WO2021155858A1 - 一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法 - Google Patents

一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法 Download PDF

Info

Publication number
WO2021155858A1
WO2021155858A1 PCT/CN2021/075728 CN2021075728W WO2021155858A1 WO 2021155858 A1 WO2021155858 A1 WO 2021155858A1 CN 2021075728 W CN2021075728 W CN 2021075728W WO 2021155858 A1 WO2021155858 A1 WO 2021155858A1
Authority
WO
WIPO (PCT)
Prior art keywords
mirna
dsn
liquid chromatography
amplification technology
detection
Prior art date
Application number
PCT/CN2021/075728
Other languages
English (en)
French (fr)
Inventor
沈薇
孙俊
祁桐
唐盛
宋畅
Original Assignee
江苏科技大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏科技大学 filed Critical 江苏科技大学
Priority to US17/797,393 priority Critical patent/US20230212654A1/en
Publication of WO2021155858A1 publication Critical patent/WO2021155858A1/zh

Links

Images

Classifications

    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/207—Modifications characterised by siRNA, miRNA
    • 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
    • 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
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/137—Chromatographic separation

Definitions

  • the invention relates to a liquid chromatography method based on a double-strand specific nucleic acid cleaving enzyme (DSN) cyclic amplification technology for simultaneous detection of multiple microRNAs, and belongs to the technical field of nucleic acid detection.
  • DSN double-strand specific nucleic acid cleaving enzyme
  • miRNA also known as microRNA
  • nt nucleotides
  • the first strategy is to design and use probes with different signals.
  • Ye and colleagues proposed the use of fluorescent Raman dual-signal switchable nanoprobe switch to simultaneously quantitatively detect miRNA-21 and miRNA-203. This strategy is suitable for simultaneous detection of multiple miRNAs.
  • the commonly proposed methods for detecting multiple miRNAs cannot be implemented in one test. For example, the same sample needs to be tested multiple times with the same instrument, or two different instruments are used to detect different miRNAs.
  • the second strategy relies on the separation of miRNAs by high performance liquid chromatography (HPLC). High performance liquid chromatography is an efficient separation technique. However, because the difference between different miRNAs is too small, the separation effect is still not ideal.
  • the present invention provides a liquid chromatography based on the DSN cycle amplification technology for simultaneous detection of multiple microRNAs, specifically a double-strand specific nucleic acid cleaving enzyme (DSN) cycle amplification technology Liquid chromatography for simultaneous detection of multiple microRNAs.
  • This method uses a combination of high performance liquid chromatography and DSN cycle amplification, which can simultaneously detect multiple miRNAs with high sensitivity.
  • a liquid chromatography method based on DSN cycle amplification technology to simultaneously detect multiple microRNAs including the following steps:
  • step (1) the ratio of the molar amount of streptavidin binding sites coated on the magnetic beads to the molar amount of DNA probe is (3-5):1.
  • step (1) the loading process is carried out in a 2 ⁇ B&W buffer, which is prepared from Tris-HCl, EDTA, and NaCl.
  • the target microRNA to be detected is selected from a combination of two, three or more different target microRNAs; the target microRNA is a microRNA with 18-25 nucleotides.
  • the target miRNA is selected from miRNA-122, miRNA-155 and miRNA-21, and the corresponding single-stranded DNA probe in step (1) is selected from P122, P155 and P21:
  • the sequence of P122 is 5'-Biotin-T 9 CAAACACCATTGTCACACTCCAC 6 -Fluorophore-3'.
  • the sequence of P155 is 5'-Biotin-T 9 ACCCCTATCACGATTAGCATTAAT 3 -Fluorophore-3'.
  • the sequence of P21 is 5'-Biotin-T 9 TCAACATCAGTCTGATAAGCTAT 25 -Fluorophore-3'.
  • step (2) the incubation is at 36-38°C for 140-160 minutes.
  • step (3) a permanent magnet is used to completely remove the magnetic beads and unreacted DNA probes.
  • step (4) the high performance liquid chromatography system adopts a C18 reverse chromatographic column and adopts a gradient elution mode.
  • the gradient elution mode is: the ratio of methanol changes from 10% to 60% within 20 minutes; the mobile phase is composed of an organic phase and an aqueous phase containing TEAA.
  • the processes of the method are all carried out under dark conditions.
  • the present invention develops a highly sensitive measurement method that can simultaneously detect multiple target miRNAs through HPLC-fluorescence by integrating DSN-assisted target cyclic amplification based on long and short probes.
  • DSN-mediated amplification combined with magnetic separation enhances the signal of the target miRNA.
  • the trace amount of target miRNA is converted into a large number of cleaved DNA probes, and then separated by HPLC (the principle is shown in Figure 1).
  • This method proposes innovations in the following aspects: (1) It solves the challenge of effectively separating miRNA by HPLC; (2) It is the first time to propose a method for sensitive detection of miRNA on a conventional HPLC fluorescence platform (without the need for expensive MS/MS systems). ); (3) Multiple miRNA detections are realized in a single run/test. Finally, the practicability and effectiveness of this method are demonstrated by simultaneously detecting miRNA-122, miRNA-155 and miRNA-21 in serum samples of cervical cancer patients.
  • a variety of single-stranded DNAs with different lengths and different base sequences modified by biotin and fluorescent groups are loaded onto the surface of magnetic beads (MBs) as detection probes.
  • the DNA probes (such as P122, P155, and P21) on the magnetic beads hybridize with the matched target miRNA to form a DNA/RNA heteroduplex.
  • DSN has a strong tendency to shear the DNA in the DNA-RNA hybrid
  • the DNA probe of the heteroduplex can be selectively sheared by the DSN, while the target miRNA strand is kept intact. The direct result is that the shearing of the DNA probe releases the target miRNA strand back into the solution for use in the next cycle.
  • double-strand specific nucleic acid cleaving enzyme DSN
  • the DNA in the double-strand is cleaved, resulting in the dissociation of DNA probes of different lengths containing fluorescent groups and the release of target miRNAs, and the released target miRNA goes to the next cycle.
  • a permanent magnet is used to completely remove unreacted DNA probes on MBs to minimize the background signal. Therefore, after multiple cycles of hybridization and DSN shearing in a sufficiently long incubation time, a large number of DNA cleavage probes and fluorescent labels were obtained.
  • FIG. 1 shows a TEM image of MBs coated with streptavidin. It can be seen that the average diameter of MBs is about 300 nm, with regular morphology and uniform size. On the surface of MBs, it can be clearly seen that the streptavidin layer with a thickness of about 20nm is a light-colored contour coating.
  • Figure 2 shows that the retention time is different when different base tails (cytosine, thymine and adenine) of different lengths are attached to the DNA probe. Therefore, by changing the length and base type of nucleotides, the retention time can be controlled.
  • Figure 2A shows that the retention time is different when different base tails (cytosine 25 (C25), thymine 25 (T25) and adenine 25 (A25)) are attached to the DNA probe. It can be seen from Figure 2A that the order of retention time is C25 ⁇ A25 ⁇ T25. Due to the limitation of synthesis technology, guanine (G) isotopic nucleotides larger than 6-mer cannot be synthesized.
  • the retention times of G6 and C6 are very close compared to C6 and the shorter T3 ( Figure 2B).
