CN115724835B - Nucleic acid dye with low concentration effect and high fluorescence efficiency and its preparation and application - Google Patents

Abstract

The invention relates to the field of nucleic acid dyes, in particular to a novel nucleic acid dye with low concentration effect and high fluorescence effect. The invention provides a nucleic acid dye, which has a structural general formula shown in a formula I, wherein A ^‑ is an anion, R is H, methyl or halogen, and X is O, S or Se. The nucleic acid dye obtained by the invention has low concentration effect and high fluorescence effect, and has the advantages of readily available raw materials, low cost, simple synthesis steps and no need of additional purification of intermediate products. In addition, the nucleic acid dye obtained by the method has low concentration effect and low sequence preference on nucleic acid molecules in multiplex PCR, and can effectively improve detection resolution.

Description

Nucleic acid dye with low concentration effect and high fluorescence effect and preparation and application thereof

Technical Field

The invention relates to the field of nucleic acid dyes, in particular to a novel nucleic acid dye with low concentration effect and high fluorescence effect.

Background

Nucleic acid molecules such as DNA or RNA are used as carriers of genetic information of living bodies, and have the characteristics of large information carrying capacity and relatively stable intracellular content, so that the nucleic acid molecules are used as conventional analysis targets. Currently fluorescence detection is the dominant method for achieving nucleic acid analysis. Among the numerous fluorescence detection means, non-covalent nucleic acid dyes are an important class of tools that recognize nucleic acid molecules and their different secondary structures through non-covalent interactions. Qualitative or quantitative analysis of nucleic acid molecules in different media, such as solutions, gels or biological tissues, can be further achieved based on the change in autofluorescence properties of dye molecules before and after binding to the nucleic acid molecules.

Compared with biochemical diagnosis and immune diagnosis, the molecular diagnosis has the advantages of short detection window period, short time, higher sensitivity, stronger specificity and the like. Technically, the molecular diagnosis field mainly comprises PCR, fluorescence In Situ Hybridization (FISH), gene chip, second generation high throughput sequencing technology (NGS) and the like, wherein the real-time quantitative PCR (q-PCR) is most widely applied. In general, in order to achieve accurate detection, to avoid generation of false positive signals or to improve detection efficiency and save cost, it is necessary to detect expression of multiple target genes simultaneously or to detect multiple targets simultaneously, i.e., multiplex q-PCR.

At present, although there are many commercially available nucleic acid dyes, the number of dyes that can be used for q-PCR or multiplex q-PCR is rare, and SYBR Green I is sold by Invitrogen corporation of America because it has low background, low toxicity and ultra-high sensitivity, has been widely used as a special nucleic acid dye for conventional detection of q-PCR.

However, SYBR Green I this dye still has the following disadvantages:

(1) SYBR Green I has a concentration effect, and dye use at high concentrations inhibits the PCR amplification process and also shows an increase in melting temperature in q-PCR (Nucleic Acids Res, 2007,35, e 127.);

(2) SYBR Green I generally only detects the melting curve of one product when subjected to double PCR amplification (whereas electrophoretic analysis clearly shows that there are two products). This is mainly due to its tendency to select long-chain DNA or DNA fragments with high g+c% content (Nucleic Acids res.2003,31, e 136.) making it difficult for another amplification product to be identified. Therefore, SYBR Green I is not suitable for multiplex q-PCR, and its detection resolution is extremely low;

(3) In addition, although SYBR Green I has excellent fluorescence performance, the synthesis is difficult, so that the SYBR Green I is expensive (the official selling price of a stock solution with the specification of 10000x and 1mL is about 1 ten thousand yuan), and the cost is high in practical application;

Although some dyes, such as SYTO 9 (100 uL, official price about 4400 Yuan) from Invitrogen and Evagreen (50 uL,2000 Xstock, official price about 1300 Yuan) from Biotum, claim to have a lower concentration effect as a substitute for SYBR Green I. However, these dyes, although having low concentration effects, tend to be accompanied by a corresponding decrease in their fluorescence efficacy (BMC Res. Notes 2011,4,263.) and are also more expensive than SYBR Green I and therefore not widely used.

Disclosure of Invention

One of the purposes of the invention is to provide another series of compounds which have high sensitivity, extremely low inhibition to PCR and are suitable for multiplex q-PCR, aiming at the defects of high-efficiency nucleic acid dye (such as SYBR Green I) in the prior art, such as concentration effect, sequence selection tendency, difficult synthesis and the like, and the dye is structurally modified.

The second purpose of the invention is to provide a synthesis method of the dye.

