Fluorescent detection reagent and preparation method and application thereof

Abstract

A fluorescent detection reagent, a method of incorporating labeled nucleotides using the reagent, and a kit or mixture system are provided. The fluorescent detection reagent comprises an enzymatic oxygen scavenging system and a plurality of reducing agents, and does not comprise DABCO. The composition is suitable for application scenarios involving detection of fluorescent signals, particularly applications involving detection of single-molecule fluorescent signals.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence detection technology, in particular to a fluorescence detection reagent and a preparation method and application thereof. BACKGROUND

[0002] The subject matter discussed in this section should not be taken as an admission that the that the subject matter is prior art merely by virtue of its inclusion in this section. Similarly, any problems discussed in this section or associated with the subject matter as provided as background should not be considered to have been appreciated in the prior art merely by virtue of their inclusion in this section. The subject matter in this section merely represents different approaches that, in and of themselves, can also correspond to embodiments of the technical solutions of the claims.

[0003] Detecting biomolecules labeled with photoluminescent molecules based on optical systems is often used in scientific research and diagnostic applications. For example, in molecular diagnostics, fluorescent molecules are often used to modify nucleotides and / or oligonucleotides to achieve detection and characterization of nucleic acid structures.

[0004] For example, in sequencing by synthesis (SBS) technology, nucleotide molecules with inhibitory groups and fluorescent labels, after being combined to the extended chain, are excited by a light source of a specific wavelength and produce fluorescence, and the signal is detected by an optical imaging system. By repeating this step, the biochemical signal is converted into an electronic signal, and the sequence of the nucleic acid is determined based on the processing of the electronic signals.

[0005] However, in the process of repeated signal collection, the general signal will become weaker, and even in the original signal position, no signal can be collected. There are many possible reasons for this situation, for example: out-of-phase amplification of the sequencing reaction, the reaction efficiency decreases as the sequencing progresses, and the light signal intensity will weaken (see WO2006 / 064199 and Lakowicz JR, Principles of fluorescence spectroscopy, 3rd edition, Springer, 2006.).

[0006] The influence of this phenomenon is particularly severe for single molecule sequencing based on SBS, especially for platforms based on imaging of the nucleic acid molecules to be sequenced on a solid surface to achieve single molecule sequencing. Generally, single molecule sequencing does not involve amplification of the nucleic acid molecules to be sequenced, i.e. does not involve amplification of the detection signal, and a nucleic acid molecule to be sequenced usually carries only one or a few fluorescent molecules, so it is understandable that for single molecule sequencing, the detection of each nucleic acid molecule to be sequenced relies on the detection of the signal of the single fluorescent molecule carried by the nucleic acid molecule to be sequenced by the imaging system, i.e. it is generally recognized that obtaining an image with a high signal-to-noise ratio is a difficulty in achieving single molecule sequencing; in other words, in the process of collecting or detecting single molecule fluorescent signals, the intensity and stability of the single molecule fluorescent signals directly affect the accuracy and apparent read length of sequencing, and directly affect whether single molecule sequencing can be achieved. Here, a series of biochemical phenomena that can occur under laser irradiation need to be considered. These include laser irradiation damage to DNA molecules, fluorescence brightness of labeled fluorescent molecules, and fluorescence stability. Understandably, when a fluorescent group is excited into an excited singlet state (S1), it can return to the ground state (S0) by releasing photons (i.e. fluorescence); it can also change state by other means, such as by intersystem crossing (ISC), to enter an excited triplet state (T1), and the triplet state returns to the ground state in a longer time (lifetime), which leads to a decrease in brightness. Moreover, photo-induced damage under light irradiation also leads to a decrease in brightness, and even the fluorescent signal is difficult to be collected. In addition, molecules in the S1 and T1 states have higher reactivity and can react with free radicals and other molecules in the solution, leading to photobleaching. At the same time, under light induction, nucleic acids can also be broken down / degraded, leading to damage to the DNA strand to be sequenced, and thus making it difficult to perform sequencing.

[0007] Thus, in the process of detecting fluorescent molecules, it is often necessary to place the nucleic acid molecules to be sequenced / fluorescent molecules in a specific solution environment to maintain the stability of the fluorescent signal, enhance the fluorescent signal, and / or reduce the damage of photochemical reactions to the solid surface, the DNA strand on the solid surface, nucleotides (bases), and enzymes and other substances involved or catalyzed by the reaction.

[0008] The so-called solution environment, i.e. reagents or formulations that are conducive to imaging and collection of fluorescent signals and suitable for detection of nucleic acid molecules to be sequenced, especially single molecule detection, include the preparation method of the reagents or formulations, which need to be provided or improved. SUMMARY

[0009] To at least partially solve at least one of the above technical problems or provide a practical commercial means, embodiments of the present application provide a method for incorporating labeled nucleotides, a fluorescent detection reagent and its use, and a method for preparing the fluorescent detection reagent.

[0010] Based on the principle of SBS, and by imaging to collect optical signals, processing and converting image signals into base / nucleotide information to realize sequencing, a sequencing platform usually includes multiple rounds of reactions / multiple repeated reactions, each round of reaction including the introduction of an optically detectable label such as a fluorescent label, the excitation of a fluorescent signal, the collection of an image, and the removal of the fluorescent label.

[0011] It can be understood that, at least due to the fact that each biochemical reaction cannot be 100% complete, after multiple rounds of reactions, the reaction conditions / environment will become increasingly unfavorable for subsequent reactions / detection, manifested by the fact that the signal-to-noise ratio of the images collected for the corresponding fields of view becomes increasingly low, and the reliability of the correspondence between the signals on the images and the nucleic acid molecules to be detected becomes increasingly low.

[0012] Although it is difficult to determine the factors that affect the above problems and the relationship between the factors, the inventors believe that if a solution environment conducive to fluorescent light emission and fluorescent signal detection can be configured, the above problems can be significantly improved.

[0013] To this end, based on the contents disclosed in the documents (US7,282,337 and US7,666,593) that the inventors believe are closest to the technology used in the development platform, including the solution environment used, the test results described and illustrated, combined with the inventors' understanding of the principles of fluorescent light emission and the factors that have been reported to affect fluorescent light emission, as well as their knowledge of oxidation or reduction reactions or substances, and their findings from multiple comparative tests, the following is summarized and the solution system is made.

[0014] Specifically, the inventors believe that, in the process of imaging a solid-phase substrate surface to achieve single-molecule sequencing, the solution system (sometimes also referred to as "imaging liquid") in which the fluorescent molecules are placed should be able to enhance the intensity of the fluorescent signal, for example, by inhibiting the ISC process, reducing the probability of quenching, avoiding or reducing photodamage, etc., to enhance the fluorescence quantum efficiency of the fluorescent molecules and prolong the fluorescence lifetime, in order to maintain the intensity (brightness) and stability of the fluorescent signal in detection.

[0015] Moreover, preferably, the imaging liquid should contain an oxygen scavenging system; this is because oxygen molecules can participate in photochemical processes causing fluorescence molecule quenching, for example, oxygen molecules can directly react with excited state fluorescence molecules leading to fluorescence molecule quenching, also can generate free radicals leading to fluorescence molecule quenching (Lakowicz JR, Principles of fluorescence spectroscopy, Springer science & business media; 2013 Apr 17; J. Vogelsang et al., A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes, Angewandte Chemie International Edition, vol. 47, no. 29, pp. 5465-5469, 2008.), at the same time, also can have obvious effect on guanine in DNA (Helmut Sies et al., Single oxygen induced DNA damage, Mutation Research / DNA aging, 1992; H. Piwonski et al., Optimal oxygen concentration for the detection of single indocarbocyanine molecules in a polymeric matrix, Chemical Physics Letters, 2005, 405(4-6):352-356.) and amino acid residues such as tryptophan, cysteine and histidine in protein (Davies M J, Reactive species formed on proteins exposed to singlet oxygen, Photochemical & Photobiological ences Official Journal of the European Photochemistry Association & the European Society for Photobiology, 2004, 3(1): 17-25.), leading to damage of important raw materials such as DNA chain, base and enzyme in sequencing reaction, thus leading to error recognition of base.

[0016] Moreover, the imaging solution should contain an effective oxygen scavenging system. It is expected that a solution containing an effective oxygen scavenging system can effectively reduce the influence of oxygen molecules on fluorescent molecules and biological macromolecules, and on the other hand, can also reduce the proportion of triplet-state fluorescent molecules (C. Steinhauer et al., Superresolution microscopy on the basis of engineered dark states, Journal of the American Chemical Society, vol. 130, pp. 16840-16841, Dec. 2008; R. Zondervan et al., Photoblinking of rhodamine 6g in poly(vinyl alcohol): Radical dark state formed through the triplet, The Journal of Physical Chemistry A, vol. 107, no. 35, pp. 6770-6776, 2003; T. Basche et al., Direct spectroscopic observation of quantum jumps of a single molecule, Nature, vol. 373, pp. 132-134, Jan. 1995; T. Ha et al., Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging, Annual review of physical chemistry, vol. 63, pp. 595-617, 2012.), which is mainly because a large number of fluorescent molecules entering the triplet state will reduce the fluorescence quantum efficiency and increase the quenching probability.