  • T due to the strong electrostatic interaction between T and the ion pair reagent and the strong hydrophobic interaction between the resulting ion pair and the RP column, T has a longer retention time. This interaction will increase with the length of the base sequence, as shown in Figure 2C. Therefore, by changing the length of nucleotides, the retention time can be controlled.
  • C and G have shorter retention times, as shown in Figure 2B.
  • T25 is selected as the tail end of the long DNA probe
  • C6 and T3 are selected as the tail end of the short DNA probe.
  • the retention time in the chromatogram is different: the shorter DNA probe P122 (with C6 sequence) has a retention time of 5.4 minutes, and P155 (with T3 sequence) is 6.4 Minutes, the longer DNA probe P21 (with T25 sequence) is 6.9 minutes, thus realizing simultaneous detection of multiple miRNAs.
  • the detection limit of this method for miRNA-122 is 0.39fM
  • the detection limit of miRNA-155 is 0.30fM
  • the detection limit of miRNA-21 is 0.26fM
  • the linear range is 1.0fM to 10pM (Figure 5), Figure 5 The correlation between the peak area and miRNA concentration is shown.
  • the calibration curve shows that in the range of 1.0fM to 100pM, the peak area is linearly related to the logarithm of the three target miRNA concentrations, and the linear coefficients (r2) are 0.991 and 0.994, respectively And 0.996.
  • miRNA-122 was 0.39fM
  • miRNA-155 was 0.30fM
  • miRNA-21 was 0.26fM.
  • LOD 3NQ/I, where Q is the injection volume, N is the noise level, and I is the fluorescence signal.
  • the relative standard deviation (RSD) is less than 5%.
  • the established method has been successfully applied to simultaneously detect miRNA-122, miRNA-155 and miRNA-21 in serum samples of patients with cervical cancer, lupus erythematosus, ovarian cancer and healthy people (see Table 1-4, Figure 7). It is inferred from this that the method is effective for two, three and more miRNAs (such as microRNA-141, let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7i, microRNA-200, microRNA-203, microRNA-223, microRNA-16, microRNA-125b, microRNA-199a, microRNA-182-5p, microRNA-210, microRNA-200b, microRNA-200c, microRNA-429, has-microRNA- A combination of two or more of 20a, has-microRNA-20b, or a combination with the aforementioned microRNA), that is, a combination of 2-24 kinds and more combinations are applicable.
  • miRNAs such as microRNA-141, let-7a, let-7b, let-7c, let-7d,
  • the present invention mixes and incubates miRNA, DNA probes and DSN in different proportions under optimal detection conditions.
  • Figure 3 in the absence of the target miRNA, almost no fluorescent signal was detected regardless of the presence of DSN ( Figures 3a and b).
  • Figure 3c When only the target miRNA and DNA probe are present, the background signal observed is negligible ( Figure 3c), which may be due to the slight separation of the DNA/RNA heteroduplex from the MB.
  • the fluorescence signal was only observed when the target miRNA, DSN, and DNA probe coexist in the solution ( Figure 3d, e, f, and g).
  • the experiment of the present invention proves the feasibility of simultaneously detecting multiple miRNAs in actual samples by using the HPLC fluorescence platform.
  • the signals of different miRNAs were separated by HPLC.
  • the introduction of DSN promotes the successful application of the isothermal target cyclic amplification method. It successfully solves the problem of low sensitivity of conventional HPLC fluorescence detection, and at the same time ensures the method's high selectivity for target miRNA (Figure 6).
  • the present invention is at the same optimal level. Under the experimental detection conditions, the fluorescence response of other mismatched miRNAs was compared, and the fluorescence response of the target miRNA was compared.
  • Figure 6 shows the target miRNA, single-base mismatch miRNA, two-base mismatch miRNA, and complete mismatch. miRNA response. Even when the concentration of the interfering substance is 100 times higher than the target miRNA, no obvious interference is observed. The peak area of single-base mismatch, double-base mismatch and complete mismatch miRNA can be ignored, and the presence of target miRNA-122, miRNA-155 and miRNA-21 significantly increases the fluorescence signal. These results show that this method is very effective for detecting target miRNA.
  • Figure 7 shows the chromatograms of the original sample and the spiked serum sample after applying this method, indicating that this assay method can conveniently detect multiple miRNAs simultaneously in a single run.
  • the analysis method of the present invention will promote the high sensitivity and high selectivity analysis of biological macromolecules (such as nucleic acids) based on conventional HPLC methods by introducing more types of bases (A and G) as the tail ends of DNA probes. , Greatly improve the separation efficiency of DNA probes after shearing. Therefore, more target miRNAs can be detected at the same time.
  • new amplification methods such as LH-PCR
  • HPLC also has the potential to analyze nucleic acid length heterogeneity (LH).
  • the method of the present invention can use a commonly used liquid chromatograph to realize simultaneous detection of multiple miRNAs, with low instrument cost, convenient operation, high sensitivity of the method, low detection limit, and wide linear range.
  • the detection limit for miRNA-122 is 0.39fM
  • the detection limit for miRNA-155 is 0.30fM
  • the detection limit for miRNA-21 is 0.26fM
  • the linear range is 1.0fM to 10pM.
  • the established method can be successfully applied to detect miRNA-122, miRNA-155 and miRNA-21 in serum samples of patients with lupus erythematosus, cervical cancer, ovarian cancer and healthy people.
  • Figure 1 The mechanism diagram of the high-performance liquid chromatography method for simultaneous detection of miRNA-122, miRNA-155 and miRNA-21 based on the DSN cycle amplification technology.
  • Figure 2 is a chromatogram of a DNA probe, where: A is a chromatogram of a DNA probe with the same length (25nt) and different base sequences; B is a DNA with different lengths and the same base sequence (cytosine C and guanine G) The chromatogram of the probe, C is the chromatogram of DNA probes with different lengths and the same base sequence (thymine T).
  • Figure 3 is a graph of fluorescence signals verifying the feasibility of simultaneous detection of miRNA-122, miRNA-155 and miRNA-21, (a) blank, (b) 0.4U DSN, (c) 100pM miRNA-122 and 100pM miRNA-155+100pM miRNA-21, (d)100pM miRNA-122+0.4U DSN, (e)100pM miRNA-155+0.4U DSN, (f)100pM miRNA-21+0.4U DSN, (g)100pM miRNA-122+100pM miRNA -155+100pM miRNA-21+0.4U DSN fluorescence signal.
  • Experimental conditions 100nM DNA probe, 25mM Mg 2+ , pH 8.0 and incubation at 40°C for 180 minutes. Error bars represent the standard deviation of three independent experiments.
  • Figure 4 is an optimized diagram of experimental conditions, 100pM target miRNA-122, miRNA-155 and miRNA-21, 100nM DNA probe: (A) DSN concentration from 0.1U to 0.5U; (B) buffer solution pH value from 7 To 9; (C) Mg 2+ concentration from 15mM to 35mM; (D) the corresponding probe chromatograms in the presence of different concentrations of Mg 2+ ; (E) incubation temperature from 30°C to 50°C; (F) incubation time Optimized graph from 60min to 210min.
  • Figure 5 shows the calibration curves of three miRNAs, (A) miRNA-122, (B) miRNA-155 and (C) miRNA-21 calibration curves.
  • Experimental conditions 100nM DNA probe, 25mM Mg 2+ , 0.4U DSN, pH 8.0 and incubation at 40°C for 180 minutes. Error bars represent the standard deviation of three independent experiments.
  • Figure 6 shows the selective detection of miRNA-122, miRNA-155 and miRNA-21: peak area of M1 (single base mismatch), M2 (double base mismatch) and NM (complete mismatch), interference
