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

Abstract

The present invention relates to the field of nucleic acid dyes, and in particular to a novel nucleic acid dye having both low concentration effect and high fluorescence efficacy. The present invention provides a nucleic acid dye, the general structural formula of which is shown in Formula I, wherein A ‑ is an anion; R is H, methyl or halogen; X is O, S or Se. The nucleic acid dye obtained by the present invention has both low concentration effect and high fluorescence efficiency, and the raw materials are easily available, the cost is low, the synthesis steps are simple, and the intermediate products do not need to be additionally purified. Moreover, in multiplex PCR, the nucleic acid dye obtained by the present invention not only has a low concentration effect but also has a low sequence preference for nucleic acid molecules, which can effectively improve the detection resolution. #imgabs0#

Description

Technical Field

[0001] The invention relates to the field of nucleic acid dyes, in particular to a novel nucleic acid dye having both low concentration effect and high fluorescence efficacy. Background Art

[0002] Nucleic acid molecules such as DNA or RNA are carriers of genetic information of living organisms. They carry a large amount of information and have relatively stable content in cells. They have been used as a conventional analytical target. Fluorescence detection is currently the mainstream method for nucleic acid analysis. Among the many fluorescence detection methods, non-covalent nucleic acid dyes are an important tool that can identify nucleic acid molecules and their different secondary structures through non-covalent interactions. According to the changes in the fluorescence properties of the dye molecules before and after binding to the nucleic acid molecules, it is possible to achieve qualitative or quantitative analysis of nucleic acid molecules in different media such as solutions, gels or biological tissues.

[0003] Compared with biochemical diagnosis and immunodiagnosis, molecular diagnosis has the advantages of short detection window, short time, higher sensitivity and stronger specificity. From a technical point of view, the field of molecular diagnosis mainly includes PCR, fluorescence in situ hybridization (FISH), gene chips and second-generation high-throughput sequencing technology (NGS), among which real-time quantitative PCR (q-PCR) is the most widely used. In general, in order to achieve accurate detection, avoid the generation of false positive signals or improve detection efficiency and save costs, it is necessary to detect the expression of multiple target genes or multiple targets at the same time, that is, multiplex q-PCR (multiplex q-PCR).

[0004] At present, although there are many nucleic acid dyes on the market, the number of dyes that can be actually used for q-PCR or multiplex q-PCR is scarce. Among them, SYBR Green I, sold by Invitrogen, USA, has been widely used as a dedicated nucleic acid dye for routine q-PCR detection because of its low background, low toxicity and ultra-high sensitivity.

[0005] However, SYBR Green I still has the following shortcomings:

[0006] (1) SYBR Green I has a concentration effect, which means that high concentrations of the dye will inhibit the PCR amplification process and increase the melting temperature in q-PCR (Nucleic Acids Res 2007, 35, e127.);

[0007] (2) When SYBR Green I is used for double PCR amplification, it can usually only detect the melting curve of one product (while electrophoresis analysis clearly shows that it contains two products). This is mainly due to its selective tendency for long-chain DNA or DNA fragments with high G+C% content (Nucleic Acids Res. 2003, 31, e136.), which makes it difficult to identify the other amplified product. Therefore, SYBR Green I is not suitable for multiplex q-PCR, and its detection resolution is extremely low;

[0008] (3) In addition, although SYBR Green I has excellent fluorescence performance, it is difficult to synthesize, which makes it expensive (specifications 10000x, 1 mL of stock solution has an official price of up to about 10,000 yuan), and the cost remains high in practical applications;

[0009] Although some dyes, such as Invitrogen's SYTO 9 (100uL, official price of about 4400 yuan) and Biotum's Evagreen (50uL, 2000× stock solution, official price of about 1300 yuan), claim to have a lower concentration effect and can be used as a substitute for SYBR Green I. However, despite the low concentration effect of these dyes, they are often accompanied by a corresponding decrease in their fluorescence efficacy (BMC Res. Notes 2011, 4, 263.), and these dyes are more expensive than SYBR Green I, so they have not been widely used. Summary of the invention

[0010] One of the purposes of the present invention is to modify the structure of the dye to address the shortcomings of the efficient nucleic acid dyes (such as SYBR Green I) in the prior art, such as concentration effect, sequence selection tendency and difficulty in synthesis, so as to provide another series of compounds with high sensitivity, extremely low inhibition on PCR and suitable for multiplex q-PCR.

[0011] A second object of the present invention is to provide a method for synthesizing the above dye.