[0017] Furthermore, the imaging solution should contain a redox system, which includes components of the oxygen scavenging system, or is cross-linked with the components of the oxygen scavenging system, or is independent of the oxygen scavenging system. A large number of publications, including textbooks, disclose various oxidizing and reducing agents, such as methyl viologen (MV), p-nitrobenzyl alcohol, water-soluble vitamin E quinone (TXQ), ascorbic acid (AA), propyl gallate (nPG), water-soluble vitamin E (Trolox), mercaptoethanol (BME), dithiothreitol (DTT), mercaptoethylamine (MEA), and the like, each of which can independently effectively eliminate or restore the triplet state fluorescent molecules to the ground state to improve the stability of the fluorescent dye (Holzmeister P et al., Geminate recombination as a photoprotection mechanism for fluorescent dyes, Angew Chem Int Ed Engl, 2014, 53(22): 5685-5688; Vogelsang J et al., A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes, Angewandte Chemie, 2010, 47(29): 5261-5261; Aitken et al., An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments, Biophysical Journal, 2008, 94(5): 1826-1835.).

[0018] In addition, the solution containing the oxygen scavenging system and / or the redox system depends on a suitable buffer system during preparation, storage and use, including pH value, buffer capacity, solution ionic strength, solution polarity, etc., which can affect the performance of the functional components, the composition, and can directly affect the quantum efficiency of the fluorescent molecules.

[0019] In summary, based on the conventional understanding in the art, literature reports, guesses and experimental tests, the inventors have found and summarized the above information, including identifying some factors and directions that need to be concerned. However, it is difficult to predict whether a fluorescent detection solution that meets the above expectations, is effective and stable, can be made, given the unknown relationship between reagents / components, whether there are other influencing factors, and the relationship between influencing factors.

[0020] To this end, first, the inventors set or formulated the same detection conditions, detection environments, and detection objects, and tested the solution system in which the fluorescent molecules are placed during imaging disclosed in the above-mentioned published documents US 7,282,337 or US 7,666,593, i.e., the following formula "134 μl HEPES / NaCl, 24 μl 100 mM aqueous vitamin E Trolox (configured using 2-(N-morpholino)ethanesulfonic acid, i.e., MES buffer system, pH 6.1), 10 μl triethylene diamine DABCO (configured using MES, pH 6.1), 8 μl 2 M glucose, 20 μl NaI (50 mM, configured using water), and 4 μl glucose oxidase" containing 30% acetonitrile (hereinafter, the formula is referred to as "base formula"); specifically, using the same optical imaging system including the same light intensity and exposure time, multiple imaging was performed on multiple fields of view of a batch of solid substrates having the same treatment mode and having surface-attached nucleic acid molecules to be tested, including optimization of the concentration of the redox system components Trolox and / or DABCO therein. As a result, the application value of the obtained sequencing results was relatively low, for example, the read length was short, and the error rate was high; for example, after changing the fluorescent dye Atto647N on the reaction substrate nucleotide to Atto532, the formula not only failed to enhance the brightness of Att532, but also reduced the brightness and stability of Att532.

[0021] Moreover, during the component concentration optimization test of the above-mentioned base formula, the inventors also found that, as the sequencing proceeds, the decay of the fluorescence signal collected each time is relatively obvious, and the inventors speculate that this is likely due to the fact that the solution is exposed to air, and O2 in the air continuously reacts with the oxygen removal system in the solution to produce substances that make the solution acidic, and if this continues, the buffer capacity of the buffer system is exceeded, resulting in a decrease in the pH value of the solution, which significantly affects the intensity of the fluorescence signal. Therefore, the inventors believe that the buffer capacity of the buffer solution and the substances or combinations of substances that affect the pH are necessary factors to be measured in formula optimization.

[0022] Further, the inventors evaluated the chemical properties of the components NaI and DABCO in the base formula and their effects / roles in the detection solution system. In the base formula, the main role of NaI should be to act as a catalyst to achieve the decomposition of H2O2, while DABCO is a commonly used quencher of singlet O, which is used in fluorescence detection. Experimental tests found that: (1) NaI is relatively unstable and easy to be oxidized, and is prone to form a precipitate during the preparation and storage process. Unexpectedly, the inventors found that removing it from the formula did not affect the working efficiency of the formula, in other words, the presence or absence of NaI did not seem to affect or did not significantly affect the function and effect of the base formula; (2) More unexpectedly, the inventors also found that the sequencing results obtained by using the base formula without DABCO of the specified concentration for fluorescence imaging were basically the same as those obtained by using the complete base formula.

[0023] And, the base formula without DABCO of the specified concentration is used as a "new base formula" for formula optimization, that is, the above-mentioned oxidizing agents or reducing agents or their analogs / derivatives are independently or based on functions, redox ability and requirements for reaction environment are combined and added to the new base formula, and whether the solution system containing a specific substance or substance combination is suitable for the mixed use of different water-soluble or electric fluorescent dyes (can make the fluorescent dyes in the system have a higher signal-to-noise ratio, increase the stability of the fluorescent dyes, etc.), whether it can reduce the degradation of nucleic acids, whether it has higher stability, etc. are considered in order to obtain a formula that can significantly outperform the base formula or the new base formula in sequencing results.

[0024] Based on the above unexpected findings and multiple experimental adjustments and optimizations, the inventors developed the desired fluorescence detection solution system based on the new base formula.

[0025] In one embodiment, the present application provides a method for incorporating a labeled nucleotide, the method comprising: (a) providing a hybridization complex, the hybridization complex being a hybrid of a primer and a template molecule, the primer being configured to hybridize to the 3' end of the template molecule, the template molecule being a single-stranded nucleic acid molecule; (b) placing a polymerase, a nucleotide analogue, and the hybridization complex under conditions suitable for a polymerization reaction, the nucleotide analogue comprising a linked sugar unit, a base, a cleavable blocking group, and a fluorescent label, to obtain an extension product by allowing the nucleotide analogue to bind to the hybridization complex; (c) replacing the solution system of (b) with a fluorescent detection reagent, the fluorescent detection reagent comprising an enzymatic oxygen scavenging system and a plurality of reducing agents, and not comprising triethylenediamine (DABCO); (d) irradiating at least a portion of the hybridization complex in the presence of the fluorescent detection reagent and collecting at least a portion of a signal from the fluorescent label; and (e) replacing the fluorescent detection reagent with a cleavage reagent to cleave the cleavable blocking group and the fluorescent label on the extension product, the cleavage reagent being used to cleave the cleavable blocking group and the fluorescent label of the nucleotide analogue.

[0026] The method comprises a step of replacing the solution system of the extension step with a specific solution system (herein referred to as a fluorescent detection reagent), and the method comprising the step is capable of placing the fluorescent label in a solution system that is conducive to its stable and efficient luminescence without affecting the template molecule / hybridization complex, which is conducive to accurate detection of the signal from the fluorescent label, and thus, is conducive to accurate identification of the type of nucleotide incorporated into the template molecule based on the detected signal.

[0027] The method is suitable for platforms that achieve sequencing-by-synthesis based on fluorescent imaging detection, in particular, single-molecule sequencing platforms based on SBS. Specifically, the solution system in which the fluorescent label is placed comprises an enzymatic oxygen scavenging system and a plurality of reducing agents and does not comprise DABCO, which is capable of rapidly and effectively scavenging oxygen, quenching triplet-state fluorescent molecules by transferring electrons, etc., avoiding the reaction of triplet-state fluorescent molecules with oxygen molecules, and helping the fluorescent molecules to recover to the ground state, thereby providing an effective and stable detection / imaging environment; and the presence of the solution system can make the fluorescent signal stable during fluorescent detection, reduce or inhibit nucleic acid damage, and prolong the quenching time of the fluorescent molecules, thereby facilitating the increase of sequencing read length and the improvement of sequencing quality.

[0028] In another embodiment, the present application also provides a composition or reagent formulation, also referred to as a fluorescent detection reagent, comprising an enzymatic oxygen scavenging system and a plurality of reducing agents, and not comprising DABCO. The composition is made by the inventors based on the above-mentioned unexpected findings, and is suitable for applications involving detection of fluorescent signals, in particular, applications involving single-molecule fluorescent signal detection.

[0029] In yet another embodiment of the present application, there is also provided use of the above-mentioned composition or reagent formulation in detecting fluorescence, in particular in detecting single-molecule fluorescence signals.

[0030] Additional aspects and advantages of embodiments of the present application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and / or can be learned by practice of embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0031] The foregoing and / or additional aspects and advantages of embodiments of the present application are also set forth in the Description which follows, and in part will become apparent to those skilled in the art upon examination of the following or can be learned by practice of embodiments of the present application.

