the production of A detectable signal (eg, fluorescence).
  • a detectable signal eg, fluorescence
  • the gene editing enzyme Ago is directed to cut the second reporter nucleic acid molecule, thereby producing a detectable signal (such as fluorescence).
  • step (c) the fluorescence detection is performed by a microplate reader or a fluorescence spectrophotometer.
  • the detection system further contains buffer or buffer.
  • the sample to be detected is a sample to be detected obtained by amplification with primers selected from the following group.
  • the primer is any primer pair selected from the following group:
  • the guide ssDNA is selected from the following group:
  • P is the phosphorylation modification of the 5' end of the oligonucleotide.
  • the sample to be detected is a nucleic acid sample prepared from a sample selected from the group consisting of throat swab, bronchoalveolar lavage fluid, nasal swab, urine, feces, body fluid, or a combination thereof.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • a second aspect of the present invention provides a detection kit for target nucleic acid molecules, the kit includes:
  • an amplification reagent for amplifying a target nucleic acid molecule comprising: a primer pair for amplifying a target nucleic acid molecule, the primer pair for performing specific amplification based on the target nucleic acid molecule Amplification reaction, thereby producing specific nucleic acid amplification products;
  • cleavage reagent or a cleavage buffer containing the cleavage reagent, wherein the cleavage reagent includes: 2 guide ssDNAs, a gene editing enzyme (Ago), and a first reporter nucleic acid with the first reporter nucleic acid
  • the fluorophore and the quencher, and the two guide ssDNAs are adjacent to each other.
  • the kit further includes a second reporter nucleic acid and a third reporter nucleic acid.
  • the kit also includes:
  • a reverse transcription reagent for reverse transcription reaction includes: reverse transcriptase or Bst enzyme (or a mutant thereof) with reverse transcriptase activity.
  • the kit contains the following preferred combination of reagents:
  • Primer pair 1 SEQ ID No: 1-6;
  • Fluorescent reporter nucleic acid SEQ ID No.59;
  • ssDNA SEQ ID Nos: 49 and 50;
  • Primer pair 3 SEQ ID No: 13-18;
  • Fluorescent reporter nucleic acid SEQ ID No.60;
  • ssDNA SEQ ID Nos: 51 and 52;
  • Primer pair 5 SEQ ID No: 25-30;
  • Fluorescent reporter nucleic acid SEQ ID No.62;
  • ssDNA SEQ ID Nos: 55 and 56;
  • Primer pair 7 SEQ ID No: 37-42;
  • Fluorescent reporter nucleic acid SEQ ID No.63 and 64;
  • ssDNA SEQ ID Nos: 57 and 58.
  • a third aspect of the present invention provides a kit for detecting target nucleic acid molecules, the kit comprising:
  • the target nucleic acid molecule includes single-stranded DNA.
  • the kit further includes a sixth container, and the second reporter nucleic acid and the third reporter nucleic acid located in the sixth container.
  • the amplification reagent includes: a primer pair for amplifying a target nucleic acid molecule, and the primer pair is used to perform a specific amplification reaction based on the target nucleic acid molecule, thereby generating a specific nucleic acid Amplification product.
  • the buffer located in the fifth container includes: a buffer for amplification reaction and a buffer for enzyme cleavage.
  • the buffer for the amplification reaction includes: Bst buffer (Tris-HCl, EDTA, NaCl or KCl), MgSO 4 , dNTP, DNase/RNase free H 2 O.
  • Bst buffer Tris-HCl, EDTA, NaCl or KCl
  • MgSO 4 MgSO 4
  • dNTP DNase/RNase free H 2 O.
  • the buffer for enzyme cleavage includes Bst buffer or Reaction buffer (Tris-HCl, EDTA, NaCl or KCl), MnCl 2 , and DNase/RNase free H 2 O.
  • any two, three, four or all of the first container, the second container, the third container, the fourth container, the fifth container and the sixth container may be the same or different containers.
  • Figure 1 shows the physical diagram of the combination of the improved liner tube and the PCR tube, including a three-dimensional perspective schematic diagram and a top cross-sectional schematic diagram; 1 is the lining tube body, 2 is the PCR tube body, 3 is the lining tube reaction chamber, and 4 is the PCR tube Tube reaction chamber, 101 is the lining tube wall (thickness 3-7mm), 102 is the bottom hole of the lining tube (aperture 3-5mm), 103 is the gap between the lining tube wall and the PCR tube (the gap is the largest 5-10mm);
  • Figure 2 shows a schematic diagram of the "one-tube" double nucleic acid rapid detection of reverse transcription LAMP-RADAR coupling
  • FIG. 3 shows the schematic diagram of reverse transcription LAMP-RADAR detection
  • Figure 4 shows the detection sensitivity of SARS-CoV-2 virus LAMP-RADAR
  • Figure 5 shows a schematic diagram of fluorescence real-time detection of some SARS-CoV-2 clinical samples
  • FIG. 6 shows the results of the SARS-CoV-2 dual LAMP-RADAR specificity experiment
  • Figure 7 shows a schematic diagram of the detection principle of SARS-CoV-2 LAMP-RADAR typing
  • Figure 8 shows the D614G mutation site of the SARS-CoV-2 gene sequence.
  • a target nucleic acid molecule such as the target nucleic acid molecule of the new coronavirus SARS-CoV-2
  • the present invention performs reverse transcription loop-mediated isothermal amplification reaction (reverse transcription LAMP) on the target nucleic acid molecule, and then utilizes the properties of Ago enzyme, that is, for the first time, it is mediated by two primary guide ssDNAs adjacent to and without spacer sequences (guide ssDNA) for the first time.
  • reverse transcription LAMP reverse transcription loop-mediated isothermal amplification reaction
  • the fragmented 5' nucleic acid fragment can be used again by Ago enzyme to shear the complementary fluorescent reporter nucleic acid chain, so as to determine whether the sample is in the sample according to the fluorescence.
  • a suitable reaction temperature such as about 90-98 degrees
  • the fragmented 5' nucleic acid fragment can be used again by Ago enzyme to shear the complementary fluorescent reporter nucleic acid chain, so as to determine whether the sample is in the sample according to the fluorescence.
  • a suitable reaction temperature such as about 90-98 degrees
  • LAMP Loop-mediated isothermal amplification, which is an isothermal nucleic acid amplification technique suitable for genetic diagnosis.
  • PCR is the polymerase chain reaction technique (Polymerase chain reaction), a technique suitable for the amplification of target nucleic acids.
  • the term "secondary cleavage" means that in the detection method of the present invention, in the presence of the primary guide ssDNAs, the Ago enzyme of the present invention cleaves the target nucleic acid sequence to form a new 5' phosphorylated nucleic acid sequence (secondary nucleotide sequence). Guide ssDNAs); then, the secondary guide ssDNA continues to cleave the fluorescent reporter nucleic acid complementary to the secondary guide ssDNAs under the action of PfAgo enzyme.
  • the specific cleavage of the target nucleic acid sequence (first cleavage) and then the specific cleavage of the fluorescent reporter nucleic acid (second cleavage) is defined as "secondary cleavage". In the present invention, both the first cleavage and the second cleavage are specific cleavage.
  • a core component is a gene editing enzyme, such as Ago enzyme.
  • the preferred Ago enzyme is PfAgo enzyme, which comes from the archaea Pyrococcus furiosus, the gene length is 2313 bp, and the amino acid sequence consists of 770 amino acids.