  • concentration of miRNA is 10 nM
  • concentration of each miRNA is 100 pM.
  • Experimental conditions 100nM DNA probe, 25mM Mg 2+ , 0.4U DSN, pH 8.0 and incubation at 40°C for 180 minutes. Error bars represent the standard deviation of three independent experiments.
  • Figure 7 is the chromatogram of the original and serum samples added with a certain amount of miRNA.
  • the chromatograms of the original and spiked serum samples were detected by the established method.
  • Experimental conditions 100nM DNA probe, 25mM Mg 2+ , 0.4U DSN , PH 8.0 and incubate at 40°C for 180 minutes.
  • a liquid chromatography method based on DSN cycle amplification technology to simultaneously detect multiple microRNAs including the following steps:
  • the supernatant was taken by magnetic separation, and fluorescence detection was performed at the excitation wavelength of 495nm and the emission wavelength of 518nm. Then the coupling efficiency between the probe and MBs was estimated based on the fluorescence data. It is estimated that about 1.16 ⁇ 10 4 DNA probes (the sum of P122, P155 and P21 are coupled to each MB, accounting for about 12% of the total capacity of MBs. Therefore, there is enough space for target miRNA hybridization and DSN cleavage Finally, the obtained long and short DNA probes and MB conjugates are washed and dispersed in the hybridization buffer, and stored at 4°C for use.
  • the working range of DSN is from 3.5 to 8.5, and it has the maximum activity at a pH of about 6.6, and a pH value of ⁇ 3.0 or >9.0 will inactivate DSN.
  • the pH of the solution also has a significant effect on the fluorescence of the fluorescein label.
  • fluorescein In acidic media, fluorescein is usually protonated. Instead, it is deprotonated in an alkaline medium. Both the protonated and deprotonated states will change the conformation of fluorescein, thereby reducing its fluorescence efficiency. According to the observation results of our experiment ( Figure 4B), it is found that pH 8.0 is the best choice for this method.
  • Mg 2+ concentration plays a key role in miRNA detection.
  • the present invention evaluates the influence of this parameter, and the results are shown in Figs. 4C and D. It can be seen that as the Mg 2+ content increases, the peak area increases, but when the Mg 2+ concentration is higher than 25 mM, the peak area decreases significantly. The possible reason is that the activity of DSN will decrease under high ionic strength. Taking these results into consideration, 25mM Mg 2+ was selected as the optimal concentration for the determination. Another important variable is the incubation temperature of the reaction.
  • the incubation temperature not only directly affects the hybridization efficiency, but also directly affects the shearing activity of DSN.
  • the melting temperature should be 10-15°C higher than the hybridization temperature to achieve high hybridization efficiency while ensuring good selectivity.
  • this experiment should be carried out at a temperature lower than 45°C.
  • DSN shear performs well at temperatures higher than 45°C, and the best shear temperature is 60°C.
  • the peak areas of the three miRNAs reached their maximum at 40°C.
  • the method adopted by optimizing all the embodiments of the present invention is: adding 44 ⁇ L of 100 nM P122, P155, and P21 to the centrifuge tube, and then adding 0.4 U of DSN and 5 ⁇ L of target miRNA. Then, a brief shaking treatment for about 2 seconds was carried out to make the reaction mixture thoroughly mixed. After incubating at 40°C for 180 minutes, the MBs and unreacted DNA probes were separated with a permanent magnet. Finally, the supernatant is injected into the high performance liquid chromatography system for separation and quantification.
  • the stationary phase of the chromatographic column described in this experiment is octadecyl (C18).
  • C18 column is a typical reverse phase (RP) column, which is often used to retain and separate hydrophobic compounds.
  • RP reverse phase
  • oligonucleotides have strong polarity and are difficult to retain in any RP column. Therefore, add 100mM TEAA as an ion pair reagent in the mobile phase to make the DNA probes on the column stay longer.
  • the gradient elution mode used in this experiment is: the ratio of methanol changes from 10% to 60% within 20 minutes, and the flow rate is 1 mL/min.
  • the mobile phase of this experiment consisted of organic phase: methanol, water phase: 100mM TEAA aqueous solution and 5% acetonitrile.
  • the method of the present invention realizes the simultaneous detection of multiple miRNAs.
  • the detection limit of miRNA-122 is 0.39fM
  • the detection limit of miRNA-155 is 0.30fM
  • the detection limit of miRNA-21 is 0.26fM
  • the linear range is 1.0fM to 10pM.
  • the established method has been successfully applied to detect miRNA-122, miRNA-155 and miRNA-21 in serum samples of patients with lupus erythematosus, cervical cancer, ovarian cancer and healthy people.
  • the above methods are used to detect miRNA-122, miRNA-155 and miRNA-21 in serum samples of healthy people, patients with lupus erythematosus, cervical cancer, and ovarian cancer. Specific examples are as follows:
  • Example 1 Detection of miRNA-122, miRNA-155 and miRNA-21 in serum samples of healthy people:
  • the detection results of miRNA-122, miRNA-155 and miRNA-21 in serum samples of healthy people are shown in Table 1.
  • serum sample 1 healthy volunteers
  • miRNA-122, miRNA-155 and miRNA-21 were detected.
  • the concentrations were 0.063pM, 0.057pM, and 0.046pM, while in serum sample 2 (healthy volunteers) they were 0.067pM and 0.053pM and 0.048pM, respectively.
  • different concentrations of miRNA-122, miRNA-155 and miRNA-21 were spiked into sample 1, and a good relative recovery rate (101.7%-104.7%) was obtained, and the relative standard deviation (RSD) was 2.4 % ⁇ 4.7%.
  • the results of the detection of miRNA-155 and miRNA-21 in the serum samples of patients with lupus erythematosus are shown in Table 2.
  • the serum samples of patients with lupus erythematosus detected 0.399pM miRNA-155 and 0.034pM miRNA-21, respectively.
  • miRNA-155 in patients with lupus erythematosus has obvious overexpression.
  • the detection results of miRNA-122, miRNA-155 and miRNA-21 in the serum samples of cervical cancer patients are shown in Table 4.
  • the concentrations of miRNA-122 detected in samples 3 and 4 (cervical cancer patients) were 0.070pM and At 0.090pM, the concentrations of miRNA-155 were 0.209pM and 0.224pM, and the concentrations of miRNA-21 were 0.115pM and 0.117pM, respectively.
  • These results indicate that miRNA-155 and miRNA-21 are up-regulated in cancer patients compared with healthy people.
  • the method of the present invention was compared with other methods for multiple detection of miRNA or HPLC-related methods reported in recent years, and the results are shown in Table 5.
  • the miRNA analysis method using fluorescence detection has high selectivity, but not high sensitivity (Wang,R.; Xu,X.; Li,X.; Zhang,N.; Jiang,W.pH-responsive ZnO nanoprobe mediated DNAzyme signal amplifier strategy for sensitive detection and live cell imaging of multiple microRNAs.Sens.Actuators,B.2019,293,93-99.Jie,G.; Zhao,Y.;Wang,X.;Ding,C.Multiplexed fluorescence detection of microRNAs based on novel distinguishable quantum dot signal probes by cycle amplification strategy.Sens.Actuators,B.2017,252,1026-1034.).
  • Electrochemiluminescence (ECL)-based miRNA assays show high sensitivity, but they have disadvantages such as the synthesis of complex materials (Feng, X.; Gan, N.; Zhang, H.; Li, T.; Cao, Y.;Hu,F.;Jiang,Q.Ratiometric biosensor array for multiplexed detection of microRNAs based on electrochemiluminescence coupled with cyclic voltammetry.Biosens.Bioelectron.2016,75,308-314.Peng,L.;Zhang,P.;Chai, Y.; Yuan, R.