[0012] The third object of the present invention is to provide the use of the above dye in q-PCR and multiplex q-PCR.

[0013] The technical solution of the present invention:

[0014] The first technical problem to be solved by the present invention is to provide a nucleic acid dye, the general structural formula of which is:

[0015]

[0016] In Formula I, A - is an anion; R is H, methyl or halogen; X is O, S or Se.

[0017] Further, the A - Selected from: Cl - Br - ,I - or

[0018] Preferably, R is hydrogen or halogen.

[0019] Preferably, X is S or O.

[0020] Furthermore, the nucleic acid dye is a compound represented by the following structural formula:

[0021]

[0022] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned nucleic acid dye, wherein the preparation method comprises: The compound with the structural formula is The compound is mixed to obtain the structural formula The compound with the structural formula The compound is reacted with N,N-dimethylethylenediamine to obtain a dye shown in formula I

[0023] Furthermore, the structural formula is The compound is prepared by the following method: 4-methylquinolin-2(1H)-one is reacted with an arylating agent to obtain a compound having a structural formula of Compound a; Compound a Reaction with halogenating agent to obtain the structural formula of compounds.

[0024] Furthermore, the arylation agent is phenylboronic acid; and the halogenation agent is selected from: POCl3 or SOCl2.

[0025] Furthermore, the structural formula is The compound of the formula is prepared by the following method: using a methylating agent to The compound is subjected to methylation reaction to obtain the structural formula of compound b.

[0026] Furthermore, the methylating agent is selected from: methyl iodide, methyl p-toluenesulfonate or dimethyl sulfate.

[0027] Furthermore, the structural formula is The compound with the structural formula is The compound is mixed under alkaline conditions containing 5% to 20% N,N-dimethylformamide (DMF) to obtain a compound having the structural formula of compounds.

[0028] The third technical problem to be solved by the present invention is to point out the application of the nucleic acid dye shown in Formula I in q-PCR (fluorescence quantitative PCR) and multiplex q-PCR.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1) The nucleic acid dye has both low concentration effect and high fluorescence efficiency, and the raw materials are easily available, the cost is low, the synthesis steps are simple, and the intermediate products do not need additional purification.

[0031] 2) In multiplex PCR, the nucleic acid dye not only has a low concentration effect but also has a low sequence preference for nucleic acid molecules, which can effectively improve the detection resolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the molecular structure of compound c of the present invention and comparative examples 1-5 in which the alkylamino side chains contain different numbers of alkyl groups.

[0033] Figure 2 The compound c of the present invention and the compound C of comparative examples 1-5 under the same conditions t A graph showing the relationship between the value and the dye concentration.

[0034] Figure 3 It is a comparison chart of the fluorescence efficacy of compound c of the present invention and comparative examples 1-5 in q-PCR under the same conditions and concentrations.

[0035] Figure 4 It is the melting curve diagram of the q-PCR amplicon of the compound c of the present invention and SYBR Green I at different concentrations under the same conditions.

[0036] Figure 5 is the T of the compound c of the present invention and SYBR Green I under the same conditions m A graph showing the relationship between the value and the dye concentration.

[0037] Figure 6 It is the gel electrophoresis imaging diagram of the q-PCR amplification products of the compound c of the present invention and SYBR Green I at different concentrations.

[0038] Figure 7 Melting curves of amplicon 1 and amplicon 2 in multiplex q-PCR.

[0039] Figure 8 For different concentrations of compound c( Figure 8 A) and SYBR Green I ( Figure 8 B) Comparison of the discrimination between amplicon 1 and amplicon 2.

[0040] Fig. 9 It is a gel electrophoresis imaging diagram of the multiplex q-PCR amplification products of compound c of the present invention and SYBR Green I at different concentrations.

[0041] Fig.10 The absorption spectrum and fluorescence spectrum of compound c of the present invention are shown in FIG.

[0042] Fig.11 The graph is a relationship between the fluorescence intensity of different concentrations of the compound c of the present invention and SYBR Green I combined with a certain concentration of double-stranded DNA and the corresponding dye concentration change.

[0043] Fig.12 It is a curve diagram showing the relationship between the fluorescence intensity of compound c of the present invention and SYBR Green I after binding to double-stranded DNA of different concentrations and the corresponding double-stranded DNA concentration.

[0044] Fig.13 is the C of the compound d of the present invention and SYBR Green I under the same conditions t A graph showing the relationship between the value and the dye concentration.