[0032] Figure 1 Fluorescence quenching time data for each experimental group and control group in Example 10;

[0033] Figure 2 Sequencing read length data for each experimental group and control group in Example 10;

[0034] Figure 3 CallRatio data for Experimental Group 1 and Experimental Group 2 in Example 12;

[0035] Figure 4 Observed read length data for each experimental group in Example 14;

[0036] Figure 5 Changes in pH and dissolved oxygen (DO) values over time (left) and imaging quality after 45 hours (right) for the control group in Example 16;

[0037] Figure 6 Changes in pH and dissolved oxygen (DO) values over time (left) and imaging quality after 45 hours (right) for Experimental Group 1 in Example 16;

[0038] Figure 7 Changes in pH and dissolved oxygen (DO) values over time (left) and imaging quality after 45 hours (right) for Experimental Group 2 in Example 16;

[0039] Figure 8 Changes in pH and dissolved oxygen (DO) values over time (left) and imaging quality after 45 hours (right) for Experimental Group 3 in Example 16;

[0040] Figure 9 Quenching curves for each experimental group with different concentrations of ascorbic acid added in Example 18;

[0041] Figure 10Quenching time data for each experimental group with different concentrations of ascorbic acid added in Example 18;

[0042] Figure 11 Sequencing read length distribution data for experimental groups with 0 mM and 20 mM ascorbic acid added in Example 18;

[0043] Figure 12 Read length distribution data for each experimental group with different concentrations of ascorbic acid added in Example 18;

[0044] Figure 13 Imaging quality comparison for experimental group 1 (right panel) and control group (left panel) in Example 22;

[0045] Figure 14 Fluorescence intensity data for fluorescent detection reagents under different pH conditions in Example 28;

[0046] Figure 15 Observed sequencing read length for fluorescent detection reagents under different pH conditions in Example 28;

[0047] Figure 16 Quenching curve for fluorescent detection reagents with different concentrations of BME added in Example 30;

[0048] Figure 17 Quenching time for fluorescent detection reagents with different concentrations of BME added in Example 30;

[0049] Figure 18 Quenching curve for fluorescent detection reagents with different concentrations of DTT added in Example 30;

[0050] Figure 19 Quenching time for fluorescent detection reagents with different concentrations of DTT added in Example 30;

[0051] Figure 20 Quenching time for fluorescent detection reagents with different concentrations of AA and MV added under the same ratio in Example 30;

[0052] Figure 21 Quenching time for fluorescent detection reagents with different ratios of AA and MV added in Example 30;

[0053] Figure 22 Quenching time for fluorescent detection reagents with different ratios of AA and MV added in Example 30;

[0054] Figure 23 Sequencing read length distribution for fluorescent detection reagents with or without AA and MV added in Example 30;

[0055] Figure 24 Fluorescence intensity of the fluorescent detection reagent added with or without AA and MV in Example 30;

[0056] Figure 25 Mapping results of the fluorescent detection reagent added with or without AA and MV in Example 30. DETAILED DESCRIPTION

[0057] Embodiments of the present application are described in detail below. The present application can be implemented in many different forms and is not limited to the embodiments described herein. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present application only, and should not be construed as limiting the present application.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0059] In this document, unless otherwise indicated, a nucleotide refers to one of the four natural nucleotides (such as dATP, dCTP, dGTP and dTTP or ATP, CTP, GTP and UTP) or a derivative thereof, and sometimes is directly referred to as the base (A, T or U, C and G) it contains. A person of ordinary skill in the art can know the nucleotide or base referred to by the expression according to the context.

[0060] In this document, the term "sequencing" refers to sequence determination, also known as "nucleic acid sequencing" or "genetic sequencing", which refers to the determination of the order of bases in a nucleic acid sequence; includes sequencing by synthesis (SBS) and / or sequencing by ligation (SBL); includes DNA sequencing and / or RNA sequencing; includes long fragment sequencing and / or short fragment sequencing, the long and short fragments being relative, such as a nucleic acid molecule longer than 1 Kb, 2 Kb, 5 Kb or 10 Kb can be referred to as a long fragment, and shorter than 1 Kb or 800 bp can be referred to as a short fragment; includes paired-end sequencing, single-end sequencing and / or mate-pair sequencing, etc., and the paired-end sequencing or mate-pair sequencing can refer to the readout of any two segments or two parts of a nucleic acid molecule that do not overlap completely.

[0061] The so-called sequencing includes the process of binding nucleotides (including nucleotide analogs) to the template and collecting the corresponding reaction signals. In some sequencing platforms in which the binding of nucleotides to the template and the collection of the corresponding reaction signals are not synchronized / real-time, the determination of the order of multiple nucleotides / bases on the template is generally achieved by multiple sequencing cycles, and a cycle is also called a sequencing round, which can be defined as one base extension of four nucleotides / bases, or in other words, the determination process of the base type at any specified position on the template.

[0062] For sequencing platforms based on controlling polymerization or ligation reactions to achieve sequencing, a cycle can include the process of binding four nucleotides to the so-called template and collecting the corresponding reaction signals; for platforms based on polymerization reactions to achieve sequencing, the reaction system includes reaction substrates nucleotides, polymerase and template, and a predetermined sequence (sequencing primer) is bound to the template, based on the principle of base pairing and polymerization reaction, the added reaction substrate (nucleotide) can be controllably connected to the 3' end of the sequencing primer under the catalysis of the polymerase, to achieve the pairing with the corresponding position base of the template; generally, a cycle can include one or more base extensions (repeats), for example, four nucleotides are added to the reaction system in turn, and base extension and collection of the corresponding reaction signals are performed respectively, a cycle includes four base extensions and four signal collections; for another example, four nucleotides are added to the reaction system in any combination, for example, two-by-two or one-three combination, two combinations are performed for base extension and collection of the corresponding reaction signals respectively, a cycle includes two base extensions and four signal collections; for another example, four nucleotides are added to the reaction system at the same time for base extension and collection of the reaction signals, a cycle includes one base extension and four signal collections.

[0063] Sequencing can be performed by a sequencing platform, which can be selected from but not limited to the Hiseq / Miseq / Nextseq / Novaseq sequencing platform of Illumina, the IonTorrent platform of Thermo Fisher / Life Technologies, the BGISEQ and MGISEQ / DNBSEQ platform of Huada Gene, and the single molecule sequencing platform; the sequencing method can be selected from single-end sequencing and double-end sequencing; the obtained sequencing results / data, i.e., the reads, are called reads, and the length of the reads is called read length.

[0064] Single molecule detection as referred to herein includes single molecule sequencing, for a detection that does not involve amplification of the signal to be detected, for example a sequencing platform that does not involve amplification of the nucleic acid molecule to be detected, the molecule to be detected exists in physical form of a single molecule or a few molecules, and is reflected in the signal acquisition result such as an image as a weak, easily disturbed / overwhelmed, and relatively unstable signal. "Single molecule" as referred to herein means one or a few molecules, generally no more than 10 molecules, for example 1, 2, 3, or 5 molecules.

[0065] In the present context, "solid substrate" as referred to herein can be any solid support that can be used to immobilize nucleic acid sequences, such as nylon membranes, glass slides, plastics, silicon wafers, magnetic beads, etc.; sometimes also referred to as reactors, chips, or flow cells.

[0066] According to an embodiment of the present application, a method of incorporating a labeled nucleotide is provided, comprising the steps of: (a) providing a hybridization complex, the hybridization complex being a hybrid of a primer and a template molecule, the primer being configured to hybridize to the 3' end of the template molecule, the template molecule being a single-stranded nucleic acid molecule; (b) placing a polymerase, a nucleotide analog, and the hybridization complex under conditions suitable for a polymerization reaction, the nucleotide analog comprising a linked sugar unit, a base, a cleavable blocking group, and a fluorescent label, to obtain an extension product by allowing the nucleotide analog to bind to the hybridization complex; (c) replacing the solution system of (b) with a fluorescent detection reagent, the fluorescent detection reagent comprising an enzymatic oxygen scavenging system and a plurality of reducing agents and not comprising triethylenediamine; (d) irradiating at least a portion of the hybridization complex in the presence of the fluorescent detection reagent and collecting at least a portion of a signal from the fluorescent label; (e) replacing the fluorescent detection reagent with a cleavage reagent to cleave the cleavable blocking group and the fluorescent label on the extension product, the cleavage reagent being used to cleave the cleavable blocking group and the fluorescent label of the nucleotide analog. Unless otherwise specified, the labeled nucleotide and the nucleotide analog herein are equivalent.