Landscapes

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

Abstract

提供了一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，该方法包括：根据待检测的目标microRNA，设计荧光基团修饰的单链DNA，然后将其装载至磁珠表面作为检测探针；在上述检测探针中加入待检测的目标miRNA样品和DSN，充分混合，孵育；孵育后，将磁珠和未反应的DNA探针完全去除，得分离液；将分离液注入到高效液相色谱系统进行分离和定量。

Description

一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法 技术领域
本发明涉及一种基于双链特异核酸切割酶(DSN)循环扩增技术对多种microRNA同时进行检测的液相色谱法，属于核酸检测技术领域。
背景技术
目前普遍认为，microRNA(miRNA，也称微RNA)是一系列内源性非编码小核糖核酸分子(18-25个核苷酸(nt))，在细胞分化、增殖、凋亡、死亡等生物进化过程中发挥重要作用。miRNA的异常表达与各种疾病的发生、发展密切相关，尤其是各种类型的人类癌症、神经系统疾病、病毒感染和糖尿病。因此，miRNA被认为是这些疾病的有价值的生物标志物。但是，通常一种疾病可能与多种miRNA相关，或者一种特异性miRNA的异常表达可能与多种疾病相关。因此，仅分析单一的miRNA生物标志物不足以为癌症的早期诊断或监测癌症的治疗效果提供有说服力的证据。为了应对这一挑战，研究人员越来越重视通过开发各种检测方法来灵敏地分析多种miRNA。
迄今为止，为了分析多种miRNA，通常根据两种策略来设计方法。第一种策略是设计使用具有不同信号的探针。例如，Ye及其同事提出了利用荧光拉曼双信号可切换纳米探针开关同时定量检测miRNA-21和miRNA-203。该策略适用于多种miRNA的同时检测。但是通常提出的检测多种miRNA的方法无法在一次测试中实现，比如，同一样本需使用相同的仪器进行多次检测，或者通过使用两个不同的仪器来检测不同的miRNA。第二种策略依赖于通过高效液相色谱(HPLC)分离miRNA。高效液相色谱法是一种高效的分离技术。但由于不同miRNA之间的差异太小，导致其分离效果仍不理想。尽管Nakayama等人成功分离了四种类型的miRNA，但这是通过纳米流动LC，使用串联质谱(MS/MS)进行miRNA检测。为了区分五种miRNA的重叠信号(保留时间非常接近)，Xu等人引入了五种DNA-肽探针作为这五种miRNA的标记物进行LC-MS/MS分析。但是，串联质谱得到的数据的过程通常是繁琐的。此外，这些检测技术的检测限与利用遗传技术的检测限是远比不上的。因此，在一次测试中对多个miRNA进行高灵敏分析是非常迫切的。
为了提高miRNA检测的灵敏度，开发了许多信号放大策略。如热循环扩增技术，包含有实时PCR扩增(qRT-PCR)技术，滚环扩增技术，催化发夹组装技术等，再比 如链置换扩增(SDA)技术，它包括酶促SDA和无酶SDA等。其中，作为一种简单有效的策略，DSN辅助目标回收实现信号循环扩增已被尝试用于miRNA的检测。在文献报道中，研究人员结合磁珠(MBs)的优良分离和DSN辅助的靶标回收，开发了一种简单、灵敏、高选择性的分析miRNA的检测方法。也有研究人员尝试将DSN与LC-MS/MS结合起来进行miRNA的检测。然而，它只适用于一种类型的miRNA，因为涉及到DSN辅助扩增的探针不能被HPLC有效地分离。
发明内容
发明目的：为了解决上述技术问题，本发明提供了一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，具体是一种基于双链特异核酸切割酶(DSN)循环扩增技术对多种microRNA同时进行检测的液相色谱法。该方法采用高效液相色谱和DSN循环扩增相结合的方法，能够对多种miRNA同时进行高灵敏度检测。
技术方案：为了实现上述目的，本发明采用以下技术方案：
一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，包括以下步骤：
(1)根据待检测的目标microRNA，设计荧光基团修饰的单链DNA探针，然后将其装载至链霉亲和素包被的磁珠(MBs)表面作为检测探针；
(2)在上述检测探针中加入待检测的目标miRNA样品和DSN，充分混合，孵育；
(3)孵育后，将磁珠和未反应的DNA探针完全去除，得分离液；
(4)将分离液注入到高效液相色谱系统进行分离和定量。
作为优选：
步骤(1)中，所述磁珠上包被的链霉亲和素结合位点的摩尔量与DNA探针摩尔量的比例为(3-5):1。
步骤(1)中，装载过程在2×B&W缓冲液中进行，所述缓冲液是由Tris-HCl和EDTA、NaCl配制成。
步骤(2)中，所述待检测的目标microRNA选自两种、三种或者多种不同目标microRNA的组合；所述目标microRNA为具有18-25个核苷酸的microRNA。
作为本发明的一种具体实施方案，所述目标miRNA选自miRNA-122、miRNA-155和miRNA-21，步骤(1)中对应的单链DNA探针选自P122、P155和P21：
P122的序列为5’-生物素-T 9CAAACACCATTGTCACACTCCAC 6-荧光基团-3’。
P155的序列为5’-生物素-T 9ACCCCTATCACGATTAGCATTAAT 3-荧光基团-3’。
P21的序列为5’-生物素-T 9TCAACATCAGTCTGATAAGCTAT 25-荧光基团-3’。
步骤(2)中，所述孵育是在36-38℃下孵育140-160分钟。
步骤(3)中，利用永磁体将磁珠和未反应的DNA探针完全去除。
步骤(4)中，所述高效液相色谱系统采用C18反向色谱柱，采用梯度洗脱模式。
进一步优选，所述梯度洗脱模式为：甲醇的比例在20分钟内从10％变化到60％；流动相由有机相和包含TEAA的水相组成。
优选，所述方法的过程均在避光条件下进行。
本发明通过集成基于长短探针的DSN辅助目标物循环扩增，开发了一种高灵敏且可通过HPLC-荧光同时检测多个目标miRNA的测定方法。在该方法中，通过DSN介导的扩增结合磁分离增强了目标miRNA的信号。扩增后，将痕量的目标miRNA转化为大量切割的DNA探针，然后通过HPLC对其进行分离(其原理如图1)。该方法从以下几个方面提出了创新：(1)解决了通过HPLC有效分离miRNA的挑战；(2)首次提出了在常规HPLC荧光平台上进行灵敏检测miRNA的方法(无需昂贵的MS/MS系统)；(3)在单次运行/测试中实现了多个miRNA检测。最后，通过同时检测宫颈癌患者血清样品中的miRNA-122、miRNA-155和miRNA-21证明了该方法的实用性和有效性。
本发明首先将生物素和荧光基团修饰的多种长度不同、碱基序列不同的单链DNA装载至磁珠(MBs)表面作为检测探针。如图1所示，磁珠上的DNA探针(例如P122、P155和P21)与匹配的目标miRNA杂交形成DNA/RNA异源双链。由于DSN对DNA-RNA杂交体中DNA具有强烈的剪切倾向，因此异源双链体的DNA探针可被DSN选择性剪切，同时目标miRNA链完整保留。直接结果是DNA探针的剪切将目标miRNA链重新释放回溶液中以供下一个循环使用。具体的，在双链特异核酸切割酶(DSN)的作用下，双链中的DNA被切割，导致含有荧光基团的不同长度的DNA探针的解离和目标miRNA的释放，所释放的目标miRNA进行下一次循环。利用永磁铁将MBs上未反应的DNA探针完全去除，使背景信号最小化。因此，在足够长的孵育时间内进行了多次杂交和DSN剪切循环之后，获得了大量的DNA裂解探针和荧光标记。孵育后通过磁分离，只有三种不同长度的含有荧光标记的DNA裂解探针保留在溶液中，而MBs和未反应的DNA探针一起用永磁体完全去除。而此时荧光信号来自三种类型的DNA 探针。为了将裂解的DNA探针与荧光素标记分开，将溶液引入HPLC系统。另外，图1中示出了被链霉亲和素包被的MBs的TEM图像。可以看出，MBs的平均直径约为300nm，其形态规则，大小均匀。在MBs表面可以清楚地看到厚度约为20nm的链霉亲和素层是一个浅色的轮廓涂层。图2显示，当DNA探针附加不同长度的不同碱基尾端(胞嘧啶，胸腺嘧啶和腺嘌呤)时保留时间不同。因此，通过改变核苷酸的长度和碱基类型，可以控制保留时间。图2A显示，当DNA探针附加不同的碱基尾端(胞嘧啶25(C25)，胸腺嘧啶25(T25)和腺嘌呤25(A25))时保留时间不同。从图2A可以看出，保留时间的顺序为C25＜A25＜T25。由于合成技术的限制，不能合成大于6聚体的鸟嘌呤(G)同位核苷酸。因此，与C6和较短的T3(图2B)相比，G6与C6的保留时间非常接近。通常，由于T与离子对试剂之间的静电相互作用较强以及所得离子对与RP柱之间有较强的疏水相互作用，使得T具有更长的保留时间。这种相互作用将随着碱基序列的长度而增加，如图2C所示。因此，通过改变核苷酸的长度，可以控制保留时间。C和G具有较短的保留时间，如图2B所示。考虑到成本和分离效率，选择T25作为长DNA探针的尾端，选择C6、T3作为短DNA探针的尾端。由于切割后的探针的长度和碱基序列不同，在色谱图中保留时间各异：较短的DNA探针P122(具有C6序列)的保留时间为5.4分钟，P155(具有T3序列)为6.4分钟，更长的DNA探针P21(具有T25序列)是6.9分钟，从而实现了多种miRNA的同时检测。该方法对miRNA-122的检测限为0.39fM，miRNA-155的检测限为0.30fM，对miRNA-21的检测限为0.26fM，线性范围均为1.0fM至10pM(如图5)，图5显示了峰面积与miRNA浓度之间的相关性，校准曲线显示，在1.0fM到100pM范围内，峰面积与三种目标miRNA浓度的对数呈线性相关，线性系数(r2)分别为0.991、0.994和0.996。以背景信号标准偏差的三倍计算该方法对三种miRNA的LOD，得miRNA-122为0.39fM，miRNA-155为0.30fM，miRNA-21为0.26fM。(根据LOD＝3NQ/I计算值，其中Q为进样量，N为噪声水平，I表示荧光信号。)在整个线性范围内，相对标准偏差(RSD)均小于5％。所建立的方法成功应用于同时检测宫颈癌、红斑狼疮、卵巢癌患者和健康人血清样本中的miRNA-122、miRNA-155和miRNA-21(见表1-4，图7)。由此推论，该方法对两种、三种及以上的miRNA(例如microRNA-141、let-7a、let-7b、let-7c、let-7d、let-7e、let-7f、let-7i、microRNA-200、microRNA-203、microRNA-223、microRNA-16、microRNA-125b、microRNA-199a、microRNA-182-5p、microRNA-210、microRNA-200b、microRNA-200c、 microRNA-429、has-microRNA-20a、has-microRNA-20b中的两种或两种以上的组合，或者与前述microRNA的组合)，即2-24种的组合以及更多的组合均适用。
