WO2019192156A1 - 基于原核Argonaute蛋白的核酸检测方法及其应用 - Google Patents

基于原核Argonaute蛋白的核酸检测方法及其应用 Download PDF

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WO2019192156A1
WO2019192156A1 PCT/CN2018/110056 CN2018110056W WO2019192156A1 WO 2019192156 A1 WO2019192156 A1 WO 2019192156A1 CN 2018110056 W CN2018110056 W CN 2018110056W WO 2019192156 A1 WO2019192156 A1 WO 2019192156A1
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nucleic acid
target nucleic
detection system
acid molecule
detection
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PCT/CN2018/110056
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French (fr)
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冯雁
荀冠华
刘倩
崇曰盛
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上海交通大学
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Priority to EP18913920.7A priority Critical patent/EP3792364A4/en
Priority to JP2020554479A priority patent/JP7224676B2/ja
Priority to US17/045,393 priority patent/US20210164024A1/en
Publication of WO2019192156A1 publication Critical patent/WO2019192156A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • 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
    • C12Q1/70—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
    • C12Q1/708—Specific hybridization probes for papilloma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a nucleic acid detection method based on prokaryotic Argonaute protein and application thereof.
  • Nucleic acid detection technology is widely used in many fields such as molecular medical diagnosis, food safety testing and environmental monitoring. Fast, inexpensive and sensitive nucleic acid detection can be widely used in pathogen detection, genotyping, disease monitoring and other aspects.
  • the object of the present invention is to provide a nucleic acid detection method based on high sensitivity, specificity and high throughput for target DNA and its application.
  • a detection system for detecting a target nucleic acid molecule comprising:
  • target nucleic acid molecule is a target DNA.
  • the guide ssDNA pair comprises a sense strand guide ssDNA and an antisense strand guide ssDNA.
  • the guide ssDNA is a 5'-phosphorylated single-stranded DNA molecule.
  • the guide ssDNA is n bases in length and n ⁇ 14.
  • said n ⁇ 100, preferably ⁇ 80, more preferably ⁇ 60.
  • the guide ssDNAs are 14-60 nt in length, preferably 16-40 nt.
  • the number of the guide ssDNAs is one or more pairs.
  • the PfAgo enzyme is derived from the archaea Pyrococcus furiosus.
  • the PfAgo comprises wild-type and mutant PfAgo.
  • the different positions of the target nucleic acid molecules correspond to mutation sites at positions 10 and 11 of the ssDNA.
  • the detection system further comprises (d) a buffer.
  • the detection system further comprises a primer for amplifying the target nucleic acid molecule.
  • the detection system further comprises a target nucleic acid molecule to be detected.
  • the target nucleic acid molecule (or an amplification product thereof) is cleaved by the PfAgo enzyme to produce a secondary guide ssDNA.
  • the secondary guide ssDNA is complementary to the sequence of the fluorescent reporter nucleic acid.
  • the PfAgo enzyme is directed to cleave the fluorescent reporter nucleic acid to generate a detectable signal (such as fluorescence). ).
  • the concentration of the target nucleic acid molecule to be detected in the detection system is 1-1000 copies/ ⁇ l or 10 3 -10 10 copies/ ⁇ L, preferably 100-1000 copies. / microliter, more preferably 1-100 copies / microliter.
  • the concentration of the target nucleic acid molecule to be detected in the detection system is from 1 fM to 200 pM, preferably from 1 to 1000 fM, more preferably from 1 to 100 fM, most preferably from 1 to 20 fM.
  • the genetically modified enzyme has an operating temperature of from 87 to 99 °C.
  • the concentration of the fluorescent reporter nucleic acid in the detection system is from 100 to 1000 nM.
  • the molar ratio of the fluorescent reporter nucleic acid to the target nucleic acid molecule is from 10 3 :1 to 10 8 :1, preferably 10 4 :1 to 10 7 : 1.
  • the target DNA comprises cDNA.
  • the target DNA is selected from the group consisting of single-stranded DNA (including cDNA), double-stranded DNA, or a combination thereof.
  • the fluorophore and the quenching group are each independently located at the 5' end and the 3' end of the fluorescent reporter nucleic acid.
  • the fluorescent reporter nucleic acid has a length of from 9 to 100 nt, preferably from 10 to 60 nt, more preferably from 15 to 40 nt.
  • the target nucleic acid molecule comprises a target nucleic acid molecule derived from a group selected from the group consisting of a plant, an animal, a microorganism, a virus, or a combination thereof.
  • the target DNA is synthetic or naturally occurring DNA.
  • the target DNA comprises wild-type or mutant DNA.
  • kits for detecting a target nucleic acid molecule comprising:
  • the kit may further comprise a buffer.
  • the kit comprises:
  • the kit further comprises:
  • the buffer for enzymatic digestion of the enzyme contains MnCl 2 .
  • the kit further comprises:
  • a method of detecting the presence or absence of a target nucleic acid molecule in a sample comprising the steps of:
  • the fluorescent signal value is detected in the first reaction solution, indicating that the target nucleic acid molecule is present in the sample; and the fluorescent signal value is not detected in the first reaction solution, indicating that the target is not present in the sample. Nucleic acid molecule.
  • 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 nucleic acid amplification method is selected from the group consisting of PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification. Addition and rolling circle amplification.
  • the PCR comprises high temperature PCR, normal temperature PCR, and low temperature PCR.
  • the method is for detecting whether a nucleic acid at a target site is at a SNP, a point mutation, a deletion, and/or an insertion.