[0045] Fig.14 This is a comparison chart of the fluorescence efficacy of the compound d of the present invention and SYBR Green I in q-PCR at different concentrations. DETAILED DESCRIPTION

[0046] The reaction formula of the nucleic acid dye of the present invention is as follows:

[0047]

[0048] The nucleic acid dye of the present invention can be prepared by the following method:

[0049] 1) 4-methylquinolin-2(1H)-one is reacted with an arylating agent to obtain a structural formula Compound a;

[0050] 2) Use a methylating agent to convert the structural formula The compound is subjected to methylation reaction to obtain the structural formula Compound b;

[0051] 3) Compound a Reaction with halogenating agent to obtain compound Then it was mixed with compound b The compound is obtained by mixing under alkaline conditions containing 5% to 20% N,N-dimethylformamide (DMF) Finally, it reacts with N,N-dimethylethylenediamine to obtain the final dye.

[0052] The nucleic acid dye prepared by the present invention has almost no fluorescence in the free state due to the strong non-radiative transition process in the molecule. After the dye is combined with the nucleic acid molecule, the non-radiative transition process of the dye itself can be effectively inhibited, thereby enhancing the fluorescence signal. In addition, more importantly, due to its unique side chain alkylamine structure Due to the presence of NH bonds and appropriate alkyl chain length (i.e., the number of alkyl groups), this series of dyes has a lower binding ratio and affinity for nucleic acid molecules than SYBR Green I, and therefore exhibits higher fluorescence efficacy and lower concentration effect.

[0053] The specific embodiments of the present invention are further described below in conjunction with the examples, but the present invention is not limited to the scope of the examples described. By detecting the fluorescence signal of the mixed system of dye and nucleic acid molecules, the dye obtained by the present invention (compound c, compound d and compound e listed below) can achieve high-sensitivity detection of nucleic acid molecules in solutions, gels, cells and environments. The following examples are convenient for better understanding of the present invention, but do not limit the present invention.

[0054]

[0055] Example 1: Preparation of Compound C

[0056] The preparation method is as follows:

[0057] 1) Dissolve the raw materials 4-methylquinolin-2(1H)-one (1.5g, 9.4mmol, 1eq), phenylboronic acid (1.2g, 9.4mmol, 1eq) and copper acetate (1.9g, 9.4mmol, 1eq) in 100mL of dichloromethane, add triethylamine (0.9g, 9.4mmol, 1eq) and pyridine (0.8g, 9.4mmol, 1eq), react at room temperature for 48-96h, and then filter under reduced pressure to collect the filtrate. The filtrate is extracted three times with a saturated ammonium chloride solution, and the dichloromethane phase extract is retained, and anhydrous sodium sulfate is added to dry for 1-2h; then the extract is concentrated and purified by a SiO2 column to obtain a white solid a:

[0058]

[0059] 2) Mix iodomethane (3.5 g, 24.9 mmol, 3 eq) and 2-methylmercaptobenzothiazole (1.5 g, 8.3 mmol, 1 eq) and heat to 100-120° C. and react for 10-30 minutes; after the reaction is completed, cool to room temperature, wash the product three times with anhydrous ether, and collect the white solid product b1:

[0060]

[0061] 3) Under nitrogen atmosphere, compound a (50 mg, 0.2 mmol, 1 eq) was dissolved in 20-30 mL of acetonitrile solution, phosphorus oxychloride (92 mg, 0.6 mmol, 3 eq) was added, and refluxed at 60-85 ° C for 12-24 h; after the reaction was completed, it was cooled to room temperature, the solvent was removed, ether was added for washing, and it was dissolved in 30-40 mL of dichloromethane together with compound b1, and then triethylamine was added dropwise to the reaction system until no obvious white smoke came out of the system, and the reaction was continued for 3-6 h. After the reaction was completed, N, N-dimethylethylenediamine (176 mg, 2 mmol, 10 eq) was added dropwise, and stirring was continued at room temperature for 6-24 h; after the reaction was completed, the solvent was removed, and the mixture was purified and separated by SiO2 column, and compound c was obtained after recrystallization.

[0062]

[0063] Performance Testing:

[0064] Application performance of compound c in q-PCR

[0065] The performance of compound c and comparative example 1 (patent number: 202110958363.6), comparative example 2, comparative example 3, comparative example 4 and comparative example 5 (SYBR Green I) which have similar structures but different numbers of alkyl groups in the alkylamine side chain in q-PCR were compared. The structures of different comparative examples are shown in FIG. Figure 1 As shown; the number of side chain alkyl groups is ranked as Comparative Example 5 (8) > Comparative Example 2 (7) > Comparative Example 4 (6) > Comparative Example 1 (5) > Compound C (4) > Comparative Example 3 (2).