为了验证测定原理的可行性，本发明按最优检测条件将不同比例的miRNA、DNA探针和DSN混合孵育。如图3所示，在不存在目标miRNA的情况下，无论是否存在DSN，都几乎没有检测到荧光信号(图3a和b)。仅存在目标miRNA和DNA探针时，观察到的背景信号可忽略不计(图3c)，这可能是由于DNA/RNA异源双链体从MB上轻微分离所致。另一方面，仅当目标miRNA、DSN和DNA探针在溶液中共存时才观察到荧光信号(图3d、e、f和g)。上述结果清楚地表明，所提出的测定方法对于同时高灵敏检测miRNA-122、miRNA-155和miRNA-21是可行的。
本发明实验证明了利用HPLC荧光平台同时检测实际样品中多种miRNA的可行性。通过引入长短DNA探针，利用HPLC对不同miRNA的信号实现了分离。DSN的引入促进了等温目标循环扩增方法的成功应用，它成功解决了常规HPLC荧光检测低灵敏度的问题，同时保证了方法对目标miRNA的高选择性(图6)，本发明在相同最优实验检测条件下通过其他错配miRNA的荧光响应，并将其与目标miRNA的荧光响应进行了比较，图6显示了目标miRNA、单碱基错配miRNA、双碱基错配miRNA和完全错配miRNA的响应。即使干扰物的浓度比目标miRNA高100倍时，仍未观察到明显的干扰。单碱基错配、双碱基错配和完全错配miRNA的峰面积可以忽略不计，而目标miRNA-122、miRNA-155和miRNA-21的存在使荧光信号显著升高。这些结果表明，该方法对于检测目标miRNA非常有效。同时，图7展示了应用本方法后的原始样品和加标血清样品的色谱图，说明该测定方法便捷地实现了在单次运行中对多种miRNA的同时检测。此外，本发明的分析方法将促进基于常规HPLC方法的生物大分子(例如核酸)的高灵敏度和高选择性分析，通过引入更多类型的碱基(A和G)作为DNA探针的尾端，大大提高剪切后DNA探针的分离效率。因此，可以同时检测更多的目标miRNA。此外将新的扩增方法(如LH-PCR)与HPLC结合使用，还具有对核酸长度异质性(LH)分析的潜力。有益效果：相对于现有技术，本发明方法能利用常用的液相色谱仪实现对多种miRNA的同时检测，仪器成本低，操作方便，方法的灵敏度高，检测限低，线性范围广。如示例中：对miRNA-122的检测限为0.39fM，对miRNA-155的检测限为0.30fM，对miRNA-21的检测限为0.26fM，线性范围均为1.0fM至10pM。所建立的方法能够成功应用于检测红斑狼疮、宫颈癌、卵巢癌患者和健康人血清样本中的miRNA-122、 miRNA-155和miRNA-21。
附图说明
图1基于DSN循环扩增技术同时检测miRNA-122、miRNA-155和miRNA-21的高效液相色谱分析方法机理图。
图2为DNA探针的色谱图，其中：A为相同长度(25nt)不同碱基序列的DNA探针的色谱图；B为不同长度相同碱基序列(胞嘧啶C和鸟嘌呤G)的DNA探针的色谱图，C为不同长度相同碱基序列(胸腺嘧啶T)的DNA探针的色谱图。
图3为验证同时检测miRNA-122、miRNA-155和miRNA-21可行性的荧光信号图，(a)空白，(b)0.4U DSN，(c)100pM miRNA-122和100pM miRNA-155+100pM miRNA-21，(d)100pM miRNA-122+0.4U DSN，(e)100pM miRNA-155+0.4U DSN，(f)100pM miRNA-21+0.4U DSN，(g)100pM miRNA-122+100pM miRNA-155+100pM miRNA-21+0.4U DSN的荧光信号。实验条件：100nM DNA探针，25mM Mg 2+，pH 8.0以及在40℃下孵育180分钟。误差线代表三个独立实验的标准偏差。
图4为实验条件的优化图，100pM的目标miRNA-122、miRNA-155和miRNA-21,100nM DNA探针：(A)DSN的浓度从0.1U到0.5U；(B)缓冲溶液pH值从7到9；(C)Mg 2+浓度从15mM到35mM；(D)不同浓度Mg 2+存在时所对应的探针色谱图；(E)孵化温度从30℃到50℃；(F)孵化时间从60min到210min的优化图。
图5为三种miRNA的校准曲线，(A)miRNA-122、(B)miRNA-155和(C)miRNA-21的校准曲线图。实验条件：100nM DNA探针，25mM Mg 2+，0.4U DSN，pH 8.0和在40℃孵育180分钟。误差线代表三个独立实验的标准偏差。
图6为对miRNA-122、miRNA-155和miRNA-21的选择性检测图：M1(单碱基错配)、M2(双碱基错配)和NM(完全错配)的峰面积，干扰miRNA的浓度为10nM，每个miRNA的浓度为100pM。实验条件：100nM DNA探针，25mM Mg 2+，0.4U DSN，pH 8.0和在40℃孵育180分钟。误差线代表三个独立实验的标准偏差。
图7为原始的和加入一定量的miRNA的血清样本的色谱图，应用所建立方法检测原始样品和加标血清样品的色谱图，实验条件：100nM DNA探针，25mM Mg 2+，0.4U DSN，pH 8.0和在40℃孵育180分钟。
具体实施方式
以下结合附图和具体实施例，对本发明方法做出进一步说明。
一种基于DSN循环扩增技术对多种microRNA同时进行检测的液相色谱方法，包 括以下步骤：
首先将40μL粒径为300nm的链霉亲和素包被的磁珠加入到1.5mL的棕色聚丙烯离心管中，磁性分离移去溶剂并将MBs保留在管中。再用1×B&W缓冲液洗涤3次后，将MBs重新分散于90μL 2×B&W缓冲液中，同时加入5μL10μM不同长短的DNA探针(本示例中，P122为37nt，P155为35nt，P21为58nt)。为了保证探针上的生物素和磁珠的链霉亲和素能充分结合，在室温下轻轻涡流15分钟。磁性分离取上清液，在激发波长495nm，发射波长518nm处进行荧光检测。然后根据荧光数据估算了探针与MBs之间的偶联效率。据估计，约有1.16×10 4个DNA探针(P122、P155和P21的总和偶联到每个MB，约占MBs总容量的12％。因此，有足够的空间进行目标miRNA杂交和DSN切割。最后，将得到的长、短DNA探针与MB偶联物在杂交缓冲液中洗涤、分散，并在4℃下储存备用。
在对目标miRNA检测前，首先对所有实验条件进行优化，包括偶联时间、DSN剂量、Mg 2+浓度、缓冲液pH、孵育温度和时间。优化结果如图4所示。
从图4A可以看出，当DSN的剂量从0.10U变为0.50U时，荧光信号的峰面积明显增大，而当DSN>0.40U时，P122、P155和P21的荧光信号的峰面积均趋于稳定。为了最大限度地提高测定灵敏度并保持低成本，采用0.4U作为最佳的DSN剂量，这足以检测低至飞摩尔水平的miRNA。pH值将强烈影响DNA探针的剪切速率，从而影响测定的扩增能力。为了最大限度地提高扩增能力，将样品溶液的pH值从7到9进行了调整。据报道，DSN的工作范围从3.5到8.5，在pH约为6.6时具有最大的活性，而pH值<3.0或>9.0会使DSN失活。此外，溶液的pH值也对荧光素标记的荧光有显著影响。在酸性介质中，荧光素通常被质子化。相反，它在碱性介质中被去质子化。质子化和去质子化状态都会改变荧光素的构象，从而减弱其荧光效率。根据我们实验的观察结果(图4B)，发现pH 8.0是本方法的最佳选择。Mg 2+的存在对于促进DSN的剪切以及miRNA/DNA异源双链的高效杂交具有重要意义。因此，Mg 2+浓度在miRNA的检测中起着关键作用。本发明评估了该参数的影响，结果如图4C和D所示。可以看出，随着Mg 2+含量的增加，峰面积增加，但当Mg 2+的浓度高于25mM时，峰面积明显减小。可能的原因是在高离子强度下DSN的活性会降低。考虑到这些结果，选择25mM的Mg 2+作为测定的最佳浓度。另一个重要的变量是反应的孵育温度。孵育温度不仅直接影响杂交效率，而且直接影响DSN的剪切活性。在实际应用中，解链温度应比杂交温度高10～15℃，以在确保良好选择性的同时实现高杂交效率。在目前的实验条件下，估计miRNA-122、miRNA-155和miRNA-21的解链温度分别约为53℃、54℃和51℃。因此，理论上本实验应在低于45℃的温度下进行。而另一方面，DSN剪切在高于45℃ 的温度下表现良好，最佳剪切温度为60℃。如图4E所示，三种miRNA的峰面积在40℃达到最大值。因此，采用40℃作为测定的孵育温度。实验中使用了从60到210分钟的一系列孵育时间。图4F显示，当孵育时间从60分钟增加到180分钟时，荧光信号的峰面积迅速增加，此后三个miRNA的谱图变得平坦。这些结果表明，孵育180分钟足以剪切DNA探针。考虑到反应效率，由于在较长的孵育时间后未观察到明显差异，因此选择180分钟作为后续实验的最佳时间。
通过优化本发明所有实施例采用的方法为：将44μL100nM的P122、P155和P21加入到离心管中，然后再加入0.4U的DSN和5μL目标miRNA。随后进行约2秒的短暂的震荡处理，使反应混合物充分混合。在40℃下孵育180分钟后，用永磁体将MBs连同未反应的DNA探针分离出来。最后，将上清液注入高效液相色谱系统进行分离和定量。