  • the fluorescent detection in step (b) is detected using a microplate reader or a fluorescence spectrophotometer.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • a nuclease Pyrococcus furiosus Argonaute
  • a reagent or kit for detecting a target nucleic acid molecule based on a secondary cleavage for the preparation of a reagent or kit for detecting a target nucleic acid molecule based on a secondary cleavage.
  • the enzyme Pyrococcus furiosus Argonaute is derived from the archaea Pyrococcus furiosus; or a homologous analog having the same or similar function.
  • the PfAgo comprises wild-type and mutant PfAgo.
  • Figure 1 shows a first cleavage of the enzyme encoding the gene of the present invention, in which a pair of guide ssDNAs are designed for the same DNA strand, the corresponding cleavage sites are indicated by black arrows, and the pfAgo enzyme acts to form a blunt end.
  • Figure 2 shows a second cleavage of the gene encoding enzyme of the present invention in which specific cleavage guided by primary guide ssDNAs produces secondary guide ssDNAs.
  • FIG. 3 shows the steps and principles of the detection method of the present invention.
  • Figure 4 shows the specificity and sensitivity of the nucleic acid detection system.
  • Figure 5 shows the results of the multiplicity experiment of the nucleic acid detection system in one embodiment of the present invention. among them
  • Figure 5A uses a CON1-1b-FAM reporter nucleic acid molecule (no template);
  • Figure 5B uses the CON1-1b-FAM reporter nucleic acid molecule (CON1-1b template);
  • Figure 5C uses the CON1-1b-FAM reporter nucleic acid molecule (JFH-1 2a template);
  • Figure 5D uses JFH-1 2a-VIC reporter nucleic acid molecules (no template);
  • Figure 5E uses JFH-1 2a-VIC reporter nucleic acid molecule (JFH-1 2a template);
  • Figure 5F uses the JFH-1 2a-VIC reporter nucleic acid molecule (CON1-1b template).
  • Figure 6 shows the multiplex detection of different combinations of HPV samples using plasmids as samples.
  • Figure 7 shows the high-risk subtype HPV16/HPV18 for multiple detection clinical samples.
  • the method of the present invention utilizes the property of the PfpAgo enzyme, that is, after the first ssDNA (guide ssDNA) mediated cleavage, the cleavable 5' nucleic acid fragment can be again used by PfAgo at a suitable reaction temperature (e.g., about 90-98 degrees).
  • the enzyme utilizes to cleave the fluorescent reporter nucleic acid strand complementary thereto.
  • detection system of the invention As used herein, the terms “detection system of the invention”, “nucleic acid detection system based on Argonaute protein” are used interchangeably and refer to the detection system described in the first aspect of the invention.
  • the terms "detection method of the present invention”, “nucleic acid detection method based on Argonaute protein” are used interchangeably and refer to the detection method described in the second aspect of the invention.
  • the terms "gene editing enzyme Pyrococcus furiosus”, “nuclease Pyrococcus furiosus”, “PfAgo enzyme” are used interchangeably and refer to the enzymes described in the first aspect of the invention.
  • the term "secondary cleavage" means that in the detection method of the present invention, the Ago enzyme of the present invention cleaves a target nucleic acid sequence in the presence of a primary guide ssDNAs to form a new 5' phosphorylated nucleic acid sequence (secondary) The guide ssDNAs); then, the secondary guide ssDNA continues to direct the PfAgo enzyme to cleave the fluorescent reporter nucleic acid complementary to the secondary guide ssDNAs under the action of the PfAgo enzyme.
  • This specific cleavage (first cleavage) of the target nucleic acid sequence followed by specific cleavage of the fluorescent reporter nucleic acid (second cleavage) is defined as "secondary cleavage". In the present invention, both the first cut and the second cut are specific cuts.
  • one core component is a gene editing enzyme such as an Ago enzyme.
  • a preferred Ago enzyme is a PfAgo enzyme derived from the archaea Pyrococcus furiosus, having a gene length of 2313 bp and an amino acid sequence consisting of 770 amino acids.
  • the enzymatic cleavage property of the PfAgo enzyme is that the enzyme can use the 5' phosphorylated oligonucleotide as a guide ssDNA to guide the precise cleavage of the target nucleic acid sequence by the enzyme; the cleavage site is located at the 10th and 11th with the guide ssDNA The phosphodiester bond between the target nucleic acid (ssDNA) corresponding to the nucleotide.
  • the preferred operating temperature of the PfAgo enzyme is 95 ⁇ 2 degrees.
  • one core component is a guide ssDNA pair.
  • preferred guide ssDNAs are oligonucleotides of 14-24 nt (e.g., 16 nt) in length, and the 5' first nucleotide is phosphorylated thymidine (T).
  • a pair of guide ssDNAs for the same DNA strand bind to the target nucleic acid molecule, the corresponding cleavage site is indicated by a black arrow, and the pfAgo enzyme acts to form a blunt end.
  • one core component is a reporter nucleic acid carrying a reporter molecule.
  • Preferred reporter molecules are fluorescent molecules or fluorophores.
  • a preferred reporter nucleic acid molecule is a nucleic acid molecule that carries a fluorophore and a quencher, respectively.
  • a fluorescent group (F) is labeled at the 5' end and a quenching group (Q) is labeled at the 3' end.
  • a fluorescent reporter nucleic acid of 17 nt in length, labeled with a fluorescent group (F) at the 5' end and a quenching group (Q) at the 3' end is shown in Figure 2.
  • the fluorescent reporter nucleic acid is determined according to the 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 a secondary guide.
  • the fluorescent reporter nucleic acid covers the corresponding position of the secondary guide ssDNAs (eg, 1-16 bases).
