WO2020133713A1 - 一种双链寡核苷酸核酸探针的结构和用途 - Google Patents

一种双链寡核苷酸核酸探针的结构和用途 Download PDF

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WO2020133713A1
WO2020133713A1 PCT/CN2019/077879 CN2019077879W WO2020133713A1 WO 2020133713 A1 WO2020133713 A1 WO 2020133713A1 CN 2019077879 W CN2019077879 W CN 2019077879W WO 2020133713 A1 WO2020133713 A1 WO 2020133713A1
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nucleic acid
double
oligonucleotide
stranded oligonucleotide
acid probe
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French (fr)
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王升启
刘琪琦
刘丽艳
张影
赵�怡
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中国人民解放军军事科学院军事医学研究院
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Priority to US17/419,329 priority Critical patent/US20220090195A1/en
Publication of WO2020133713A1 publication Critical patent/WO2020133713A1/zh

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q2533/00—Reactions characterised by the enzymatic reaction principle used
    • C12Q2533/10—Reactions characterised by the enzymatic reaction principle used the purpose being to increase the length of an oligonucleotide strand
    • C12Q2533/107—Probe or oligonucleotide ligation
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence

Definitions

  • the present disclosure relates to a biological detection technology tool, in particular to a double-stranded oligonucleotide nucleic acid probe and its use method and its application in life science research such as gene fluorescence characterization, quantitative analysis, medical diagnosis, etc. It belongs to genetic detection Technology area.
  • fluorescent probe methods are generally used in molecular diagnosis of infectious diseases and personalized concomitant diagnosis and other disease diagnosis applications.
  • the fluorescent probe method relies on Fluorescent Resonance Energy Transfer (FRET) to achieve detection, including TaqMan probes, molecular beacons, and Scorpion probes. This method only detects specific amplification products and is therefore highly specific.
  • FRET Fluorescent Resonance Energy Transfer
  • the most widely used probe technique is TaqMan, which mainly utilizes the 5′ exonuclease activity of Taq enzyme.
  • the 5'end of the probe is labeled with a fluorescent molecule, and the 3'is labeled with a corresponding fluorescence quenching molecule.
  • the 3'end quenching molecule can absorb the 5'end fluorescent molecule.
  • fluorescence Under normal circumstances, the probe does not emit fluorescence theoretically, but when there is a PCR product in the solution, the probe binds to the PCR product, activates the 5′ exonuclease activity of the Taq enzyme, and cleaves the probe into single nucleotides. At the same time, the fluorescent group labeled on the probe is released, causing fluorescence, and the number of cleaved fluorescent molecules is proportional to the number of PCR products. Therefore, the initial template can be calculated according to the intensity of the fluorescent signal in the PCR reaction solution concentration.
  • TaqMan probe technology is widely used, there are still many defects.
  • the purpose of the present disclosure is to solve at least one problem existing in the prior art, to provide a new double-stranded oligonucleotide nucleic acid probe structure, method of use and its application in gene fluorescence qualitative and quantitative analysis, medical diagnosis and life science research Applications in other fields.
  • two probes are first synthesized, and both probes are labeled with a fluorescent group at the 5′ end as a reporter molecule (F1/F2), two The probes are labeled at the 3′ end with corresponding fluorescence quenching molecules (Q1/Q2) at the 5′ end, and the two probes are completely or partially complementary to each other.
  • F1/F2 fluorescent group at the 5′ end as a reporter molecule
  • Q1/Q2 fluorescence quenching molecules
  • Fluorescence is generated; when the two probes are separated, the probe is combined with the PCR product, and the 5′ exonuclease activity of the Taq enzyme cleaves the probe into a single nucleotide, and F1 and F2 are released and emit fluorescence.
  • the advantage of double-stranded probes is that quenching is more thorough and the fluorescence background is lower. Based on this concept, the present disclosure synthesizes a double-stranded oligonucleotide nucleic acid probe.
  • the two probes When there is no template in the PCR amplification system, the two probes are complementary and no fluorescence is generated in the solution; when there is a template in the amplification system At higher temperatures, both probes preferentially bind to the template, so the two probes separate and produce fluorescence.
  • the fluorescence intensity is proportional to the concentration of the template in the solution, and quantitative determination of the template can be performed based on this.
  • oligonucleotide nucleic acid probe which is composed of two completely or partially complementary oligonucleotide chains, wherein two probes, each oligonucleotide A fluorescent group or a corresponding fluorescence quenching group can be connected to the end of the chain. Both oligonucleotide probe chains can be hybridized with some fragments of the target DNA or RNA nucleic acid sequence to be tested according to the principle of base pairing.
  • Double-stranded probes Each probe is independently composed of 6-50 oligonucleotides, the preferred long-stranded probe in the double-stranded probe is composed of 25-30 nucleotides, and the short-stranded probe is composed of 15 -25 nucleotides.
  • the number of fluorescent molecules and quenching molecules connected to the probe can be 1-5. Generally, it is 1 considering the cost and convenience of synthesis.
  • the fluorophore can be one or more of FAM, HEX, TET, ROX, CY3, CY5, VIC, JOE, SIMA, AlexaFluor488, TexasRed, or Quasar670, the quencher group connected to the other end of the chain
  • the group may be one or more of TAMRA, Dabcyl, BHQ-1, BHQ-2, BHQ-3, MGB, or Eclipse.
  • the present disclosure discloses a method for using a double-stranded oligonucleotide nucleic acid probe in gene detection, which includes the following steps:
  • the length of the nucleic acid as a template is preferably 60-500 bases, and more preferably 70-150 bases. Record the fluorescence value during each cycle of annealing or extension;
  • Threshold fluorescence refers to the fluorescence intensity of the gene to be tested which is twice the background fluorescence coefficient of variation.
  • the double-stranded oligonucleotide nucleic acid probe of the present disclosure can be widely used in the field of gene detection such as gene fluorescence characterization, quantitative analysis and medical diagnosis, especially in the simultaneous detection and typing of multiple genes and high gene There is a greater advantage in sensitivity detection.
  • the double-stranded probes are each other's fluorescent probe and quenching probe.
  • the distance between the fluorescent group and the quenching group is closer, and the quenching is more thorough.
  • Reduced fluorescence background does not completely rely on exonuclease activity; can label two or more fluorescent molecules, and both probes are combined with the template, which improves detection sensitivity.