[0066] Compound c and comparative examples 1-5 were used as fluorescent signal indicator dyes, and StepOnePlus TM q-PCR instrument (ABI, USA) was used to amplify the invA gene (M90846) of Salmonella as a template. The invA gene in Salmonella was amplified. The primers used were as follows:

[0067] Upstream primer: 5'-CTTGATTGAAGCCGATGC-3'

[0068] Downstream primer: 5'-GATAAACTGGACCACGGTG-3'

[0069] Amplification conditions: 95°C for 20 s, 60°C for 30 s, and 72°C for 30 s, for a total of 30 to 40 cycles; the fluorescence signal was collected at 60°C in each cycle.

[0070] The q-PCR system included TaKaRa Taq HS Low DNA (2x) reaction solution, 10 μL; upstream primer and downstream primer, 0.4 μL each (the final concentration of each primer was 200 nmol / L); compound c or comparative examples 1-5 for comparison, 1 μL; Salmonella genome extracted by IANampBacteria DNA Kit (TIANGEN, Beijing), 1 μL (final concentration was 10 5 Sterile water was added to make up the final volume to 20 μL.

[0071] 1) Comparison of the concentration effect of compound c and comparative examples 1-5 in q-PCR

[0072] First, when the genome concentration remains constant, as the dye concentration increases, C t The phenomenon that the value shows an increase is the concentration effect. t The increase in the value indicates that the amplification process is gradually inhibited until C t The value cannot be determined (generally q-PCR amplification does not exceed 40 cycles). t The greater the change in the value with the increase in dye concentration, the stronger the concentration effect of the dye, and vice versa. Keeping other conditions the same, the final concentrations of compound c and comparative examples 1-5 were changed to 0.5 μmol / L, 1 μmol / L, 3 μmol / L, 5 μmol / L, 7 μmol / L and 11 μmol / L to obtain q-PCR amplification curves at different concentrations (the horizontal axis is the dye concentration, and the vertical axis is C at different concentrations). t value). Figure 2 As shown, on the one hand, the rule of dye concentration effect follows Comparative Example 5≈Comparative Example 2>Comparative Example 4>Comparative Example 1>>Compound C>Comparative Example 3, which is positively correlated with the number of alkyl groups in the side chain alkylamine group. On the other hand, when the number of side chain alkyl groups is less than or equal to 4, compared with Comparative Example 1, the concentration effects of Compound C and Comparative Example 3 show a trend of significant decline, and Compound C still maintains a relatively good amplification efficiency when the concentration is greater than 10 μmol / L.

[0073] 2) Comparison of the fluorescence efficacy of compound c and comparative examples 1-5 at the same concentration in q-PCR

[0074] Under the same conditions, the dye concentration was kept constant (3 μM) to obtain the q-PCR fluorescence amplification curves of different dyes (the horizontal axis is the number of cycles, and the vertical axis is the fluorescence intensity). Figure 3As shown, the order of the fluorescence signal increase with the increase in the number of cycles or the fluorescence efficacy when the signal is saturated follows Comparative Example 2>Compound C>Comparative Example 1>Comparative Example 5>Comparative Example 4>>Comparative Example 3. This order is positively correlated with the number of alkyl groups in the side chain to a certain extent. Combined with the comparison results of the concentration effect, it can be found that although the concentration effect of Comparative Example 3, which has the least number of alkyl groups in the side chain, is low, its fluorescence intensity when the signal is saturated has a fault-like decrease, and the substantial decrease in the intensity of the fluorescence signal will undoubtedly affect the sensitivity of q-PCR detection. It is also reported that dyes with low concentration effects are often accompanied by a decrease in their fluorescence efficacy in q-PCR (compared to Comparative Example 5, BMC Res. Notes 2011, 4, 263.). Compared with other comparative examples, compound c not only has an extremely low concentration effect, but also has a fluorescence intensity that is almost equivalent to that of comparative example 1 under the same conditions, and is significantly better than comparative example 5 (commercially available q-PCR-specific nucleic acid dye). The above experiments confirm that compound c can be used for real-time quantitative detection of target nucleic acid molecules within a large concentration range, and the results are more accurate and sensitive.

[0075] Comparison of different concentrations of compound c and SYBR Green I in monitoring the melting temperature of the amplicon

[0076] In q-PCR, the unique function of the SYBR Green I dye method is that it can monitor the melting temperature of the amplicon and provide information such as whether there is nonspecific amplification in the system. In addition, monitoring the melting temperature of different amplicons can realize multiplex q-PCR.