  • the present invention provides a detection system for detecting a target nucleic acid molecule, which comprises:
  • target nucleic acid molecule is a target DNA.
  • Also provided in the present invention is a nucleic acid detection method based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo).
  • a series of guide ssDNAs can be designed according to the target nucleic acid sequence, and these guide ssDNAs are targeted to the nucleic acid to be detected and mediate the PfAgo enzyme to cut the target fragment to form.
  • New secondary guide ssDNA The secondary guide ssDNA continues to direct the PfAgo enzyme to cleave the fluorescent reporter nucleic acid complementary to the secondary guide ssDNAs under the action of the PfAgo enzyme to achieve detection of the target nucleic acid.
  • the PfAgo enzyme can selectively selectively cleave the nucleic acid sequence having a difference in some sites by special design, thereby realizing the 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 The point mutation can inhibit the shear activity, thus achieving the detection of different types.
  • primers, guide ssDNAs, and fluorescent reporter nucleic acids for nucleic acid detection are provided in the present invention, for example, for detecting the target gene PIK3CA E545K, or for detecting two types of HCV virus, JFH-1 2a, respectively. And CON1-1b.
  • multiple detections of the nucleic acid of interest can be achieved by simultaneously adding a plurality of target nucleic acids to be detected and guide ssDNAs in the cleavage system of the PfAgo enzyme, and combining the reporter nucleic acids having different fluorophores.
  • the method of the invention is very suitable for the detection of trace nucleic acids.
  • the present invention can detect target nucleic acids down to the fM concentration by combining PCR and a guide ssDNA pair having a specific sequence.
  • the detection method of the present invention comprises the following steps:
  • Step 1 Designing amplification primers, specific oligonucleotide guide ssDNAs and fluorescent reporter nucleic acids for different target nucleic acid sequences to be tested;
  • Step 2 collecting the sample to be inspected and extracting the nucleic acid complex containing the target sequence
  • Step 3 adding the obtained sample to be tested as a template to different amplification primer pairs for pre-amplification reaction;
  • Step 4 In the step 3 pre-amplification reaction system, a specific oligonucleotide is added to the ssDNAs, and the corresponding fluorescent reporter nucleic acid and the PfAgo enzyme are specifically sheared under the condition of continuous incubation at 95 degrees;
  • Step 5 Perform quantitative real-time PCR quantitative analysis on the system of step 4;
  • Step 6 After analyzing the image, adjust the Start value, End value and threshold line of the Baseline to determine the result.
  • the amplification primer used for the amplification reaction has a Tm value of usually about 60 ⁇ 3 degrees and an amplified fragment size of about 90 to 120 bp.
  • the amplification primer is designed to avoid the segment to be detected.
  • the invention also provides a kit for use in the detection method of the invention.
  • the kit comprises:
  • the kit further comprises:
  • the buffer for enzymatic cleavage of the gene editing enzyme contains MnCl 2 .
  • the kit further comprises:
  • the invention is particularly suitable for detecting micro-target nucleic acid molecules, as well as 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 detected by reverse transcription into DNA.
  • the target nucleic acid molecule comprises a target nucleic acid molecule derived from a group selected from the group consisting of a plant, an animal, a microorganism, a virus, or a combination thereof.
  • the target DNA is synthetic or naturally occurring DNA.
  • the target DNA comprises wild-type or mutant DNA.
  • the invention can actively control the disease prevention and prevention in the aspect of disease monitoring, and realize early detection of early treatment or early prediction of early prevention. Because the detection sensitivity of the invention is very high, it is suitable for early diagnosis, the right medicine, saving the treatment time of the patient and improving the success rate of the treatment. The invention reduces the waste of high medical costs and strives to treat the golden opportunity.
  • the invention can accurately and quickly identify nucleic acid molecules in environmental pollutants and provide effective environmental detection data.
  • the nucleic acid detection method based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo) of the present invention fully exerts the characteristic shearing property of the enzyme, making it a highly specific detection means;
  • the reaction system of the present invention can simultaneously add a plurality of target nucleic acids to be detected and corresponding primary guide ssDNAs and fluorescent reporter nucleic acids to realize single tube multiple detection;
  • the nucleic acid detection method of the present invention has high sensitivity, and the detection limit for nucleic acid is aM-fM level;
  • the nucleic acid detection method of the present invention has good specificity and is capable of distinguishing nucleic acid sequences of different types
  • the nucleic acid detection method of the invention has convenient operation, simple design and low price.
  • kits for use in the nucleic acid detection method based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo) of the present invention and a method of using the same are provided.
  • the E545K mutation of the PIK3CA gene is taken as an example, and the corresponding specific target nucleic acid sequence is 5'-CTGTGACTCCATAGAAAATCTTTCTCCTGCTCAGTGATTTCAGAGAGAGGATCTCGTGTAGAAATTGCTTTGAGCTGTTCTTTGTCATTTTCCCT-3', SEQ ID No.: 1.
  • the corresponding detection reagents include the following:
  • amplification primers F-primer and R-primer the specific sequence is as follows:
  • F-primer 5'-CTGTGACTCCATAGAAAATCTTTCTCC-3' (SEQ ID No. 2)
  • R-primer 5'-AGGGAAAATGACAAAGAACAGCTC-3' (SEQ ID No. 3)
  • the sense strand guide ssDNA 5'P-TTCTCCTGCTCAGTGA-3' (SEQ ID No. 4)
  • Nonsense strand guide ssDNA 5'P-TGAAATCACTGAGCAG-3' (SEQ ID No. 5)
  • Fluorescent reporter nucleic acid corresponding to the secondary guide ssDNA the specific sequence is as follows:
  • Fluorescent reporter nucleic acid 5' FAM (fluorescent group) - CTCGTCCTCTTTCTAAA-BHQ1 (quenching group) 3' (SEQ ID No. 6).
