WO2023019983A1 - 一种核酸荧光染料及其制备和应用 - Google Patents

一种核酸荧光染料及其制备和应用 Download PDF

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WO2023019983A1
WO2023019983A1 PCT/CN2022/086524 CN2022086524W WO2023019983A1 WO 2023019983 A1 WO2023019983 A1 WO 2023019983A1 CN 2022086524 W CN2022086524 W CN 2022086524W WO 2023019983 A1 WO2023019983 A1 WO 2023019983A1
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fluorescent dye
formula
compound
preparation
compound shown
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French (fr)
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吴鹏
王彦莹
董真
李显明
杨琴
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四川大学
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur

Definitions

  • the invention relates to the field of fluorescent dyes, in particular to a novel nucleic acid fluorescent dye.
  • Fluorescent dye refers to a fluorescent substance that can emit a specific wavelength after absorbing excitation light of a certain wavelength. Combining a fluorescent dye with a substance that does not emit fluorescence in a certain way can selectively convert the chemical signal of the substance into a fluorescent signal that can be easily measured by an analytical instrument, thereby realizing the fluorescence detection of a specific target.
  • the detection method has the advantages of high sensitivity, good selectivity, visualization, and real-time, non-destructive online monitoring in vivo, so it has been widely used in the field of biological analysis.
  • Nucleic acid such as DNA and RNA
  • Nucleic acid is the carrier of the genetic information of living organisms. Because of its large amount of information and its relatively stable content in cells, it is often used as a target analyte.
  • fluorescence detection is the mainstream method for nucleic acid analysis.
  • non-covalently bound "luminescent probes” ie, nucleic acid fluorescent dyes
  • Fluorescent dyes can be non-covalently bound to nucleic acid molecules, and the detection of target nucleic acid molecules can be achieved through the huge difference in autofluorescence properties before and after binding.
  • fluorescent dyes are widely used in various states, such as solution, gel electrophoresis, nucleic acid analysis in cells and the environment. At present, there are many fluorescent dyes for nucleic acid analysis on the market. Among them, the SYBR series of dyes sold by Invitrogen of the United States have excellent performance and occupy most of the international nucleic acid dye market.
  • SYBR Green I is the most widely used, because of its low background, low toxicity and ultra-high sensitivity, it has been widely used in routine DNA analysis, DNA gel staining and as a real-time quantitative PCR (qPCR) Nucleic acid dye for routine detection.
  • qPCR real-time quantitative PCR
  • the dyes applied to qPCR should have excellent chemical stability during PCR (105°C-55°C) and during storage (-20°C-4°C). This requires that the dye and its complex with nucleic acid molecules should have certain thermal stability.
  • qPCR mostly uses Tris as a buffer, and the low temperature is alkaline and the high temperature is slightly acidic. Therefore, dyes are required to have better acid-base stability.
  • SYBR Green I does not have good stability under the above conditions;
  • SYBR Green I has a concentration-dependent inhibitory effect on the PCR process, and the use of high-concentration dyes will inhibit the PCR process;
  • SYBR Green I can maintain good stability in DMSO, but has poor stability in aqueous solution;
  • SYBR Green I has excellent fluorescence performance, according to retrosynthesis analysis, its synthesis is extremely difficult, and the yield is extremely low (less than 2%). In addition, due to the high price of its raw materials, SYBR Green I is expensive (with a specification of 10000x, the official price of 1mL stock solution is as high as 10,000 yuan), and the cost remains high in practical applications.
  • Invitrogen also sells another dye, SYBR Safe, which is less expensive and has low mutagenicity, it can be used as a substitute for SYBR Green I, but the water solubility and sensitivity of this alternative dye are not ideal.
  • the present invention carries out structural transformation on the dye to provide another high-sensitivity, good-stability, raw material A compound that is easy to obtain, easy to synthesize, low in sequence preference, and less inhibitory to PCR.
  • the first technical problem to be solved in the present invention is to provide a kind of fluorescent dye, the structural formula of described fluorescent dye is as shown in formula I:
  • A- is chloride ion, bromide ion or iodide ion.
  • the second technical problem to be solved by the present invention is to provide the preparation method of the above-mentioned fluorescent dye.
  • the preparation method is: stirring and reacting the compound shown by formula II and monomer M in a solvent at room temperature for 6 to 24 hours to obtain a mixture, and then Purify the mixture to obtain the fluorescent dye shown in formula I; wherein, the monomer M is propylamine substituted with alkylamino group.
  • the equivalent ratio of the compound represented by formula II to the monomer M is 1:1 ⁇ 1:5.
  • the monomer M is selected from: N,N-dimethyl-1,3-diaminopropane or 3-diethylaminopropylamine.
  • the solvent is ethanol or dichloromethane.
  • the stirring time is 6-12 hours.
  • the solvent 1 is selected from at least one of acetonitrile, dichloroethane or dimethylformamide;
  • the compound shown in formula V and the compound shown in formula III are mixed in solvent 2 with an equivalent ratio of 1:1 to 1:3 at room temperature to obtain the compound shown in formula II; the solvent 2 is ethanol or dichloro methane.
  • the compound shown in formula III is prepared by the following method: 2-methylmercaptobenzothiazole (with or without substituents) is methylated with a methylating reagent (such as methyl iodide) to obtain formula III Compounds shown.
  • a methylating reagent such as methyl iodide
  • the compound shown in formula IV is prepared by the following method: 2-hydroxy-4-methylquinoline and iodobenzene or phenylboronic acid are used as a catalyst to prepare the compound shown in formula IV; wherein, the The catalysts are copper acetate, potassium carbonate and copper powder.
  • the third technical problem to be solved by the present invention is to provide the use of the above-mentioned fluorescent dye, which can be used in qPCR analysis or gel electrophoresis imaging.
  • the present invention finds for the first time that when the compound represented by formula II and monomers such as N,N-dimethyl-1,3-diaminopropane are used as raw materials, a class of A new type of fluorescent dye (Formula I), and the resulting fluorescent dye has higher nucleic acid molecule response sensitivity than SYBR Green I, has higher thermal stability after binding to nucleic acid, and can also increase the melting temperature of nucleic acid molecules.