[0077] Melting temperature determination conditions: maintain at 95°C for 20 seconds, anneal to 60°C for 15 minutes, then heat to 95°C at intervals of +0.5°C and collect fluorescence signals once, and maintain at 95°C for 30 seconds.

[0078] Keeping other conditions the same, the final concentrations of compound c and SYBR Green I for comparison were changed to 0.5 μmol / L, 1 μmol / L, 3 μmol / L, 5 μmol / L, 7 μmol / L and 11 μmol / L, and the melting curves of the amplicons at different concentrations were obtained (the abscissa is the temperature, the ordinate is the normalized fluorescence intensity, Figure 4 ). First, compound c can fully realize the function of SYBRGreen I and complete the detection of the melting temperature of the amplicon. The results of the amplification curve show that the melting temperature measured is significantly increased when the concentration of SYBRGreen I is greater than 1μmol / L, and the function of monitoring the melting temperature is lost when the concentration is greater than 5μmol / L. Compound c can monitor the melting temperature in a larger concentration range. With the dye concentration as the horizontal axis, the Tm The value is used as the ordinate to compare the monitoring function of compound c and SYBR Green I on the melting temperature ( Figure 5 The results showed that compound c not only had a smaller change in melting temperature with increasing concentration, but also its actual concentration could be much higher than SYBR Green I.

[0079] Comparison of gel electrophoresis images of compound c and SYBR Green I q-PCR amplification products

[0080] In q-PCR, in addition to monitoring the melting temperature of the amplicon to identify the amplicon information, gel electrophoresis analysis of the amplified product is another effective means (gel electrophoresis can obtain information such as the length of the amplicon fragment). The final concentrations of compound c and SYBR Green I used for comparison were changed to 1μmol / L, 3μmol / L, 5μmol / L, 7μmol / L and 11μmol / L to obtain q-PCR amplification products under different dye concentrations (the amplification product is mainly a complex formed by the dye and the amplicon). The loading amount of 20uL of q-PCR amplification product was 2uL, and a 25-500bp DNA molecular weight standard Marker (BBI, USA) was used as a standard. The results are shown in Figure 2. Figure 6 As shown. The results show that when the concentration of SYBR Green I is greater than 3μmol / L, the migration rate of the target amplicon band slows down, accompanied by the disappearance of the amplicon band, which means that high concentrations of SYBRGreen I inhibit the amplification process, which also shows that it has a high concentration effect. Compared with SYBR GreenI, compound c can also achieve gel electrophoresis identification of amplicons in a very wide concentration range, and there is no slowing of the amplicon migration rate and inhibition of the amplification process at high concentrations.

[0081] Application performance of compound c in multiplex PCR amplification

[0082] The unique function of the SYBR Green I dye method is that it can monitor the melting temperature of the amplicon. By taking advantage of the fact that different amplicons have different melting temperatures, it is possible to simultaneously analyze multiple amplicons of different lengths or different G+C% contents, i.e., dye-based multiplex q-PCR. Based on this, the invA gene of Salmonella was used as a template, and two groups of fragments of different lengths and G+C% contents, amplicon 1 (length: 78 bp, G+C% = 39.7%) and amplicon 2 (length: 290 bp, G+C% = 50.0%), were selected. The primers used are as follows:

[0083] Upstream primer (amplicon 1): 5'-TGATTTGATGCGAGTGGTAA-3'

[0084] Downstream primer (amplicon 1): 5'-CTACCTTGCTGATGGATTGT-3'

[0085] Upstream primer (amplicon 2): 5'-GCCGGTGAAATTATCGCCAC-3'

[0086] Downstream primer (amplicon 2): 5'-TTCATCGCACCGTCAAAGGA-3'

[0087] The amplification system was composed of the same composition as in Example 6, and the final concentration of the Salmonella genome was 10 4 copies.

[0088] Amplification conditions: 95°C for 20 s, 50°C for 30 s, and 72°C for 30 s, for a total of 30-40 cycles; fluorescence signals were collected at 72°C in each cycle.

[0089] Melting temperature determination conditions: maintain at 95°C for 20 seconds, anneal to 50°C for 15 minutes, then heat to 95°C at intervals of +0.5°C and collect fluorescence signals once, and maintain at 95°C for 30 seconds.