  • Amplification reaction enzyme preparation for example, AceQ qPCR Probe Master Mix (Vazyme).
  • FIG. 3 A schematic diagram of the nucleic acid detection method based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo) of the present invention is shown in FIG. 3, and the specific operation steps are as follows:
  • the amplification primers F-primer and R-primer dry powder are dissolved in ultrapure water to prepare a 10 uM stock solution; the sense strand guide ssDNA and the nonsense strand guide ssDNA dry powder are dissolved in ultrapure water. Preparing a storage solution of 100 uM; the fluorescent powder of the fluorescent reporter nucleic acid is dissolved in ultrapure water to prepare a storage solution of 10 uM;
  • the sample to be tested is added to the amplification reaction premix, the reaction system is 20uL;
  • the amplification system is placed in a PCR machine for amplification reaction (95-degree pre-denaturation for 5 min, 95-degree denaturation for 15 sec, 60-degree extension for 15 sec, 30 cycles);
  • step (4) After the completion of the amplification reaction, PfAgo enzyme, MnCl 2 , sense strand guide ssDNA and nonsense strand guide ssDNA, fluorescent reporter nucleic acid are added to step (4) to make 25uL reaction system (final concentration of PfAgo enzyme) 200 nM, the final concentration of MnCl 2 is 500 uM, the final concentration of the guide ssDNAs is 2 uM, and the final concentration of the fluorescent reporter nucleic acid is 400 nM);
  • the reaction system in the step (5) is placed on a real-time PCR instrument for detection (95-degree incubation for 30 minutes, fluorescent signal is detected once every minute).
  • the specific target nucleic acid (SEQ ID NO.: 1) was diluted according to the 10-fold dilution method and diluted to standard stock solutions of 200 pM, 20 pM, 2 pM, 200 fM, 20 fM, 2 fM and 0 fM, respectively.
  • Different concentrations of the nucleic acid standard mother liquid were separately added to the reaction system described in Example 1, and the sample loading reaction was carried out in steps, and the fluorescence signal value at the wavelength of the corresponding fluorescent group was detected by real-time quantitative PCR.
  • FIGS. 4A-4H The results are shown in Figures 4A-4H.
  • the concentration of the target nucleic acid in FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G is 0fM, 2fM, 20fM, 200fM, 2pM, 20pM, 200pM, respectively, and FIG. 4H is a non-target nucleic acid (20 ng is added to the system) .
  • the non-target nucleic acid is total DNA extracted from normal human serum. The results showed that even if 20 ng of unrelated non-target nucleic acid was added to the system, no positive results were produced.
  • the method of the present invention can detect target nucleic acid molecules of the fM level with minimal detection.
  • a component of the detection system is added, a positive signal is not generated, and thus the specificity of the method of the present invention is very high.
  • Target nucleic acid solutions with a concentration of 200 pM were configured, which were the two types of HCV virus, JFH-1 2a and CON1-1b, respectively.
  • the multiple mixed detection system is configured: two pairs of specific amplification primers are simultaneously added to the detection amplification system, and the target detection nucleic acid is added to the detection amplification system as follows: (1) blank control; (2) JFH- 1 2a; (3) CON1-1b; (4) JFH-1 2a and CON1-1b. (The specificity of the fluorescent reporter nucleic acid was verified in Figure 5, respectively, and did not affect each other in one reaction system)
  • HPV-6 SEQ ID No. 19
  • HPV-11 SEQ ID No. 20
  • HPV-16 SEQ ID No. 21
  • HPV- 18 SEQ ID No. 22
  • PCR amplification was performed on 16 combined template DNAs followed by multiplex fluorescence detection.
  • PfAgo enzyme MnCl 2 , 4 specific ssDNAs, and 4 fluorescent reporter nucleic acids with different fluorophores (HPV-6 corresponds to NED fluorescence, HPV-11 corresponds to ROX fluorescence, HPV- The type 16 corresponds to FAM fluorescence, and the HPV-18 type corresponds to JOE fluorescence) was added to the same reaction tube, and the reaction was carried out in accordance with the reaction procedure in Example 1.
  • the clinical HPV16/18 sample was used in the system for verification, and the sample was reacted and reacted according to the above reaction system.
  • the detection system can be used for actual clinical detection, and has high sensitivity, high specificity, and rapid effect.