  • the technology of double-stranded oligonucleotide nucleic acid probes has great value in popularization and application.
  • Fig. 1a shows that when the two oligonucleotide chains provided by the present invention are not of equal length, a relatively short chain is reversely complementary starting from the 5'end of a relatively long chain, and a relatively short probe is relatively
  • Fig. 1b shows that when the two oligonucleotide chains provided by the present invention are not of equal length, a relatively short chain is reversely complementary starting from 3′ of a relatively long chain, a relatively short probe is relatively long A schematic diagram of the reverse complementary regions of the probes in the longer probe region;
  • Fig. 1c shows that when the two oligonucleotide chains provided by the present invention are not of equal length, a relatively short chain is reversely complementary starting from the middle of a relatively long chain, a relatively short probe and a relatively long
  • the schematic diagram of the reverse complementary regions of the probe are in the longer probe region;
  • FIG. 1d shows that when the two oligonucleotide chains provided by the present invention are not of equal length, a relatively short chain is reversely complementary starting from the 5′ end of the relatively long chain, and the Schematic diagram of the reverse complementary region beyond the 5'end of the relatively long probe;
  • Figure 1e shows that when the two oligonucleotide chains provided by the present invention are not of equal length, a relatively short chain is reversely complementary starting from the 3'end of a relatively long chain, and the Schematic diagram of the reverse complementary region beyond the 3'end of the relatively long probe;
  • FIG. 1f is a schematic diagram of the presence of mutant bases that are not completely reverse complementary on the two oligonucleotide chains when the two oligonucleotide chains are unequal in length;
  • FIG. 1g is a schematic diagram of two oligonucleotide chains provided by the present invention with equal lengths, and there are mutant bases that are not completely reverse complementary on the two chains;
  • Example 2 is a probe specific analysis diagram provided by Example 1 of the present invention.
  • FIG. 3 is a background analysis diagram of the probe provided in Example 1 of the present invention.
  • FIG. 4 is an analysis diagram of the probe detection range provided by Embodiment 1 of the present invention.
  • FIG. 5 is a detection diagram of a double-stranded oligonucleotide nucleic acid probe 10IU/mL HBV nucleic acid quantitative detection standard provided by Example 1 of the present invention
  • FIG. 6 is a detection diagram of a double-stranded oligonucleotide nucleic acid probe 10IU/mL HBV nucleic acid quantitative detection standard provided by Example 1 of the present invention
  • Example 7 is a detection diagram of a Taqman probe 10IU/mL HBV nucleic acid quantitative detection standard provided by Example 1 of the present invention.
  • Example 8 is a detection diagram of a double-stranded oligonucleotide nucleic acid probe sample provided by Example 1 of the present invention.
  • Example 9 is a Taqman probe sample detection diagram provided in Example 1 of the present invention.
  • FIG. 10 is a result diagram of the detection of the 2C19*2 gene mutant A/A by the double-stranded oligonucleotide probe provided in Example 2 of the present invention.
  • FIG. 11 is a result diagram of the detection of the 2C19*2 gene mutant G/G by the double-stranded oligonucleotide probe provided in Example 2 of the present invention.
  • FIG. 12 is a graph showing the detection result of the 2C19*2 gene mutant G/A by the double-stranded oligonucleotide probe provided in Example 2 of the present invention.
  • the upstream primer F has a total of 21 nucleotides, 17 nucleotides from the long-chain oligonucleotide probe, and 22 nucleotides from the short-chain oligonucleotide probe.
  • the downstream primer R has a total of 21 nucleotides, 27 nucleotides from the long-chain oligonucleotide probe, and 32 nucleotides from the short-chain oligonucleotide probe.
  • the long-chain oligonucleotide probe P1 is complementary to the negative strand of the target sequence and is composed of 27 nucleotides, of which a fluorescein molecule at the 5′ end and a quencher molecule at the 3′ end;
  • the needle P2 is complementary to the positive strand of the target sequence, and is composed of 17 nucleotides, of which a fluorescein molecule at the 5′ end and a quencher molecule at the 3′ end are located 5 bases away from the probe P1 and are closer to P1 Short 10 bases;
  • Taqman probe P3 is complementary to the negative strand of the target sequence and consists of 25 nucleotides, including a fluorescein molecule at the 5′ end and a quencher molecule at the 3′ end;
  • probes with mutant bases P4 has a mutant base that is not completely reverse complementary to the positive strand of the target sequence; it consists of 27 nucleot
  • PCR reaction system including: 10 ⁇ PCR buffer 4 ⁇ L, dNTPs 0.2 mmol/L, upstream and downstream primers 0.55 ⁇ mol/L, Taq DNA polymerase 2.5U, long-chain oligonucleotide probe 0.275 ⁇ mol/L, Short-chain oligonucleotide probe 0.330 ⁇ mol/L, and HBV template extracted by nucleic acid extraction kit 20 ⁇ L, the total reaction volume is 40 ⁇ L;
  • Reaction conditions 50°C, 2min; 94°C, 2min; 94°C, 15s, 55°C, 45s, 45 cycles in total, fluorescence collected during annealing.
  • PCR reaction system including: 10 ⁇ PCR buffer 4 ⁇ L, dNTPs 0.2 mmol/L, upstream and downstream primers 0.55 ⁇ mol/L, Taq DNA polymerase 2.5U, long-chain oligonucleotide probe 0.275 ⁇ mol/L, Incomplete reverse complementary oligonucleotide probe 0.330 ⁇ mol/L, and HBV template extracted by nucleic acid extraction kit 20 ⁇ L, the total reaction volume is 40 ⁇ L;
  • Reaction conditions 50°C, 2min; 94°C, 2min; 94°C, 15s, 55°C, 45s, 45 cycles in total, fluorescence collected during annealing.
  • the prepared PCR reaction system includes: 10 ⁇ PCR buffer 4 ⁇ L, dNTPs 0.2 mmol/L, upstream and downstream primers 0.55 ⁇ mol/L, Taq DNA polymerase 2.5U, Taqman probe 0.275 ⁇ mol/L, and nucleic acid extraction kit
  • the extracted HBV template is 20 ⁇ L, and the total reaction system is 40 ⁇ L;
  • Reaction conditions 50°C, 2min; 94°C, 2min; 94°C, 15s, 55°C, 45s, 45 cycles in total, fluorescence collected during annealing.