  • the benzothiazole and quinoline rings in the fluorescent dye of the present invention are connected by a methine bond, and the single bond can rotate freely, resulting in strong intramolecular torsional charge transfer (TICT) in the free state of the dye molecule.
  • TCT intramolecular torsional charge transfer
  • the tightness of aggregation and stacking of the dye molecules is reduced, resulting in almost no fluorescence of the dye monomer itself.
  • the methine bond connecting the benzothiazole and the quinoline ring is fixed by the structure of the nucleic acid molecule, which facilitates the formation of a conjugated structure, thereby obtaining an enhanced fluorescent signal.
  • the present invention provides the design of a novel nucleic acid fluorescent dye.
  • the raw materials for preparing this type of dye are easy to obtain, there are many purchase channels, and the cost is low. Simultaneously, the synthesis conditions are simple and easy to synthesize (the reaction can be completed in a short time at room temperature), and No additional purification of intermediates was required.
  • the fluorescent dye obtained in the present invention has excellent fluorescent properties and good stability, and can be directly used in common PCR buffers (even in pure water), without adding protective reagents; it can specifically recognize the secondary structure of nucleic acid molecules ;
  • the dye has good thermal stability after interacting with nucleic acid molecules, and can effectively increase the melting temperature of nucleic acid molecules.
  • the fluorescent dye obtained in the present invention has low sequence preference for nucleic acid molecules and high sensitivity. Highly sensitive detection of nucleic acid molecules in solutions, gels, cells and environments can be achieved by detecting the fluorescent signal of the mixed system of dyes and nucleic acid molecules.
  • Fig. 1 is the absorption spectrum and fluorescence spectrum of compound c of Example 1 of the present invention.
  • Fig. 2 is the relationship between the fluorescence intensity and the corresponding double-stranded DNA concentration after compound c (- - -) of the present invention, SYBR Green I (- - -) and SYBR Green I analog (- ⁇ -) are combined with different concentrations of double-stranded DNA of the graph.
  • Fig. 3A is a graph showing the relationship between fluorescence intensity and temperature after compound c (solid line) of the present invention and SYBR Green I (dotted line) are combined with double-stranded DNA sample dsDNA-2
  • Fig. 3B is the corresponding melting curve of dsDNA-2.
  • Figure 4A is a graph showing the relationship between the fluorescence intensity of compound c (solid line) of the present invention and SYBR Green I (dotted line) in double-stranded DNA sample Harpin-1 versus temperature
  • Figure 4B is the corresponding melting curve of Harpin-1.
  • Figure 5A is a graph showing the relationship between the fluorescence intensity of the compound c (solid line) of the present invention and SYBR Green I (dotted line) double-stranded DNA sample dsDNA-3 versus temperature
  • Figure 5B is the corresponding melting curve of dsDNA-3.
  • Fig. 6 is a histogram of the fluorescence response changes of compound c of the present invention and SYBR Green I to double-stranded DNA with different d(A-T) contents.
  • the left figure of Fig. 7 is the qPCR fluorescence amplification curve of compound c of the present invention under 0.5, 1, 2, 4 ⁇ mol/L concentration
  • the right figure of Fig. 7 is SYBR Green I at 0.5 (0.55x), 1 (1.1x), 2 qPCR fluorescence amplification curves at (2.2x), 4(4.4x) ⁇ mol/L concentrations.
  • the upper figure of Figure 8 is the qPCR amplification curve of 1 ⁇ mol/L compound c of the present invention at primer concentrations of 50, 100, 200, and 300 nmol/L concentrations
  • the lower figure of Figure 8 is the qPCR amplification curve of 1 ⁇ mol/L (1.1x) SYBR Green I in the same qPCR amplification curve under the conditions.
  • the upper figure in Figure 9 is the melting curve of the amplification product of 1 ⁇ mol/L compound c of the present invention at primer concentrations of 50, 100, 200, and 300 nmol/L, and the lower figure in Figure 8 is 1 ⁇ mol/L (1.1x) SYBR Green I in Melting curves of amplification products under the same conditions.
  • Figure 10 is a comparison diagram of the gel imaging effect of standard molecular weight DNA in the pre-staining mode, the left picture is 1 ⁇ mol/L (1.1x) SYBR Green I and the right picture is 1 ⁇ mol/L Compound c of the present invention.
  • Figure 11 is a comparison diagram of the gel imaging effect of standard molecular weight DNA in post-staining mode.
  • the left picture is 1 ⁇ mol/L (1.1x) SYBR Green I and the right picture is 1 ⁇ mol/L Compound c of the present invention.
  • the left figure in Figure 12 is a comparison of gel imaging effects of 1 ⁇ mol/L (1.1x) SYBR Green I and 1 ⁇ mol/L Compound c of the present invention on PCR amplification products in the pre-staining mode, and the right figure in Figure 12 is in the post-staining mode Comparison of gel imaging effects.
  • Fig. 13 is a graph showing the relationship between the fluorescence intensity detected at 55°C and the number of thermal cycles experienced by compound c of the present invention.
  • Figure 14A is a graph showing the relationship between the absorbance of the compound c (- ⁇ -) of the present invention and SYBR Green I (- - -) and time in pure water
  • Figure 14B is the relationship between the compound c (- ⁇ -) of the present invention and SYBR Green I ( - ⁇ -) The relationship between absorbance and time in Tris-HCl buffer solution.
  • Fluorescent dye of the present invention can adopt following preparation method to make, and preparation method comprises the following steps:
  • Embodiment 1 Preparation of nucleic acid fluorescent dye of the present invention
  • the preparation method is as follows:
  • Embodiment 2 Absorption spectrum and fluorescence spectrum of compound c
  • DNA samples were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., and were two complementary oligonucleotide chains, as shown in Table 1.