采用岛津LC-20A系统，配备岛津RP-20A荧光检测器进行高效液相色谱分析。数据采集和处理使用LCsolution数据分析软件(免费版)完成。使用phenomenonex的clarity反向色谱柱(50×4.6mm(内部直径)，3μm粒径)用于miRNA的分离。柱温保持在35℃。采用梯度洗脱模式。荧光检测器的参数设置为：激发波长为495nm，发射波长为518nm。
所述1×B&W缓冲液是由5.0mM Tris-HCl和0.5mM EDTA、1.0M NaCl配制成实验所需的pH＝7.5的1×B&W缓冲液。所述2×B&W缓冲液是由10.0mM Tris-HCl和1.0mM EDTA、2.0M NaCl配制成实验所需的pH＝7.5的2×B&W缓冲液。所述杂交缓冲液是由50mM Tris-HCl和25mM MgCl 2配制成实验所需的pH＝8的杂交缓冲液。
本实验所述色谱柱的固定相为十八烷基(C18)。C18柱是典型的反相(RP)柱，常用于保留和分离疏水化合物。然而，寡核苷酸具有很强的极性，很难保留在任何RP柱中。因此，在流动相中加入100mM TEAA作为离子对试剂，使该柱上的DNA探针保留时间更长。
本实验为了防止暴露于可能对荧光基团的荧光性质产生不利影响的光照下，所有与荧光基团相关的步骤均在铝箔包裹的离心管中进行。
本实验采用的梯度洗脱模式为：甲醇的比例在20分钟内从10％变化到60％，流速为1mL/min。
本实验的流动相由有机相：甲醇，水相：100mM TEAA水溶液和5％乙腈组成。
通过上述方法检测，本发明方法实现了对多种miRNA的同时检测。示例中，对 miRNA-122的检测限为0.39fM，miRNA-155的检测限为0.30fM，对miRNA-21的检测限为0.26fM，线性范围均为1.0fM至10pM。所建立的方法成功应用于检测红斑狼疮、宫颈癌、卵巢癌患者和健康人血清样本中的miRNA-122、miRNA-155和miRNA-21。
采用上述方法，对健康人血清样本、红斑狼疮、宫颈癌、卵巢癌患者血清样本中的miRNA-122、miRNA-155和miRNA-21进行检测，具体实例如下：
实施例1、健康人血清样本中miRNA-122、miRNA-155和miRNA-21的检测：
对健康人血清样本中miRNA-122、miRNA-155和miRNA-21的检测结果如表1所示，在血清样品1(健康志愿者)中检测到miRNA-122、miRNA-155和miRNA-21的浓度分别0.063pM、0.057pM和0.046pM，而在血清样本2(健康志愿者)中分别为0.067pM 0.053pM和0.048pM。为了评估基质效应，将不同浓度的miRNA-122、miRNA-155和miRNA-21加标至样品1中，获得了良好的相对回收率(101.7％～104.7％)，相对标准偏差(RSD)为2.4％～4.7％。这些结果与qRT-PCR的结果一致。以上结果清楚地表明，本文提出的方法对于实际样品中多个miRNA的分析具有良好的实用性。
表1健康人血清样本中miRNA-122、miRNA-155和miRNA-21的检测
Figure PCTCN2021075728-appb-000001
a相对回收率＝(总浓度-空白浓度)/掺入浓度
实施例2、红斑狼疮患者血清样本中miRNA-155和miRNA-21的检测：
对红斑狼疮患者血清样本中miRNA-155和miRNA-21的检测结果如表2所示，红斑狼疮患者血清样本中分别检测到0.399pM的miRNA-155和0.034pM的miRNA-21。与健康人相比，红斑狼疮患者的miRNA-155有明显的过度表达。这些结果与qRT-PCR的结果一致。以上结果清楚地表明，本文提出的方法对于实际样品中多个miRNA的分析具有良好的实用性。
表2红斑狼疮患者血清样本中miRNA-155和miRNA-21的检测
Figure PCTCN2021075728-appb-000002
a相对回收率＝(总浓度-空白浓度)/掺入浓度
实施例3、卵巢癌患者血清样本中miRNA-155和miRNA-21的检测：
对卵巢癌患者血清样本中miRNA-155和miRNA-21的检测结果如表3所示，卵巢癌患者血清样本中分别检测到0.090pM的miRNA-155和0.137pM的miRNA-21。与健康人相比，卵巢癌患者的miRNA-21有明显的过度表达。这些结果与qRT-PCR的结果一致。以上结果清楚地表明，本文提出的方法对于实际样品中多个miRNA的分析具有良好的实用性。
表3卵巢癌患者血清样本中miRNA-155和miRNA-21的检测
Figure PCTCN2021075728-appb-000003
实施例4、宫颈癌患者血清样本中miRNA-122、miRNA-155和miRNA-21的检测：
对宫颈癌患者血清样本中miRNA-122、miRNA-155和miRNA-21的检测结果如表4所示，在样品3和4(宫颈癌患者)中检测到miRNA-122的浓度分别为0.070pM和0.090pM，miRNA-155的浓度分别为0.209pM和0.224pM以及miRNA-21的浓度分别为0.115pM和0.117pM。这些结果表明，与健康人相比，miRNA-155和miRNA-21在癌症患者中表达上调。这些结果与qRT-PCR的结果一致。以上结果清楚地表明，本文提出的方法对于实际样品中多个miRNA的分析具有良好的实用性。
表4宫颈癌患者血清样本中miRNA-122、miRNA-155和miRNA-21的检测
Figure PCTCN2021075728-appb-000004
a相对回收率＝(总浓度-空白浓度)/掺入浓度
实施例5、比较检测miRNA的不同方法
采用本发明的方法与近年来报道的其他多重检测miRNA的方法或与HPLC相关的方法进行了比较，结果如表5所示。使用荧光检测的miRNA分析方法具有很高的选择性，但灵敏度却不高(Wang,R.；Xu,X.；Li,X.；Zhang,N.；Jiang,W.pH-responsive ZnO nanoprobe mediated DNAzyme signal amplification strategy for sensitive detection and live cell imaging of multiple microRNAs.Sens.Actuators,B.2019,293,93-99.Jie,G.；Zhao,Y.；Wang,X.；Ding,C.Multiplexed fluorescence detection of microRNAs based on novel distinguishable quantum dot signal probes by cycle amplification strategy.Sens.Actuators,B.2017,252,1026-1034.)。基于电化学发光(ECL)的miRNA测定法显示出很高的灵敏度，但它们有诸如合成复杂材料等缺点(Feng,X.；Gan,N.；Zhang,H.；Li,T.；Cao,Y.；Hu,F.；Jiang,Q.Ratiometric biosensor array for multiplexed detection of microRNAs based on electrochemiluminescence coupled with cyclic voltammetry.Biosens.Bioelectron.2016,75,308-314.Peng,L.；Zhang,P.；Chai,Y.；Yuan,R.Bi-directional DNA Walking Machine and Its Application in an Enzyme-Free Electrochemiluminescence Biosensor for Sensitive Detection of MicroRNAs.Anal.Chem.2017,89(9),5036-5042.)。基于HPLC-MS/MS的分析显示出高选择性，但通常涉及繁琐的数据分析(Kuang,Y.；Cao,J.；Xu,F.；Chen,Y.Duplex-Specific Nuclease-Mediated Amplification Strategy for Mass Spectrometry Quantification of MiRNA-200c in Breast Cancer Stem Cells.Anal.Chem.2019,91(14),8820-8826.Liu,L.；Xu,Q.；Hao,S.；Chen,Y.A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide  Probe.Sci.Rep.2017,7(1),5669.)。此外，这些分析依赖于肽链的检测，这是间接依赖于将靶向蛋白质组学插入miRNA中进行定量。更重要的是，这些分析方法中的大多数无法在一次运行中进行多个miRNA检测。此外，可以看出，本实验测定的LOD远低于文献中报道的LOD。尽管由Nakayama等人(Nakayama,H.；Yamauchi,Y.；Taoka,M.；Isobe,T.Direct Identification of Human Cellular MicroRNAs by Nanoflow Liquid Chromatography–High-Resolution Tandem Mass Spectrometry and Database Searching.Anal.Chem.2015,87(5),2884-2891.)在单个非靶向纳米流LC-MS/MS中鉴定了超过十几种人类细胞miRNA，但该方法仍需要更复杂的仪器，并且LOD低于其他基因学方法。由此可见，本发明方法具有较好的同时检测多种miRNA的优势。
表5.检测miRNA的不同方法比较
Figure PCTCN2021075728-appb-000005
Figure PCTCN2021075728-appb-000006