It is a further object of the present invention to provide the use of the above-described dyes in q-PCR, multiplex q-PCR.

The technical scheme of the invention is as follows:

The first technical problem to be solved by the invention is to provide a nucleic acid dye, which has the following structural general formula:

In the formula I, A ^- is an anion, R is H, methyl or halogen, and X is O, S or Se.

Further, the A ^- is selected from Cl ^-、Br^-、I^- or

Preferably, R is hydrogen or halogen.

Preferably, X is S or O.

Further, the nucleic acid dye is a compound shown in the following structural formula:

The second technical problem to be solved by the invention is to provide a preparation method of the nucleic acid dye, which comprises the following steps of The compound and the structural formula are as followsIs mixed with the compound to obtain the structural formulaAnd then the structural formula is shown asThe compound of (C) reacts with N, N-dimethylethylenediamine to obtain the dye shown in the formula I

Further, the structural formula isThe compound of (2) is prepared by adopting the following method, 4-methylquinoline-2 (1H) -ketone is subjected to the action of an arylating reagent to obtain the compound with the structural formula ofCompound a of (2)React with halogenating reagent to obtain the structural formulaIs a compound of (a).

Further, the arylating reagent is phenylboronic acid, and the halogenating reagent is selected from POCl ₃ or SOCl ₂.

Further, the structural formula isThe compounds of formula (I) are prepared by using methylating agentsMethylation reaction is carried out on the compound to obtain the compound with the structural formula ofCompound b of (a).

Further, the methylating agent is selected from methyl iodide, methyl p-methylsulfonate or dimethyl sulfate.

Further, the structural formula isThe compound and the structural formula are as followsThe compound of (2) is mixed under the alkaline condition containing 5% -20% of N, N-Dimethylformamide (DMF) to obtain the compound with the structural formula ofIs a compound of (a).

The third technical problem to be solved by the invention is to indicate the application of the nucleic acid dye shown in the formula I in q-PCR (fluorescence quantitative PCR) and multiplex q-PCR.

Compared with the prior art, the invention has the following advantages:

1) The nucleic acid dye has low concentration effect and high fluorescence effect, and has the advantages of readily available raw materials, low cost, simple synthesis steps and no need of additional purification of intermediate products.

2) The nucleic acid dye has low concentration effect and low sequence preference to nucleic acid molecules in multiplex PCR, and can effectively improve detection resolution.

Drawings

FIG. 1 is a schematic diagram of the molecular structure of comparative examples 1-5 in which the side chains of compound c and alkylamino groups of the present invention contain different numbers of alkyl groups.

FIG. 2 is a graph showing the relationship between the C _t value and the dye concentration of the compound C of the present invention and comparative examples 1 to 5 under the same conditions.

FIG. 3 is a graph showing comparison of fluorescence efficacy in q-PCR of the compound c of the present invention with comparative examples 1 to 5 under the same conditions and concentrations.

FIG. 4 is a graph showing the melting curves of q-PCR amplicons of different concentrations of compound c of the invention and SYBR Green I under the same conditions.

FIG. 5 is a graph showing the T _m value of the compound c and SYBR Green I of the present invention under the same conditions, as a function of the dye concentration.

FIG. 6 is a gel electrophoresis image of q-PCR amplification products of different concentrations of compound c of the present invention and SYBR Green I.

FIG. 7 is a graph showing melting curves of amplicon 1 and amplicon 2 in multiplex q-PCR.

FIG. 8 is a graph comparing the discrimination of amplicon 1 and amplicon 2 for different concentrations of compound c (FIG. 8A) versus SYBR Green I (FIG. 8B).

FIG. 9 is a gel electrophoresis image of multiplex q-PCR amplification products of compound c of the invention and SYBR Green I at different concentrations.

FIG. 10 shows the absorption spectrum and fluorescence spectrum of the compound (c) of the present invention.

FIG. 11 is a graph showing the change in fluorescence intensity with respect to the concentration of dye after a certain concentration of double-stranded DNA was bound to SYBR Green I by the compound c of the present invention at various concentrations.

FIG. 12 is a graph showing the relationship between fluorescence intensity and the concentration of double-stranded DNA after the compound c of the present invention binds to SYBR Green I at different concentrations.

FIG. 13 is a graph showing the relationship between the C _t values of the compound d and SYBR Green I of the present invention and the change in dye concentration under the same conditions.

FIG. 14 is a graph comparing the fluorescence efficacy of compound d of the present invention with SYBR Green I in q-PCR at various concentrations.