  • DNA samples were purified by HPLC, and an appropriate amount of DNA was dissolved in TE buffer (pH 7.4, 10mmol/L Tris, 1mmol/L EDTA) according to the instructions to prepare a stock solution of corresponding concentration, and stored at 4°C.
  • dsDNA-1 can be formed by adding the above two single strands (Pro-20 and Tar-20) at the same concentration respectively.
  • the absorption spectrum of compound c (1 ⁇ mol/L) and the fluorescence emission spectrum combined with dsDNA-1 (20 bp, 100 nmol/L) were detected in TE buffer (shown in FIG. 1 ).
  • the maximum absorption peak of compound c is 467nm (blue light), and the maximum fluorescence emission peak after binding to DNA is located at 505nm (green light).
  • Compound c can be excited by a light source in the 400nm-500nm region, which matches the optical channel of the mainstream qPCR and chemiluminescence imagers currently on the market, which indicates that the dye can be well adapted to existing instruments.
  • Embodiment 3 compound c is used for conventional nucleic acid molecular quantification
  • the DNA sample is the same as in Example 2.
  • the saturation fluorescence intensity of compound c is significantly higher than that of SYBR Green I and SYBR Green I analogs.
  • the above results show that compared with SYBR Green I and SYBR Green I analogs, the structure of compound c has unique advantages, and it has better binding ability to DNA molecules and higher detection sensitivity.
  • Embodiment 4 The thermostability after compound c is combined with nucleic acid molecule
  • the preparation of the DNA sample was the same as in Example 2, and the DNA sequence used is shown in Table 2.
  • the Tm value detected by compound c is also greater than SYBR Green I (as shown in Table 3), further It was confirmed that the complex of compound c and DNA has good binding ability and thermal stability.
  • Embodiment 5 the sequence selectivity of compound c
  • the preparation of the DNA sample was the same as in Example 2, and the DNA sequence used is shown in Table 4.
  • the signal response difference of compound c to A-T sequence preference is only 15%, which is significantly lower than that of SYBR Green I (40%), indicating that compound c has a low sequence preference for nucleic acid molecules and is suitable for routine detection of nucleic acid molecules.
  • Embodiment 6 the application of compound c in qPCR amplification
  • the invA gene in Salmonella was amplified using StepOnePlus TM qPCR instrument (ABI, USA).
  • the primer sequences for qPCR amplification are as follows:
  • Upstream primer 5'-TCCCTTTCCAGTACGCTTCG-3'
  • Template gene acquisition use bacterial DNA extraction kit (TIANamp Bacteria DNA Kit, product number: DP302, TIANGEN, Beijing), and extract Salmonella DNA according to the instructions.
  • qPCR conditions keep at 95°C for 20s, then hold at 55°C for 30s, set the test fluorescence intensity at 55°C, then extend at 72°C for 30s, a total of 30-40 cycles.
  • a PCR kit (TaKaRa Taq HS Low DNA, Cat. No.: R090A, TaKaRa, Japan) was selected to prepare a reaction system with a volume of 20 ⁇ L according to the instruction manual, including Taq TM HS Low DNA (2x), 10 ⁇ L; each of the upstream primer and the downstream primer was 0.4 ⁇ L (with final concentrations of 50nmol/L, 100nmol/L, 200nmol/L and 300nmol/L, respectively, DNA); compound c or SYBR Green I for comparison, 2 ⁇ L (with final concentrations of 0.5 ⁇ mol/L, 1 ⁇ mol /L, 2 ⁇ mol/L and 4 ⁇ mol/L); template gene, 2 ⁇ L; add sterile water to make up to a final volume of 20 ⁇ L.
  • SYBR Green I is a favorable tool for monitoring the amplified product (i.e., the melting temperature of the amplicon) after qPCR amplification is completed. Determining the melting temperature of the amplicon can provide valuable information such as whether there is non-specific amplification and whether the amplicon is single. Accordingly, at the same time, it was determined to change the primer concentration, monitor the melting curve of the amplicon with compound c of the present invention (Fig. 9A), to test the effect of monitoring the melting peak of the amplicon with compound c, and compared with SYBR Green I (Fig. 9B )comparing. Firstly, the test results of the melting temperature of the two amplicons have a good correlation, and the determination results are consistent. Secondly, compared with SYBR Green I, the test baseline of compound c is lower, indicating that compound c provided in Example 1 of the present invention can detect the melting temperature of the amplicon.
  • Embodiment 7 Comparative experiment of gel staining effect
  • gel staining is currently mainly achieved in two ways: (1) before separation, the dye is added to the DNA mixture in advance for electrophoresis separation and then fluorescent imaging, that is, the pre-staining method; (2) the DNA mixture is electrophoresed After separation, soak the gel in the aqueous solution or buffer of the fluorescent dye for a period of time before performing fluorescence imaging, that is, the post-staining method; both methods of use are very common.
  • the compound c (final concentration is 1 ⁇ mol/L) and SYBR Green I (final concentration is 1 ⁇ mol/L, 1.1x) prepared in Example 1 were respectively carried out on the effects of pre-staining method ( Figure 10) and post-staining method ( Figure 11).
  • the DNA sample used was 25-500bp DNA Molecular Weight Standard Marker (Product No.: B600303, BBI, USA). According to the instruction manual, the sample loading was controlled in order: 10ng, 5ng, 2ng, 1ng and 0.5ng.
  • Embodiment 8 Intrinsic stability of compound c
  • the temperature range of qPCR is 55°C-105°C, and the qPCR reagents should be stored at -20°C-4°C.
  • nucleic acid molecules need to be in pure aqueous or buffered solutions. This requires that the dye molecule is stable in water or buffer, and is also stable in the temperature range of 55°C to 105°C and -20°C to 4°C.
  • compound c is very stable in pure water, which indicates that the storage environment of compound c is not as harsh as SYBR Green I.
  • the post-staining method is used for gel staining, it can be prepared directly in pure water without additional buffer preparation , which is more convenient to use.
  • Embodiment 9 Comparing compound c and SYBR Green I synthetic cost
  • SYBR Green I is the most widely used and relatively cost-effective dye in the market.