Claims (10)

  1. 一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，包括以下步骤：
    (1)根据待检测的目标microRNA，设计荧光基团修饰的单链DNA探针，然后将其装载至链霉亲和素包被的磁珠(MBs)表面作为检测探针；
    (2)在上述检测探针中加入待检测的目标microRNA样品和DSN，充分混合，孵育；
    (3)孵育后，将磁珠和未反应的DNA探针完全去除，得分离液；
    (4)将分离液注入到高效液相色谱系统进行分离和定量。
  2. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，步骤(1)中，所述磁珠上包被的链霉亲和素结合位点的摩尔量与DNA探针的摩尔量的比例为(3-5):1。
  3. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，步骤(1)中，装载过程在2×B&W缓冲液中进行，所述缓冲液是由Tris-HCl和EDTA、NaCl配制成。
  4. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，步骤(2)中，所述待检测的目标microRNA选自两种、三种或者多种不同目标microRNA的组合；所述目标microRNA为具有18-25个核苷酸的microRNA。
  5. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，步骤(2)中，所述目标microRNA为miRNA-122、miRNA-155和miRNA-21，步骤(1)中对应的单链DNA探针选自P122、P155和P21。
  6. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，步骤(2)中，所述孵育是在36-38℃下孵育140-160分钟。
  7. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，步骤(3)中，利用永磁体将磁珠和未反应的DNA探针完全去除，以降低背景干扰。
  8. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，步骤(4)中，所述高效液相色谱系统采用C18反向色谱柱，采用梯度洗脱模式。
  9. 根据权利要求8所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，所述梯度洗脱模式为：甲醇的比例在20分钟内从10％变化到60％；流动相由有机相和包含TEAA的水相组成。
  10. 根据权利要求1所述的基于DSN循环扩增技术同时检测多种microRNA的液相色谱法，其特征在于，所述方法的过程均在避光条件下进行。
PCT/CN2021/075728 2020-02-07 2021-02-07 一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法 WO2021155858A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/797,393 US20230212654A1 (en) 2020-02-07 2021-02-07 Liquid chromatography method for simultaneously detecting multiple micrornas based on duplex-specific nuclease (dsn) cyclic amplification technology