  • the primers are F and R
  • the probes are double-stranded oligonucleotide nucleic acid probes (P1/P2) and Taqman probes (P3)
  • the templates are 10 5 IU/mL hepatitis B virus (HBV), 10 5 IU/mL Hepatitis C Virus (HCV), 10 5 IU/mL Hepatitis A Virus (HAV), 10 5 IU/mL Human Cytomegalovirus (CMV), 10 5 IU/mL Single-pass Herpes Virus Type I (HSV- 1).
  • ddH 2 O is a negative control
  • PCR detection is performed according to the operation in step 2.
  • Double-stranded oligonucleotide nucleic acid probes and Taqman probes were tested against 10 5 IU/mL hepatitis B virus (HBV), 10 5 IU/mL hepatitis C virus (HCV), 10 5 IU/mL hepatitis A virus (HAV), 10 5 PFU/mL human cytomegalovirus (CMV), 10 5 PFU/mL single-pass herpes virus type I (HSV-1), 10 5 PFU/mL single-pass herpes virus Type II (HSV-2) and negative control ddH 2 O were tested.
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HAV hepatitis A virus
  • CMV human cytomegalovirus
  • HSV-1 single-pass herpes virus type I
  • HSV-2 single-pass herpes virus Type II
  • negative control ddH 2 O were tested.
  • the "S" curve 1 is the amplification curve of the double-stranded oligonucleotide nucleic acid probe for 10 5 IU/mL hepatitis B virus detection
  • the "S” curve 2 is the Taqman probe for 10 5 IU/mL
  • the fluorescence intensity of the two types of probes did not change with the increase of the number of cycles, and the results were all negative and no a
  • the primers are F and R
  • the probes are double-stranded oligonucleotide nucleic acid probes (P1/P2) and Taqman probes (P3)
  • ddH 2 O is the template
  • the amplification is performed for 15 cycles according to the operation in step 2 to detect Quenching efficiency of double-stranded oligonucleotide nucleic acid probe and Taqman probe.
  • the flat line of the "1" group with a fluorescence value between 900-1700 is the background of the amplified fluorescent signal of the double-stranded oligonucleotide nucleic acid probe (P1/P2), and the fluorescence value is 3500-
  • the "2" set straight line between 5300 is the background of the Taqman probe amplified fluorescent signal, and the background of the Taqman probe amplified fluorescent signal is more than 3 times that of the double-stranded oligonucleotide nucleic acid probe.
  • the primers are F and R
  • the probes are double-stranded oligonucleotide nucleic acid probe (P1/P2) and Taqman probe (P3)
  • the template is: HBV nucleic acid quantitative detection standard with a concentration of 10 9 IU/mL, 10 Dilute the gradient to 10 9 IU/mL ⁇ 10IU/mL, and amplify according to the operation in step 2 to detect the quantitative range and sensitivity of the double-stranded oligonucleotide nucleic acid probe and Taqman probe.
  • the double-stranded oligonucleotide nucleic acid probe set (P1/P2) and Taqman probe set change with the cycle number. Visible corresponding fluorescence response.
  • It is the graph of the amplification of the Taqman probe to the HBV nucleic acid quantitative detection standard from 10 9 IU/mL to 10 2 IU/mL, R 2 0.9985, 18 is the 10IU/ml HBV nucleic acid quantitative detection standard, and 19 is negative.
  • the fluorescence response intensity of the double-stranded oligonucleotide nucleic acid probe set is significantly higher than that of the Taqman probe set.
  • the primers are F and R
  • the probes are double-stranded oligonucleotide nucleic acid probe (P1/P2), double-stranded oligonucleotide nucleic acid probe (P1/P4) and Taqman probe (P3)
  • the template is the concentration 10IU/mL HBV nucleic acid quantitative detection standard, repeat the detection 8 times, and perform the amplification according to the operation in step 2 to detect the minimum detection limit of the double-stranded oligonucleotide nucleic acid probe and Taqman probe.
  • the double-stranded oligonucleotide nucleic acid probe can accurately and quantitatively detect samples in the concentration range of 10 9 IU/mL ⁇ 10IU/mL, and provide reference for the detection results of samples with a concentration of 10IU/mL.
  • the Taqman probe can accurately and quantitatively detect samples with a concentration ranging from 10 9 IU/mL to 10 2 IU/mL.
  • the primers are F and R
  • the probes are double-stranded oligonucleotide nucleic acid probes (P1/P2) and Taqman probes (P3)
  • the template is 18 cases of HBV DNA positive sera that have been valued.
  • follow step 2 Perform amplification.
  • Fig. 8 double-stranded oligonucleotide nucleic acid probe sample detection chart
  • 9 Taqman probe sample detection chart
  • Table 5 Table 5. It can be seen from the results that the double-stranded oligonucleotide nucleic acid probe can be effectively detected in each concentration range, and the Taqman probe for low concentration samples has poor detection effect. The results show that the double-stranded probe can effectively detect HBV clinical samples.
  • the double-stranded oligonucleotide nucleic acid probe detects 18 HBV clinical samples that have been set, and all 18 samples can be detected, which is a typical “S” curve.
  • the sample concentration of curve 1-2 is 6.31 ⁇ 10 9 , 5.30 ⁇ 10 9
  • the sample concentration of curve 3-4 is 1.65 ⁇ 10 8 , 2.60 ⁇ 10 8
  • the sample concentration of curve 5-6 is 2.77 ⁇ 10 7 , 2.38 ⁇ 10 7
  • Curve 7-8 sample concentration is 1.50 ⁇ 10 6 , 1.12 ⁇ 10 6
  • curve 9-10 sample concentration is 1.50 ⁇ 10 5 , 1.80 ⁇ 10 5
  • curve 11-12 sample concentration is 3.17 ⁇ 10 4 , 5.56 ⁇ 10 4
  • the sample concentration of curve 13-14 is 5.26 ⁇ 10 3 , 3.16 ⁇ 10 3
  • the sample concentration of curve 15-16 is 3.14 ⁇ 10 2 , 1.95 ⁇ 10 2
  • the sample concentration of curve 17-18 is 7.10 ⁇ 10, 4.90 ⁇ 10
  • Curve 19 is negative,
  • the Taqman probe detects 18 HBV clinical samples that have been set, and 14 of them can be detected, which is a typical "S" curve. 4 samples failed to be detected, which were flat and straight lines.