  • the reaction conditions of compound c are low, and there are many raw material purchase channels, no need for self-synthesis, and the price is cheap (reagent price query website: https://www.bidepharm.com; https://www.bidepharm.com; ://www.chemicalbook.com/ProductIndex.aspx); only three-step reaction is required, no intermediate product purification is required, the key step reaction does not require heating, and the yield is higher, and the above experiments have shown that the performance of compound c is better than SYBR Green I.
  • SYBR Green I is a highly sensitive nucleic acid dye, it is expensive.
  • the official price is about 10,000 yuan per mL (10000x), and the cost of the reagent itself is too high, especially for large-scale nucleic acid detection and gel electrophoresis imaging.
  • the staining effect of compound c is not only equivalent to that of SYBR Green I, but even better, but the synthesis cost of compound c is much lower than that of SYBR Green I. This shows that compound c can greatly reduce the matching production cost while maintaining high sensitivity.

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Abstract

一种新型的核酸荧光染料，所述荧光染料的结构式如式I所示，其中，A -为氯离子、溴离子或碘离子。通过对染料进行结构改造，提供另一种灵敏度高、稳定性好、原料易得、合成简单、序列偏好低、对PCR的抑制性小的化合物。

Description

一种核酸荧光染料及其制备和应用 技术领域
本发明涉及荧光染料领域，特别是涉及一种新型的核酸荧光染料。
背景技术
荧光染料，指吸收某一波长的激发光后可以发射出特定波长的荧光物质。将荧光染料与不发射荧光的物质以某种方式结合，可以有选择性地将该物质的化学信号转变为易被分析仪器测量到的荧光信号，从而实现对特定目标的荧光检测。该检测方法具有灵敏性高，选择性好，可实现可视化以及生物体内实时、非破坏性在线监测等优势，因此在生物分析领域得到广泛的应用。
核酸(如DNA及RNA)是生命体遗传信息的载体，由于其携带信息量大，并且在细胞内含量相对稳定经常被作为目标分析物。目前，荧光检测是实现核酸分析的主流方法。除通过共价偶联标记荧光染料外，非共价结合的“发光探针”(即核酸荧光染料)是核酸定性定量分析最有利的手段。荧光染料可与核酸分子非共价结合，通过结合前后自身荧光性质的巨大差异实现对靶标核酸分子的检测。这类荧光染料被广泛用于各种状态下，如溶液、凝胶电泳、细胞内及环境中等的核酸分析。目前，市售的核酸分析荧光染料较多，其中由美国Invitrogen公司销售的SYBR系列染料性能优异，占据了国际上大部分的核酸染料市场。
在SYBR系列的染料中，应用最广的是SYBR Green I，因其具有低背景、低毒性及超高的灵敏度已被广泛应用于DNA常规分析、DNA凝胶染色以及作为实时定量PCR(qPCR)常规检测专用的核酸染料。然而，SYBR Green I仍有如下不足：
(1)首先，应用于qPCR的染料在PCR(105℃-55℃)和保存期间(-20℃-4℃)应具有优异的化学稳定性。这就要求染料及其与核酸分子的复合物应具备一定的热稳定性。此外，qPCR多使用Tris作为缓冲液，低温为碱性高温为微酸性。因此，要求染料也具备较好的酸碱稳定性。然而，在上述条件下SYBR Green I并不具备良好的稳定性；
(2)SYBR Green I对PCR过程具有浓度依赖的抑制效果，高浓度的染料使用会抑制PCR过程；
(3)在核酸的常规分析中(包括qPCR以及凝胶电泳成像)，染料结合DNA的序列应偏好低或没有。但据文献报道，SYBR Green I存在较明显的A-T序列偏好(参见Zipper H,Brunner H,Bernhagen J,Vitzthum F(2004)Nucleic Acids Res 32:103e)；
(4)根据Invitrogen公司提供的使用说明，SYBR Green I在DMSO中可保持良好的稳定性，在水溶液中稳定性差；
(5)另外，虽然SYBR Green I荧光性能优异，但根据逆合成分析，其合成极其困难，并且收率极低(小于2％)。此外，由于其原料的价格较高致使SYBR Green I价格昂贵(规格10000x，1mL的储备液官方售价高达1万元左右)，在实际应用中成本居高不下。
尽管Invitrogen公司还售有另一种染料SYBR Safe，价格较低且诱变性低，可以作为SYBR Green I的替代品，但是这种替代的染料水溶性和灵敏度不够理想。
发明内容
针对现有技术中高效的核酸荧光染料(如SYBR Green I)不稳定、高浓度影响PCR过程、合成困难等缺点，本发明对染料进行结构改造，提供另一种灵敏度高、稳定性好、原料易得、合成简单、序列偏好低、对PCR的抑制性小的化合物。