[0067] Nucleotides used in SBS sequencing generally include the use of modified nucleotides, which are often also referred to as "nucleotide analogs". Nucleotide analogs used in SBS sequencing are often also referred to as terminators, and in the current stage of development, reversible terminators are generally used, i.e. the so-called reversible terminators, which in addition to the phosphate group, the pentose sugar, and the base that are generally included in nucleotides, generally also include a cleavable blocking group and a cleavable detectable label, the cleavable blocking group being able to reversibly prevent the subsequent incorporation / binding of a subsequent reversible terminator or nucleotide to the next position on the template, and the cleavable detectable label being able to generate a signal after the reversible terminator is incorporated / bound to the current position on the template to be detected.

[0068] Based on the position of the blocking group, the reported reversible terminator can be divided into three categories: one is that the blocking group is located at the 3'-OH of the pentose, i.e., the 3'-O-blocking group of the pentose, so that the phosphodiester bond cannot be formed, and the blocking group is, for example, azide; two is that the blocking group is located at the phosphate side, so that the phosphodiester bond cannot be formed; three is that the blocking group is located at the base side of the nucleotide, which blocks the formation of the phosphodiester bond in the solution environment based on the charge and / or steric hindrance to achieve polymerization blocking, and this kind of reversible terminator is also commonly referred to as a virtual terminator.

[0069] In one specific example, the nucleotide analogues include four nucleotide analogues of dATP, dUTP or dTTP, dCTP and dGTP, two of the four nucleotide analogues carry fluorescent labels X, and the other two carry fluorescent labels Y, the fluorescent labels X and the fluorescent labels Y are two fluorescent labels with different emission wavelengths, and the polymerization reaction in (b) includes two of the four nucleotide analogues with different fluorescent labels.

[0070] In one specific example, the nucleotide analogues include four nucleotide analogues of dATP, dUTP or dTTP, dCTP and dGTP, and the four nucleotide analogues carry four fluorescent labels with different emission wavelengths, and the polymerization reaction in (b) includes four nucleotide analogues.

[0071] In one specific example, the blocking group and the fluorescent label on the nucleotide analogue are located on the same side of the base. More specifically, in one example, the 3' position of the sugar unit on the nucleotide analogue is -OH, i.e., the 3' position of the pentose is in the natural state. Such a nucleotide analogue can block the incorporation / binding of nucleotides to the next position of the template through non-physical blocking such as steric hindrance and / or charge of the molecule. For specific structures, for example, refer to the content disclosed in WO2019105421A1.

[0072] In one specific example, the blocking group and the fluorescent label on the nucleotide analogue are located on different sides of the base. More specifically, the blocking group on the nucleotide analogue is located at the 3' position of the sugar unit, i.e., the 3'-OH of the pentose of the nucleotide analogue is modified to a 3'-O-blocking group. For specific structures, for example, refer to the content disclosed in US7057026B2.

[0073] In one specific example, the above method of incorporating labeled nucleotides further comprises the following steps: (f) performing (b)-(e) at least once, and the specific number of times can be determined as needed. Generally, performing (b)-(e) means performing a base extension or a sequencing reaction for no less than 20, 30, 50, 100 or 150 times, etc., to read a sequence (reads) with a certain length, so as to meet the requirements of various application detection purposes.

[0074] In one specific example, the hybridization complex is attached to the surface of the substrate, (d) comprises illuminating the surface of the substrate with light of a specific wavelength to excite the fluorescent label on the surface of the substrate to emit fluorescence, and collecting at least a portion of the fluorescence from the fluorescent label. It is understood that the specific wavelength described above can be adjusted according to the excitation wavelength of different fluorescent labels. For example, the wavelength range commonly used in fluorescent signal imaging detection is 500-700 nm, and the fluorescent dye or dye combination that is optional and suitable for this excitation wavelength range can be selected from Cy3, Alexafluor 532, HEX, Atto 532, ROX, Alexafluorosis 630, Cy5, Atto647N, BODIPY650, Cy 5.5, IF700 and Alex680. More specifically, for example, the excitation wavelength of ATTO647N is 646 nm, and the emission wavelength is 664 nm; the excitation wavelength of ATTO532 is 532 nm, and the emission wavelength is 552 nm; the excitation wavelength of CY5 is 651 nm, and the emission wavelength is 670 nm; the excitation wavelength of IF700 is 690 nm, and the emission wavelength is 713 nm; the excitation wavelength of ROX is 578 nm, and the emission wavelength is 604 nm; the excitation wavelength of Alexa Fluor 532 is 534 nm, and the emission wavelength is 553 nm.

[0075] In one specific example, the fluorescent label comprises at least one of the combination of ROX, ATTO532 and Alexa fluor 532, and at least one of the combination of CY5, IF700 and ATTO647N. That is, one base extension or one round of sequencing involves collecting signals from two or more fluorescent labels.

[0076] In one specific example, the fluorescent label is selected from a combination of fluorescent dyes with excitation wavelengths near 550 nm and near 660 nm. For example, the fluorescent label comprises ATTO532 and ATTO647N.

[0077] In one specific example, the enzymatic oxygen removal system is selected from combination I, combination II or combination III, combination I comprises glucose and glucose oxidase, combination II comprises glucose, glucose oxidase and catalase, and combination III comprises protocatechuic acid and protocatechuate 3,4-dioxygenase. The detection solution containing any of the above enzymatic oxygen removal systems can effectively remove oxygen in the solution, and can reduce the effect of singlet oxygen on the luminescence of the fluorescent dye.

[0078] In one specific example, the reducing agent is selected from at least two of ascorbic acid, gallic acid, an analog or derivative of gallic acid, cyanuric acid, and a water-soluble vitamin E or a derivative of water-soluble vitamin E. The so-called analog or derivative of gallic acid is, for example, a lower alkyl ester of gallic acid, such as methyl gallate, ethyl gallate, propyl gallate, or a combination thereof. In this way, by using a combination of multiple reducing agents, fluorescent dyes with different properties can be affected. At the same time, other reducing agents in the reagent composition can be protected and / or the stability of the nucleic acid molecules can be enhanced.

[0079] In some examples, when the fluorescent signal is detected, an image is obtained by capturing the signal from the fluorescent label by a camera; it can be understood that at least part of the signal from the fluorescent label appears as a bright spot on the image.

[0080] The so-called "bright spot" (dots, spots, or peaks) on the image, also known as "bright dot" or "light dot", refers to a position on the image where the signal is relatively strong, for example, the signal at this position is stronger than the surrounding, which appears as a relatively bright spot or dot on the image. A bright spot occupies one or more pixels. The signal corresponding to the bright spot can come from the target molecule or from non-target substances. The detection of "bright spot" includes the detection of the optical signal of the target molecule such as the extended base or base cluster.

[0081] In one specific example, the fluorescent detection reagent comprises combination I, ascorbic acid, and gallic acid or an analog or derivative of gallic acid. The combination of ascorbic acid and gallic acid can improve the intensity of the detected fluorescent signal and the image score, and make the imaging quality more stable during the signal acquisition process, reducing the decline in imaging quality during the signal acquisition process, thus obtaining a better detection result. The inventors speculate that the fluorescent detection reagent of the present embodiment uses glucose and glucose oxidase as the oxygen removal system, which has a faster oxygen removal speed, and combines ascorbic acid as a reducing agent to quench the triplet state fluorescent dye by transferring electrons, avoiding its reaction with oxygen molecules, which helps the fluorescent dye to return to the ground state, thereby forming an effective and stable detection solution system, which can stabilize the fluorescent signal, reduce or inhibit nucleic acid damage, prolong the quenching time of the fluorescent molecule, and thus increase the sequencing read length and improve the sequencing quality during the fluorescent detection process.

[0082] The imaging quality refers to the observation of the image after the fluorescent detection reagent is introduced, and the preliminary analysis of the brightness and image score value is performed by the applet. The evaluation method of the imaging quality (image quality) in this embodiment is not limited. Based on prior data or based on the establishment of expectations, a relatively high score is given to the one that meets or is closer to the expectation, and a relatively low score is given to the one that does not meet or is far from the expectation. For example, the identification or detection of bright spots on the image and the evaluation of the image quality can be performed according to the method disclosed in CN112285070A.

[0083] In one specific example, the fluorescent detection reagent comprises 50mM-300mM glucose, 2U / mL-20U / mL glucose oxidase, 1mM-200mM ascorbic acid, and 1mM-20mM gallic acid or an analog or derivative thereof.

[0084] Preferably, in one specific example, the fluorescent detection reagent comprises 80mM-150mM glucose, 8U / mL-12U / mL glucose oxidase, 1mM-50mM ascorbic acid, and 1mM-10mM gallic acid or an analog or derivative thereof.

[0085] In one example, the fluorescent detection reagent further comprises water-soluble vitamin E (Trolox) or a derivative thereof, or a combination of water-soluble vitamin E or a derivative thereof and a quinone derivative. It has been found that water-soluble vitamin E or a derivative thereof has a protective effect on the fluorescent group, but the fluorescent detection reagent freshly prepared with the addition of water-soluble vitamin E or a derivative thereof has poor imaging performance for fluorescent dyes such as Atto532, while the fluorescent detection reagent after storage does not have this problem. The inventors speculate that this may be due to the fact that the protective effect of water-soluble vitamin E or a derivative thereof on the fluorescent group requires the cooperation of reduced Trolox and its oxidized form TX-quinone (TQ). Therefore, it is speculated that in the newly prepared fluorescent detection reagent, Trolox is mostly in the reduced state, and TQ is lacking, which cannot create good conditions for the excitation of the fluorescent dye.