  • the cleavage characteristics of PfAgo enzyme are: the enzyme can use the 5' phosphorylated oligonucleotide as the guide ssDNA to guide the enzyme to precisely cut the target nucleic acid sequence; the cleavage site is located at the 10th and 11th positions of the guide ssDNA The phosphodiester bond between the target nucleic acid (ssDNA) corresponding to the nucleotide at the position.
  • the preferred working temperature for PfAgo enzymes is 95 ⁇ 2 degrees.
  • a core component is a guide ssDNA, especially two ssDNAs, which are adjacent to each other without spacer bases or spacer sequences between each other.
  • the preferred guide ssDNAs are all oligonucleotides with a length of 10-60nt, preferably 10-40nt, more preferably 13-20nt, and the first 5' nucleotides are both Thymine (T), can be modified by phosphorylation.
  • a core component is a reporter nucleic acid carrying a reporter molecule.
  • the reporter nucleic acid molecule of the present invention is a nucleic acid molecule carrying a fluorescent group and a quencher group, respectively.
  • a fluorophore (F) is labeled at the 5' end and a quencher (Q) is labeled at the 3' end.
  • the fluorescent reporter nucleic acid molecule is determined according to the production position of the secondary guide ssDNAs; the target nucleic acid sequence is cleaved by the primary guide ssDNAs to form a new 5' phosphorylated nucleic acid sequence, which is called secondary The guide ssDNAs, fluorescent reporter nucleic acid covers all positions of the secondary guide ssDNAs.
  • the present invention also provides a nucleic acid detection method based on gene editing enzyme Ago, such as Pyrococcus furiosus Argonaute (PfAgo).
  • Ago such as Pyrococcus furiosus Argonaute (PfAgo).
  • two guide ssDNAs can be designed according to the target nucleic acid molecule (such as single-stranded DNA, preferably the amplified target nucleic acid molecule), and the two guide ssDNAs are adjacent to each other.
  • the guide ssDNA targets the target nucleic acid molecule and mediates the cleavage of the target nucleic acid molecule by the PfAgo enzyme to form a new secondary guide ssDNA.
  • the secondary guide ssDNA guides the PfAgo enzyme to cleave the fluorescent reporter nucleic acid complementary to the secondary guide ssDNAs, so as to detect the target nucleic acid molecule.
  • the method of the present invention can greatly improve the sensitivity and accuracy of target nucleic acid detection.
  • the PfAgo enzyme can selectively cut nucleic acid sequences with partial site differences through special design, so as to realize typing detection.
  • the mutation sites corresponding to different types are placed in the 10th and 11th positions of the guide ssDNAs. Due to the selection specificity of the PfAgo enzyme, two consecutive Point mutation can inhibit the splicing activity, thus achieving the detection of different types.
  • multiple target nucleic acid molecules and guide ssDNAs can be simultaneously added to the cleavage system of PfAgo enzyme, combined with reporter nucleic acids with different fluorescent groups, to achieve multiple detection of target nucleic acids.
  • the method of the present invention is very suitable for the detection of trace amounts of nucleic acids.
  • the present invention can detect target nucleic acid molecules of low concentration nucleic acid template (220 copies/mL).
  • the amplification primer used in the amplification reaction generally has a Tm value of about 65 ⁇ 10 degrees, and the amplified fragment size is about 90-200bp.
  • amplification primers should be designed to avoid the segment to be detected.
  • the present invention also provides a detection kit for target nucleic acid molecules.
  • the kit of the present invention comprises: (a) an amplification reagent for amplifying a target nucleic acid molecule, the amplification reagent comprising: a primer pair for amplifying a target nucleic acid molecule, the primers for performing a specific amplification reaction based on the target nucleic acid molecule, thereby producing a specific nucleic acid amplification product;
  • cleavage reagent or a cleavage buffer containing the cleavage reagent, wherein the cleavage reagent includes: 2 guide ssDNAs, a gene editing enzyme (Ago), and a first reporter nucleic acid, the fluorescent reporter nucleic acid has fluorescence group and quencher group, and the two guide ssDNAs are adjacent to each other.
  • the cleavage reagent includes: 2 guide ssDNAs, a gene editing enzyme (Ago), and a first reporter nucleic acid
  • the fluorescent reporter nucleic acid has fluorescence group and quencher group
  • the two guide ssDNAs are adjacent to each other.
  • the kit of the present invention comprises:
  • the target nucleic acid molecule includes single-stranded DNA.
  • the present invention is particularly suitable for detection of trace target nucleic acid molecules and multiplex detection, and has wide applicability.
  • the target nucleic acid molecule may be DNA or RNA.
  • the target nucleic acid molecule is RNA, it can be converted into cDNA by reverse transcription and then detected.
  • the present invention can achieve active and active management of disease prediction, prevention, etc., achieve early detection and early treatment, or early prediction and early prevention. Because the detection sensitivity and accuracy of the invention are very high, it is suitable for early diagnosis, prescribes the right medicine, saves the treatment time of patients, and improves the success rate of treatment. The present invention reduces waste of high medical costs and strives for a golden opportunity for treatment.
  • the present invention can conveniently and rapidly identify nucleic acid molecules in environmental pollutants and provide effective environmental detection data.
  • Extensive detection spectrum the present invention has a wide nucleic acid detection spectrum, which can realize efficient detection of virus, bacteria, and genetic disease genes;
  • the present invention avoids false positives caused by non-specific pairing between loop primers in the LAMP technology, and improves detection accuracy;
  • POCT system "one-tube” one-step operation detection of target nucleic acids such as new coronavirus, using a portable isothermal fluorescence system, in line with the POCT concept;
  • the present invention can also realize multiple detection in a single-tube reaction system, which significantly reduces the complexity of the system and the use cost.
  • High error tolerance of mutant bases Viruses with single-base or double-base mutations in the amplified region can be detected.
  • kit for isothermal rapid detection of SARS-CoV-2 virus nucleic acid of the present invention and the method for using it are provided.
  • the corresponding specific target nucleic acid sequence is:
  • the corresponding detection reagents include:
  • amplification primer the specific sequence is as follows:
  • 2019-nCoV ORF 1b-gDNA 1 5'-P-TTGATGAGGTTCCACC-3' SEQ ID NO.51
  • 2019-nCoV ORF 1b-gDNA 4 5'-P-TCAGTTGTGGCATCTC-3' SEQ ID NO.52
  • LAMP amplification buffer Bst buffer (Tris-HCl, EDTA, NaCl or KCl), MgSO 4 , dNTP, DNase/RNase free H 2 O.
  • Ago digestion buffer Bst buffer or Reaction buffer (Tris-HCl, EDTA, NaCl or KCl), MnCl 2 , DNase/RNase free H 2 O.
  • Amplification enzymes Bst polymerase, reverse transcriptase.
  • the specific operation steps of the detection method of the SARS-CoV-2 virus nucleic acid isothermal rapid detection kit of the present invention are as follows:
  • gDNA dry powder are dissolved with DNase/RNase free H 2 O to make 100uM storage solution; Described fluorescent reporter nucleic acid dry powder is dissolved with DNase/RNase free H 2 O to make 10uM storage solution liquid;
  • kit for isothermal rapid detection of SARS-CoV-2 virus nucleic acid of the present invention and the method for using it are provided.