本发明的技术方案：
本发明要解决的第一个技术问题是提供一种荧光染料，所述荧光染料的结构式如式I所示：
Figure PCTCN2022086524-appb-000001
其中，A -为氯离子、溴离子或碘离子。
本发明要解决的第二个技术问题是提供上述荧光染料的制备方法，所述制备方法为：将式II所示的化合物与单体M室温下在溶剂中搅拌反应6～24h得混合物，然后将混合物纯化处理得式I所示的荧光染料；其中，所述单体M为烷胺基取代的丙胺。
Figure PCTCN2022086524-appb-000002
进一步，上述制备方法中，式II所示的化合物与单体M的当量比为1:1～1:5。
进一步，上述制备方法中，单体M选自：N,N-二甲基-1,3-二氨基丙烷或3-二乙胺基丙胺。
进一步，上述制备方法中，所述溶剂为乙醇或二氯甲烷。
进一步，上述反应中，搅拌时间为6～12小时。
进一步，所述式II所示的化合物采用下述方法制得：
先制得式III所示的化合物
Figure PCTCN2022086524-appb-000003
再制得式Ⅳ所示的化合物
Figure PCTCN2022086524-appb-000004
将式Ⅳ所示的化合物与三氯氧磷在溶剂1中于65℃～85℃回流反应得式Ⅴ所示的化合物
Figure PCTCN2022086524-appb-000005
其中，所述溶剂1选自乙腈、二氯乙烷或二甲基甲酰胺中的至少一种；
式Ⅴ所示的化合物与式III所示的化合物在溶剂2中，以当量比为1:1～1:3于室温下混合得式II所示的化合物；所述溶剂2为乙醇或二氯甲烷。
进一步，式III所示的化合物采用下述方法制得：用甲基化试剂(如碘甲烷)将2-甲巯基苯并噻唑(含有或不含有取代基)通过甲基化反应即得式III所示的化合物。
进一步，式Ⅳ所示的化合物采用下述方法制得：将2-羟基-4-甲基喹啉与碘苯或苯硼酸 在催化剂的作用下制得式Ⅳ所示的化合物；其中，所述催化剂为醋酸铜、碳酸钾和铜粉。
本发明要解决的第三个技术问题式提供上述荧光染料的用途，其可用在qPCR分析或凝胶电泳成像。
本发明首次发现，当使用式II所示的化合物与N,N-二甲基-1,3-二氨基丙烷等单体为原料时，仅在室温下搅拌反应6～24h就能够得到一类新型荧光染料(式I)，并且所得荧光染料较SYBR Green I具有更高的核酸分子响应灵敏度，与核酸结合之后具有更高的热稳定性，同时还能够提高核酸分子的熔解温度。
另外，本发明荧光染料中的苯并噻唑和喹啉环通过甲川键连接，其中的单键可自由转动，导致该染料分子在自由状态下存在强烈的分子内扭转电荷转移(TICT)。并且，由于喹啉环上苯基取代基和烷胺基取代基的位阻作用，降低了染料分子聚集堆积的紧密性，进而导致染料单体本身几乎没有荧光。当该荧光染料与核酸分子结合后，其中连接苯并噻唑和喹啉环的甲川键受到核酸分子结构的限制而固定，便于共轭结构的形成，从而可以获得增强的荧光信号。
本发明的有益效果：
1)本发明提供了一种新型核酸荧光染料的设计，制备该类染料的原料易得、购买渠道众多、成本低廉，同时合成条件简单、容易合成(室温下短时间即可完成反应)，并且无需对中间产物进行额外纯化。
2)本发明所得荧光染料具有优异的荧光性能和良好的稳定性，可以直接用于普通的PCR缓冲液(甚至纯水中)，不需要添加保护试剂；可特异性识别核酸分子的二级结构；该染料在与核酸分子作用后具有良好热稳定性，可以有效提高核酸分子的熔解温度。
3)本发明所得荧光染料对核酸分子具有低的序列偏好性，并且具有高灵敏度。通过探测染料与核酸分子混合体系的荧光信号可实现对溶液、凝胶、细胞及环境中核酸分子的高灵敏检测。
附图说明：
图1为本发明实施例1的化合物c的吸收光谱及荧光光谱。
图2为本发明化合物c(-■-)，SYBR Green I(-●-)及SYBR Green I类似物(-▲-)结合不同浓度双链DNA后的荧光强度与相应的双链DNA浓度关系的曲线图。
图3A为本发明化合物c(实线)与SYBR Green I(虚线)结合双链DNA样品dsDNA-2后的荧光强度对温度关系的曲线图，图3B为相应的dsDNA-2的熔解曲线。
图4A为本发明化合物c(实线)与SYBR Green I(虚线)结合双链DNA样品Harpin-1 荧光强度对温度关系的曲线图，图4B为相应的Harpin-1的熔解曲线。
图5A为本发明化合物c(实线)与SYBR Green I(虚线)结合双链DNA样品dsDNA-3荧光强度对温度关系的曲线图，图5B为相应的dsDNA-3的熔解曲线。
图6为本发明化合物c与SYBR Green I对不同d(A-T)含量的双链DNA荧光响应变化的柱状图。
图7左图为本发明化合物c在0.5、1、2、4μmol/L浓度下的qPCR荧光扩增曲线，图7右图为SYBR Green I在0.5(0.55x)、1(1.1x)、2(2.2x)、4(4.4x)μmol/L浓度下的qPCR荧光扩增曲线。
图8上图为1μmol/L本发明化合物c在引物浓度为50、100、200、300nmol/L浓度下的qPCR扩增曲线，图8下图为1μmol/L(1.1x)SYBR Green I在相同条件下的qPCR扩增曲线。
图9上图为1μmol/L本发明化合物c在引物浓度为50、100、200、300nmol/L浓度下的扩增产物熔解曲线，图8下图为1μmol/L(1.1x)SYBR Green I在相同条件下的扩增产物熔解曲线。
图10为标准分子量DNA在前染方式下的凝胶成像效果对比图，左图为1μmol/L(1.1x)SYBR Green I以及右图为1μmol/L本发明化合物c。
图11为标准分子量DNA在后染方式下的凝胶成像效果对比图，左图为1μmol/L(1.1x)SYBR Green I以及右图为1μmol/L本发明化合物c。
图12左图为1μmol/L(1.1x)SYBR Green I以及1μmol/L本发明化合物c对PCR扩增产物在前染方式下的凝胶成像效果对比图，图12右图为后染方式下的凝胶成像效果对比图。
图13为本发明化合物c在55℃检测的荧光强度与经历热循环次数的关系图。