  • the sample concentration of curve 1-2 is 6.31 ⁇ 10 9 , 5.30 ⁇ 10 9
  • the sample concentration of curve 3-4 is 1.65 ⁇ 10 8 , 2.60 ⁇ 10 8
  • the sample concentration of curve 5-6 is 2.77 ⁇ 10 7 , 2.38 ⁇ 10 7
  • Curve 7-8 sample concentration is 1.50 ⁇ 10 6 , 1.12 ⁇ 10 6
  • curve 9-10 sample concentration is 1.50 ⁇ 10 5 , 1.80 ⁇ 10 5
  • curve 11-12 sample concentration is 3.17 ⁇ 10 4 , 5.56 ⁇ 10 4
  • the sample concentration of curve 13-14 is 5.26 ⁇ 10 3 , 3.16 ⁇ 10 3
  • the sample concentration of curve 15-16 is 3.14 ⁇ 10 2 , 1.95 ⁇ 10 2
  • the sample concentration of curve 17-18 is 7.10 ⁇ 10, 4.90 ⁇ 10
  • Curve 19 is negative, flat and straight, without
  • PCR reaction system including: 10 ⁇ Buffer 2.5 ⁇ L, upstream and downstream primers 1.2 ⁇ L (10 ⁇ M), double-stranded oligonucleotide probe 0.6 ⁇ L (10 ⁇ M), Mg 2+ 2.5 ⁇ L, 50 ⁇ ST enzyme 0.5 ⁇ L (BIORI, China), nucleic acid extraction kit to extract human DNA template 3 ⁇ L, total reaction volume is 25 ⁇ L.
  • Reaction conditions 50 °C, 2 min; 95 °C, 5 min; 95 °C, 20 s, 60 °C, 45 s, 40 cycles in total, collecting fluorescence during annealing.
  • Figure 10 shows the results of the detection of mutant A/A samples, where the black line represents the mutant chain probe labeled with FAM fluorophore, and the detection of mutant sites, except for negative, the fluorescence signal intensity increases with the increase of the number of cycles;
  • the gray line represents the wild-chain probe labeled with the HEX fluorescent group. No wild-type sites were detected, and no amplification was detected.
  • Figure 11 shows the results of the detection of wild-type G/G samples, where the black line represents the FAM fluorophore-labeled mutant chain probe, the mutant site is detected, and no amplification is detected; the gray line represents the HEX fluorophore-labeled Wild-strand probes detect wild-type sites.
  • Figure 12 shows the results of the detection of hybrid G/A samples, in which the black line represents the FAM fluorescent group labeled mutant chain probe to detect the mutant site; the gray line represents the HEX fluorescent group labeled wild chain probe, Examining wild-type loci, except for negative, the intensity of both fluorescent signals increases with the number of cycles.
  • the double-stranded probe can detect the genotype of the single nucleotide polymorphism site, and accurately detect the 2C19*2 gene mutant A/A, wild-type G/G, and heterozygous G/A And negative samples for testing.
  • the mutant chain probe labeled with FAM fluorescent group increases the fluorescence signal intensity as the number of cycles increases, and the wild-chain probe does not change;
  • the wild chain probe labeled with HEX fluorescent group increases with the cycle number Increasing fluorescence signal intensity also increases, and the mutant strand probe has no change; both the hybrid samples and the fluorescence signal increase with the increase of the cycle number, and there is no change in the negative.