  • Example 1 the detection of the ORF1b gene of SARS-CoV-2 virus is taken as an example, and the corresponding specific target nucleic acid sequence is referred to in Example 1.
  • the corresponding detection reagents include:
  • amplification primer the specific sequence is as follows:
  • 2019-nCoV ORF 1b-gDNA 1 5'-P-TTGATGAGGTTCCACC-3' SEQ ID NO.51
  • 2019-nCoV ORF 1b-gDNA 4 5'-P-TCAGTTGTGGCATCTC-3' SEQ ID NO.52
  • LAMP amplification buffer Bst buffer (Tris-HCl, EDTA, NaCl or KCl), MgSO 4 , dNTP, DNase/RNase free H 2 O.
  • Ago digestion buffer Bst buffer or Reaction buffer (Tris-HCl, EDTA, NaCl or KCl), MnCl 2 , DNase/RNase free H 2 O.
  • Amplification enzymes Bst polymerase, reverse transcriptase.
  • the specific operation steps of the detection method of the SARS-CoV-2 virus nucleic acid isothermal rapid detection kit of the present invention are as follows:
  • gDNA dry powder are dissolved with DNase/RNase free H 2 O to make 100uM storage solution; Described fluorescent reporter nucleic acid dry powder is dissolved with DNase/RNase free H 2 O to make 10uM storage solution liquid;
  • test standard SEQ ID NO.: 65
  • pipette 140 ⁇ l of standard dilutions for nucleic acid extraction QIAamp Viral RNA Mini Kit.
  • 15 ⁇ l of nucleic acid extraction sample, negative control (H2O) and positive control (target fragment plasmid) were added to the amplification system in Example 2, and each concentration gradient was divided into 3 groups, each group was repeated 7 times, and each group was calculated.
  • the detection rate of the experiment was carried out according to the steps of Example 3.
  • the results show that the minimum detection limit of the SARS-CoV-2 virus of the present invention can reach 670 copies/mL.
  • RNase P reference gene is a part of the human genome as an internal quality control.
  • the configuration of the double reaction system the RNase P internal reference gene amplification primer (primer pair 7) was added to the amplification system in Example 2, and the RNase P gDNA (SEQ ID NO.57 and SEQ ID NO. 58) and RNase P fluorescent reporter nucleic acid (SEQ ID NO.64), the reaction conditions refer to Example 2.
  • D614G mutation appears in the new crown cases in Beijing, and the infectivity may be increased by nearly 10 times.
  • D614A>G means A changes to G.
  • the mutant strain has strong pathogenicity, and the possibility of outbreak cannot be ruled out.
  • the present invention designs a typing test. Because RT-PCR is difficult to do single-base typing, ddPCR and sequencing methods are time-consuming and expensive.
  • the present invention can be flexibly designed to distinguish and detect known mutations, and the specific principle is shown in FIG. 7 .
  • the D614G mutation of SARS-CoV-2 virus and the nucleic acid sequence of SARS-CoV-2 virus are differentiated and detected, and the specific design is as follows:
  • two additional Reporters are designed.
  • One set of gDNA retains the design of the original SARS-CoV-2 detection site, and the other two Reporters introduce one or two consecutive mismatches relative to the 10th or 11th position of the secondary gDNA, thus ensuring that the secondary gDNA is relative to the newly designed Reporter There is at least one mismatch.
  • Reporter takes FAM probe and VIC probe as an example:
  • the design of D614 (wild type) secondary gDNA and FAM probe has only one mismatch, and there are 2 consecutive mismatches with VIC probe, so that in D614 (wild type) G614 (mutant type) secondary gDNA and FAM probe have two consecutive mismatches, and only one mismatch with VIC probe, so Under the mediation of G614 (mutant) secondary gDNA, PfAgo only cleaved the VIC probe, but not the FAM probe.
  • D614 type and G614 type are judged according to the fluorescence signal generation of the two Reporters, and the type detection of D614G is achieved.
  • D614-FAM-11G 5'FAM-CTGTGCAGTTAACGTCCTGATAAAGAACAG (SEQ ID NO. 66)-BHQ1 3'
  • D614-FAM-11C 5'FAM-CTGTGCAGTTAACCTCCTGATAAAGAACAG (SEQ ID NO. 67)-BHQ1 3'
  • D614-FAM-11T 5'FAM-CTGTGCAGTTAACTTCCTGATAAAGAACAG (SEQ ID NO. 68)-BHQ1 3'
  • G614-VIC-11T 5'VIC-CTGTGCAGTTAACTCCCTGATAAAGAACAG (SEQ ID NO. 69)-BHQ1 3'
  • G614-VIC-11G 5'VIC-CTGTGCAGTTAACGCCCTGATAAAGAACAG (SEQ ID NO. 70)-BHQ1 3'
  • G614-VIC-11C 5'VIC-CTGTGCAGTTAACCCCCTGATAAAGAACAG (SEQ ID NO. 71)-BHQ1 3'

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Abstract

本发明提供SARS-CoV-2病毒核酸等温快速检测试剂盒及检测方法，所述检测方法包括：（a）提供一含靶标核酸分子的待检测的样本；（b）将所述待检测的样本与切割试剂或含所述切割试剂的切割缓冲液进行混合，从而形成一检测体系，其中，所述的切割试剂包括：2个向导ssDNA、基因编辑酶（Ago）、和第一报告核酸分子，所述第一报告核酸分子带有荧光基团和淬灭基团，并且，所述的2个向导ssDNA之间彼此相邻；和（c）对所述检测体系进行荧光检测，从而获得荧光信号值，其中，所述检测体系中检测到荧光信号值，则表示所述样本中存在靶标核酸分子；而所述检测体系中没有检测到荧光信号值，则表示所述样本中不存在靶标核酸分子。

Description

SARS-CoV-2病毒核酸等温快速检测试剂盒及检测方法 技术领域
本发明属于生物技术领域，具体地说，本发明涉及SARS-CoV-2病毒核酸等温快速检测试剂盒及检测方法。
背景技术
新型冠状病毒(2019-nCoV，以下简称“新冠病毒”)，感染爆发至今，疫情已波及全国，WHO也宣布将2019-nCoV疫情列为“国际关注公共卫生突发事件”。中国政府积极行动，启动紧急防控方案。疫情当前，从传染病人确诊到治疗都需要高新技术的投入。目前，新冠病毒，尤其是低丰度病毒的精准和定量检测对于病人确诊、后续控制和治疗都具有积极影响。自病毒基因组测序结果推出后，针对特定核酸序列开发检测产品成为关注点。到目前为止，已有七家体外诊断公司的产品投入市场，还有二十余家企业也相继推出检测产品。实时荧光PCR(逆转录PCR)法是检测COVID-19病毒核酸的一种临床标准。逆转录PCR即逆转录-聚合酶链反应，其原理是，提取组织或细胞中的总RNA，以其中的mRNA作为模板，采用Oligo(dT)或随机引物利用逆转录酶逆转录成cDNA，再以cDNA为模板进行PCR扩增，而获得目的基因或检测基因表达。从临床应用效果看，核酸诊断试剂盒比常规基因组测序分析具有成本和时间上的优势，但因其多基于荧光PCR方法，仍然还存在检测时间较长(2小时左右)、价格高(200元/次)、设备昂贵(Q-PCR仪器30-60万元)，及操作人员专业要求高等问题，因此，目前确诊和疑似病人的诊断仍是后续防控和治疗的局限因素。开发新型传染病的诊断新技术、研制与其配套的POCT(即时现场检测)仪器，实现快速(低于1小时)、低价、灵敏等基层也能推广应用的产品，是急需攻克的科研难题。
一步法逆转录LAMP核酸扩增技术的原理:逆转录LAMP即逆转录和Loop-Mediated Isothermal Amplification(环介导等温扩增)的结合，专门用于快速扩增RNA。LAMP是2000年才出现的一种新颖的等温核酸扩增方法。它利用4或6条模板专一的特异引物和具有链置换能力的DNA聚合酶，在等温条件下(63℃左右)15-60分钟完成扩增。该技术在灵敏度、特异性和检测范围等指标上能媲美甚至优于逆转录PCR技术，同时还不需要模板的热变性、温度循环、电泳及紫外观察等过程，不依赖任何专门的仪器设备。目前已成功应用于人类及动植物、细菌、病毒、寄生虫、真菌等病原体的快速检测。近日，逆转录LAMP法(逆转 录环介导等温扩增法)也应用到了SARS-CoV-2病毒核酸检测中。但逆转录LAM P法检测新型冠状病毒还存在假阳性高、可靠性差等问题。
并且基于CRISPR***的核酸检测技术也仍存在许多限制因素，例如CRI SPR对检测基因序列的限制、多重检测难以实现、crRNA设计相对复杂且合成价格昂贵、crRNA易降解等，仍然成为其进入市场的限制。
因此，本领域迫切需要开发快速、简便、高效、高灵敏并且高准确性的检测SARS-CoV-2的方法。
发明内容
本发明的目的在于提供一种快速、简便、高效、高灵敏并且高准确性的检测SARS-CoV-2的方法。
在本发明第一方面，提供了一种靶标核酸分子的检测方法，包括步骤：
(a)提供一含靶标核酸分子的待检测的样本，所述靶标核酸分子包括单链DNA；
(b)将所述待检测的样本与切割试剂或含所述切割试剂的切割缓冲液进行混合，从而形成一检测体系，其中，所述的切割试剂包括：2个向导ssDNA、基因编辑酶(Ago)、和第一报告核酸分子，所述第一报告核酸分子带有荧光基团和淬灭基团，并且，所述的2个向导ssDNA之间彼此相邻；和
(c)对所述检测体系进行荧光检测，从而获得荧光信号值，其中，所述检测体系中检测到荧光信号值，则表示所述样本中存在靶标核酸分子；而所述检测体系中没有检测到荧光信号值，则表示所述样本中不存在靶标核酸分子。
在另一优选例中，所述的待检测的样本包括未经扩增的样本以及经过扩增(或核酸扩增)的样本。
在另一优选例中，所述的待检测的样本是经过扩增而获得的样本。
在另一优选例中，所述扩增选自下组：PCR扩增、LAMP扩增、RPA扩增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增、滚环扩增、HDA,SPIA,NEAR,TMA、SMAP2、EXPAR、或其组合。
在另一优选例中，所述各向导ssDNA中的5'端的第一位核苷酸为T。
在另一优选例中，所述向导ssDNA的长度各自独立地为10-60nt，较佳地，10-40nt，更佳地，13-20nt。
在另一优选例中，所述向导ssDNA可相同或不同。
在另一优选例中，所述向导ssDNA的长度可相同或不同。
在另一优选例中，所述向导ssDNA为磷酸化的单链DNA分子。
在另一优选例中，所述的向导ssDNA为5'-磷酸化的单链DNA分子；和/或3'-磷酸化的单链DNA分子。