Abstract

提供了一种基于原核Argonaute蛋白的核酸检测方法及其应用。具体地，提供了一种用于检测靶标核酸分子的检测体系，该体系包含向导ssDNAs，基因编辑酶Pyrococcus furiosus Argonaute(PfAgo)和荧光报告核酸。

Description

基于原核Argonaute蛋白的核酸检测方法及其应用 技术领域
本发明属于生物技术领域，具体涉及一种基于原核Argonaute蛋白的核酸检测方法及其应用。
背景技术
核酸检测技术广泛应用于分子医学诊断、食品安全检测以及环境监测等诸多领域。快速、廉价和灵敏的核酸检测可广泛应用于病原体检测、基因分型、病程监测等方面。
一些传统的核酸检测方法(qPCR、测序、核酸印记等)虽然已经有广泛应用，然而，仍存在一些缺点。例如qPCR和高通量测序等存在耗时长，费用高，设计原理复杂等缺点。此外，对于数量极其微少的待检测核酸，本领域尚缺乏令人满意的检测方法。
目前，利用基因编辑酶作为核酸检测的方法有利用C2C2酶介导的旁系剪切效应对目标RNA进行检测。但是尚缺乏对目标DNA(尤其是微量DNA)进行快速、有效检测的方法。
因此，本领域迫切需要开发针对目标DNA的灵敏度高、特异性好、通量高的核酸检测方法。
发明内容
本发明的目的就是提供一种基于对目标DNA的灵敏度高、特异性好、通量高的核酸检测方法及其应用。
在本发明的第一方面，提供了一种用于检测靶标核酸分子的检测体系，该体系包含：
(a)向导ssDNA对；
(b)基因编辑酶Pyrococcus furiosus(PfAgo)；和
(c)荧光报告核酸，所述荧光报告核酸带有荧光基团和淬灭基团；
其中，所述的靶标核酸分子为靶标DNA。
在另一优选例中，所述的向导ssDNA对包括有义链向导ssDNA和反义链向导ssDNA。
在另一优选例中，所述的向导ssDNA为5’-磷酸化的单链DNA分子。
在另一优选例中，所述的向导ssDNA的长度为n个碱基，且n≥14。
在另一优选例中，所述的n≤100，较佳地≤80，更佳地≤60。
在另一优选例中，所述的向导ssDNAs的长度为14-60nt，较佳地16-40nt。
在另一优选例中，所述的向导ssDNAs的数量为一对或多对。
在另一优选例中，所述PfAgo酶来源于古菌Pyrococcus furiosus。
在另一优选例中，所述的PfAgo包括野生型和突变型的PfAgo。
在另一优选例中，所述靶标核酸分子的不同分型对应的突变位点在向导ssDNA的第10、11位。
在另一优选例中，所述的检测体系还含有(d)缓冲液。
在另一优选例中，所述的检测体系还包括用于对靶标核酸分子进行扩增的引物。
在另一优选例中，所述的检测体系还含有待检测的靶标核酸分子。
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PfAgo酶切割后，产生次级向导ssDNA。
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的序列互补结合后，引导所述PfAgo酶对所述荧光报告核酸进行切割，从而产生可检测的信号(如荧光)。
在另一优选例中，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1-1000拷贝/微升或10 3-10 10拷贝/微升，较佳地100-1000拷贝/微升，更佳地1-100拷贝/微升。
在另一优选例中，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1fM-200pM，较佳地1-1000fM，更佳地1-100fM，最佳地1-20fM。
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，所述的检测体系中，所述荧光报告核酸的浓度为100-1000nM。
在另一优选例中，所述的检测体系中，所述荧光报告核酸与所述靶标核酸分子的摩尔比为10 3：1至10 8：1，较佳地10 4：1至10 7：1。
在另一优选例中，所述的靶标DNA包括cDNA。
在另一优选例中，所述的靶标DNA选自下组：单链DNA(包括cDNA)、双链DNA、或其组合。
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，所述的荧光报告核酸的长度为9-100nt，较佳地10-60nt，更佳地15-40nt。
在另一优选例中，所述靶标核酸分子包括来源于选自下组的靶标核酸分子：植物、动物、微生物、病毒、或其组合。
在另一优选例中，所述的靶标DNA是人工合成或天然存在的DNA。
在另一优选例中，所述的靶标DNA包括野生型或突变型的DNA。
在本发明的第二方面，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)本发明第一方面所述的检测体系或用于配制所述检测体系的试剂；和
(ii)使用说明书，所述说明书描述了用所述的检测体系检测靶标核酸分子的方法。
在另一优选例中，所述试剂盒还可以包括缓冲液。
在另一优选例中，所述的试剂盒包括：
(a)第一容器以及位于所述第一容器的向导ssDNA；
(b)第二容器以及位于第二容器的酶Pyrococcus furiosus Argonaute(PfAgo)；和
(c)第三容器以及位于第三容器的荧光报告核酸。
在另一优选例中，所述的试剂盒还含有：
(d)第四容器以及位于第四容器的用于酶进行酶切的缓冲液。
在另一优选例中，所述的用于酶进行酶切的缓冲液含有MnCl 2。
在另一优选例中，所述的试剂盒还包括：
(f)第五容器以及位于第五容器的用于扩增靶标核酸分子的引物或引物对；
(g)任选的第六容器以及位于第六容器的用于扩增反应的聚合酶；和
(h)任选的第七容器以及位于第七容器的用于扩增反应的扩增缓冲液。
在本发明的第三方面，提供了一种检测样本中是否存在靶标核酸分子的方法，包括以下步骤：
(a)提供本发明第一方面所述的用于检测靶标核酸分子的检测体系；和
(b)将所述检测体系与待检测的样本在一定温度下进行反应，从而形成第一反应溶液；
(c)对所述第一反应溶液进行荧光检测，从而获得荧光信号值；
其中，所述第一反应溶液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；而所述第一反应溶液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，所述核酸扩增的方法选自下组：PCR扩增、LAMP扩增、RPA扩增、连接酶链式反应、分支DNA扩增、NASBA、SDA、转录介导扩增和滚环扩增。
在另一优选例中，所述的PCR包括高温PCR、常温PCR、低温PCR。