[0086] Based on the above test findings and speculations, the inventors adjust the method of configuring a solution containing Trolox or a derivative thereof, for example, by increasing ultraviolet irradiation or increasing the standing time in air; for example, by using a combination of Trolox and a quinone compound, and adjusting the ratio of trolox or a derivative thereof and a quinone compound, etc., to enhance the brightness of the dye in the detection solution, control the stability of the detection solution system, and enhance the realization of the function / role of the detection solution.

[0087] In one specific example, a certain proportion of a quinone derivative such as p-benzoquinone is added to a freshly prepared fluorescent detection reagent containing Trolox or its derivative, so that the p-benzoquinone can simulate the oxidized Trolox, thereby achieving the purpose of enhancing the signal intensity of the fluorescent group, and further helping to improve the imaging quality, sequencing read length and reduce the error rate.

[0088] Preferably, in one example, the fluorescent detection reagent further comprises water-soluble vitamin E and p-benzoquinone.

[0089] In one specific example, the concentration of water-soluble vitamin E is 6mM-12mM, and the concentration of p-benzoquinone is 0.36mM-0.96mM.

[0090] In one specific example, the fluorescent detection reagent further comprises cyanuric acid. In this way, a better quality image can be obtained. The inventors speculate that cyanuric acid, free in solution, can react with oxygen radicals in preference to the molecules to be tested, such as nucleic acids on the chip surface, can consume oxygen radicals generated after laser irradiation, and weaken or avoid damage to the nucleic acid chain, thereby reducing the error recognition of bases.

[0091] In one specific example, the mass percentage of cyanuric acid is 0.0001%-0.001%.

[0092] Preferably, in one example, the mass percentage of cyanuric acid is 0.0003%-0.0009%.

[0093] In one specific example, the fluorescent detection reagent further comprises at least one of adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate and thymidine monophosphate. The inventors speculate that, similar to cyanuric acid, these components, independently or in combination, can be free in solution, can react with oxygen radicals in preference to the molecules to be tested, can consume oxygen radicals generated after laser irradiation, and weaken or avoid damage to the nucleic acid chain, thereby helping to reduce the error rate of sequencing.

[0094] In one specific example, the fluorescent detection reagent further comprises 1μM-50μM 5'-adenosine acid and 1μM-50μM guanosine-5'-monophosphate. In this way, a better quality image can be obtained, and a better quality sequencing result can be obtained based on these images.

[0095] In one specific example, the reagent component in the fluorescent detection reagent contains Tris (tris(hydroxymethyl aminomethane) buffer system or HEPES buffer system, and the pH of the fluorescent detection reagent is 6.5-8.5. By detecting the physicochemical properties of the fluorescent detection reagent at different times, it is found that the pH value gradually decreases and the dissolved oxygen value gradually increases. According to the fact that the fluorescent detection reagent uses glucose oxidase and glucose as the deoxidation system, it is determined that the oxidation of glucose to gluconic acid is the reason for the decrease in pH, and the dissolved oxygen increases when the glucose is consumed at the late stage of sequencing. A certain concentration of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) can maintain the pH above 7 for a long time, thereby improving the stability of the fluorescent detection reagent. In addition, it is further found that using tris(hydroxymethyl aminomethane) (Tris) instead of HEPES is more helpful to improve the stability of the fluorescent detection reagent.

[0096] In one specific example, the fluorescent detection reagent also includes acetonitrile (ACN), and the volume percentage of acetonitrile is 5%-40%, preferably 18%-35%. It is found in tests that the solubility of some tested reducing substances is poor in a solution system without acetonitrile; for example, water-soluble vitamin E or some quinone derivatives are difficult to dissolve in a solution without acetonitrile. By adding more than 10% acetonitrile, the solubility of these functional substances in the solution can be enhanced.

[0097] Tests have found that the combination of Tris and ACN can make the collected images perform better in terms of fluorescence signal intensity, imaging quality, and error rate of detection results.

[0098] Specifically, in one example, Trolox is dissolved in acetonitrile, placed in air for more than 10h, and then other components are added to prepare a fluorescent detection solution I without p-benzoquinone. In another example, a fluorescent detection solution II is prepared by adding the same concentration of Trolox, 0.36mM-0.96mM, for example, 0.52mM of p-benzoquinone, and the same concentration of other components as I without placing in air. Both fluorescent detection solutions I and II are used for sequencing, and the results show that both formulations can obtain good sequencing results and have comparable effects.

[0099] In some tests, for example, sequencing of a reaction system containing multiple fluorescent dyes, the formulations in some of the above examples can enhance the signal of some of the fluorescent dyes while weakening the signal of another part of the fluorescent dyes. However, from the sequencing data of each example, it can be seen that the SBS sequencing of multiple fluorescent dyes can be effectively realized, and relatively speaking, the read length with application value can be basically obtained and the error rate is not high.

[0100] In one specific example, the fluorescent detection reagent includes the following components: 50 mM to 200 mM glucose, 2 U / mL to 20 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 1 mM to 10 mM gallic acid, 6 mM to 12 mM water-soluble vitamin E, 0.1 mM to 1.0 mM p-benzoquinone, 0.0001 wt% to 0.001 wt% cyanuric acid, 1 mM to 20 μΜ 5'-adenylic acid, 1 μΜ to 20 μΜ guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 10 v / v% to 40 v / v% acetonitrile.

[0101] Preferably, in one example, the fluorescent detection reagent includes the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 50 mM ascorbic acid, 1 mM to 10 mM gallic acid, 6 mM to 12 mM water-soluble vitamin E, 0.36 mM to 0.96 mM p-benzoquinone, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 μΜ 5'-adenylic acid, 4 μΜ to 12 μΜ guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

[0102] More preferably, the volume percentage of acetonitrile therein is 28% to 35% to obtain high quality detection results.

[0103] In some preferred examples, the fluorescent detection reagent includes the following components 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 50 mM ascorbic acid, 1 mM to 15 mM gallic acid or gallic acid ethyl ester or gallic acid propyl ester, 6 mM to 15 mM water-soluble vitamin E, 0.36 mM to 0.96 mM p-benzoquinone, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 μΜ 5'-adenylic acid, 4 μΜ to 12 μΜ guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

[0104] Alternatively, the fluorescent detection reagent includes the following components 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 10 mM to 100 mM ascorbic acid, 10 mM to 15 mM gallic acid ethyl ester, 6 mM to 12 mM water-soluble vitamin E, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 μΜ 5'-adenylic acid, 4 μΜ to 12 μΜ guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

[0105] or, comprising the following components 80mM-150mM glucose, 8U / mL-12U / mL glucose oxidase, 50mM-150mM ascorbic acid, 10mM-15mM propyl gallate, 3-7mM hydroquinone, 6mM-12mM water-soluble vitamin E, 0.0003wt%-0.0009wt% cyanuric acid, 4mM-12mM 5'-adenylic acid, 4mM-12mM guanosine-5'-monophosphate, 100mM-300mM tris-hydroxymethyl aminomethane and 18v / v%-35v / v% acetonitrile.

[0106] It is found that the above-mentioned three preferred fluorescent detection reagent formulations can be applied to nucleic acid sequencing, especially sequencing involving detection of multiple fluorescent dyes, and that each of the formulations can obtain good sequencing results and the quality of the sequencing results obtained by each of the formulations is comparable.

[0107] It is understandable that, in a sequencing process of sequencing-by-synthesis, the reaction time usually lasts for several hours, for example, 12 hours or more, and the mixed reagents are used for sequencing after being mixed in advance. As the sequencing proceeds, the performance of the mixed reagents exposed to air gradually deteriorates, which makes it difficult to meet the requirements of sequencing. Moreover, as a premixed reagent kit, the components thereof are gradually oxidized during storage, and finally the reducing components in the components are completely or partially oxidized, which makes it difficult for the reagent kit to be used for detection after being stored for a certain period of time while still being able to perform its performance.

[0108] In some examples, the fluorescent detection reagent further comprises liquid paraffin and / or silicone oil. Sealed storage with liquid paraffin or silicone oil can ensure the stability of the fluorescent detection reagent for a longer period of time. Moreover, it is beneficial to the transportation of the reagent.