在另一优选例中，所述基因编辑酶Ago选自下组：PfAgo(Pyrococcus furiosus Ago)、MfAgo(Methanocaldococcus fervens Ago)、TcAgo(Thermogladius calderae Ago)、TfAgo(Thermus filiformis Ago)、AaAgo(Aquifex aeolicus Ago)、TpAgo(Thermus parvatiensis Ago)、或其组合。
在另一优选例中，所述基因编辑酶Ago包括PfAgo(Pyrococcus furiosus Ago)。
在另一优选例中，所述的Ago包括野生型和突变型的Ago。
在另一优选例中，所述基因编辑酶的工作温度为87-99℃。
在另一优选例中，所述的靶标核酸分子(或其扩增产物)被所述基因编辑酶Ago切割后，产生次级向导ssDNA。
在另一优选例中，所述次级向导ssDNA的长度为10-60nt，较佳地，10-40nt，更佳地，15-17nt。
在另一优选例中，所述的次级向导ssDNA与所述的荧光报告核酸(第一报告核酸分子)的序列是互补的。
在另一优选例中，所述的次级向导ssDNA与所述的荧光报告核酸(第一报告核酸分子)的序列互补结合后，引导所述基因编辑酶Ago对所述荧光报告核酸(第一报告核酸分子)进行切割，从而产生可检测的信号(如荧光)。
在另一优选例中，所述的荧光基团和淬灭基团各自独立地位于所述荧光报告核酸的5'端、3'端。
在另一优选例中,所述荧光基团选自下组：FAM、HEX、CY3、CY5、ROX、VIC、JOE、TET、Texas Red、NED、TAMRA、或其组合。
在另一优选例中，所述淬灭基团选自下组：TAMRA、BHQ、DABSYL、或其组合。
在另一优选例中，所述的荧光报告核酸(第一报告核酸分子)的长度为9-100nt，较佳地10-60nt，更佳地15-40nt。
在另一优选例中，所述的靶标核酸分子选自下组：病原微生物的核酸分子、基因突变的核酸分子、和特异靶标核酸分子。
在另一优选例中，所述的病原微生物包括病毒、细菌、衣原体、支原体。
在另一优选例中，所述病毒包括植物病毒或动物病毒。
在另一优选例中，所述的病毒包括：冠状病毒、流感病毒、HIV、肝炎病毒、副流感病毒。
在另一优选例中，所述病毒为冠状病毒。
在另一优选例中，所述病毒选自下组：SARS、SARS-CoV-2(COVID-19)、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、Mers-CoV、或其组合。
在另一优选例中，所述的靶标核酸分子包括野生型或突变型的DNA。
在另一优选例中，所述的靶标核酸分子包括单链cDNA。
在另一优选例中，所述的靶标核酸分子包括由RNA逆转录或扩增而获得的DNA，如cDNA等。
在另一优选例中，所述靶标核酸分子包括SARS-CoV-2病毒中存在单碱基突变。
在另一优选例中，所述靶标核酸分子包括SARS-CoV-2病毒ORF1ab,N,E,S基因等的单核苷酸多态性(Single Nucleotide Polymorphism,SNP)突变型。
在另一优选例中，所述靶标核酸分子包括SARS-CoV-2病毒S基因表达的刺突蛋白(spike protein)序列的第614位的突变，氨基酸突变类型为D614G，即第614位氨基酸由天冬氨酸(D)变成了甘氨酸(G)。
在另一优选例中，所述靶标核酸分子还包括在核苷酸水平，SARS-CoV-2病毒的第23,403位核苷酸从A突变为G的突变型。
在另一优选例中，所述靶标核酸分子还包括在核苷酸水平，SARS-CoV-2病毒的T8782C,G11083T,G26144T,C28144T的突变型。
在另一优选例中，所述靶标核酸分子还包括在氨基酸水平，SARS-CoV-2病毒的N501Y,E484Q,L452R的突变型。
在另一优选例中，所述方法用于检测靶位点处的核酸是否在SNP、点突变、缺失、和/或***。
在另一优选例中，所述方法用于对病毒进行分型检测，即判断病毒是野生型还是突变型。
在另一优选例中，当用于对病毒进行分型检测时，所述切割试剂还包括额外的2个报告核酸分子(第二报告核酸分子和第三报告核酸分子)。
在另一优选例中，所述的第二报告核酸分子、第三报告核酸分子的长度各自独立地为9-200nt，较佳地10-60nt，更佳地15-40nt。
在另一优选例中，所述第二报告核酸分子与野生型的病毒所产生的次级向导ssDNA的序列互补结合后，引导基因编辑酶Ago对第二报告核酸分子进行切割， 从而产生可检测的信号(如荧光)。
在另一优选例中，所述第三报告核酸分子与野生型的病毒所产生的次级向导ssDNA的序列不能互补结合，因此基因编辑酶Ago对第三报告核酸分子不进行切割，从而不能产生可检测的信号(如荧光)。
在另一优选例中，所述第二报告核酸分子与突变型的病毒所产生的次级向导ssDNA的序列不能互补结合，因此基因编辑酶Ago对第二报告核酸分子不进行切割，从而不能产生可检测的信号(如荧光)。
在另一优选例中，所述第三报告核酸分子与突变型的病毒所产生的次级向导ssDNA的序列互补结合后，引导基因编辑酶Ago对第二报告核酸分子进行切割，从而产生可检测的信号(如荧光)。
在另一优选例中，在步骤(c)中所述荧光检测采用酶标仪或者荧光分光光度计进行检测。
在另一优选例中，所述的检测体系中还含有缓冲液或缓冲剂。
在另一优选例中，所述的待检测的样本是用选自下组的引物进行扩增所获得的待检测的样本。
在另一优选例中，所述的引物是选自下组的任一引物对：
Figure PCTCN2021113348-appb-000001
Figure PCTCN2021113348-appb-000002
Figure PCTCN2021113348-appb-000003
Figure PCTCN2021113348-appb-000004
Figure PCTCN2021113348-appb-000005
在另一优选例中，所述的向导ssDNA选自下组：
Figure PCTCN2021113348-appb-000006
Figure PCTCN2021113348-appb-000007
其中P为寡核苷酸5'端磷酸化修饰。
在另一优选例中，所述的待检测的样本是用选自下组样本制备的核酸样本：咽喉拭子、肺泡灌洗液、鼻拭子、尿液、粪便、体液、或其组合。
在另一优选例中，所述的方法是体外方法。
在另一优选例中，所述的方法是非诊断性和非治疗性的。
本发明第二方面提供了一种用于靶标核酸分子的检测试剂盒，所述试剂盒包括：
(a)用于扩增靶标核酸分子的扩增试剂，所述扩增试剂包括：用于扩增靶标核酸分子的引物对，所述引物对用于进行基于所述靶核酸分子的特异性扩增反应，从而产生特异的核酸扩增产物；
(b)切割试剂或含所述切割试剂的切割缓冲液，其中所述的切割试剂包括：2个向导ssDNA、基因编辑酶(Ago)、和第一报告核酸，所述第一报告核酸带有荧光基团和淬灭基团，并且，所述的2个向导ssDNA之间彼此相邻。
在另一优选例中，所述试剂盒还包括第二报告核酸、第三报告核酸。
在另一优选例中，所述的试剂盒还包括：
(c)用于逆转录反应的逆转录试剂，所述的逆转录试剂包括：逆转录酶或具备逆转录酶活性的Bst酶(或其突变体)。
在另一优选例中，所述试剂盒含有以下优选的试剂组合：
针对SARS-CoV-2ORF1a：
引物对1:SEQ ID No:1-6；
荧光报告核酸：SEQ ID No.59；
ssDNA：SEQ ID No:49和50；
针对SARS-CoV-2ORF1b基因：
引物对3:SEQ ID No:13-18；
荧光报告核酸：SEQ ID No.60；
ssDNA：SEQ ID No:51和52；
针对SARS-CoV-2N基因：
引物对5:SEQ ID No:25-30；
荧光报告核酸：SEQ ID No.62；
ssDNA：SEQ ID No:55和56；
针对RNase P内参基因：
引物对7:SEQ ID No:37-42；
荧光报告核酸：SEQ ID No.63和64；
ssDNA：SEQ ID No:57和58。
本发明第三方面提供了一种用于检测靶标核酸分子的试剂盒，所述试剂盒包括：
(i)第一容器以及位于第一容器内的向导ssDNA，所述向导ssDNA为2个，并且所述的2个向导ssDNA之间彼此相邻；