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/或插入。
在另一优选例中，在步骤(b)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。
在本发明的第四方面，提供了一种核酸酶Pyrococcus furiosus Argonaute的用途，用于制备基于二次切割检测靶标核酸分子的试剂或试剂盒。
在另一优选例中，所述酶Pyrococcus furiosus Argonaute来源于古菌Pyrococcus furiosus；或是其具备相同或相似功能的同源类似物。
在另一优选例中，所述的PfAgo包括野生型和突变型的PfAgo。
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显示了本发明基因编码酶的第一次切割示意图，其中针对同一条DNA链设计一对向导ssDNAs，其对应的剪切位点由黑色箭头标出，pfAgo酶作用后形成一个平末端。
图2显示了本发明基因编码酶的第二次切割示意图，其中由初级向导ssDNAs引导的特异性切割产生了次级向导ssDNAs。
图3显示了本发明检测方法的步骤和原理。
图4显示了核酸检测体系的特异性及灵敏度。
图5显示了本发明一个实施例中的核酸检测体系的多重性实验结果。其中
图5A采用CON1-1b-FAM报告核酸分子(无模板)；
图5B采用CON1-1b-FAM报告核酸分子(CON1-1b模板)；
图5C采用CON1-1b-FAM报告核酸分子(JFH-1 2a模板)；
图5D采用JFH-1 2a-VIC报告核酸分子(无模板)；
图5E采用JFH-1 2a-VIC报告核酸分子(JFH-1 2a模板)；
图5F采用JFH-1 2a-VIC报告核酸分子(CON1-1b模板)。
图6显示了以质粒为样本多重检测HPV不同组合样本。
图7显示了多重检测临床样本高危型亚型HPV16/HPV18。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种针对目标DNA的检测限低、灵敏度高、操作简便、检测成本低、耗时短、通量高的核酸检测方法。本发明方法利用PfpAgo酶的特性，即在首次由初级向导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PfAgo酶利用去剪切与之互补的荧光报告核酸链。结果表明，本发明方法不仅可以对痕量核酸分子进行快速地而高通量地检测，而且可以准确地给出检测 结果，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
如本文所用，术语“本发明检测体系”、“基于Argonaute蛋白的核酸检测体系”可互换使用，指本发明第一方面中所述的检测体系。
如本文所用，术语“本发明检测方法”、“基于Argonaute蛋白的核酸检测方法”可互换使用，指本发明第二方面中所述的检测方法。
如本文所用，术语“基因编辑酶Pyrococcus furiosus”、“核酸酶Pyrococcus furiosus”、“PfAgo酶”可互换使用，指本发明第一方面中所述的酶。
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
在本发明的检测体系和检测方法中，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对
在本发明的检测体系和检测方法中，一个核心成分是向导ssDNA对。
在本发明中，优选的向导ssDNAs均为长度为14-24nt(如16nt)的寡聚核苷 酸，其5’第一个核苷酸均为磷酸化修饰的胸腺嘧啶(T)。
如图1所示，针对同一条DNA链的一对向导ssDNAs结合于靶标核酸分子，其对应的剪切位点由黑色箭头标出，pfAgo酶作用后形成一个平末端。
报告核酸分子
在本发明的检测体系和检测方法中，一个核心成分是携带报告分子的报告核酸。
优选的报告分子是荧光分子或荧光基团。一种优选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)。图2中示出了一个荧光报告核酸，其长度为17nt，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的相应位置(例如1-16位碱基)。
检测体系
本发明提供了用于检测靶标核酸分子的检测体系，它包括：
(a)向导ssDNA对；
(b)基因编辑酶Pyrococcus furiosus Argonaute(PfAgo)；和
(c)荧光报告核酸，所述荧光报告核酸带有荧光基团和淬灭基团；
其中，所述的靶标核酸分子为靶标DNA。
检测方法
在本发明还提供了基于基因编辑酶Pyrococcus furiosus Argonaute(PfAgo)的核酸检测方法。
为了便于理解，本发明人提供了本发明检测方法的原理。应理解，本发明的保护范围并不受所述原理的限制。
参见图2和图3。在本发明方法中，基于PfAgo酶的剪切活性，可根据目标核酸序列的不同设计出一系列向导ssDNAs,这些向导ssDNAs靶向于待检测核酸并介导PfAgo酶对目的片段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，从而达到对目标核酸的检测。
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。
在优选例中，本发明中提供了用于核酸检测的引物、向导ssDNAs及荧光报告核酸，例如，分别用于检测目标基因PIK3CA E545K、或用于检测HCV病毒的两种分型JFH-1 2a和CON1-1b。
在本发明中，可以在PfAgo酶的剪切体系中同时加入多种目标待检核酸和向导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PCR以及具有特定序列的向导ssDNA对，本发明可以检测低至fM浓度的靶核酸。
在一个优选例中，本发明检测方法包括如下步骤：
步骤1：针对不同的目标待检核酸序列设计扩增引物、特异性的寡核苷酸向导ssDNAs和荧光报告核酸；
步骤2：采集待检样本，提取含目标序列的核酸复合物；
步骤3：将获取的待检样本作为模板加入不同扩增引物对进行预扩增反应；
步骤4：在步骤3预扩增反应体系中加入特异性的寡核苷酸向导ssDNAs、与之相应的荧光报告核酸及PfAgo酶在95度持续保温的条件下进行特异性剪切；
步骤5：对步骤4的体系进行quantitativereal-timePCR定量分析；
步骤6：分析图像后调节Baseline的Start值、End值和阈值线，判定结果。