Detailed Description

The nucleic acid dye of the invention has the following reaction formula:

the nucleic acid dye can be prepared by the following modes:

1) 4-methylquinoline-2 (1H) -ketone is subjected to the action of an arylating reagent to obtain a structural formula of A compound a;

2) Using methylating agents to give structural formula Methylation reaction is carried out on the compound to obtain the compound with the structural formula ofCompound b of (a);

3) Compound a With halogenating reagent to obtain the compoundIt is then combined with compound bMixing under alkaline condition containing 5% -20% of N, N-Dimethylformamide (DMF) to obtain compoundFinally, reacting with N, N-dimethyl ethylenediamine to obtain the final dye

The nucleic acid dye prepared by the invention has almost no fluorescence in a free state because of the strong non-radiative transition process in the molecule. After the dye is combined with nucleic acid molecules, the non-radiative transition process of the dye can be effectively inhibited, and further fluorescence signals are enhanced. In addition, and more importantly, due to its unique pendant alkylamino structureThe series of dyes, because of containing N-H bonds and suitable alkyl chain lengths (i.e., alkyl numbers), have lower binding ratios and affinities for nucleic acid molecules than SYBR Green I, and thus exhibit higher fluorescence efficacy and lower concentration effects.

The following describes the invention in further detail with reference to examples, which are not intended to limit the invention thereto. The dyes obtained according to the present invention (compound c, compound d and compound e listed below) allow for highly sensitive detection of nucleic acid molecules in solution, gel, cells and environment by detecting fluorescent signals of a mixed system of the dye and the nucleic acid molecule, and the present invention is better understood in the following examples, but is not limited thereto.

EXAMPLE 1 preparation of Compound c

The preparation method comprises the following steps:

1) Raw material 4-methylquinolin-2 (1H) -one (1.5 g,9.4mmol,1 eq) and phenylboronic acid (1.2 g,9.4mmol,1 eq) are dissolved in 100mL of dichloromethane, triethylamine (0.9 g,9.4mmol,1 eq) and pyridine (0.8 g,9.4mmol,1 eq) are added thereto, and after reaction at room temperature for 48 to 96 hours, the solution is filtered under reduced pressure, and the filtrate is collected. Extracting the filtrate with saturated ammonium chloride solution for three times, collecting dichloromethane phase extract, adding anhydrous sodium sulfate, drying for 1-2 hr, concentrating the extract, and purifying with SiO ₂ column to obtain white solid a:

2) Methyl iodide (3.5 g,24.9mmol,3 eq) and 2-methyl mercapto benzothiazole (1.5 g,8.3mmol,1 eq) are mixed and heated to 100-120 ℃ for reaction for 10-30 minutes, the reaction is cooled to room temperature, the product is washed three times with anhydrous diethyl ether, and the white solid product b1 is collected:

3) Under the nitrogen atmosphere, dissolving a compound a (50 mg,0.2mmol,1 eq) in 20-30 mL of acetonitrile solution, adding phosphorus oxychloride (92 mg,0.6mmol,3 eq), refluxing at 60-85 ℃ for 12-24 h, cooling to room temperature after the reaction is finished, removing a solvent, adding diethyl ether for cleaning, dissolving the mixture and the compound b1 in 30-40 mL of dichloromethane, and then dropwise adding triethylamine into a reaction system until no obvious white smoke appears in the system, and reacting for 3-6h. After the reaction is completed, dropwise adding N, N-dimethyl ethylenediamine (176 mg,2mmol,10 eq), continuously stirring at room temperature for 6-24 h, removing the solvent after the reaction is completed, purifying and separating by a SiO ₂ column, and recrystallizing to obtain the compound c.

Performance test:

application of Compound c in q-PCR

Comparative example 1 (patent number: 202110958363.6), comparative example 2, comparative example 3, comparative example 4 and comparative example 5 (SYBR Green I) having similar structures but having different numbers of alkyl groups in the alkylamino side chains exhibited q-PCR, the structures of the different comparative examples are shown in FIG. 1, wherein the number of alkyl groups in the side chains was ordered as comparative example 5 (8) > comparative example 2 (7) > comparative example 4 (6) > comparative example 1 (5) > compound c (4) > comparative example 3 (2).

Compounds c and comparative examples 1-5 were used as fluorescent signal indicating dyes and amplified using StepOnePlus ^TM q-PCR apparatus (ABI, USA) using the invA (M90846) gene of Salmonella as template. The invA gene in Salmonella was amplified. The primers used were as follows:

upstream primer 5'-CTTGATTGAAGCCGATGC-3'

Downstream primer 5'-GATAAACTGGACCACGGTG-3'

The amplification conditions are that the temperature is kept at 95 ℃ for 20s, the temperature is kept at 60 ℃ for 30s, the temperature is kept at 72 ℃ for 30s, the total time is 30-40 cycles, and fluorescent signals are collected at 60 ℃ in each cycle.