(ii)第二容器以及位于第二容器内的基因编辑酶(Ago)；
(iii)第三容器以及位于第三容器内的第一报告核酸，所述第一报告核酸带有荧光基团和淬灭基团；
(iv)第四容器以及位于第四容器内的用于扩增靶标核酸分子的扩增试剂；和
(v)任选的第五容器以及位于第五容器内的缓冲液；
(vi)任选的PCR管和与PCR对应的内衬管；
其中，所述的靶标核酸分子包括单链DNA。
在另一优选例中，所述试剂盒还包括第六容器，以及位于第六容器的第二报告核酸和第三报告核酸。
在另一优选例中，所述扩增试剂包括：用于扩增靶标核酸分子的引物对，所述引物对用于进行基于所述靶核酸分子的特异性扩增反应，从而产生特异的核酸扩增产物。
在另一优选例中，所述的位于第五容器内的缓冲液包括：用于扩增反应的缓冲液和用于酶进行酶切的缓冲液。
在另一优选例中，所述用于扩增反应的缓冲液包括：Bst buffer(Tris-Hcl，EDTA，NaCl或KCl)、MgSO 4、dNTP、DNase/RNase free H 2O。
在另一优选例中，所述的用于酶进行酶切的缓冲液包括Bst buffer或Reaction buffer(Tris-Hcl，EDTA，NaCl或KCl)、MnCl 2、DNase/RNase free H 2O。
在另一优选例中，所述的第一容器、第二容器、第三容器、第四容器、第五 容器、第六容器中的任何二个、三个、四个或全部可以是相同或不同容器。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了改良内衬管和PCR管组合的实物图，包括立体透视示意图，俯视截面示意图；1为内衬管管体，2为PCR管体，3为内衬管反应腔，4为PCR管反应腔，101为内衬管管壁(厚度3-7mm)，102为内衬管管底孔(孔径3-5mm)，103为内衬管管壁与PCR管之间的空隙(空隙最大处为5-10mm)；
图2显示了逆转录LAMP-RADAR偶联的“一管式”双重核酸快速检测示意图；
图3显示了逆转录LAMP-RADAR检测原理图；
图4显示了SARS-CoV-2病毒LAMP-RADAR检测灵敏度；
图5显示了部分SARS-CoV-2临床样品荧光实时检测示意图；
图6显示了SARS-CoV-2双重LAMP-RADAR特异性实验结果；
图7显示了SARS-CoV-2LAMP-RADAR分型检测原理示意图；
图8显示了SARS-CoV-2基因序列D614G突变位点。
具体实施方式
本发明人通过广泛而深入的研究，首次开发了一种针对靶标核酸分子(比如新冠病毒SARS-CoV-2的靶标核酸分子)的灵敏度高、操作简便、检测成本低、耗时短、准确性高的核酸检测方法。本发明对靶标核酸分子进行逆转录环介导等温扩增反应(逆转录LAMP)，然后利用Ago酶的特性，即在首次由2个邻近且无间隔序列的初级向导ssDNA(guide ssDNA)介导剪切后，在合适的反应温度(如约90-98度)下，断裂的5'核酸片段可再次被Ago酶利用去剪切与之互补的荧光报告核酸链，从而根据荧光来判断样本中是否含有靶标核酸分子。结果表明，本发明方法可以对靶标核酸分子进行快速且高灵敏度、高准确度的检测，从而为病原体检测、基因分型、病程监测等提供帮助。在此基础上完成了本发明。
术语
术语“LAMP”是环介导等温扩增技术(Loop-mediated isothermal amplification),是一种适用于基因诊断的恒温核酸扩增技术。
术语“PCR”是聚合酶链式反应技术(Polymerase chain reaction)，是一种适用于靶标核酸扩增的技术。
如本文所用，术语“二次切割”指在本发明检测方法中，在初级向导ssDNAs存在下，本发明Ago酶对目标核酸序列进行剪切，形成新的5'磷酸化的核酸序列(次级向导ssDNAs)；然后，次级向导ssDNA继续在PfAgo酶的作用下，引导PfAgo酶对与次级向导ssDNAs互补的荧光报告核酸进行剪切。这种先针对目标核酸序列进行特异性切割(第一次切割)，后对荧光报告核酸进行特异性切割(第二次切割)，被定义为“二次切割”。在本发明中，第一次切割和第二次切割都是特异性切割。
Ago酶
在本发明和检测方法中，一个核心成分是基因编辑酶，例如Ago酶。
在本发明中，优选的Ago酶是PfAgo酶，其来自于古菌Pyrococcus furiosus，基因长度2313bp，氨基酸序列由770个氨基酸组成。
PfAgo酶的酶切特性为：该酶可利用5'磷酸化的寡聚核苷酸作为向导ssDNA指导该酶对目标核酸序列的精确剪切；剪切位点位于与向导ssDNA的第10与11位核苷酸对应的目标核酸(ssDNA)之间的磷酸二酯键。
通常，PfAgo酶的优选工作温度为95±2度。
向导ssDNA
在本发明的检测方法中，一个核心成分是向导ssDNA，尤其是2个ssDNA，并且彼此之间邻近，且彼此之间无间隔碱基或间隔序列。
在本发明中，优选的向导ssDNAs均为长度为10-60nt，较佳地，10-40nt，更佳地，13-20nt的寡聚核苷酸，其5'第一个核苷酸均为胸腺嘧啶(T)，可被磷酸化修饰。
报告核酸分子
在本发明的检测方法中，一个核心成分是携带报告分子的报告核酸。
在一优选实施方式中，本发明的报告核酸分子是分别携带荧光基团和淬灭基团的核酸分子。例如，在5'端标记荧光基团(F)，3'端标记淬灭基团(Q)。
在本发明中，荧光报告核酸分子是根据次级向导ssDNAs的产生位置所决 定的；由初级向导ssDNAs对目标核酸序列进行剪切，形成新的5'磷酸化的核酸序列，称之为次级向导ssDNAs，荧光报告核酸覆盖次级向导ssDNAs的所有位置。
检测方法
在本发明还提供了基于基因编辑酶Ago，比如Pyrococcus furiosus Argonaute(PfAgo)的核酸检测方法。
在本发明方法中，基于PfAgo酶的剪切活性，可根据靶标核酸分子(比如单链DNA，优选经扩增后的靶标核酸分子)设计出2个向导ssDNA，这2个向导ssDNA彼此相邻，并且无间隔序列，向导ssDNA靶向于靶标核酸分子并介导PfAgo酶对靶标核酸分子进行剪切以形成新的次级向导ssDNA。次级向导ssDNA继续在PfAgo酶的作用下，引导PfAgo酶对与次级向导ssDNAs互补的荧光报告核酸进行剪切，从而达到对靶标核酸分子的检测。本发明的方法可大幅度提高靶标核酸检测的灵敏度和准确性。
在本发明中，根据向导ssDNAs的设计要求，可通过特殊设计使PfAgo酶可以选择性地对存在部分位点存在差异的核酸序列进行选择性剪切，从而实现分型检测。
在本发明中，当用于区分不同分型，在向导ssDNAs设计时，将不同分型对应的突变位点置于向导ssDNAs的第10、11两位，由于PfAgo酶的选择特异性，连续两点突变时可抑制剪切活性，从而达到了对不同分型的检测。
在本发明中，可以在PfAgo酶的剪切体系中同时加入多种靶标核酸分子和向导ssDNAs，结合带有不同荧光基团的报告核酸，能达到对目的核酸的多重检测。
本发明方法非常适合用于检测痕量核酸。通过结合逆转录LAMP以及具有特定序列的向导ssDNA，本发明可以检测低浓度核酸模板(220copies/mL)的靶标核酸分子。
在本发明中，所用于扩增反应的扩增引物，其Tm值通常65±10度左右，扩增片段大小约为90-200bp。优选地，扩增引物设计时应避开待检测区段。
试剂盒
本发明还提供了一种用于靶标核酸分子的检测试剂盒。
在一优选实施方式中，本发明的试剂盒包括：(a)用于扩增靶标核酸分子的 扩增试剂，所述扩增试剂包括：用于扩增靶标核酸分子的引物对，所述引物对用于进行基于所述靶核酸分子的特异性扩增反应，从而产生特异的核酸扩增产物；
(b)切割试剂或含所述切割试剂的切割缓冲液，其中所述的切割试剂包括：2个向导ssDNA、基因编辑酶(Ago)、和第一报告核酸，所述荧光报告核酸带有荧光基团和淬灭基团，并且，所述的2个向导ssDNA之间彼此相邻。
在一优选实施方式中，本发明的试剂盒包括：
(i)第一容器以及位于第一容器内的向导ssDNA，所述向导ssDNA为2个，并且所述的2个向导ssDNA之间无间隔序列；