在本发明中，所用于扩增反应的扩增引物，其Tm值通常60±3度左右，扩增片段大小约为90-120bp。优选地，扩增引物设计时应避开待检测区段。
试剂盒
本发明还提供了用于一种用于本发明检测方法的试剂盒。
典型地，所述的试剂盒包括：
(a)第一容器以及位于所述第一容器的向导ssDNA；
(b)第二容器以及位于第二容器的基因编辑酶Pyrococcus furiosus Argonaute(PfAgo)；和
(c)第三容器以及位于第三容器的荧光报告核酸。
在另一优选例中，所述的试剂盒还含有：
(d)第四容器以及位于第四容器的用于基因编辑酶进行酶切的缓冲液。
在另一优选例中，所述的用于基因编辑酶进行酶切的缓冲液含有MnCl 2。
在另一优选例中，所述的试剂盒还包括：
(f)第五容器以及位于第五容器的用于扩增靶标核酸分子的引物或引物对；
(g)任选的第六容器以及位于第六容器的用于扩增反应的聚合酶；和
(h)任选的第七容器以及位于第七容器的用于扩增反应的扩增缓冲液。
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DNA再进行检测。
在另一优选例中，所述靶标核酸分子包括来源于选自下组的靶标核酸分子：植物、动物、微生物、病毒、或其组合。
在另一优选例中，所述的靶标DNA是人工合成或天然存在的DNA。
在另一优选例中，所述的靶标DNA包括野生型或突变型的DNA。
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)本发明所述的基于基因编辑酶Pyrococcus furiosus Argonaute(PfAgo)的 核酸检测方法充分发挥了该酶的特性剪切特性，使之成为一种特异性高的检测手段；
2)本发明所述的反应体系中可同时加入多种待检测的目标核酸以及对应的初级向导ssDNAs和荧光报告核酸，实现单管多重检测；
3)本发明所述的核酸检测方法具有较高的灵敏度，对核酸的检测限为aM-fM级别；
4)本发明所述的核酸检测方法具有很好的特异性，能够区分不同分型的核酸序列；
5)本发明所述的核酸检测方法操作便捷，设计简单，价格低廉。
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。
序列信息
实施例中涉及3组目标基因富集及扩增寡核酸序列如下表所示。
Figure PCTCN2018110056-appb-000001
Figure PCTCN2018110056-appb-000002
Figure PCTCN2018110056-appb-000003
实施例1
检测试剂的制备和检测方法
在本实施例中，提供了用于本发明基于基因编辑酶Pyrococcus furiosus Argonaute(PfAgo)的核酸检测方法的试剂盒及其使用方法。
1.1检测试剂和试剂盒
在本实施例中，以检测PIK3CA基因的E545K突变为例子，相应的特异性目标核酸序列为5’-CTGTGACTCCATAGAAAATCTTTCTCCTGCTCAGTGATTTCAGAGAGAGGATCTCGTGTAGAAATTGCTTTGAGCTGTTCTTTGTCATTTTCCCT-3’，SEQ ID No.：1。
基于本发明方法，相应的检测试剂包括以下：
(1)、扩增引物F-primer和R-primer，具体序列如下：
F-primer：5’-CTGTGACTCCATAGAAAATCTTTCTCC-3’(SEQ ID No.2)
R-primer：5’-AGGGAAAATGACAAAGAACAGCTC-3’(SEQ ID No.3)
(2)、特异性向导ssDNAs对，包括有义链向导ssDNA和无义链向导ssDNA,具体序列如下：
有义链向导ssDNA:5’P-TTCTCCTGCTCAGTGA-3’(SEQ ID No.4)
无义链向导ssDNA:5’P-TGAAATCACTGAGCAG-3’(SEQ ID No.5)
(3)、与次级向导ssDNA相应的荧光报告核酸，具体序列如下：
荧光报告核酸：5’FAM(荧光基团)-CTCGTCCTCTTTCTAAA-BHQ1(淬灭基团)3’(SEQ ID No.6)。
(4)、扩增反应酶制剂：例如AceQ qPCR Probe Master Mix(Vazyme)。
(5)、MnCl 2溶液：10mM MnCl 2溶液
1.2检测方法
本发明的基于基因编辑酶Pyrococcus furiosus Argonaute(PfAgo)的核酸检测方法的示意图如图3所示，具体操作步骤如下：
(1)、所述的扩增引物F-primer和R-primer干粉用超纯水溶解制成10uM的储存液；所述的有义链向导ssDNA和无义链向导ssDNA干粉用超纯水溶解制成100uM的储存液；所述的荧光报告核酸干粉用超纯水溶解制成10uM的储存液；
(2)、用AceQ qPCR Probe Master Mix(2X)酶制剂与超纯水、扩增引物制成扩增反应预混液(扩增引物终浓度为500nM)；
(3)、将待检测样品加入扩增反应预混液，反应体系为20uL；
(4)、将扩增体系放入PCR仪中进行扩增反应(95度预变性5min，95度变性15sec，60度延伸15sec，30个循环)；
(5)、扩增反应结束后，向步骤(4)中加入PfAgo酶、MnCl 2、有义链向导ssDNA和无义链向导ssDNA、荧光报告核酸，使之成为25uL反应体系(PfAgo酶终浓度为200nM,MnCl 2终浓度为500uM,向导ssDNAs终浓度为2uM,荧光报告核酸终浓度为400nM)；
(6)、将步骤(5)中的反应体系放置在荧光定量PCR仪上进行检测(95度保温 30分钟，每分钟检测一次荧光信号)。
实施例2
针对不同浓度的待检核酸进行检测
按10倍稀释法的原则对特异性目标核酸(SEQ ID NO.：1)进行稀释，分别稀释成200pM、20pM、2pM、200fM、20fM、2fM和0fM的标准母液。将不同浓度的核酸标准母液分别加入到实施例1所述的反应体系中，按步骤进行加样反应，并通过荧光定量PCR检测对应荧光基团波长处的荧光信号值。
结果如图4A-4H所示。其中，图4A、4B、4C、4D、4E、4F、4G中的目标核酸的浓度分别为0fM、2fM、20fM、200fM、2pM、20pM、200pM，图4H为非目标核酸(体系中加入20ng)。
非目标核酸为抽提自正常人血清的总DNA。结果表明，即使在体系中加入20ng的无关的非目标核酸，仍不会产生阳性结果。
结果表明，本发明的方法可最低检测至fM级别的目标核酸分子。另外，对于非目标核酸，即使加入检测体系的成分，也不会产生阳性信号，因此本发明方法的特异性非常高。
实施例3.