[0109] In a specific example, an inert substance such as silicone oil or liquid paraffin is added, for example, 8-20v / v%. Since the density of any of the two substances is less than that of an aqueous solution, the substance(s) can separate air from the solution system, thus avoiding or reducing the entry of oxygen in the air into the solution to consume the oxygen scavenging reagents therein, causing changes in the reducing substances and / or undesired reactions with the substances or intermediates in the solution, changing the pH of the solution and / or affecting the performance of the solution system, etc. Therefore, the stability of the detection solution when in use can be increased, and the shelf life of the detection solution can be extended.

[0110] It is found through tests that, within 12 hours after the preparation of the three preferred fluorescent detection reagent formulations of the above-mentioned examples, 10v / v% of silicone oil is added, and the performance does not decrease when the mixture is exposed to air for 48h and then applied to sequencing, and good sequencing results can still be obtained. Moreover, the shelf life test results prove that the shelf life of the fluorescent detection reagent containing a certain amount of silicone oil or liquid paraffin can reach 6 months. It can be seen that the formulations can be produced in batches and have high industrial applicability.

[0111] In one specific example, the time of irradiation is 10-100 milliseconds, preferably, the time of irradiation is 50-100 milliseconds.

[0112] In one specific example, in the step (d), the light intensity for irradiating ATTO532 is 30-40 milliwatts, and the light intensity for irradiating ATTO647N is 60-80 milliwatts; and / or, the time for irradiating ATTO532 or ATTO647N in one field of view is 50-500 milliseconds. Preferably, the time for irradiating ATTO532 or ATTO647N in one field of view is 50-100 milliseconds.

[0113] The fluorescent detection reagent of one embodiment of the present application comprises an enzymatic oxygen removal system and a plurality of reducing agents, and does not comprise triethylenediamine.

[0114] In one specific example, the enzymatic oxygen removal system is selected from combination I, combination II or combination III, combination I comprises glucose and glucose oxidase, combination II comprises glucose, glucose oxidase and catalase, and combination III comprises protocatechuic acid and protocatechuate 3,4-dioxygenase.

[0115] In one specific example, the plurality of reducing agents is selected from at least two of ascorbic acid, gallic acid, an analog or derivative of gallic acid, cyanuric acid and water-soluble vitamin E.

[0116] In one specific example, the fluorescent detection reagent comprises combination I, ascorbic acid and gallic acid or an analog or derivative of gallic acid. The fluorescent detection reagent of this embodiment uses glucose and glucose oxidase as the oxygen removal system, which has a faster oxygen removal speed, and combines ascorbic acid as the reducing agent to quench the triplet state fluorescent dye by electron transfer method, avoid its reaction with oxygen molecules, help the fluorescent dye to return to the ground state, so as to form an effective and stable detection solution system, which can stabilize the fluorescent signal in the process of fluorescent detection, reduce or inhibit the damage of nucleic acid, prolong the quenching time of fluorescent molecules, and further increase the sequencing read length and improve the sequencing quality. The combination of ascorbic acid and gallic acid can improve the detected fluorescent signal intensity and imaging quality score, and make the imaging quality more stable during the signal acquisition process, and reduce the decline amplitude of the imaging quality during the signal acquisition process. The imaging quality refers to the observation of single molecule image after the fluorescent detection reagent is introduced, and the preliminary analysis of brightness, image score value and the like through a small program.

[0117] In one specific example, the fluorescent detection reagent comprises 50mM-200mM glucose, 2U / mL-20U / mL glucose oxidase, 1mM-200mM ascorbic acid, and 1mM-10mM gallic acid or an analog or derivative of gallic acid.

[0118] Preferably, in one example, the fluorescent detection reagent comprises 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 50 mM ascorbic acid, and 1 mM to 10 mM gallic acid or an analog or derivative of gallic acid.

[0119] In one specific example, the fluorescent detection reagent further comprises water-soluble vitamin E (Trolox) and p-benzoquinone. It is found that water-soluble vitamin E has a protective effect on the fluorescent group, but the fluorescent detection reagent freshly prepared with water-soluble vitamin E has poor imaging performance for fluorescent dyes such as Atto532, while the fluorescent detection reagent after storage does not have this problem. It is possible that the protective effect of water-soluble vitamin E on the fluorescent group requires the joint action of reduced Trolox and its oxidized form TX-quinone (TQ). Therefore, it is speculated that in the newly prepared fluorescent detection reagent, Trolox is mostly in the reduced state, lacking TQ, and cannot create good conditions for the excitation of fluorescent dyes. Based on the above speculation, an attempt is made to add a certain proportion of quinone derivatives, such as p-benzoquinone, to the freshly prepared fluorescent detection reagent to simulate the oxidized form of Trolox, so as to achieve the purpose of enhancing the signal intensity of the fluorescent group, which helps to improve the imaging quality, sequencing read length and reduce the error rate.

[0120] Optionally, the concentration of water-soluble vitamin E is 6 mM to 12 mM, and the concentration of p-benzoquinone is 0.1 mM to 1 mM; preferably, the concentration of p-benzoquinone is 0.36 mM to 0.96 mM.

[0121] In one specific example, the fluorescent detection reagent further comprises cyanuric acid. In this way, cyanuric acid is free in the solution, preferentially reacts with oxygen free radicals, and can consume oxygen free radicals generated after laser irradiation, reducing or avoiding damage to nucleic acid chains, so as to reduce the incorrect identification of nucleic acids in sequencing technology. In some examples, the mass percentage of cyanuric acid is 0.0001% to 0.001%; preferably, the mass percentage of cyanuric acid is 0.0003% to 0.0009%.

[0122] In one specific example, the fluorescent detection reagent further comprises at least one of adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, and thymidine monophosphate. Similar to cyanuric acid, the inventors speculate that these components are free in the solution, preferentially react with oxygen free radicals, and can consume oxygen free radicals generated after laser irradiation, reducing or avoiding damage to nucleic acid chains, which helps to reduce the error rate of sequencing.

[0123] In one specific example, the fluorescent detection reagent further comprises 5'-adenylic acid and guanosine-5'-monophosphate. Optionally, the fluorescent detection reagent further comprises 1 mM to 20 μM 5'-adenylic acid and 1 μM to 20 μM guanosine-5'-monophosphate. Preferably, the fluorescent detection reagent further comprises 4 mM to 12 μM 5'-adenylic acid and 4 μM to 12 μM guanosine-5'-monophosphate.

[0124] In one specific example, the fluorescent detection reagent adopts Tris (tris-hydroxymethyl aminomethane) buffer system or HEPES buffer system, and the pH of the fluorescent detection reagent is 6.5-8.5, preferably, the pH of the fluorescent detection reagent is 8.5. Optionally, the concentration of tris-hydroxymethyl aminomethane or HEPES is 80 mM to 120 mM. In sequencing, it is found that the imaging quality gradually deteriorates as the sequencing proceeds, which seriously affects the data quality in the later stage of sequencing. By detecting the physicochemical properties of the fluorescent detection reagent at different times, it is found that the pH gradually decreases and the dissolved oxygen gradually increases. According to the fact that the fluorescent detection reagent uses glucose oxidase and glucose as the oxygen removal system, it is determined that the oxidation of glucose to gluconic acid is the cause of the decrease in pH, and by the time of the later stage of sequencing, the glucose is almost consumed and the dissolved oxygen increases. By using a certain concentration of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), the pH can be maintained above 7 for a long time, thereby improving the stability of the fluorescent detection reagent. In addition, it is further found that using tris-hydroxymethyl aminomethane (Tris) instead of HEPES is more helpful to improve the stability of the fluorescent detection reagent.

[0125] In one specific example, the fluorescent detection reagent further comprises acetonitrile, and the volume percentage of acetonitrile is 10% to 40%; preferably, the volume percentage of acetonitrile is 18% to 35%; more preferably, the volume percentage of acetonitrile is 28% to 35%. The combination of Tris and ACN performs better in terms of fluorescent signal intensity, imaging quality and error rate.

[0126] In one specific example, the fluorescent detection reagent comprises the following components: 50 mM to 200 mM glucose, 2 U / mL to 20 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 1 mM to 10 mM gallic acid, 6 mM to 12 mM water-soluble vitamin E, 0.1 mM to 1.0 mM p-benzoquinone, 0.0001 wt% to 0.001 wt% cyanuric acid, 1 mM to 20 μM 5'-adenylic acid, 1 μM to 20 μM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile. Optionally, the fluorescent detection reagent further comprises a monovalent soluble salt such as sodium chloride, which cooperates with the buffer components.

[0127] In a preferred example, the fluorescent detection reagent comprises the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 50 mM ascorbic acid, 1 mM to 10 mM gallic acid, 6 mM to 12 mM water-soluble vitamin E, 0.36 mM to 0.96 mM p-benzoquinone, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 mM 5'-adenylic acid, 4 mM to 12 mM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

[0128] The method for preparing the fluorescent detection reagent described above according to an embodiment of the present application comprises the following steps: adding liquid paraffin and / or silicone oil before packaging the fluorescent detection reagent.