[0090] 1) Comparison of the resolution of different amplicons between compound c and SYBR Green I in multiplex q-PCR

[0091] First, SYBR Green I (final concentration of 1 μmol / L) was used to measure the expression of amplicon 1 (T m =78.4°C) and amplicon 2 (T m = 86.5 ° C) respective melting temperatures, such as Figure 7 As shown. Then, keeping other conditions the same, the final concentrations of compound c and SYBR Green I for comparison were changed to 0.05 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L and 4 μmol / L, and the melting curves containing both amplification products of amplification 1 and amplification products of amplification 2 at different concentrations were obtained (the horizontal axis is the temperature, and the vertical axis is the normalized fluorescence intensity). Figure 8As shown. In the figure, (1) represents amplicon 1, and (2) represents amplicon 2. It is reported that due to the selective tendency of SYBR Green I for amplified fragments with high G+C% content in multiplex q-PCR, it shows a high affinity for amplicon 2, and a lower affinity for amplicon 1. Therefore, SYBR Green I (SG in the figure) cannot distinguish amplicon 1 at low concentrations (0.05μmol / L), and only the melting temperature measurement peak of amplicon 2 is obtained. As the concentration increases, the resolution of amplicon 1 and amplicon 2 increases. However, when the concentration is greater than 3μmol / L, the melting temperature peak of amplicon 2 cannot be distinguished at high concentrations, 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, which causes the concentration effect to appear earlier on amplicon 2, inhibiting the amplification of amplicon 2. Compared with SYBR Green I, at the same concentration, compound c can completely distinguish the melting temperature peaks of amplicon 1 and amplicon 2 at a low concentration (0.05 μmol / L), which indicates that compound c has a lower sequence selection tendency. And the resolution is significantly improved with the increase of concentration, but there is no inhibition of amplicon 2, which also shows the advantage of compound c in low concentration effect in multiplex q-PCR.

[0092] 2) Comparison of gel electrophoresis images of multiple q-PCR amplification products of compound c and SYBR Green I

[0093] The final concentrations of compound c and SYBR Green I for comparison were changed to 1 μmol / L, 2 μmol / L, 3 μmol / L and 4 μmol / L to obtain multiplex q-PCR amplification products containing different concentrations of compound c or SYBR Green I. The loading amount of 20 uL of multiplex q-PCR amplification products was 2 uL, and a 25-500 bp DNA molecular weight standard Marker (BBI, USA) was used as a standard. The results are shown in FIG. Fig. 9 As shown. The results show that when the concentration of SYBR Green I is greater than 1μmol / L, the migration rate of the target amplicon 2 tends to slow down, and when the concentration is greater than 2μmol / L, the migration band of amplicon 2 disappears, that is, the gel electrophoresis imaging results also show that the concentration effect of SYBR Green I inhibits the amplification of amplicon 2, making it impossible to distinguish amplicon 2. Compound c not only shows a lower sequence selection tendency in multiplex q-PCR, but also has a low concentration effect, so it does not interfere with the normal amplification process, so that it can achieve high resolution of amplicon 1 and amplicon 2 in a very wide concentration range, showing the huge application potential of compound c in multiplex q-PCR.

[0094] Absorption and fluorescence spectra of compound c

[0095] The DNA sample was calf thymus DNA (ctDNA) purchased from Sigma-Aldrich (Shanghai) Co., Ltd., which is a natural double-stranded DNA. The absorption spectrum of compound c (1 μmol / L) and the fluorescence emission spectrum after binding to ctDNA (500 ng / mL) were measured in Tris-HCl buffer. Fig.10 As shown, the maximum absorption peak of compound c is 468nm (blue light), and the maximum fluorescence emission peak after binding to DNA is located at 499nm (cyan light). Compound c has a wide absorption range and can be excited by light sources in the 280-320nm and 400nm-510nm regions. It can fully adapt to the optical channels of q-PCR and chemiluminescence imagers currently on the market, indicating the good compatibility of the dye with existing instruments.

[0096] Comparison of the fluorescence efficacy of compound c and SYBR Green I

[0097] The DNA samples are the same as those used in the absorption spectrum and fluorescence spectrum. The changes in the fluorescence response amplitudes of compound c and SYBR Green I at different concentrations to a certain amount of nucleic acid molecules were compared. In a 1 mL Tris-HCl buffer, a mixture containing a final concentration of compound c or SYBR Green I of 0-2 μmol / L and a final concentration of ctDNA of 500 ng / mL was prepared with a dye concentration gradient of 0.2 μmol / 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. Fig.11 As shown in the figure, for the same equivalent of dye (0.2μmol / L), the fluorescence signal increase caused by compound c is significantly stronger than SYBR Green I. Secondly, at the dye concentration where the fluorescence signal tends to saturate, the fluorescence signal of compound c is 1.5 times that of SYBR Green I. In addition, from the trend of the curve, it can be seen that the concentration at which the compound c signal reaches saturation is significantly higher than SYBR Green I, which indirectly reflects that the binding ratio of compound c to nucleic acid molecules is greater than SYBR Green I, that is, the effective number of nucleic acid molecules that compound c can bind to is greater than SYBR Green I. At the same time, within the same dye concentration range, the fluorescence of free compound c can be ignored. The above results show that the unique side chain amine structure of compound c helps to improve the fluorescence performance of the dye after binding to nucleic acid molecules.