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010082179.5A CN111118120B (zh) 2020-02-07 2020-02-07 一种基于DSN循环扩增技术对多种microRNA同时进行检测的液相色谱法
CN202010082179.5 2020-02-07

Publications (1)

Publication Number Publication Date
WO2021155858A1 true WO2021155858A1 (zh) 2021-08-12

Family

ID=70491809

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/075728 WO2021155858A1 (zh) 2020-02-07 2021-02-07 一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法

Country Status (3)

Country Link
US (1) US20230212654A1 (zh)
CN (1) CN111118120B (zh)
WO (1) WO2021155858A1 (zh)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111118120B (zh) * 2020-02-07 2022-09-23 江苏科技大学 一种基于DSN循环扩增技术对多种microRNA同时进行检测的液相色谱法
CN111549100A (zh) * 2020-05-15 2020-08-18 中国科学院重庆绿色智能技术研究院 一种检测miRNA分子的磁性微球DNA探针的构建方法及其产品和应用
CN112538521A (zh) * 2020-12-09 2021-03-23 澳门科技大学 一种检测miRNA的方法及一种用于检测miRNA的生物探针
CN112626209A (zh) * 2020-12-22 2021-04-09 绵竹市人民医院 用于卵巢癌诊断的miRNA标志物、其应用及诊断试剂盒
CN112662777B (zh) * 2021-01-19 2022-06-24 山东师范大学 一种同时检测多种长链非编码rna的复合物及方法
CN112961907B (zh) * 2021-03-04 2022-11-15 中南大学 一种同时检测两种rna的荧光生物传感器及其制备和使用方法
CN112813170B (zh) * 2021-04-09 2021-12-10 江苏达伯药业有限公司 一种用于宫颈癌筛查的试剂盒及其使用方法
CN115896248A (zh) * 2022-11-24 2023-04-04 四川大学 一种基于石墨烯纳米传感器的卵巢癌液态活检方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103882132A (zh) * 2014-03-27 2014-06-25 中国人民解放军第三军医大学第一附属医院 痕量rna实时动态检测方法
CN107385037A (zh) * 2017-07-24 2017-11-24 广州海力特生物科技有限公司 一种miRNA间接实时荧光定量PCR检测方法
CN108841923A (zh) * 2018-06-07 2018-11-20 南京邮电大学 一种基于DSN酶的量子点-磁珠miRNA传感器及其制备方法和检测方法
CN111118120A (zh) * 2020-02-07 2020-05-08 江苏科技大学 一种基于DSN循环扩增技术对多种microRNA同时进行检测的液相色谱法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103160611B (zh) * 2013-04-12 2015-07-15 武汉大学 一种microRNA检测探针和检测microRNA的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103882132A (zh) * 2014-03-27 2014-06-25 中国人民解放军第三军医大学第一附属医院 痕量rna实时动态检测方法
CN107385037A (zh) * 2017-07-24 2017-11-24 广州海力特生物科技有限公司 一种miRNA间接实时荧光定量PCR检测方法
CN108841923A (zh) * 2018-06-07 2018-11-20 南京邮电大学 一种基于DSN酶的量子点-磁珠miRNA传感器及其制备方法和检测方法
CN111118120A (zh) * 2020-02-07 2020-05-08 江苏科技大学 一种基于DSN循环扩增技术对多种microRNA同时进行检测的液相色谱法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
QI TONG, SONG CHANG, HE JING, SHEN WEI, KONG DEZHAO, SHI HAIWEI, TAN LI, PAN RUIRONG, TANG SHENG, LEE HIAN KEE: "Highly Sensitive Detection of Multiple MicroRNAs by High-Performance Liquid Chromatography Coupled with Long and Short Probe-Based Recycling Amplification", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 92, no. 7, 7 April 2020 (2020-04-07), US, pages 5033 - 5040, XP055833640, ISSN: 0003-2700, DOI: 10.1021/acs.analchem.9b05301 *

Also Published As

Publication number Publication date
US20230212654A1 (en) 2023-07-06
CN111118120A (zh) 2020-05-08
CN111118120B (zh) 2022-09-23

Similar Documents

Publication Publication Date Title
WO2021155858A1 (zh) 一种基于DSN循环扩增技术同时检测多种microRNA的液相色谱法
Zhang et al. Hairpin DNA-templated silver nanoclusters as novel beacons in strand displacement amplification for microRNA detection
Wang et al. Surface plasmon resonance biosensor for enzyme-free amplified microRNA detection based on gold nanoparticles and DNA supersandwich
Qi et al. Highly sensitive detection of multiple microRNAs by high-performance liquid chromatography coupled with long and short probe-based recycling amplification
Ge et al. A highly sensitive target-primed rolling circle amplification (TPRCA) method for fluorescent in situ hybridization detection of microRNA in tumor cells
Fang et al. Ultrasensitive electrochemical detection of miRNA-21 using a zinc finger protein specific to DNA–RNA hybrids
Kim et al. Advances in aptamer screening and small molecule aptasensors
Liao et al. Target-triggered enzyme-free amplification strategy for sensitive detection of microRNA in tumor cells and tissues
Gao et al. A label-free biosensor for electrochemical detection of femtomolar microRNAs
Labib et al. Electrochemical sensing of microRNAs: avenues and paradigms
D’Agata et al. Advanced methods for microRNA biosensing: a problem-solving perspective
Pan et al. Lighting up fluorescent silver clusters via target-catalyzed hairpin assembly for amplified biosensing
Xu et al. Quantification of microRNA by DNA–peptide probe and liquid chromatography–tandem mass spectrometry-based quasi-targeted proteomics
Fan et al. Chemiluminescence platforms in immunoassay and DNA analyses
WO2007093050A1 (en) Gene expression assays conducted by elemental analysis
Jirakova et al. Multiplexed immunosensing platform coupled to hybridization chain reaction for electrochemical determination of microRNAs in clinical samples
Liu et al. A label-free sensitive method for membrane protein detection based on aptamer and AgNCs transfer
Zhang et al. A DNA tetrahedral structure-mediated ultrasensitive fluorescent microarray platform for nucleic acid test
Tran et al. Recent trends in application of nanomaterials for the development of electrochemical microRNA biosensors
Zhou et al. Expressed peptide assay for DNA detection
Zouari et al. Ultrasensitive determination of microribonucleic acids in cancer cells with nanostructured-disposable electrodes using the viral protein p19 for recognition of ribonucleic acid/microribonucleic acid homoduplexes
Zhu et al. Highly sensitive and specific mass spectrometric platform for miRNA detection based on the multiple-metal-nanoparticle tagging strategy
Zhou et al. In situ amplified electrochemical aptasensing for sensitive detection of adenosine triphosphate by coupling target-induced hybridization chain reaction with the assembly of silver nanotags
Song et al. Recent advances in the detection of multiple microRNAs
Li et al. Mass spectrometric quantification of microRNAs in biological samples based on multistage signal amplification

Legal Events

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

Ref document number: 21750226

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21750226

Country of ref document: EP

Kind code of ref document: A1