[0129] The kit according to an embodiment of the present application comprises the fluorescent detection reagent described above and the nucleotide analog described above. It can be understood that the fluorescent detection reagent described above can be used not only for sequencing but also for other methods or products that require fluorescent detection.

[0130] The present application is described in detail below through specific examples. It should be understood that the examples are only illustrative. The materials, reagents, and sequences involved in the examples, unless otherwise specified, can be prepared, synthesized, or obtained through commercial channels.

[0131] Example 10

[0132] The fluorescent detection reagents of each experimental group and the control group were prepared according to the formulations shown in the following table. The difference between experimental group 1 and experimental group 3 is the enzymatic oxygen removal system. Experimental group 1 uses glucose and glucose oxidase, and experimental group 3 uses protocatechuate-3,4-dioxygenase (PCD) and 3,4-dihydroxybenzoic acid (protocatechuic acid / PCA). The difference between experimental group 2 and experimental group 1 is that the buffer system replaces HEPES with Tris. Sequencing was performed using the fluorescent detection reagents of each experimental group, and the fluorescence quenching time of the Atto647N-labeled terminator under a specific laser working intensity was compared.

[0133]

[0134] When the oxygen removal system is added to the solution, the dissolved oxygen content in the system can be ensured to be undetectable by the dissolved oxygen meter. However, the quenching times of different systems are different when they are actually used. Figure 1The detection data of different enzymatic oxygen removal systems are shown. The average fluorescence quenching time of Atto647N under the glucose and glucose oxidase oxygen removal system is 278 s, and the average fluorescence quenching time under the PCD-PCA oxygen removal system is 11 s, and the number of experimental repetitions N = 9. Under the glucose and glucose oxidase oxygen removal system, the fluorescence quenching time of Atto647N is longer. If the exposure time is 0.5 s, 0.2% of the fluorescent molecules will be quenched under the glucose and glucose oxidase oxygen removal system, and 4.5% of the fluorescent molecules will be quenched under the PCD-PCA oxygen removal system. Therefore, the glucose and glucose oxidase oxygen removal system using glucose and glucose oxidase as the fluorescent detection reagent can prolong the quenching time of the fluorescent molecules.

[0135] In addition, HEPES is easy to produce hydrogen peroxide under light, which may accelerate the quenching of fluorescent molecules and increase the photochemical reaction of biological macromolecules, so it is more reasonable to replace HEPES with Tris in principle. We also compared the apparent sequencing read length under the two different buffer systems. Figure 2 It is shown that there is no obvious difference in the sequencing read length between experimental group 1 and experimental group 2, Figure 1 It is shown that the average quenching time of experimental group 2 is 99 s. Under the extreme 0.5 s exposure time, the fluorescent detection reagent using Tris instead of HEPES will have 0.6% of the fluorescent molecules quenched, and the quenching ratio is small.

[0136] Example 12

[0137] In order to simplify the formula, we use 100 mM Tris to replace the combination of 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), and Tris in the previous formula, as shown in Figure 3 As shown in the figure, the CallRatio (CallRatio represents the proportion of bases that can be recognized, which can reflect the sequencing performance of the imaging liquid to a certain extent) of 100 mM Tris gradually outperforms that of the combination buffer system of 77 mM HEPES + 25 mM MES + 3 mM Tris, especially in the later stage of sequencing, which is more obvious, indicating that 100 mM Tris is more helpful to improve the stability of the performance of the fluorescent detection reagent.

[0138] In addition, we also compared 1,3-bis((trishydroxymethyl)methylamino) propane (Bis-tris propane), 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), Tris, and acetonitrile (ACN) combination and combined with paraffin or silicone oil preservation, phosphate buffered saline solution (PBS), among which the combination of Tris and acetonitrile performs better in fluorescence signal intensity, imaging quality, and error rate.

[0139]

[0140]

[0141] Example 14

[0142] The fluorescence quantum efficiency of the fluorescent molecules is also affected by the polarity of the solvent, so different proportions of acetonitrile can cause different fluorescence quantum efficiencies, and thus different fluorescence signal intensities. As shown in the following table, the final concentration of acetonitrile in the fluorescent detection reagent was adjusted, and the effects of different concentrations of acetonitrile on sequencing read length, imaging quality, and sequencing error rate were compared. The sequencing here involves two fluorescent dyes, Atto532 and Atto647N.

[0143] Component Experiment 1 Experiment 2 Experiment 3 Sodium chloride 20mM 20mM 20mM Water-soluble vitamin E 6mM 6mM 6mM p-Benzoquinone 0.96mM 0.96mM 0.96mM Buffer system Tris 100mM Tris 100mM Tris 100mM Glucose 100mM 100mM 100mM Glucose oxidase 10.4U / mL 10.4U / mL 10.4U / mL Acetonitrile 30.25％ 35.15％ 18％ Triethylene diamine (DABCO) 5mM 5mM 5mM Sodium iodide 5.25mM 5.25mM 5.25mM Ascorbic acid 50mM 50mM 50mM Gallic acid 20mM 20mM 20mM AMP 8uM 8uM 8uM GMP 8uM 8uM 8uM Cyanuric acid 0.0003％ 0.0003％ 0.0003％

[0144] The results, as shown in Figure 4 , show that different concentrations of acetonitrile in the fluorescent detection reagent do not cause significant differences in apparent read length, while different concentrations of acetonitrile in the fluorescent detection reagent result in different signal intensities, different imaging quality scores, and different error rates, as shown in the following table.

[0145]

[0146] Therefore, experiments have shown that acetonitrile in the concentration range of 10% to 40%, especially in the range of 28-35%, can obtain better sequencing read length, and the fluorescent detection reagent containing 30% acetonitrile has better performance in signal intensity, imaging quality score, and error rate.

[0147] Example 16

[0148] As shown in Figure 5 , it was found during sequencing that the imaging quality gradually deteriorated as the sequencing progressed, severely affecting the data quality in the later stages of sequencing. By detecting the physicochemical properties of the fluorescent detection reagent at different times, it was found that the pH value gradually decreased and the dissolved oxygen value gradually increased. According to the glucose oxidase and glucose as an oxygen removal system, it was determined that the oxidation of glucose to gluconic acid was the cause of the decrease in pH. In the later stages of sequencing, glucose was almost completely consumed and the dissolved oxygen increased.

[0149] Therefore, the following experiments were conducted to increase the stability of the fluorescent detection reagent. 25 mL of imaging solution was prepared in the reagent kit and placed at 4 degrees for 40 hours. The pH and dissolved oxygen of the solution were measured at different time points, and the variables were the concentrations of glucose and HEPES (pH 8.8), as shown in the following table.

[0150]

[0151]

[0152] Results as shown in Figures 5-8 Figure 6, increasing the concentration of HEPES (pH 8.8) can maintain the pH above 7 within 40 hours, and increasing the concentration of glucose can slow the rate of increase of dissolved oxygen in the fluorescent detection reagent. Therefore, the range of HEPES concentration (pH 8.8) and glucose concentration is 80-150 mM, which has a better stabilizing effect on the fluorescent detection reagent.

[0153] Example 18

[0154] The fluorescent detection reagents of each group were prepared according to the formulations shown in the table below, with the difference being that different concentrations of ascorbic acid (AA) were added to each group or no ascorbic acid was added. Sequencing was performed using the fluorescent detection reagents of each group, and the fluorescence quenching time of the Atto532-labeled terminator under the working intensity of the laser was compared.

[0155]

[0156] Figure 9 The quenching curves of Atto532 after adding different concentrations of AA are shown. As can be seen from the graph, as the concentration of AA increases, the quenching curve decreases more slowly, and the quenching curve of the 50 mM AA group decreases most slowly. Combined with the quenching time Figure 10 , the quenching time of the control group without AA is within 5 s, while the quenching time is prolonged after adding AA. As the concentration of AA increases, the quenching time of the 50 mM AA group reaches 30 s, that is, AA helps to prolong the quenching time of Atto532. Moreover, as the concentration of AA increases, the sequencing read length increases, as shown in Figure 11 The distribution of sequencing read length of the 20 mM AA group and the control group (0 mM AA) is shown. As can be seen from the read length peak, after adding 20 mM AA, the main peak is higher and the short fragments are reduced. Figure 12 The read length distribution of the group with different concentrations of ascorbic acid is shown. It can be seen that the 40 mM AA group and the 50 mM AA group are the better concentration range, the read length is increased by 1 bp, the main peak is more prominent, and the small fragments are significantly reduced. From the above research, it can be seen that when the concentration of ascorbic acid is continuously increased, the stability of the system can be further increased. Through the above results, it can be verified that the addition of AA helps to prolong the quenching time of the fluorescent molecule, increase the sequencing read length, and 40 mM-50 mM is the better concentration range.

[0157] In other test tests, it was found that the concentration of AA in the solution reached 100-200 mM, which can further reduce the quenching time (data not shown here).