  • the double-stranded oligonucleotide nucleic acid probe provided by the present disclosure has a more thorough quenching of fluorescence, greatly reducing the fluorescence background; and does not completely depend on exonuclease activity; in addition, the double-stranded oligonucleotide nucleic acid probe can be labeled Two or more fluorescent molecules improve the detection sensitivity and have great value in popularization and application.

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Abstract

本申请公开一种双链寡核苷酸核酸探针结构、使用方法及其在核酸荧光定性、定量分析、医学诊断和生命科学研究中的用途。双链寡核苷酸核酸探针是由两条完全或部分碱基互补的寡核苷酸链组成，每条寡核苷酸链的末端可以连接荧光基团或对应的荧光淬灭基团，两条寡核苷酸探针链均可与待测目的核酸序列杂交结合。

Description

一种双链寡核苷酸核酸探针的结构和用途
相关申请的交叉引用
本申请要求于2018年12月29日提交中国专利局的申请号为201811643407.0、名称为“一种双链寡核苷酸核酸探针的结构和用途”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及一种生物检测技术工具，具体是一种双链寡核苷酸核酸探针及其使用方法和它在基因荧光定性、定量分析、医学诊断等生命科学研究中的应用，属于基因检测技术领域。
背景技术
目前在传染病等的分子诊断和个体化伴随诊断等疾病诊断应用中一般采用荧光探针法。荧光探针法依赖荧光共振能量转移(FRET，Fluorescent Resonance Energy Transfer)实现检测，包括TaqMan探针、分子信标、蝎型(Scorpion)探针等。该方法仅检测特异性扩增产物，因此特异性强。目前应用最广泛的为TaqMan探针技术，该技术主要利用了Taq酶的5′外切酶活性。首先合成一个能与PCR产物杂交的探针，探针的5′端标记一种荧光分子，3′标记一种相应的荧光淬灭分子，3′端淬灭分子能够吸收5′端荧光分子发出的荧光。正常情况下该探针理论上不发出荧光，但当溶液中有PCR产物时，探针与PCR产物结合，激活Taq酶的5′端外切酶活性，将探针切割成单核苷酸，与此同时，标记在探针上的荧光基团游离出来，导致发出荧光，且切割的荧光分子数与PCR产物的数量成正比，因此，根据PCR反应液中荧光信号强度即可计算出初始模板的浓度。TaqMan探针技术尽管应用广泛，但仍存在很多缺陷。
公开内容
本公开的目的在于解决现有技术中存在的至少一个问题，提供一种新的双链寡核苷酸核酸探针结构、使用方法及其在基因荧光定性、定量分析，医学诊断和生命科学研究等领域的应用。
本公开的基本原理如下：
如图1双链寡核苷酸核酸探针定性定量分析原理图所示，首先合成两条探针，两条探针均在5′端标记荧光基团作为报告分子(F1/F2)，两条探针均在3′ 端标记与5′端相应的荧光淬灭分子(Q1/Q2)，两条探针完全或部分碱基互补。两条探针结合时，探针发出的荧光可同时被同一链及互补链上的淬灭基团吸收，如F1可被Q1和Q2同时吸收，F2可被Q2和Q1同时吸收，溶液中没有荧光产生；两条探针分离时，探针与PCR产物结合，Taq酶的5′端外切酶活性将探针切割成单核苷酸，F1和F2游离出来，发出荧光。双链探针的优势在于淬灭更彻底，荧光本底更低。基于这一构思，本公开合成了双链寡核苷酸核酸探针，当PCR扩增体系中没有模板时，两条探针互补结合，溶液中没有荧光产生；当扩增体系中有模板时，在较高温度下两条探针都优先与模板结合，因此两条探针分离，产生荧光，荧光强度与溶液中模板浓度成正比，据此可进行模板的定量测定。
本公开的目的是这样实现的：制备双链寡核苷酸核酸探针，它由两条完全或部分碱基互补的寡核苷酸链组成，其中两条探针，每条寡核苷酸链的末端可以连接荧光基团或对应的荧光淬灭基团，两条寡核苷酸探针链均可与待测目的DNA或者RNA核酸序列的部分片段按碱基配对原理杂交结合。双链探针每条探针均独立地由6-50个寡核苷酸组成，优选的双链探针中的长链探针由25-30个核苷酸组成，短链探针由15-25个核苷酸组成。探针上连接的荧光分子和淬灭分子数可以是1-5个，考虑到成本及合成方便性，一般是1个。荧光基团可以是FAM、HEX、TET、ROX、CY3、CY5、VIC、JOE、SIMA、Alexa Fluor488、TexasRed或Quasar 670中的一种或多种，该条链对应的另一端连接的淬灭基团可以是TAMRA、Dabcyl、BHQ-1、BHQ-2、BHQ-3、MGB或Eclipse中的一种或多种。
本公开公开了双链寡核苷酸核酸探针在基因检测中的使用方法，它包括以下步骤：
(1)制备双链寡核苷酸核酸探针；
(2)根据待检基因序列，设计并合成一对上下游引物，引物的Tm值低于长链探针的Tm值，引物不与探针发生重叠且邻近探针的两端，距离探针两端1-150个核苷酸；
(3)将模板加入含有探针、引物、PCR缓冲液、镁离子或锰离子、dNTPs、Taq DNA聚合酶的反应混合物中进行常规PCR，扩增25-60个循环。作为模板的核酸长度优选为60-500个碱基，更优选为70-150个碱基。在每个循环退火或延伸时记录荧光值；
(4)以模板起始浓度的对数对门槛荧光的循环数作回归分析，制作标准曲线，对待检基因的浓度进行定量分析。门槛荧光是指2倍于本底荧光变异系数的待检基因的荧光强度。
按照上述检测步骤，本公开所述的双链寡核苷酸核酸探针可以广泛地应用于基因荧光定性、定量分析和医学诊断等基因检测领域，尤其在多基因同时检 测及分型、基因高灵敏度检测中具有更大的优势。
本公开所述的双链寡核苷酸核酸探针中双链探针互为彼此的荧光探针和淬灭探针，荧光基团和淬灭基团距离更近，淬灭更彻底，大大降低了荧光本底；不完全依赖外切酶活性；可标记两个或多个荧光分子，两条探针都与模板结合，提高了检测灵敏度。综上所述，双链寡核苷酸核酸探针技术具有较大的推广应用价值。