特异性试验及多重检测
配置浓度为200pM的不同类型目标核酸溶液，分别为HCV病毒的两种分型JFH-1 2a和CON1-1b。
需要说明的是，这两种分型的区别仅在于：序列上存在连续两点差异。
配置多重混合检测体系：将2对特异性扩增引物同时加入到检测扩增体系中，将目标检测核酸按如下4组加入到检测扩增体系中：(1)空白对照；(2)JFH-1 2a；(3)CON1-1b；(4)JFH-1 2a和CON1-1b。(图5中分别验证了荧光报告核酸的特异性，在一个反应体系中互相不影响)
扩增反应结束后将PfAgo酶,MnCl 2,2组特异性的ssDNAs,2对带有不同荧光基团的荧光报告核酸(JFH-1 2a型对应VIC荧光，CON1-1b型对应FAM荧光)加入到同一反应管中，按实施例1中的反应步骤进行检测。
结果如图5A-5F所示。结果表明：当检测目标核酸为空白对照时，无荧光信号值产生；当检测目标核酸为JFH-1 2a时，只有VIC荧光产生；当检测目标核 酸为CON1-1b时，只有FAM荧光产生；当检测目标核酸为JFH-1 2a和CON1-1b时，FAM和VIC两种荧光同时产生。由此说明本发明核酸检测方法可用于单管的多重检测。
实施例4.
选取人乳头瘤病毒(HPV)中的四种亚型:HPV-6(SEQ ID No.19),HPV-11(SEQ ID No.20),HPV-16(SEQ ID No.21),HPV-18(SEQ ID No.22)进行多重检测。构建包含上述四种基因的质粒进行多重实验。
设计多重检测所需的四种gDNA，四种报告核酸以及一对简并扩增引物(SEQ ID No.17；SEQ ID No.18)。
经过对16种组合的模版DNA进行PCR扩增，随后进行多重荧光检测。
扩增反应结束后将PfAgo酶,MnCl 2,4种特异性的ssDNAs,4种带有不同荧光基团的荧光报告核酸(HPV-6型对应NED荧光，HPV-11型对应ROX荧光,HPV-16型对应FAM荧光，HPV-18型对应JOE荧光)加入到同一反应管中，按实施例1中的反应步骤进行检测。
结果如图6所示。结果表明：当检测目标核酸为空白对照时，无荧光信号值产生；当检测目标核酸为中的一种或多种时，产生对应的荧光信号采集并检测。由此说明本发明核酸检测方法可达到单管的四重检测。
将临床HPV16/18样本用于本体系进行验证，按上述反应体系进行加样、反应。
结果如图7所示。结果表现，该检测体系可用于实际临床检测，并具有高灵敏度、高特异性、快速的效果。
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. 一种用于检测靶标核酸分子的检测体系，其特征在于，该体系包含：
    (a)向导ssDNA对；
    (b)基因编辑酶Pyrococcus furiosus(PfAgo)；和
    (c)荧光报告核酸，所述荧光报告核酸带有荧光基团和淬灭基团；
    其中，所述的靶标核酸分子为靶标DNA。
  2. 如权利要求1所述的检测体系，其特征在于，所述的向导ssDNAs的长度为14-60nt，较佳地16-40nt。
  3. 如权利要求1所述的检测体系，其特征在于，所述PfAgo酶来源于古菌Pyrococcus furiosus。
  4. 如权利要求1所述的检测体系，其特征在于，所述靶标核酸分子的不同分型对应的突变位点在向导ssDNA的第10、11位。
  5. 如权利要求1所述的检测体系，其特征在于，所述的靶标核酸分子(或其扩增产物)被所述PfAgo酶切割后，产生次级向导ssDNA。
  6. 如权利要求1所述的检测体系，其特征在于，所述的待检测的靶标核酸分子在所述检测体系中的浓度为1fM-200pM，较佳地1-1000fM，更佳地1-100fM，最佳地1-20fM。
  7. 如权利要求1所述的检测体系，其特征在于，所述的靶标DNA选自下组：单链DNA(包括cDNA)、双链DNA、或其组合。
  8. 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)权利要求1所述的检测体系或用于配制所述检测体系的试剂；和
    (ii)使用说明书，所述说明书描述了用所述的检测体系检测靶标核酸分子的方法。
  9. 一种检测样本中是否存在靶标核酸分子的方法，其特征在于，包括以下步骤：
    (a)提供权利要求1所述的用于检测靶标核酸分子的检测体系；和
    (b)将所述检测体系与待检测的样本在一定温度下进行反应，从而形成第一反应溶液；
    (c)对所述第一反应溶液进行荧光检测，从而获得荧光信号值；
    其中，所述第一反应溶液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；而所述第一反应溶液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。
  10. 一种核酸酶Pyrococcus furiosus Argonaute的用途，其特征在于，用于制备基于二次切割检测靶标核酸分子的试剂或试剂盒。
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