The q-PCR system included TAKARA TAQ HS Low DNA (2X) reaction, 10uL, 0.4 uL each of the upstream and downstream primers (primer final concentration 200nmol/L each), 1uL (final concentration 10 ⁵ copies) of the Salmonella genome extracted from Compound c or comparative examples 1-5,1uL;IANamp Bacteria DNA Kit (TIANGEN, beijing) for comparison, and 20 uL of sterile water was added to make up to a final volume.

1) Comparison of the concentration Effect of Compound c in q-PCR with comparative examples 1-5

First, when the genome concentration is unchanged, the phenomenon that the C _t value increases with the increase of the dye concentration is the concentration effect. An increase in the C _t value represents a gradual inhibition of the amplification process until the C _t value is undetectable (typically no more than 40 cycles of q-PCR amplification). Wherein a larger variation of the C _t value with increasing dye concentration means a stronger dye concentration effect, whereas a smaller dye concentration effect. The other conditions were kept the same, and the final concentrations of the compound C and comparative examples 1 to 5 were changed to 0.5. Mu. Mol/L, 1. Mu. Mol/L, 3. Mu. Mol/L, 5. Mu. Mol/L, 7. Mu. Mol/L and 11. Mu. Mol/L, to obtain q-PCR amplification curves at different concentrations (dye concentration on the abscissa and C _t value at different concentrations on the ordinate). As shown in fig. 2, the law of dye concentration effect on the one hand follows that comparative example 5≡comparative example 2> comparative example 4> comparative example 1> > compound c > comparative example 3, and is positively correlated with the number of alkyl groups of the side chain alkylamino group. On the other hand, when the number of side chain alkyl groups was 4 or less by using the compound c as a boundary, the concentration effect of the compound c was significantly reduced compared to that of comparative example 1 and comparative example 3, and when the concentration of the compound c was more than 10. Mu. Mol/L, the amplification efficiency was still relatively good.

2) Comparison of the fluorescence efficacy of Compound c at the same concentration in q-PCR with comparative examples 1 to 5

Under the same conditions, q-PCR fluorescence amplification curves (the abscissa indicates the number of cycles, and the ordinate indicates the fluorescence intensity) of different dyes were obtained with the dye concentration kept uniform (3. Mu.M). As shown in fig. 3, the order of fluorescence efficacy, whether with increasing cycle number, the magnitude of increase in fluorescence signal or reaching signal saturation, followed comparative example 2> compound c > comparative example 1> comparative example 5> comparative example 4> > comparative example 3. The ordering is in a positive correlation with the number of side chain alkyl groups to some extent. As a result of comparison of the effect of the binding concentration, it was found that, although the effect of the concentration of comparative example 3 in which the side chain had the least number of alkyl groups was lower, the fluorescence intensity at which saturation of the signal was achieved was reduced in a broken layer manner, and that a large reduction in the fluorescence signal intensity clearly affected the sensitivity of the q-PCR detection. And it has been reported that dyes with low concentration effects tend to be accompanied by a decrease in their fluorescence efficacy in q-PCR (compared to comparative example 5,BMC Res.Notes 2011,4,263). In contrast, compound c not only had an extremely low concentration effect compared with other comparative examples, but also had a fluorescence intensity almost equivalent to that of comparative example 1 under the same conditions, and was significantly superior to that of comparative example 5 (commercially available q-PCR-specific nucleic acid dye). The experiment proves that the compound c can be used for real-time quantitative detection of target nucleic acid molecules in a larger concentration range, and the result is more accurate and sensitive.

Comparison of the melting temperature of amplicon for different concentrations of Compound c and SYBR Green I

In q-PCR, the SYBR Green I dye method has the unique functions of monitoring the melting temperature of the amplicon and providing information of whether nonspecific amplification exists in a system, and the like, and in addition, the multiple q-PCR can be realized by monitoring the melting temperatures of different amplicons.

Melting temperature measurement conditions 95 ℃ were kept annealed to 60 ℃ for 15min, followed by heating and acquisition of fluorescent signals to 95 ℃ at intervals of +0.5 ℃ and incubation at 95 ℃ for 30s.