结合附图对本公开的具体实施方式作进一步详细说明。
附图说明
图1a为本发明提供的两条寡核苷酸链不等长时，相对较短的一条链从相对较长的一条链的5′端开始反向互补，相对较短的探针与相对较长的探针的反向互补区域都在相对较长的探针区域内的示意图；
图1b为本发明提供的两条寡核苷酸链不等长时，相对较短的一条链从相对较长的一条链的3′开始反向互补，相对较短的探针与相对较长的探针的反向互补区域都在较长探针区域内的示意图；
图1c为本发明提供的两条寡核苷酸链不等长时，相对较短的一条链从相对较长的一条链的中间开始反向互补，相对较短的探针与相对较长的探针的反向互补区域都在较长探针区域内的示意图；
图1d为本发明提供的两条寡核苷酸链不等长时，相对较短的一条链从相对较长的一条链的5′端开始反向互补，两条寡核苷酸链的非反向互补区域在相对较长的探针的5′端以外的示意图；
图1e为本发明提供的两条寡核苷酸链不等长时，相对较短的一条链从相对较长的一条链的3′端开始反向互补，两条寡核苷酸链的非反向互补区域在相对较长的探针的3′端以外的示意图；
图1f为本发明提供的两条寡核苷酸链不等长时，两条寡核苷酸链上存在不完全反向互补的突变碱基的示意图；
图1g为本发明提供的两条寡核苷酸链等长时，两条链上存在不完全反向互补的突变碱基的示意图；
图2为本发明实施例1提供的探针特异性分析图；
图3为本发明实施例1提供的探针本底分析图；
图4为本发明实施例1提供的探针检测范围分析图；
图5为本发明实施例1提供的双链寡核苷酸核酸探针10IU/mL HBV核酸定量检测标准品检测图；
图6为本发明实施例1提供的双链寡核苷酸核酸探针10IU/mL HBV核酸定量检测标准品检测图；
图7为本发明实施例1提供的Taqman探针10IU/mL HBV核酸定量检测标准 品检测图；
图8为本发明实施例1提供的双链寡核苷酸核酸探针样本检测图；
图9为本发明实施例1提供的Taqman探针样本检测图；
图10为本发明实施例2提供的双链寡核苷酸探针对2C19*2基因突变型A/A进行检测的结果图；
图11为本发明实施例2提供的双链寡核苷酸探针对2C19*2基因突变型G/G进行检测的结果图；
图12为本发明实施例2提供的双链寡核苷酸探针对2C19*2基因突变型G/A进行检测的结果图。
具体实施方式
为进一步说明双链寡核苷酸核酸探针技术及应用，参照下列实施例进行说明，下列实施例旨在举例说明而不是以任何方式限制本公开。
实施例1乙型肝炎病毒双链寡核苷酸核酸探针检测
1.HBV检测引物探针设计
根据双链寡核苷酸核酸探针定性定量分析原理，依照待测靶分子HBV的DNA序列，设计合成引物F、R，长链寡核苷酸探针P1，短链寡核苷酸探针P2，Taqman探针P3，含突变碱基寡核苷酸探针P4，引物及探针序列见表1。
Figure PCTCN2019077879-appb-000001
(1)引物
上游引物F，共21个核苷酸，与长链寡核苷酸探针相距17个核苷酸，与短链寡核苷酸探针相距22个核苷酸。
下游引物R，共21个核苷酸，与长链寡核苷酸探针相距27个核苷酸，与短链寡核苷酸探针相距32个核苷酸。
(2)探针
长链寡核苷酸探针P1与靶序列负链互补，由27个核苷酸组成，其中5′端带一荧光素分子，3′端带一淬灭分子；短链寡核苷酸探针P2与靶序列正链互补，由17个核苷酸组成，其中5′端带一荧光素分子，3′端带一淬灭分子，位置上距 探针P1 5个碱基，且较P1短10个碱基；Taqman探针P3与靶序列负链互补，由25个核苷酸组成，其中5′端带一荧光素分子，3′端带一淬灭分子；含突变碱基探针P4与靶序列正链存在不完全反向互补的突变碱基；由27个核苷酸组成，其中5′端带一荧光素分子，3′端带一淬灭分子。
2.PCR检测
(1)双链寡核苷酸核酸探针PCR检测
配制PCR反应体系，包括：10×PCR缓冲液4μL，dNTPs 0.2mmol/L，上、下游引物各0.55μmol/L，Taq DNA聚合酶2.5U，长链寡核苷酸探针0.275μmol/L，短链寡核苷酸探针0.330μmol/L，以及经核酸提取试剂盒提取的HBV模板20μL，总反应体积为40μL；
反应条件：50℃，2min；94℃，2min；94℃，15s，55℃，45s，共45个循环，退火时采集荧光。
(2)双链寡核苷酸核酸不完全反向互补探针荧光PCR检测
配制PCR反应体系，包括：10×PCR缓冲液4μL，dNTPs 0.2mmol/L，上、下游引物各0.55μmol/L，Taq DNA聚合酶2.5U，长链寡核苷酸探针0.275μmol/L，不完全反向互补寡核苷酸探针0.330μmol/L，以及经核酸提取试剂盒提取的HBV模板20μL，总反应体积为40μL；
反应条件：50℃，2min；94℃，2min；94℃，15s，55℃，45s，共45个循环，退火时采集荧光。
(3)Taqman探针荧光PCR检测
配制PCR反应体系包括：10×PCR缓冲液4μL，dNTPs 0.2mmol/L，上、下游引物各0.55μmol/L，Taq DNA聚合酶2.5U，Taqman探针0.275μmol/L，以及经核酸提取试剂盒提取的HBV模板20μL，总反应体系为40μL；
反应条件：50℃，2min；94℃，2min；94℃，15s，55℃，45s，共45个循环，退火时采集荧光。
3.双链寡核苷酸核酸探针的特异性
引物为F与R，探针分别为双链寡核苷酸核酸探针(P1/P2)和Taqman探针(P3)，模板分别为10 5IU/mL乙型肝炎病毒(HBV)、10 5IU/mL丙型肝炎病毒(HCV)、10 5IU/mL甲型肝炎病毒(HAV)、10 5IU/mL人巨细胞病毒(CMV)、10 5IU/mL单传疱疹病毒Ⅰ型(HSV-1)、单传疱疹病毒Ⅱ型(HSV-2)，ddH 2O为阴性对照，按照步骤2中操作进行PCR检测。
实验结果如图2所示，双链寡核苷酸核酸探针和Taqman探针，分别对10 5IU/mL乙型肝炎病毒(HBV)、10 5IU/mL丙型肝炎病毒(HCV)、10 5IU/mL甲型肝炎病毒(HAV)、10 5PFU/mL人巨细胞病毒(CMV)、10 5PFU/mL单传疱疹病毒Ⅰ型(HSV-1)、10 5PFU/mL单传疱疹病毒Ⅱ型(HSV-2)，及阴性对照ddH 2O进行检测。其中，“S”型曲线1为双链寡核苷酸核酸探针对10 5 IU/mL乙型肝炎病毒检测的扩增曲线，“S”型曲线2为Taqman探针对10 5IU/mL乙型肝炎病毒检测的扩增曲线，二者荧光强度均随循环数增加而变化；两组探针对10 5IU/mL丙型肝炎病毒(HCV)、10 5IU/mL甲型肝炎病毒(HAV)、10 5PFU/mL人巨细胞病毒(CMV)、10 5PFU/mL单传疱疹病毒Ⅰ型(HSV-1)、10 5PFU/mL单传疱疹病毒Ⅱ型(HSV-2)，及阴性对照ddH 2O，两类探针的荧光强度均未随循环数的增加而变化，结果全部为阴性均无扩增，为平直线。由此证明双链寡核苷酸核酸探针与Taqman探针都具有良好的特异性。