图14A为本发明化合物c(-●-)与SYBR Green I(-■-)在纯水中吸光度与时间变化的关系图，图14B为本发明化合物c(-●-)与SYBR Green I(-■-)在Tris-HCl缓冲溶液中吸光度与时间变化的关系图。
图15化合物c与SYBR Green I的合成路线、成本对比图。
具体实施方式
本发明的荧光染料可采用下述制备方式制得，制备方法包括以下步骤：
1)先用甲基化试剂(如碘甲烷)将2-甲巯基苯并噻唑(含有或不含有取代基)进行甲基化反应，得到化合物a
Figure PCTCN2022086524-appb-000006
2)将2-羟基-4-甲基喹啉与苯硼酸在醋酸铜的催化下得到化合物b
Figure PCTCN2022086524-appb-000007
3)将
Figure PCTCN2022086524-appb-000008
与三氯氧磷混合，得到化合物
Figure PCTCN2022086524-appb-000009
随后将其与化合物
Figure PCTCN2022086524-appb-000010
混合得到化合物
Figure PCTCN2022086524-appb-000011
其与N,N-二甲基-1,3-二氨基丙烷反应，可得到最终的染料结构
Figure PCTCN2022086524-appb-000012
具体的反应式如下：
Figure PCTCN2022086524-appb-000013
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实例范围之中。
实施例1：本发明核酸荧光染料的制备
制备方法如下：
1)将原料2-甲巯基苯并噻唑(1.5g，8.3mmol，1eq)溶于30mL的干燥乙醇中，加入碘甲烷(1.4g，10mmol，1.2eq)，60℃下反应5小时。反应完成后，移除溶剂，使用石油醚多次清洗，利用抽滤的方法收集米白色固体产物a：
Figure PCTCN2022086524-appb-000014
2)将原料2-羟基-4-甲基喹啉(3.0g，18.8mmol，1eq)及苯硼酸(2.3g，18.8mmol，1eq)和醋酸铜(3.75g，18.8mmol，1eq)溶于150-250mL的二氯甲烷中，常温搅拌反应 48-96h后，减压抽滤。将过滤液用水萃取三次，留取二氯甲烷相萃取液，加入无水氯化钙干燥1-2h。浓缩萃取液，通过SiO 2柱纯化后得到白色固体b：
Figure PCTCN2022086524-appb-000015
3)将产物b(100mg，0.4mmol，1eq)溶于30mL乙腈中，加入三氯氧磷(184mg，1.2mmol，3eq)，在氮气氛围下60-85℃回流12-24h。反应完成后，冷却至室温，移除溶剂，与化合物a一起溶于20-30mL二氯甲烷，室温下边搅拌边滴加三乙胺至无明显白烟冒出。室温下搅拌5h后滴加原料N,N-二甲基-1,3-二氨基丙烷(122mg，1.2mmol，3eq)，室温下继续搅拌6-24h，浓缩反应混合物，通过SiO 2柱纯化分离，在二氯甲烷和石油醚溶剂中重结晶得到纯的化合物c。
Figure PCTCN2022086524-appb-000016
实施例2：化合物c的吸收光谱及荧光光谱
DNA样品购买自生工生物工程(上海)股份有限公司，为两条互补的寡聚核苷酸链，如表1所示。DNA样品经HPLC纯化，按照说明书将DNA适量溶于TE缓冲(pH 7.4，10mmol/L Tris，1mmol/L EDTA)中配置相应浓度的储备液，4℃储存。使用时，分别加入相同浓度的上述两条单链(Pro-20与Tar-20)即可形成双链DNA(dsDNA-1)。
表1
Figure PCTCN2022086524-appb-000017
在TE缓冲中检测化合物c(1μmol/L)的吸收光谱及与dsDNA-1(20bp，100nmol/L)结合后的荧光发射光谱(图1所示)。化合物c的吸收峰最大值为467nm(蓝光)，结合DNA后的荧光最大发射峰位于505nm(绿光)。化合物c可被400nm-500nm区域内的光源激发，与目前市面上主流的qPCR以及化学发光成像仪的光学通道匹配，这表明该染料可良好的适配于现有仪器。
实施例3：化合物c用于常规核酸分子定量
DNA样品同实施例2。
对比了化合物c与化合物c的N-H键被正丙基封闭后的结构SYBR Green I
Figure PCTCN2022086524-appb-000018
以及被甲基封闭后的SYBR Green I类似物
Figure PCTCN2022086524-appb-000019
对不同浓度核酸分子的荧光响应灵敏度。在体积为200μL，TE缓冲液中，以5nmol/L为浓度梯度，分别配置含有0-75nmol/L终浓度的dsDNA-1与终浓度为0.5μmol/L的化合物c、SYBR Green I(0.55x)及SYBR Green I类似物的混合液。使用荧光光谱仪Fluorolog-3(HORIBA，美国)检测了上述混合物的荧光，如图2所示，以探测到的荧光强度对dsDNA-1浓度作图。首先可以看出，dsDNA-1浓度为0时，几乎没有检测到荧光信号响应。随着dsDNA浓度的增加，在一定范围内荧光对DNA浓度变化呈线性；而DNA浓度较高时，响应变为非线性，信号逐渐趋于饱和。线性变化范围内的上升区域斜率代表染料分子对DNA浓度变化的响应灵敏度。结果显示，化合物c对5nmol/L的dsDNA-1的响应灵敏度明显高于SYBR Green I及SYBR Green I的类似物。其次，对比相同浓度的染料与dsDNA-1结合饱和后的荧光强度，化合物c的饱和荧光强度要明显高于SYBR Green I和SYBR Green I类似物。以上结果表明：相比于SYBR Green I以及SYBR Green I类似物，化合物c的结构具有独特的优势，对DNA分子具有更好的结合能力和更高的检测灵敏度。
实施例4：化合物c与核酸分子结合后的热稳定性
DNA样品制备同实施例2，DNA使用序列如表2所示。
表2
Figure PCTCN2022086524-appb-000020