The melting curves of amplicons at different concentrations (temperature on the abscissa, normalized fluorescence intensity on the ordinate, FIG. 4) were obtained by changing the final concentrations of compound c and SYBR Green I for comparison to 0.5. Mu. Mol/L, 1. Mu. Mol/L, 3. Mu. Mol/L, 5. Mu. Mol/L, 7. Mu. Mol/L and 11. Mu. Mol/L, keeping the other conditions consistent. Firstly, the compound c can completely realize the function of SYBR Green I and finish the detection of the melting temperature of the amplicon. As a result of measurement of the amplification curve, the phenomenon that the measured melting temperature is obviously increased is shown when the SYBR Green I concentration is larger than 1 mu mol/L, and the function of monitoring the melting temperature is lost after the SYBR Green I concentration is larger than 5 mu mol/L. And the compound c can monitor the melting temperature in a larger concentration range. The measured T _m values of the dye are plotted on the abscissa against the melting temperature of compounds c and SYBR Green I (FIG. 5). The results show that compound c not only varies less in melting temperature with increasing concentration, but also can be used at concentrations far higher than SYBR Green I.

Gel electrophoresis imaging of q-PCR amplification product of comparative compound c and SYBR Green I

In q-PCR, in addition to the identification of amplicon information by monitoring the melting temperature of the amplicon, gel electrophoresis analysis of the amplified product is another effective means (gel electrophoresis can obtain amplicon fragment length information). The final concentrations of compound c and SYBR Green I for comparison were changed to 1. Mu. Mol/L, 3. Mu. Mol/L, 5. Mu. Mol/L, 7. Mu. Mol/L and 11. Mu. Mol/L, to obtain q-PCR amplification products (the amplification products were mainly complexes of dye and amplicon) at different dye concentrations, the loading amounts of 20. Mu.L of the q-PCR amplification products were 2. Mu.L, and 25-500bp DNA molecular weight standard Marker (BBI, U.S.) was used as a standard, and the results are shown in FIG. 6. The result shows that when SYBR Green I concentration is more than 3 mu mol/L, the migration rate of the target amplicon strip is slow, and the target amplicon strip disappears, which means that the high-concentration SYBR Green I inhibits the amplification process, and also shows that the SYBR Green I has higher concentration effect. Compared with SYBR Green I, the compound c can realize gel electrophoresis identification of the amplicon in a very wide concentration range, and the phenomena of slow amplicon migration rate and inhibition of the amplification process do not occur in high concentration.

Application of Compound c in multiplex PCR amplification

The SYBR Green I dye method has the unique function of monitoring the melting temperature of the amplicon, and can realize simultaneous analysis of a plurality of amplicons with different lengths or different G+C% contents by utilizing the characteristic that different amplicons have different melting temperatures, namely the dye method multiplex q-PCR. Based on this, two sets of fragments of amplicon 1 (length: 78bp, g+c% = 39.7%) and amplicon 2 (length: 290bp, g+c% = 50.0%) were selected with the invA gene of salmonella as template. The primers used were as follows:

Upstream primer (amplicon 1): 5'-TGATTTGATGCGAGTGGTAA-3'

Downstream primer (amplicon 1): 5'-CTACCTTGCTGATGGATTGT-3'

Upstream primer (amplicon 2): 5'-GCCGGTGAAATTATCGCCAC-3'

Downstream primer (amplicon 2): 5'-TTCATCGCACCGTCAAAGGA-3'

The amplification system consisted of 10 ⁴ copies of Salmonella genome at the final concentration as in example 6.

The amplification conditions were 95℃for 20s,50℃for 30s,72℃for 30s, and a total of 30-40 cycles, each cycle collecting fluorescent signals at 72 ℃.

Melting temperature measurement conditions 95 ℃ were kept annealed to 50 ℃ for 15min, followed by heating and acquisition of fluorescent signals to 95 ℃ at intervals of +0.5 ℃ and incubation at 95 ℃ for 30s.

1) Resolution of different amplicons in multiplex q-PCR by comparison of Compound c with SYBR Green I