(ii)第二容器以及位于第二容器内的基因编辑酶(Ago)；
(iii)第三容器以及位于第三容器内的第一报告核酸，所述第一报告核酸带有荧光基团和淬灭基团；
(iv)第四容器以及位于第四容器内用于扩增靶标核酸分子的扩增试剂；和
(v)任选的第五容器以及位于第五容器内的缓冲液；
(vi)任选的第六容器以及位于第六容器内的第二报告核酸和第三报告核酸，所述第二报告核酸、第三报告核酸带有荧光基团和淬灭基团；
(vi)任选的PCR管和与PCR对应的内衬管(内衬管如图1-3所示)；
其中，所述的靶标核酸分子包括单链DNA。
应用
本发明特别适合检测微量靶标核酸分子，以及多重检测，具有广泛的应用性。
在本发明中，靶标核酸分子可以是DNA，也可以是RNA。当靶标核酸分子是RNA时，可通过逆转录转变为cDNA再进行检测。
本发明在疾病监控方面，可对疾病做到预测、预防等积极主动管理，做到早期发现早期治疗，或提早预测提早预防。由于本发明的检测灵敏度和准确性非常高，适合进行早期诊断，对症下药，节省患者治疗时间，提高治疗成功率。本发明减少高额医疗成本浪费，和争取治疗黄金时机。
在环境监控方面，本发明可便捷、快速的对环境污染物中的核酸分子进行准确鉴定，提供有效的环境检测数据。
本发明的主要优点包括：
(1)检测谱系广泛：本发明具有广泛的核酸检测谱，可实现对病毒、细菌、遗传病疾病基因的高效检测；
(2)灵敏度高：PfAgo特异性核酸级联反应体系，可在低浓度核酸模板(100copies/mL)下检测，具有稳定性、可靠性；
(3)时间短：45分钟内即可实现目标基因的检测；
(4)可靠性高：本发明避免了LAMP技术中环引物间的非特异性配对导致的假阳性，提高检测准确性；
(5)POCT***：新型冠状病毒等目标核酸的“一管式”一步操作检测，应用便携式等温荧光***，符合POCT理念；
(6)多重检测：本发明还可在单管反应体系中实现多重检测，显著降低体系复杂性和使用成本。
(7)突变碱基容错度高：可检测出扩增区域出现单碱基或双碱基突变的病毒。
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。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明中所涉及的实验材料如无特殊说明均可从市售渠道获得。
在本发明中，所有序列的编号均指具体的序列，其不带有任何修饰。
实施例1
检测试剂的制备和检测方法
在本实施例中，提供了本发明SARS-CoV-2病毒核酸等温快速检测试剂盒的及其使用方法。
1.1检测试剂和试剂盒
在本实施例中，以检测SARS-CoV-2病毒的ORF1b基因为例，相应的特异性目标核酸序列为：
Figure PCTCN2021113348-appb-000008
Figure PCTCN2021113348-appb-000009
基于本发明方法，相应的检测试剂包括：
(1)、扩增引物，具体序列如下：
Figure PCTCN2021113348-appb-000010
(2)、特异性guide ssDNA(gDNA)，具体序列如下：
2019-nCoV ORF 1b-gDNA 1 5'-P-TTGATGAGGTTCCACC-3' SEQ ID NO.51
2019-nCoV ORF 1b-gDNA 4 5'-P-TCAGTTGTGGCATCTC-3' SEQ ID NO.52
(3)、与特异性guide ssDNA(gDNA)相应的荧光报告核酸，具体序列如下：
Figure PCTCN2021113348-appb-000011
(4)、LAMP扩增缓冲液：Bst buffer(Tris-Hcl，EDTA，NaCl或KCl)，MgSO 4，dNTP,DNase/RNase free H 2O。
(5)、Ago酶切缓冲液：Bst buffer或Reaction buffer(Tris-Hcl，EDTA，NaCl或KCl)，MnCl 2，DNase/RNase free H 2O。
(6)、扩增酶：Bst聚合酶、反转录酶。
(7)、切割酶：PfAgo。
(7)、PCR管和与PCR管对应的内衬管。
1.2检测方法
本发明SARS-CoV-2病毒核酸等温快速检测试剂盒的检测方法具体操作步骤如下：
(1)、所述的扩增引物、gDNA干粉用DNase/RNase free H 2O溶解制成100uM的储存液；所述的荧光报告核酸干粉用DNase/RNase free H 2O溶解制成10uM的储存液；
(2)、用Bst酶、Ago酶切缓冲液扩增引物配制成25μl扩增反应预混液；
(3)、将15μl待检核酸样品加入扩增反应预混液，扩增反应体系为40μl；
(4)、用PfAgo、LAMP扩增缓冲液、gDNA、荧光报告核酸配制成20μl酶切反应体系；
(5)、分别将扩增体系和酶切体系分别转移至PCR管反应腔和内衬管反应腔中，因液体表面张力，内衬管反应腔中的酶切体系不会掉入PCR管反应腔，放入荧光定量PCR仪中进行反应(65℃扩增30min，瞬时离心后，再95℃酶切30min，每分钟检测一次信号)。
实施例2
检测试剂的制备和检测方法(不使用内衬管)
在本实施例中，提供了本发明SARS-CoV-2病毒核酸等温快速检测试剂盒的及其使用方法。
1.1检测试剂和试剂盒
在本实施例中，以检测SARS-CoV-2病毒的ORF1b基因为例，相应的特异性目标核酸序列参照实施例1。
基于本发明方法，相应的检测试剂包括：
(1)、扩增引物，具体序列如下：
Figure PCTCN2021113348-appb-000012
(2)、特异性guide ssDNA(gDNA)，具体序列如下：
2019-nCoV ORF 1b-gDNA 1 5'-P-TTGATGAGGTTCCACC-3' SEQ ID NO.51
2019-nCoV ORF 1b-gDNA 4 5'-P-TCAGTTGTGGCATCTC-3' SEQ ID NO.52
(3)、与特异性guide ssDNA(gDNA)相应的荧光报告核酸，具体序列如下：
Figure PCTCN2021113348-appb-000013
(4)、LAMP扩增缓冲液：Bst buffer(Tris-Hcl，EDTA，NaCl或KCl)，MgSO 4， dNTP,DNase/RNase free H 2O。
(5)、Ago酶切缓冲液：Bst buffer或Reaction buffer(Tris-Hcl，EDTA，NaCl或KCl)，MnCl 2，DNase/RNase free H 2O。
(6)、扩增酶：Bst聚合酶、反转录酶。
(7)、切割酶：PfAgo。
(8)、PCR管。
1.2检测方法
本发明SARS-CoV-2病毒核酸等温快速检测试剂盒的检测方法具体操作步骤如下：
(1)、所述的扩增引物、gDNA干粉用DNase/RNase free H 2O溶解制成100uM的储存液；所述的荧光报告核酸干粉用DNase/RNase free H 2O溶解制成10uM的储存液；
(2)、用Bst酶、Ago酶切缓冲液扩增引物配制成25μl扩增反应预混液；
(3)、将15μl待检核酸样品加入扩增反应预混液，扩增反应体系为40μl；
(4)、将扩增体系放入荧光定量PCR仪中进行反应，65℃扩增30min；
(5)、用PfAgo、LAMP扩增缓冲液、gDNA、荧光报告核酸配制成20μl酶切反应体系，将酶切反应体系加入到反应完成的扩增体系中，再放入荧光定量PCR仪中，95℃酶切30min，每分钟检测一次信号。
实施例3
针对不同浓度的待检标准品进行检测
按3倍倍比稀释的原则对待检标准品(SEQ ID NO.：65)进行稀释，分别稀释成18000copies/mL，6000copies/mL，2000copies/mL，670copies/mL，220copies/mL，70copies/mL的标准品稀释液，分别吸取140μl标准品稀释液进行核酸提取(QIAamp Viral RNA Mini Kit)。将15μl核酸提取样品、阴性对照(H2O)和阳性对照(目标片段质粒)加入到实施例2中的扩增体系中，每个浓度梯度分别做3组，每组做7个重复，计算每组的检出率，实验按实施例3步骤进行操作。