4.双链寡核苷酸核酸探针淬灭效率
引物为F与R，探针分别为双链寡核苷酸核酸探针(P1/P2)和Taqman探针(P3)，ddH 2O为模板，按照步骤2中操作扩增15个循环，检测双链寡核苷酸核酸探针及Taqman探针的淬灭效率。
如图3所示，图中荧光值在900-1700之间的“1”组平直线为双链寡核苷酸核酸探针(P1/P2)扩增荧光信号本底，荧光值在3500-5300之间的“2”组平直线为Taqman探针扩增荧光信号本底，Taqman探针扩增荧光信号本底是双链寡核苷酸核酸探针的3倍以上。结果表明，双链寡核苷酸核酸探针淬灭较彻底、本底较低，可以解决本底过高对扩增的影响问题。
5.双链寡核苷酸核酸探针的检测范围与灵敏度
(1)线性范围检测
引物为F与R，探针分别为双链寡核苷酸核酸探针(P1/P2)和Taqman探针(P3)，模板为：浓度10 9IU/mL的HBV核酸定量检测标准品，10倍梯度稀释至10 9IU/mL～10IU/mL，按照步骤2中操作扩增，检测双链寡核苷酸核酸探针及Taqman探针的定量范围与灵敏度。
如图4及表2所示，模板浓度在10 9IU/mL～10IU/mL之间时，随循环数改变，双链寡核苷酸核酸探针组(P1/P2)和Taqman探针组可见相应的荧光响应。图中曲线1-9分别是双链寡核苷酸核酸探针对10 9IU/mL～10IU/mL的HBV核酸定量检测标准品扩增的曲线图，R 2＝0.9999，曲线10-17分别是Taqman探针对10 9IU/mL～10 2IU/mL的HBV核酸定量检测标准品扩增的曲线图，R 2＝0.9985，18为10IU/ml HBV核酸定量检测标准品，19为阴性，均无扩增，为平直线。其中，双链寡核苷酸核酸探针组荧光响应强度明显高于Taqman探针组。
表2探针检测范围分析表
Figure PCTCN2019077879-appb-000002
(2)灵敏度检测
引物为F与R，探针分别为双链寡核苷酸核酸探针(P1/P2)、双链寡核苷酸核酸探针(P1/P4)和Taqman探针(P3)，模板为浓度10IU/mL的HBV核酸定量检测标准品，重复检测8次，按照步骤2中操作扩增，检测双链寡核苷酸核酸探针及Taqman探针的最低检测限。
如图5(双链寡核苷酸核酸探针(P1/P2)10IU/mL HBV核酸定量检测标准品检测图)、图6(双链寡核苷酸核酸探针(P1/P4)10IU/mL HBV核酸定量检测标准品检测图)、图7(Taqman探针10IU/mL HBV核酸定量检测标准品检测图)及表3、表4所示，模板量为10IU/mL时，双链寡核苷酸核酸探针组(P1/P2)8次重复检测8次有荧光响应，有典型的“S”型曲线；双链寡核苷酸核酸探针组(P1/P4)8次重复检测8次有荧光响应，有典型的“S”型曲线；Taqman探针组对10IU/mL的HBV核酸定量检测标准品重复检测8次，均无荧光响应。以上阴性均无扩增，为平直线。
表3双链寡核苷酸核酸探针(P1/P2)与Taqman探针
10IU/mL的HBV核酸定量检测标准品检测结果
Figure PCTCN2019077879-appb-000003
表4双链寡核苷酸核酸探针(P1/P4)与Taqman探针
10IU/mL HBV核酸定量检测标准品检测结果
Figure PCTCN2019077879-appb-000004
由此表明双链寡核苷酸核酸探针可对浓度为10 9IU/mL～10IU/mL范围内的样本进行准确定量检测，并为浓度为10IU/mL的样本提供检测结果参考。Taqman探针可对浓度为10 9IU/mL～10 2IU/mL范围内的样本进行准确定量检测。
6.HBV临床样本定量检测分析
引物为F与R，探针分别为双链寡核苷酸核酸探针(P1/P2)和Taqman探针(P3)，模板为18例已定值的HBV DNA阳性血清，按照步骤2中操作进行扩增。
结果如图8(双链寡核苷酸核酸探针样本检测图)、9(Taqman探针样本 检测图)及表5所示。从结果中可以看出，各浓度范围内，双链寡核苷酸核酸探针均能有效检出，低浓度样本Taqman探针检测效果不佳。结果表明，该双链探针能够对HBV临床样本进行有效检测。
其中，在图8中，双链寡核苷酸核酸探针对18份已定值的HBV临床样本进行检测，18份样本均能够检出，为典型的“S”型曲线。曲线1-2样本浓度为6.31×10 9、5.30×10 9，曲线3-4样本浓度为1.65×10 8、2.60×10 8，曲线5-6样本浓度为2.77×10 7、2.38×10 7，曲线7-8样本浓度为1.50×10 6、1.12×10 6，曲线9-10样本浓度为1.50×10 5、1.80×10 5，曲线11-12样本浓度为3.17×10 4、5.56×10 4，曲线13-14样本浓度为5.26×10 3、3.16×10 3，曲线15-16样本浓度为3.14×10 2、1.95×10 2，曲线17-18样本浓度为7.10×10、4.90×10，曲线19为阴性，平直线，无扩增。在图9中，Taqman探针对18份已定值的HBV临床样本进行检测，其中14份样本均能够检出，为典型的“S”型曲线。4份样本未能检出，为平直线。曲线1-2样本浓度为6.31×10 9、5.30×10 9，曲线3-4样本浓度为1.65×10 8、2.60×10 8，曲线5-6样本浓度为2.77×10 7、2.38×10 7，曲线7-8样本浓度为1.50×10 6、1.12×10 6，曲线9-10样本浓度为1.50×10 5、1.80×10 5，曲线11-12样本浓度为3.17×10 4、5.56×10 4，曲线13-14样本浓度为5.26×10 3、3.16×10 3，曲线15-16样本浓度为3.14×10 2、1.95×10 2，曲线17-18样本浓度为7.10×10、4.90×10，曲线19为阴性，平直线，无扩增。
表5双链寡核苷酸核酸探针与Taqman探针临床样本检测结果表
Figure PCTCN2019077879-appb-000005
实施例2双链寡核苷酸探针2C19*2(681G>A)单核甘酸多态性位点的基因型检测
1.2C19*2基因检测引物探针设计
根据双链寡核苷酸核酸探针定性定量分析原理，依照待测靶分子2C19*2基因的DNA序列，设计合成上游引物和下游引物，突变链寡核苷酸探针和野生链寡核苷酸探针，引物及探针序列见表6。
Figure PCTCN2019077879-appb-000006
2.PCR检测
(1)配制PCR反应体系，包括：10×Buffer缓冲液2.5μL，上、下游引 物各1.2μL(10μM)，双链寡核苷酸探针0.6μL(10μM)，Mg 2+2.5μL，50×ST酶0.5μL(BIORI,China)，核酸提取试剂盒提取人DNA模板3μL，总反应体积为25μL.