First, melting temperatures of each of amplicon 1 (T _m =78.4 ℃) and amplicon 2 (T _m =86.5 ℃) were measured using SYBR Green I (final concentration of 1 μmol/L), as shown in fig. 7. Then, keeping other conditions consistent, changing the final concentrations of compound c and SYBR Green I for comparison to 0.05. Mu. Mol/L, 1. Mu. Mol/L, 2. Mu. Mol/L, 3. Mu. Mol/L and 4. Mu. Mol/L, and obtaining melting curves (temperature on the abscissa and fluorescence intensity on the ordinate) of the amplification products containing both amplicon 1 and amplicon 2 at different concentrations. As shown in fig. 8. In the figure, (1) represents amplicon 1, and (2) represents amplicon 2. It was reported that SYBR Green I shows high affinity for amplicon 2 and lower affinity for amplicon 1 due to its propensity to select for high G+C% content amplified fragments in multiplex q-PCR. Therefore, SYBR Green I (SG in the figure) cannot distinguish amplicon 1 at low concentration (0.05. Mu. Mol/L), only the melting temperature measurement peak of amplicon 2 is obtained, and the resolution of amplicon 1 and amplicon 2 increases with increasing concentration, whereas when the concentration is more than 3. Mu. Mol/L, the melting temperature peak of amplicon 2 cannot be distinguished at high concentration, and only the melting temperature measurement peak of amplicon 1 exists. This is due to the higher affinity of SYBR Green I for amplicon 2, resulting in a concentration effect that appears earlier on amplicon 2, inhibiting amplification of amplicon 2. In contrast, compound c at the same concentration, at a low concentration of (0.05. Mu. Mol/L), the peak of the solubility temperature of amplicon 1 and amplicon 2 was completely distinguishable, indicating that compound c has a lower propensity for sequence selection. And the resolution ratio is obviously improved along with the increase of the concentration, but the inhibition phenomenon of the amplicon 2 does not appear, and the advantage of the low-concentration effect of the compound c in the multiplex q-PCR is also shown.

2) Gel electrophoresis imaging image of multiple q-PCR amplification products of comparative compound c and SYBR Green I

The final concentrations of compound c and SYBR Green I for comparison were changed to 1. Mu. Mol/L, 2. Mu. Mol/L, 3. Mu. Mol/L and 4. Mu. Mol/L, and multiplex q-PCR amplification products containing different concentrations of compound c or SYBR Green I were obtained, and the loading amounts of 20uL of the multiplex q-PCR amplification products were all 2uL, and 25-500bp DNA molecular weight standard Marker (BBI, U.S.A.) was used as a standard, and the results are shown in FIG. 9. The result shows that the migration rate of the band of the target amplicon 2 tends to be slow after the SYBR Green I concentration is more than 1 mu mol/L, and the migration band of the amplicon 2 disappears after the SYBR Green I concentration is more than 2 mu mol/L, namely the effect of the SYBR Green I concentration is also shown on the gel electrophoresis imaging result, so that the amplification of the amplicon 2 is inhibited, and the amplicon 2 cannot be distinguished. And the compound c not only shows a lower tendency to sequence selection in the multiplex q-PCR, but also has a low concentration effect, so that the normal amplification process is not interfered, the high resolution of the amplicon 1 and the amplicon 2 can be realized in an extremely wide concentration range, and the great application potential of the compound c in the multiplex q-PCR is shown.

Absorption spectrum and fluorescence spectrum of compound c

The DNA sample was calf thymus DNA (ctDNA) purchased from Sigma Aldrich (Shanghai) Inc., and was naturally double-stranded DNA. The absorbance spectrum of compound c (1. Mu. Mol/L) and the fluorescence emission spectrum after binding to ctDNA (500 ng/mL) were measured in Tris-HCl buffer. As shown in FIG. 10, the maximum absorption peak of the compound c was 468nm (blue light), and the maximum fluorescence emission peak after DNA binding was 499nm (blue light). The absorption range of the compound c is wide, the compound c can be excited by light sources in the areas of 280-320nm and 400-510 nm, and the compound c can be completely matched with the optical channels of the q-PCR and chemiluminescent imager on the market at present, so that the dye has good compatibility with the existing instrument.

Comparison of the fluorescence potency of Compound c and SYBR Green I

The DNA sample is the same as that used in the absorption spectrum and the fluorescence spectrum. The change of fluorescence response amplitude of the compound c with different concentrations and SYBR Green I on a certain content of nucleic acid molecules is compared. A mixture containing 0-2. Mu. Mol/L of compound c or SYBR Green I in a final concentration and ctDNA in a final concentration of 500ng/mL was prepared in a 1mL Tris-HCl buffer at a dye concentration gradient of 0.2. Mu. Mol/L. The fluorescence signal of the above mixed system was measured using a Fluorolog-3 (HORIBA usa) fluorometer, and the measured fluorescence signal was plotted against the corresponding dye concentration. As shown in FIG. 11, the same equivalent of dye (0.2. Mu. Mol/L), the increase of fluorescence signal by compound c was significant in intensity for SYBR Green I, and then at the dye concentration where the fluorescence signal tends to saturate, the fluorescence signal of compound c was 1.5 times that of SYBR Green I. In addition, as can be seen from the trend of the curve, the concentration of compound c signal reaching saturation is significantly higher than that of SYBR Green I, which laterally reflects that the binding ratio of compound c to nucleic acid molecules is greater than that of SYBR Green I, i.e. the effective number of compounds c on a nucleic acid molecule of a defined length can bind to the nucleic acid molecule is greater than that of SYBR Green I. At the same time, the fluorescence of the free compound c is negligible in the same dye concentration range. The above results indicate that the unique side chain amine structure of compound c helps to enhance the fluorescence properties of the dye after binding to the nucleic acid molecule.