[0098] Compound c is used for routine nucleic acid molecule quantitative sensitivity analysis

[0099] The DNA samples were the same as those used in the absorption spectrum and fluorescence spectrum. The analytical sensitivity and minimum detection limit of compound c and SYBR Green I for nucleic acid molecules under the same conditions were compared. In a 1 mL Tris-HCl buffer, a gradient of ctDNA base concentration of 0.3 μmol / L was used to prepare a mixture containing ctDNA with a final concentration of 0-1.2 μmol / L and a compound or SYBR Green I with a final concentration of 1 μmol / L. The amplitude of the fluorescence signal change of the above mixed system was measured under the same measurement conditions as in Example 3. The measured fluorescence signal was plotted against the corresponding dye concentration. Fig.12 As shown in the figure, within the measured concentration range, the change in the fluorescence signal of the dye is linear to the change in the concentration of ctDNA. Linear fitting of the change in fluorescence signal can obtain the slope (k) of the calibration curve, that is, for ctDNA with a concentration change of 0.3 μmol / L, the change in the fluorescence signal of the dye is significantly higher than that of SYBR Green I, indicating that compound c has a higher response sensitivity. The calculation formula for the limit of detection (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 calculation results of LOD are also shown in Table 1. It can be seen from Table 1 that the detection limit of compound c for DNA samples is lower than that of SYBR Green I.

[0100] Table 1

[0101] SYBR Green I Compound c σ 21.4 26.1 k 58.7 86.8 LOD / nmol / L 0.4 0.3

[0102] Example 2: Preparation of Compound d

[0103] The preparation method is as follows:

[0104] 1) The same preparation method as that of compound a in Example 1.

[0105] 2) Dissolve iodomethane (2.4 g, 16.6 mmol, 3 eq) and 6-bromo-2-methylmercaptobenzothiazole (1.0 g, 5.53 mmol, 1 eq) in 40 mL of acetonitrile solution containing 1%-5% dimethyl sulfoxide. Mix and heat to 60-85°C for 12-48 h; after the reaction is completed, cool to room temperature, collect the precipitated white solid and wash it with anhydrous ether several times, and collect the light yellow solid product b2:

[0106]

[0107] 3) Under nitrogen atmosphere, compound a (50 mg, 0.2 mmol, 1 eq) was dissolved in 20-30 mL of acetonitrile solution, phosphorus oxychloride (92 mg, 0.6 mmol, 3 eq) was added, and refluxed at 60-85 ° C for 12-24 h; after the reaction was completed, the solution was cooled to room temperature, the solvent was removed, ether was added for washing, and the solution was dissolved in 30-40 mL of dichloromethane containing 5%-20% N, N-dimethylformamide together with compound b2, and then triethylamine was added dropwise to the reaction system until no obvious white smoke was emitted from the system, and the reaction was carried out for 3-6 h; after the reaction was completed, N, N-dimethylethylenediamine (176 mg, 2 mmol, 10 eq) was added dropwise, and stirring was continued at room temperature for 6-12 h. After the reaction was completed, the solvent was removed, and the solution was purified and separated by SiO2 column, and recrystallized to obtain compound d.

[0108] Performance Testing:

[0109] Comparison of the concentration effect and fluorescence efficacy of compound d and SYBR Green I

[0110] The performance of compound d and SYBR Green I in q-PCR was compared. The number of side chain alkyl groups in compound d was consistent with that in compound c, except for the different substituents on the benzothiazole ring. The number of side chain alkyl groups was ranked as SYBR Green I (8) > compound d (4).

[0111] Compound d or SYBR Green I was used as the fluorescent signal indicator dye and StepOnePlus TM q-PCR instrument (ABI, USA) was used to amplify the invA gene (M90846) of Salmonella as a template. The invA gene in Salmonella was amplified. The primers used were the same as those in Example 1.