反应条件：50℃，2min；95℃，5min；95℃，20s，60℃，45s，共40个循环，退火时采集荧光。
(2)模板：2C19*2基因突变型A/A，野生型G/G，杂合型G/A各12例，ddH 2O为阴性对照，按照步骤(1)中操作进行PCR检测。
3.实验结果
如表6及图10-12所示：
表6双链探针检测36份2C19*2突变、野生及杂合型样本结果表
Figure PCTCN2019077879-appb-000007
Figure PCTCN2019077879-appb-000008
图10为对突变型A/A样本检测的结果，其中黑线代表FAM荧光基团标记的突变链探针，检测突变型位点，除阴性外，荧光信号强度均随循环数增加而增加；灰线代表HEX荧光基团标记的野生链探针，检测野生型位点，均未检测到扩增。图11为对野生型G/G样本检测的结果，其中黑线代表FAM荧光基团标记的突变链探针，检测突变型位点，未检测到扩增；灰线代表HEX荧光基团标记的野生链探针，检测野生型位点，除阴性外，荧光信号强度均随循环数增加而增加。图12为对杂合型G/A样本检测的结果，其中黑线代表FAM荧光基团标记的突变链探针，检测突变型位点；灰线代表HEX荧光基团标记的野生链探针，检测野生型位点，除阴性外，两种荧光信号强度均随循环数增加而增加。
从上述结果中可以看出，双链探针可以检测单核甘酸多态性位点的基因型，准确对2C19*2基因突变型A/A，野生型G/G，杂合型G/A及阴性样本进行检测。突变型样本，标有FAM荧光基团的突变链探针随循环数增加荧光信 号强度也增加，野生链探针无变化；野生型样本，标有HEX荧光基团的野生链探针随循环数增加荧光信号强度也增加，突变链探针无变化；杂合型样本二者均随循环数增加荧光信号也增加，阴性均无变化。
工业实用性
本公开提供的双链寡核苷酸核酸探针，荧光淬灭更彻底，大大降低了荧光本底；并且不完全依赖外切酶活性；此外，该双链寡核苷酸核酸探针可标记两个或多个荧光分子，提高了检测灵敏度，具有较大的推广应用价值。

Claims (19)

  1. 一种双链寡核苷酸核酸探针结构，其特征在于，它由两条完全或部分碱基互补的寡核苷酸链组成，其中两条探针，均独立地由6-50个寡核苷酸组成，每条寡核苷酸链的末端可以连接荧光基团或对应的荧光淬灭基团，两条寡核苷酸探针链均可与待测目的DNA或者RNA核酸序列的部分片段按碱基配对原理杂交结合。
  2. 根据权利要求1所述的双链寡核苷酸核酸探针结构，其特征在于，荧光基团或荧光淬灭基团分别连接在每条寡核苷酸链的5′或者3′末端，当一条寡核苷酸链的5′或者3′末端标记荧光基团时，与其结合的对应另一条寡核苷酸链的3′或者5′末端标记相应的荧光淬灭基团。
  3. 根据权利要求1所述的双链寡核苷酸核酸探针结构，其特征在于，所述双链寡核苷酸核酸探针中的一条寡核苷酸链的两端均标记荧光基团，另一条寡核苷酸链的两端均标记荧光淬灭基团。
  4. 根据权利要求1-3任一项所述的双链寡核苷酸核酸探针结构，其特征在于，所述荧光基团可以是FAM、HEX、TET、ROX、CY3、CY5、VIC、JOE、SIMA、Alexa Fluor 488、TexasRed或Quasar 670中的一种或多种。
  5. 根据权利要求1-3任一项所述的双链寡核苷酸核酸探针结构，其特征在于，所述荧光淬灭基团可以是TAMRA、Dabcyl、BHQ-1、BHQ-2、BHQ-3、MGB或Eclipse中的一种或多种。
  6. 根据权利要求1-5任一项所述的双链寡核苷酸核酸探针结构，其特征在于，每条寡核苷酸链连接的荧光基团和荧光淬灭基团的数量分别为1个或多个。
  7. 根据权利要求1所述的双链寡核苷酸核酸探针结构，其特征在于，两条寡核苷酸链的长度相等或不等。
  8. 根据权利要求7所述的双链寡核苷酸核酸探针结构，其特征在于，当两条寡核苷酸链不等长时，相对较短的寡核苷酸链从相对较长的寡核苷酸链的5′端、3′端以及中间开始反向互补，相对较短的寡核苷酸链与相对较长的寡核苷酸链的反向互补区域都在相对较长的寡核苷酸链区域内，或两条寡核苷酸链的非反向互补区域在相对较长的寡核苷酸链的5′端或3′端以外。
  9. 根据权利要求8所述的双链寡核苷酸核酸探针结构，其特征在于，两条寡核苷酸链反向互补的长度一般为8-35个核苷酸。
  10. 根据权利要求8或9所述的双链寡核苷酸核酸探针结构，其特征在于，所述相对较长的寡核苷酸链有25-30个核苷酸组成，所述相对较短的寡核苷酸链由15-25个核苷酸组成。
  11. 根据权利要求8-10任一项所述的双链寡核苷酸核酸探针结构，其 特征在于，两条寡核苷酸链上存在不完全反向互补的突变碱基。
  12. 根据权利要求11所述的双链寡核苷酸核酸探针结构，其特征在于，所述突变碱基的数量为1-10个。
  13. 根据权利要求7所述的双链寡核苷酸核酸探针结构，其特征在于，当两条寡核苷酸链等长时，两条寡核苷酸链上存在不完全反向互补的突变碱基。
  14. 根据权利要求13所述的双链寡核苷酸核酸探针结构，其特征在于，所述突变碱基的数量为1-10个。
  15. 根据权利要求1-14任一项所述的双链寡核苷酸核酸探针结构在核酸检测技术中的应用。
  16. 根据权利要求15所述的应用，其特征在于，所述核酸检测技术包括实时荧光PCR、基因芯片以及膜杂交中的一种或多种。
  17. 根据权利要求1-14任一项所述的双链寡核苷酸核酸探针结构在以核酸为基础的诊断与检测中的应用。
  18. 根据权利要求17所述的应用，其特征在于，所述以核酸为基础的诊断与检测包括临床疾病医学诊断、病原体检测、耐药分析以及实验研究。
  19. 根据权利要求1-14任一项所述的双链寡核苷酸核酸探针结构在实时荧光PCR或者逆转录实时荧光PCR核酸检测技术中的使用方法，包括以下步骤：
    (1)根据检测目标DNA或RNA核酸序列，设计制备双链寡核苷酸核酸探针和对应的上、下游扩增引物；
    (2)在离心管中配制PCR扩增反应液，包括引物、双链寡核苷酸核酸探针、模板、PCR缓冲体系、镁离子或锰离子、dNTPs和Taq DNA聚合酶；
    (3)将含有PCR反应液的离心管置于热循环仪上，进行实时荧光PCR扩增反应，循环数25-50个，每个循环退火或延伸时读取荧光值；
    (4)以模板起始浓度的对数值对门槛荧光的循环数做回归分析，制作标准曲线，对待检测DNA或RNA进行定性或者定量分析。
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CN113789324B (zh) * 2021-08-17 2023-08-25 广东省大湾区华南理工大学聚集诱导发光高等研究院 一种aie探针及其制备方法与在荧光定量pcr方法中的应用

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