Compound c for routine nucleic acid molecule quantitative sensitivity analysis

The DNA sample is the same as that used in the absorption spectrum and the fluorescence spectrum. The sensitivity of compound c to nucleic acid molecules and the minimum limit of detection of SYBR Green I under the same conditions were compared. A mixture containing ctDNA at a final concentration of 0 to 1.2. Mu. Mol/L and a compound or SYBR Green I at a final concentration of 1. Mu. Mol/L was prepared in a Tris-HCl buffer having a volume of 1mL at a base concentration of 0.3. Mu. Mol/L. The change in fluorescence signal of the above-described mixed system was measured under the same measurement conditions as in example 3. And the measured fluorescence signal is plotted against the corresponding dye concentration. As shown in fig. 12, the change in fluorescence signal of the dye was linear to the change in concentration of ctDNA over the measured concentration range. The slope (k) of the calibration curve can be obtained by linear fitting the change of the fluorescence signal, namely, the change amplitude of the dye fluorescence signal is obviously higher than SYBR Green I for ctDNA with the change concentration of 0.3 mu mol/L, which shows that the compound c has higher response sensitivity. The calculation of the detection Limit (LOD) can be expressed as lod=3σ/k, where σ represents the standard deviation of 11 blank samples and k represents the slope of the calibration curve. The results of LOD calculation are also shown in Table 1, and it is clear from Table 1 that the detection limit of the compound c on the DNA sample is lower than that of SYBR Green I.

TABLE 1

	SYBR Green I	Compound c
			σ	21.4	26.1
k	58.7	86.8
			LOD/nmol/L	0.4	0.3

EXAMPLE 2 preparation of Compound d

The preparation method comprises the following steps:

1) The preparation of compound a was carried out as in example 1.

2) Methyl iodide (2.4 g,16.6mmol,3 eq) and 6-bromo-2-methylthiobenzothiazole (1.0 g,5.53mmol,1 eq) were dissolved in 40mL acetonitrile containing 1% -5% dimethyl sulfoxide. Mixing and heating to 60-85 ℃ for reaction for 12-48h, cooling to room temperature after the reaction is finished, collecting white solid precipitated by the reaction, washing the white solid by using anhydrous diethyl ether for a plurality of times, and collecting a pale yellow solid product b2:

3) Compound a (50 mg,0.2mmol,1 eq) is dissolved in 20-30mL acetonitrile solution under nitrogen atmosphere, phosphorus oxychloride (92 mg,0.6mmol,3 eq) is added, reflux is carried out for 12-24h at 60-85 ℃, after the reaction is completed, the mixture is cooled to room temperature, after the solvent is removed, the mixture is washed by adding diethyl ether, the mixture and compound b2 are dissolved in 30-40mL dichloromethane containing 5% -20% of N, N-dimethylformamide, then triethylamine is dropwise added into the reaction system until no obvious white smoke is generated in the system, the reaction is carried out for 3-6h, after the reaction is completed, N-dimethylethylenediamine (176 mg,2mmol,10 eq) is dropwise added, and stirring is continued at room temperature for 6-12h. After the reaction is completed, the solvent is removed, the mixture is purified and separated through a SiO ₂ column, and the compound d is obtained through recrystallization.

Performance test:

Comparison of the concentration Effect of Compound d and SYBR Green I and fluorescence potency

The performance of compound d in q-PCR was compared with SYBR Green I. The number of side chain alkyl groups of the compound d is consistent with that of the compound c and different from that of the substituent group on the benzothiazole ring, wherein the number of the side chain alkyl groups is sequenced as SYBR Green I (8) > the compound d (4).

Amplification was performed using compound d or SYBR Green I as fluorescent signal indicator dye, using StepOnePlus ^TM q-PCR instrument (ABI, USA) with the Salmonella invA (M90846) gene as template. The invA gene in Salmonella was amplified. The primers used were the same as in example 1.

The amplification conditions were the same as in example 1.