热稳定性测定方案：将含有1μmol/L化合物c和100nmol/L DNA样品，与体积为20μL，pH＝8.3的Tris-HCl缓冲液(含Mg 2+)在95℃保持15s随后退火；在45℃保温15min后，使用StepOnePlus TMqPCR仪(ABI，美国)每隔+0.5℃采集一次荧光信号(包括45℃)；升温至95℃，保温30s(SYBR Green I的热稳定性测试条件与化合物c保持一致)。绘制化合物c及SYBR Green I与上述不同DNA样品形成复合物的荧光强度对温度的曲线(图3A、图4A及图5A)，并对每一点的斜率取负倒数后绘制不同DNA样品的熔解曲线(图3B、图4B及图5B)。测试结果表明：一方面本发明实施例1制备的化合物c与DNA结合后在升温过程中荧光强度的下降速度(在荧光强度变化发生陡降之前较为平缓的变化区域)明显慢于SYBR Green I，这表明化合物c与DNA形成的复合物热稳定性较SYBR Green I有显著提升。另一方面对比不同DNA样品熔解曲线的峰值处对应的横坐标(即熔解温度值，T m值)，化合物c探测到的T m值也要大于SYBR Green I(如表3所示)，进一步证实证明化合物c与DNA的复合物具有良好的结合能力和热稳定性。
表3
Figure PCTCN2022086524-appb-000021
实施例5：化合物c的序列选择性
DNA样品制备同实施例2，DNA使用序列如表4所示。
表4
Figure PCTCN2022086524-appb-000022
在体积为1mL，pH＝7.4TE缓冲液中分别配置浓度为100nmol/L，含10％腺嘌呤(A)-胸腺嘧啶(T)的DNA样品(AT-1)和含100％腺嘌呤(A)-胸腺嘧啶(T)的DNA样品(AT-2)，并在其中加入终浓度为1μmol/L的化合物c。孵育10min后使用荧光光谱仪Fluorolog-3(HORIBA，美国)测定了化合物c与两种不同DNA样品形成复合物的荧光强度，用于对比的SYBR Green I的序列选择性测试条件与之保持一致，结果如图6所示。化合物c对A-T序列偏好的信号响应差异仅为15％，明显低于SYBR Green I(40％)，表明化合物c对核酸分子的序列偏好性很低，适用于核酸分子的常规检测。
实施例6：化合物c在qPCR扩增中的应用
以实施例1中制备的化合物c为荧光染料，使用StepOnePlus TMqPCR仪(ABI，美国)，对沙门氏菌中的invA基因进行扩增。qPCR扩增的引物序列如下：
上游引物：5’-TCCCTTTCCAGTACGCTTCG-3’
下游引物：5’-TCTGGATGGTATGCCCGGTA-3’
模板基因获取：使用细菌DNA提取试剂盒(TIANamp Bacteria DNA Kit，货号：DP302，TIANGEN，北京)，按照说明书步骤提取沙门氏菌DNA。
qPCR条件：95℃保持20s后55℃保温30s，并在55℃时设定测试荧光强度，接着在72℃延伸30s，共30-40个循环。
选用PCR试剂盒(TaKaRa Taq TM HS Low DNA，货号：R090A，TaKaRa，日本)按照使用说明书配制体积为20μL的反应体系，包括Taq TM HS Low DNA(2x)，10μL；上游引物及下游引物各0.4μL(分别含终浓度为50nmol/L、100nmol/L、200nmol/L及300nmol/L，DNA)；化合物c或用于对比的SYBR Green I，2μL(分别含终浓度为0.5μmol/L、1μmol/L、2μmol/L及4μmol/L)；模板基因，2μL；加入无菌水补齐至终体积20μL。
1)比较qPCR中化合物c与SYBR Green I的信号强度
保持其他条件一致，仅改变化合物c浓度的qPCR荧光扩增曲线(荧光强度对循环个数)如图7A所示，用于对比的不同浓度的SYBR Green I如图7B所示，结果表明不同浓度的 化合物c所监测到的荧光信号变化幅度和强度都要高于相应浓度下的SYBR Green I。另外，在较高浓度时，SYBR Green I出现了较明显的浓度抑制效应，相比于相同浓度下的化合物c，出现C T值滞后(约1个循环)。实验证实了相比于SYBR Green I，化合物c在较大的浓度范围内均能用于核酸的实时扩增检测，并能够实现更加灵敏的检测。
2)比较qPCR中化合物c与SYBR Green I的扩增曲线
保持其他条件一致(化合物c与SYBR Green I的浓度均为1μmol/L)，仅改变引物浓度的qPCR扩增曲线(纵坐标ΔR n对横坐标循环个数)如图8A所示，用于对比的不同浓度的SYBR Green I如图8B所示。结果表明：本发明所合成的化合物c具有较低的扩增背景，并且在低引物浓度(50nmol/L)时，相比于SYBR Green I也没有明显的浓度依赖性。
3)化合物c监测PCR扩增子熔解温度
据报道，SYBR Green I是监测qPCR扩增完成后扩增产物(即扩增子的熔解温度)的有利工具。测定扩增子的熔解温度能提供是否存在非特异性扩增、扩增子是否单一等有价值的信息。据此，同时测定了改变引物浓度，以本发明中化合物c监测扩增子的熔解曲线(图9A)，以测试以化合物c监测扩增子熔解峰的效果，并与SYBR Green I(图9B)进行对比。首先，二者扩增子熔解温度测试结果具有良好的相关性，测定结果一致。其次，相较于SYBR Green I，化合物c的测试基线更低，表明本发明中实施例1提供的化合物c能够实现对于扩增子熔解温度的检测。
实施例7：凝胶染色效果对比实验
据报道，凝胶染色目前主要通过两种方式实现：(1)进行分离前，将染料预先加入DNA混合液中进行电泳分离后进行荧光成像，即前染法；(2)DNA混合液进行电泳分离后，将凝胶至于荧光染料的水溶液或缓冲液中浸泡一段时间后进行荧光成像，即后染法；两种使用方式应用都非常普遍。将实施例1制备的化合物c(终浓度为1μmol/L)与SYBR Green I(终浓度为1μmol/L，1.1x)分别对前染法(图10)与后染法(图11)效果进行对比，使用的DNA样品为25-500bp DNA分子量标准Marker(货号：B600303，BBI，美国)按照使用说明书控制上样量依次为：10ng、5ng、2ng、1ng及0.5ng。