结果如图4所示，18000copies/mL，6000copies/mL，2000copies/mL，670copies/mL均稳定检出，阴性对照和阳性对照成立。
结果表明，本发明SARS-CoV-2病毒最低检测限可达670copies/mL。
实施例4
临床样品的双重检测
使用6个经2种荧光RT-PCR方法确诊的临床样品。
需要说明的是，RNase P内参基因是作为内部质控的人类基因组的部分基因。
双重反应体系的配置：向实施例2中的扩增体系中加入RNase P内参基因扩增引物(引物对7)，向酶切体系中加入RNase P gDNA(SEQ ID NO.57和SEQ ID NO.58)和RNase P荧光报告核酸(SEQ ID NO.64)，反应条件参照实施例2。
Figure PCTCN2021113348-appb-000014
Figure PCTCN2021113348-appb-000015
结果如图5所示，当检测目标核酸为ORF1b基因和RNase P基因时，FAM和VIC同时产生荧光信号。结果表明本发明的方法可用于单管双重检测。
实施例5
特异性检测
以21种常见呼吸道病原体全长核酸样品、人类基因组全长核酸样品和SARS-CoV-2全长核酸样品为待检核酸样品，参照实施例4进行体系配置和扩增、酶切反应。
结果如图6所示，21种常见呼吸道病原体全长核酸样品均未产生信号，人类基因组全长核酸样品产生VIC信号，SARS-CoV-2全长核酸样品产生FAM信号。结果表明本发明的方法特异性良好。
实施例6
突变毒株的分型检测
北京新冠病例出现D614G突变，感染力或增强近10倍，如图8所示，D614A>G 即A变G。
该突变毒株具有较强的致病性，不排除有爆发可能性，对此，本发明设计分型检测。因为RT-PCR做单碱基分型比较困难，ddPCR和测序方法时间较长、成本昂贵等。本发明可灵活设计对已知突变进行区分检测，具体原理如图7所示。
结合本发明的技术(LAMP-RADAR)对SARS-CoV-2病毒D614G突变与SARS-CoV-2病毒核酸序列进行区分检测，具体设计如下：
在原有设计(双guide DNA)基础上，另外设计两个Reporter。一组gDNA保留对原SARS-CoV-2检测位点的设计，另两个Reporter相对于二级gDNA的10或11位引入一个或连续两个mismatch，这样保障二级gDNA相对与新设计的Reporter至少有一个mismatch。Reporter详细设计如下(Reporter以FAM探针和VIC探针为例)：设计D614(野生型)次级gDNA与FAM探针只有一个mismatch，与VIC探针有连续2点mismatch，从而在D614(野生型)次级gDNA介导下，PfAgo只切FAM探针，不切VIC探针；而G614(突变型)次级gDNA与FAM探针有连续2点mismatch，与VIC探针只有一个mismatch，从而在G614(突变型)次级gDNA介导下，PfAgo只切VIC探针，不切FAM探针。基于以上设计，根据两个Reporter的荧光信号产生情况，来判断D614型和G614型，达到对D614G的分型检测。
合成reporter对应序列设计如下：
D614-FAM-11G：5'FAM-CTGTGCAGTTAACGTCCTGATAAAGAACAG(SEQ ID NO.66)-BHQ1 3'
D614-FAM-11C:5'FAM-CTGTGCAGTTAACCTCCTGATAAAGAACAG(SEQ ID NO.67)-BHQ1 3'
D614-FAM-11T:5'FAM-CTGTGCAGTTAACTTCCTGATAAAGAACAG(SEQ ID NO.68)-BHQ1 3'
G614-VIC-11T:5'VIC-CTGTGCAGTTAACTCCCTGATAAAGAACAG(SEQ ID NO.69)-BHQ1 3'
G614-VIC-11G:5'VIC-CTGTGCAGTTAACGCCCTGATAAAGAACAG(SEQ ID NO.70)-BHQ1 3'
G614-VIC-11C:5'VIC-CTGTGCAGTTAACCCCCTGATAAAGAACAG(SEQ ID NO.71)-BHQ1 3'
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种靶标核酸分子的检测方法，其特征在于，包括步骤：
    (a)提供一含靶标核酸分子的待检测的样本，所述靶标核酸分子包括单链DNA；
    (b)将所述待检测的样本与切割试剂或含所述切割试剂的切割缓冲液进行混合，从而形成一检测体系，其中，所述的切割试剂包括：2个向导ssDNA、基因编辑酶(Ago)、和第一报告核酸分子，所述第一报告核酸分子带有荧光基团和淬灭基团，并且，所述的2个向导ssDNA之间彼此相邻；和
    (c)对所述检测体系进行荧光检测，从而获得荧光信号值，其中，所述检测体系中检测到荧光信号值，则表示所述样本中存在靶标核酸分子；而所述检测体系中没有检测到荧光信号值，则表示所述样本中不存在靶标核酸分子。
  2. 如权利要求1所述的方法，其特征在于，所述的待检测的样本包括未经扩增的样本以及经过扩增(或核酸扩增)的样本。
  3. 如权利要求1所述的方法，其特征在于，所述各向导ssDNA中的5'端的第一位核苷酸为T。
  4. 如权利要求1所述的方法，其特征在于，所述向导ssDNA的长度各自独立地为10-60nt，较佳地，10-40nt，更佳地，13-20nt。
  5. 如权利要求1所述的方法，其特征在于，所述向导ssDNA为磷酸化的单链DNA分子。
  6. 如权利要求1所述的方法，其特征在于，所述基因编辑酶Ago选自下组：PfAgo(Pyrococcus furiosus Ago)、MfAgo(Methanocaldococcus fervens Ago)、TcAgo(Thermogladius calderae Ago)、TfAgo(Thermus filiformis Ago)、AaAgo(Aquifex aeolicus Ago)、TpAgo(Thermus parvatiensis Ago)、或其组合。
  7. 如权利要求1所述的方法，其特征在于，所述的第一报告核酸分子的长度为9-100nt，较佳地10-60nt，更佳地15-40nt。
  8. 如权利要求1所述的方法，其特征在于，所述的靶标核酸分子选自下组：病原微生物的核酸分子、基因突变的核酸分子、和特异靶标核酸分子。
  9. 一种用于靶标核酸分子的检测试剂盒，其特征在于，所述试剂盒包括：
    (a)用于扩增靶标核酸分子的扩增试剂，所述扩增试剂包括：用于扩增靶标核酸分子的引物对，所述引物对用于进行基于所述靶核酸分子的特异性扩增反应，从而产生特异的核酸扩增产物；
    (b)切割试剂或含所述切割试剂的切割缓冲液，其中所述的切割试剂包括：2 个向导ssDNA、基因编辑酶(Ago)、和第一报告核酸，所述第一报告核酸带有荧光基团和淬灭基团，并且，所述的2个向导ssDNA之间彼此相邻。
  10. 一种用于检测靶标核酸分子的试剂盒，其特征在于，所述试剂盒包括：
    (i)第一容器以及位于第一容器内的向导ssDNA，所述向导ssDNA为2个，并且所述的2个向导ssDNA之间彼此相邻；
    (ii)第二容器以及位于第二容器内的基因编辑酶(Ago)；
    (iii)第三容器以及位于第三容器内的第一报告核酸，所述第一报告核酸带有荧光基团和淬灭基团；
    (iv)第四容器以及位于第四容器内的用于扩增靶标核酸分子的扩增试剂；和
    (v)任选的第五容器以及位于第五容器内的缓冲液；
    (vi)任选的PCR管和与PCR对应的内衬管；
    其中，所述的靶标核酸分子包括单链DNA。
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