The q-PCR system included TAKARA TAQ HS Low DNA (2X) reaction, 10uL, 0.4 uL each of the upstream and downstream primers (primer final concentration 200nmol/L each), 1uL (final concentration 10 ⁷ copies) of Salmonella genome extracted with Compound c or SYBR Green I,1uL;IANamp Bacteria DNAKit (TIANGEN, beijing) for comparison, and 20 uL of sterile water was added to make up to final volume.

1) Comparison of Compound d and SYBR Green I concentration Effect in q-PCR

The other conditions were kept consistent, and the final concentrations of compound d and SYBR Green I were varied to 1. Mu. Mol/L, 3. Mu. Mol/L, 5. Mu. Mol/L and 7. Mu. Mol/L to give a q-PCR amplification curve with C _t values varying with dye concentration. As shown in FIG. 13, the concentration effect of the assimilated compound c is positively correlated with the number of alkyl groups of the side chain alkylamino group, and the concentration effect of the compound d is significantly smaller than that of SYBR Green I.

2) Comparison of the fluorescence potency of Compound d and SYBR Green I at the same concentration in q-PCR

Under the same conditions, the fluorescence intensities of compound d and SYBR Green I at different concentrations (i.e., the fluorescence intensities at 40 cycles of cycle number) were compared when the fluorescence signal was saturated in q-PCR. FIG. 14 shows the fluorescence intensities of Compound d and SYBR Green I on the abscissa for 40 cycles. Firstly, at different concentrations, the fluorescence signal of the compound d is stronger than SYBR Green I, and the fluorescence intensity is in a linear increasing trend along with the increase of the dye concentration, and the SYBR Green I has a phenomenon that the fluorescence signal intensity is greatly reduced after the concentration is more than 5 mu M, which corresponds to the higher concentration effect. The compound d is similar to the compound c, has low concentration effect and high fluorescence effect, and the experiment further proves that the influence of the alkyl number of the side chain alkylamino group on the concentration effect and the fluorescence effect is small in correlation with the change of the substituent groups on the positions of the main structure of the aromatic ring, such as benzothiazole ring or quinoline ring.

Claims (12)

1. The nucleic acid dye is characterized by having a structural general formula shown in formula I:

in the formula I, A ^- is selected from Cl ^-、Br^-、I^- or R is H, methyl or halogen, and X is O, S or Se.

2. The nucleic acid dye of claim 1, wherein R is hydrogen or halogen and X is S or O.

3. A nucleic acid dye according to claim 1 or 2, wherein the nucleic acid dye is a compound of the formula:

4. The method for preparing a nucleic acid dye according to any one of claims 1 to 3, characterized in that the method comprises the steps of:

The structural formula is The compound and the structural formula are as followsIs mixed with the compound to obtain the structural formulaA compound of (a);

Then the structural formula is as The compound of (C) reacts with N, N-dimethylethylenediamine to obtain the dye shown in the formula I

5. The method for preparing nucleic acid dye according to claim 4, wherein the structural formula isThe compound of (2) is prepared by adopting the following method, 4-methylquinoline-2 (1H) -ketone is subjected to the action of an arylating reagent to obtain the compound with the structural formula ofThe compound is reacted with a halogenating reagent to obtain the compound

6. The method for preparing a nucleic acid dye according to claim 5, wherein the arylating reagent is phenylboronic acid, and the halogenating reagent is selected from POCl ₃ and SOCl ₂.

7. The method for preparing nucleic acid dye according to claim 4, wherein the structural formula isThe compounds of formula (I) are prepared by using methylating agentsMethylation reaction is carried out on the compound to obtain the compound with the structural formula ofIs a compound of (a).

8. The method for preparing a nucleic acid dye according to claim 5 or 6, wherein the nucleic acid dye has the structural formulaThe compounds of formula (I) are prepared by using methylating agentsMethylation reaction is carried out on the compound to obtain the compound with the structural formula ofIs a compound of (a).

9. The method for preparing a nucleic acid dye according to claim 7, wherein the methylating agent is selected from methyl iodide, methyl p-methylsulfonate or dimethyl sulfate.

10. The method for preparing a nucleic acid dye according to claim 8, wherein the methylating agent is selected from methyl iodide, methyl p-methylsulfonate or dimethyl sulfate.

11. The method for preparing nucleic acid dye according to claim 4, wherein the structural formula isThe compound and the structural formula are as followsThe compound of (2) is mixed under the alkaline condition containing 5% -20% of N, N-dimethylformamide to obtain the compound with the structural formula ofIs a compound of (a).

12. Use of a nucleic acid dye according to any one of claims 1 to 3 or prepared by a method according to any one of claims 4 to 11 in q-PCR or multiplex q-PCR.