从前染法(图10)和后染法的效果对比(图11)可以看出，本发明实施例1制备的化合物c两种染色方式都与SYBR Green I效果相当，并且在前染法中，与SYBR Green I对比，化合物c的染色结果没有出现明显的拖尾现象，条带更干净。另外，也对比了化合物c(终浓度为1μmol/L)与SYBR Green I(终浓度为1μmol/L，1.1x)对实施例6中产生的扩增子样品进行前染法与后染法的效果对比(图12)，结果表明在实际应用的过程中，化 合物c与SYBR Green I相比无论前染还是后染，效果相当。并且更明显地，相比于SYBR Green I化合物c前染实际样品没有的拖尾现象，条带更干净清晰，对不同DNA片段的区分更加明显。
实施例8：化合物c的本征稳定性
一般情况qPCR的温度范围为55℃-105℃，qPCR试剂储存要在-20℃-4℃。另外，核酸分子需要在在纯水溶液或者缓冲溶液的环境。这就要求染料分子在水中或缓冲液中稳定，并且同样在温度范围为55℃-105℃及-20℃-4℃是稳定的。
1)热稳定性
在体积为20μL，含有终浓度为10μmol/L的化合物c，pH＝8.3的Tris-HCl缓冲液(含Mg 2+)的体系中，化合物c在经历95℃保持20s、55℃保温30s、72℃保温30s的40次热循环后，如图13所示，依旧保持稳定(55℃时设定测试荧光强度)，信号无明显降低，证实化合物c在qPCR中的耐用性。
2)溶液中的稳定性
在体积为3mL，含有终浓度为1μmol/L的化合物c或SYBR Green I(1.1x)的pH＝8.3的Tris-HCl缓冲液(含Mg 2+)或纯水的体系中，每隔12-24h监测化合物c及SYBR Green I在各自最大吸收波长处的信号变化，纯水中为图14A，缓冲液中为图14B。结果表明：纯水中SYBR Green I相比于缓冲中极不稳定，这与已报道的现象是一致的，在经历72h已有近一半的分子分解；而经历相同时间化合物c无论在纯水中还是缓冲液中都要较SYBR Green I稳定。更为重要的是，化合物c在纯水中非常稳定，这表明化合物c的存储环境不如SYBR Green I苛刻，在凝胶染色采用后染法时，可以直接使用纯水配置无需额外再配置缓冲液，使用更为方便。
实施例9：比较化合物c与SYBR Green I合成成本
目前，SYBR Green I是目前市场上应用最为广泛、且相对性价比较高的染料。然而根据对SYBR Green I合成路径分析(图15)，化合物c反应条件要求低，原料购买渠道众多，无需自行合成，且价格便宜(试剂价格查询网站：https://www.bidepharm.com；https://www.chemicalbook.com/ProductIndex.aspx)；仅需三步反应，无需中间产物纯化，关键步骤反应无需加热，且产率更高，并且上述实验已表明化合物c的性能要优于SYBR Green I。
更为重要的是，在实际的核酸分子检测中，染料成本是影响实验总成本的最重要一环。虽然SYBR Green I是高灵敏的核酸染料，但是其价格昂贵，官方售价约1万元1mL (10000x)，其试剂本身成本过高，特别是投放于大规模核酸检测和凝胶电泳成像中。如实施例7的凝胶染色实验中已证实了化合物c不仅染色效果与SYBR Green I相当，甚至效果更优，但化合物c合成成本远远低于SYBR Green I。这表明了化合物c能够在保持高灵敏的条件下，能够极大地降低与之匹配的生产成本。

Claims (10)

  1. 一种荧光染料，其特征在于，所述荧光染料的结构式如式I所示：
    Figure PCTCN2022086524-appb-100001
    其中，A -为氯离子、溴离子或碘离子。
  2. 权利要求1所述的荧光染料的制备方法，其特征在于，所述制备方法为：将式II所示的化合物与单体M室温下在溶剂中搅拌反应6～24h得混合物，然后将混合物纯化处理得式I所示的荧光染料；其中，所述单体M为烷胺基取代的丙胺；
    Figure PCTCN2022086524-appb-100002
  3. 根据权利要求2所述的荧光染料的制备方法，其特征在于，式II所示化合物与单体M的当量比为1：1～1：5。
  4. 根据权利要求2或3所述的荧光染料的制备方法，其特征在于，所示单体M选自：N,N-二甲基-1,3-二氨基丙烷或3-二乙胺基丙胺。
  5. 根据权利要求2或3所述的荧光染料的制备方法，其特征在于，所述溶剂为乙醇或二氯甲烷。
  6. 根据权利要求2～5任一项所述的荧光染料的制备方法，其特征在于，所述搅拌时间为6～12小时。
  7. 根据权利要求2～6任一项所述的荧光染料的制备方法，其特征在于，所述式II所示的化合物采用下述方法制得：
    先制得式III所示的化合物
    Figure PCTCN2022086524-appb-100003
    再制得式Ⅳ所示的化合物
    Figure PCTCN2022086524-appb-100004
    将式Ⅳ所示的化合物与三氯氧磷在溶剂1中于65℃～85℃回流反应得式Ⅴ所示的化合物
    Figure PCTCN2022086524-appb-100005
    其中，所述溶剂1选自乙腈、二氯乙烷或二甲基甲酰胺中的至少一种；
    式Ⅴ所示的化合物与式III所示的化合物在溶剂2中，以当量比为1：1～1：3于室温下混合得式II所示的化合物；所述溶剂2为乙醇或二氯甲烷。
  8. 根据权利要求7所述的荧光染料的制备方法，其特征在于，式III所示的化合物采用下述方法制得：用甲基化试剂将2-甲巯基苯并噻唑通过甲基化反应即得式III所示的化合物。
  9. 根据权利要求7所述的荧光染料的制备方法，其特征在于，式Ⅳ所示的化合物采用下述方法制得：将2-羟基-4-甲基喹啉与碘苯或苯硼酸在催化剂的作用下制得式Ⅳ所示的化合物；其中，所述催化剂为醋酸铜、铜粉。
  10. 荧光染料在qPCR分析或凝胶电泳成像中的用途，所述荧光染料为权利要求1所述的荧光染料，或为采用权利要求2～9任一项所述的方法制得的荧光染料。
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