[0112] The amplification conditions were the same as in Example 1.

[0113] The q-PCR system included TaKaRa Taq HS Low DNA (2x) reaction solution, 10 μL; upstream primer and downstream primer, 0.4 μL each (the final concentration of each primer was 200 nmol / L); compound c or SYBR Green I for comparison, 1 μL; Salmonella genome extracted by IANampBacteria DNA Kit (TIANGEN, Beijing), 1 μL (final concentration was 10 7 Sterile water was added to make up the final volume to 20 μL.

[0114] 1) Comparison of the concentration effects of compound d and SYBR Green I in q-PCR

[0115] Keeping other conditions the same, the final concentrations of compound d and SYBR Green I were changed to 1 μmol / L, 3 μmol / L, 5 μmol / L and 7 μmol / L to obtain C t q-PCR amplification curve showing the value of the dye concentration changing. Fig.13 As shown, similar to compound c, the concentration effect is positively correlated with the number of alkyl groups in the side chain alkylamine groups, and the concentration effect of compound d is significantly smaller than that of SYBR Green I.

[0116] 2) Comparison of the fluorescence efficacy of compound d and SYBR Green I at the same concentration in q-PCR

[0117] Under the same conditions, the fluorescence intensity of compound d and SYBR Green I at different concentrations when the fluorescence signal reached saturation in q-PCR (i.e., the fluorescence intensity when the cycle number reached 40 cycles) was compared. The dye concentration was used as the horizontal axis, and the fluorescence intensity of compound d and SYBR Green I at 40 cycles was used as the vertical axis to obtain Fig.14 . First, at different concentrations, the fluorescence signal of compound d is stronger than that of SYBR Green I, and the fluorescence intensity increases linearly with the increase of dye concentration. However, when the concentration of SYBR Green I is greater than 5 μM, the fluorescence signal intensity decreases significantly, which corresponds to its high concentration effect. Compound d is similar to compound c, with both low concentration effect and high fluorescence efficacy. The above experiments further confirm the influence of the number of alkyl groups of the side chain alkylamine on the concentration effect and fluorescence efficacy, and have little correlation with the changes of substituents in the main structure of the aromatic ring, such as the benzothiazole ring or quinoline ring.

Claims

1. A nucleic acid dye, characterized in that The general structural formula of the nucleic acid dye is shown in Formula I: In Formula I, A - Selected from: Cl - Br - ,I - or R is H, methyl or halogen; X is O, S or Se.

2. A nucleic acid dye according to claim 1, characterized in that: The R is hydrogen or halogen; the X is S or O.

3. A nucleic acid dye according to claim 1 or 2, characterized in that: The nucleic acid dye is a compound represented by the following structural formula:

4. The method for preparing the nucleic acid dye according to any one of claims 1 to 3, characterized in that: The preparation method is: The structural formula is The compound with the structural formula is The compound is mixed to obtain the structural formula Compounds of Then the structural formula is The compound is reacted with N,N-dimethylethylenediamine to obtain a dye shown in formula I 5. The method for preparing the nucleic acid dye according to claim 4, characterized in that: The structural formula is The compound is prepared by the following method: 4-methylquinolin-2(1H)-one is reacted with an arylating agent to obtain a compound having a structural formula of Compound, and then react with a halogenating agent to obtain a compound 6. The method for preparing the nucleic acid dye according to claim 5, characterized in that: The arylating agent is phenylboronic acid; the halogenating agent is selected from: POCl3 or SOCl2.

7. The method for preparing the nucleic acid dye according to claim 4, characterized in that: The structural formula is The compound of the formula is prepared by the following method: using a methylating agent to The compound is subjected to methylation reaction to obtain the structural formula of compounds.

8. The method for preparing the nucleic acid dye according to claim 5 or 6, characterized in that: The structural formula is The compound of the formula is prepared by the following method: using a methylating agent to The compound is subjected to methylation reaction to obtain the structural formula of compounds.

9. The method for preparing a nucleic acid dye according to claim 7, characterized in that: The methylating agent is selected from: methyl iodide, methyl p-toluenesulfonate or dimethyl sulfate.

10. The method for preparing a nucleic acid dye according to claim 8, characterized in that: The methylating agent is selected from: methyl iodide, methyl p-toluenesulfonate or dimethyl sulfate.

11. The method for preparing a nucleic acid dye according to claim 4, characterized in that: The structural formula is The compound with the structural formula is The compound is mixed under alkaline conditions containing 5% to 20% N,N-dimethylformamide to obtain a compound with the structural formula of compounds.

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

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