[0158] Example 20

[0159] Fluorescence detection reagents were prepared for the experimental and control groups. The difference between the two groups was that the experimental group contained both ascorbic acid and gallic acid, while the control group contained ascorbic acid but not gallic acid (GA). Sequencing was performed using the fluorescence detection reagents for both the experimental and control groups, and data such as fluorescence signal intensity and image quality scores were compared.

[0160]

[0161]

[0162] Gradient testing with AA showed that the quenching time of Atto532 increased significantly with increasing AA concentration, but the fluorescence signal intensity decreased with increasing AA concentration. Furthermore, combined testing with GA, based on AA, yielded the following results. The results indicate that the combination of ascorbic acid and gallic acid improved the detected fluorescence signal intensity and imaging quality score, stabilized the image quality during signal acquisition, and minimized the degradation of image quality during signal acquisition. Therefore, the addition of gallic acid in the presence of ascorbic acid enhances the stability of image quality.

[0163]

[0164] Example 22

[0165] Fluorescence detection reagents were prepared for each experimental and control group according to the recipes shown in the table below. Each experimental group included varying concentrations of both water-soluble vitamin E and p-benzoquinone, while the control group included only water-soluble vitamin E without p-benzoquinone. Sequencing was performed using the fluorescence detection reagents for each experimental and control group, and data such as image quality, read length, and error rate were compared.

[0166]

[0167] like Figure 13 As shown in the figure, the fluorescence detection reagent of experimental group 1 (right figure) has significantly better imaging quality for Atto532 than the fluorescence detection reagent of the control group (left figure). As shown in the table below, from the comprehensive evaluation of imaging quality score, read length and error rate, 6-12mM water-soluble vitamin E combined with 0.36-0.96mM p-benzoquinone can achieve good imaging quality, read length and relatively low error rate, and 6mM water-soluble vitamin E combined with 0.96mM p-benzoquinone is the optimal condition. Therefore, the experiment proves that the combination of water-soluble vitamin E and p-benzoquinone helps to improve imaging quality, sequencing read length and reduce error rate, and 6-12mM water-soluble vitamin E combined with 0.36-0.96mM p-benzoquinone is the optimal concentration range.

[0168] Experimental conditions Imaging quality score_CT Imaging quality score_GA Read length Error rate 12mM Trolox + 0.6mM BQ 3.20 2.43 41.16 5.97％ 12mM Trolox + 0.36mM BQ 2.38 2.32 41.87 6.77％ 6mM Trolox + 0.96mM BQ 3.38 2.43 41.34 5.53％ 6mM Trolox + 0.6mM BQ 3.24 2.43 41.4 5.63％ 6mM Trolox + 0.36mM BQ 2.87 2.33 41.51 5.82％ 3mM Trolox + 0.6mM BQ 2.40 2.25 40.87 7.52％ 3mM Trolox + 0.36mM BQ 2.35 2.25 40.99 6.90％ 3mM Trolox + 0.18mM BQ 2.26 2.15 41.71 7.33％

[0169] Example 24

[0170] The fluorescent detection reagents of each group were prepared according to the formulations shown in the table below, with the difference being that different proportions of cyanuric acid were added to each group. Sequencing was performed using the fluorescent detection reagents of each group, respectively, and error rate data was compared.

[0171] Component Control Experiment 1 Experiment 2 Experiment 3 Sodium chloride 20mM 20mM 20mM 20mM Water-soluble vitamin E 12mM 12mM 12mM 12mM Buffer system Tris 100mM Tris 100mM Tris 100mM Tris 100mM Glucose 100mM 100mM 100mM 100mM Glucose oxidase 10.4U / mL 10.4U / mL 10.4U / mL 10.4U / mL Acetonitrile 30.25％ 30.25％ 30.25％ 30.25％ Triethylene diamine (DABCO) 5mM 5mM 5mM 5mM Sodium iodide 5.25mM 5.25mM 5.25mM 5.25mM Ascorbic acid 50mM 50mM 50mM 50mM AMP 8μM 8μM 8μM 8μM GMP 8μM 8μM 8μM 8μM Cyanuric acid 0 0.0003％ 0.0006％ 0.0009％

[0172] The results are shown in the table below. The addition of different proportions of cyanuric acid to the fluorescent detection reagent helps to reduce the error rate of sequencing. Therefore, the experiment proves that the addition of cyanuric acid can significantly reduce the error rate, and a concentration range of 0.0001% to 0.001% can achieve good results. The inventors speculate that cyanuric acid is free in the solution, and can react with oxygen free radicals on the surface of the chip nucleic acid in preference, consume oxygen free radicals generated after laser irradiation, weaken or avoid nucleic acid chain damage, and thus reduce the error recognition of nucleic acid in sequencing technology.

[0173] Experimental conditions Error rate No cyanuric acid added 6.53％ 0.0005% cyanuric acid 6.25％ 0.0001% cyanuric acid 6.26％ 0.001% cyanuric acid 6.23％

[0174] Example 26

[0175] The fluorescent detection reagents of each experimental group and the control group were prepared according to the formulations shown in the table below. The difference between the two is that different concentrations of 5'-adenylic acid (AMP) and guanosine-5'-monophosphate (GMP) were added to each experimental group, while the control group did not add 5'-adenylic acid (AMP) and guanosine-5'-monophosphate (GMP). Sequencing was performed using the fluorescent detection reagents of each experimental group and the control group, respectively, and error rate data was compared.

[0176] Component Control Experiment 1 Experiment 2 Experiment 3 Sodium chloride 20mM 20mM 20mM 20mM Water-soluble vitamin E 12mM 12mM 12mM 12mM Buffer system Tris 100mM Tris 100mM Tris 100mM Tris 100mM Glucose 100mM 100mM 100mM 100mM Glucose oxidase 10.4U / mL 10.4U / mL 10.4 U / mL 10.4 U / mL Acetonitrile 30.25％ 30.25％ 30.25％ 30.25％ Triethylenediamine (DABCO) 5 mM 5 mM 5 mM 5 mM Sodium iodide 5.25 mM 5.25 mM 5.25 mM 5.25 mM Ascorbic acid 50 mM 50 mM 50 mM 50 mM AMP 0 4 μM 8 μM 12 μM GMP 0 4 μM 8 μM 12 μM

[0177] The results are shown in the table below. The addition of different concentrations of monophosphate to the fluorescent detection reagent helps to reduce the error rate of sequencing. Therefore, the experiment proves that the addition of a combination of 5'-adenylic acid (AMP) and guanosine-5'-monophosphate (GMP) can reduce the error rate, with a significant effect of 1 μM to 20 μM. The inventors speculate that monophosphate is free in the solution, and can react with oxygen free radicals on the surface of the chip nucleic acid in preference, consume oxygen free radicals generated after laser irradiation, weaken or avoid nucleic acid chain damage, and thus reduce the error recognition of bases. In other words, the addition of nucleotide analogues without reactivity can be used as a sacrificial or competitive agent to replace or protect the target reaction substrate nucleotide.

[0178] Experimental conditions Error rate No addition 6.81％ 1 μM AMP + 1 μM GMP 6.78％ 10 μM AMP + 10 μM GMP 6.53％ 20 μM AMP + 20 μM GMP 6.53％

[0179] Example 28

[0180] Since the pH value can affect the electron transfer of the fluorescent molecule, different pH values can cause different fluorescence quantum efficiencies of the fluorescent molecule, and then cause different fluorescence brightness. In this embodiment, the Atto647N labeled G terminator is dissolved in Tris solutions with different pH values, and different solutions are placed in a quartz cavity. A 640 nm laser with constant light intensity is used for excitation and the fluorescence intensity is tested. As shown in Figure 14 , in the range of pH 6.5-8.5, the higher the pH value, the higher the fluorescence intensity of Atto647N. By sequencing, the apparent sequencing read length corresponding to the fluorescence detection reagent after increasing the pH is compared with that without increasing the pH, as shown in Figure 15 , the sequencing read length corresponding to the fluorescence detection reagent at pH 8.5 has a slight increase, and the advantage is not obvious. However, considering that the deoxygenation system will cause the pH of the solution to further decrease during the deoxygenation process, thereby affecting the deoxygenation rate, it is recommended to increase the pH of the fluorescence detection reagent to 8.5. Therefore, experiments prove that in the range of pH 6.5-8.5, the higher the pH value, the higher the fluorescence intensity of Atto647N, and it is more conducive to the stability of the deoxygenation system.

[0181] Example 30

[0182] In this embodiment, the influence of other additives on the performance of the fluorescence detection reagent is further detected, and the specific steps are as follows.

[0183] 1. Quenching curve

[0184] The quenching curve reflects the quenching of a base fluorescent dye. The faster the quenching curve decreases, the shorter the quenching time, and the better the stability of the fluorescent dye in the imaging system. Conversely, the stability is better. Quenching time is defined as the time corresponding to the quenching of the point number (fluorescent signal) to 50%.

[0185] 1.1. Selection principle of target sequence

[0186] Select a target sequence of the base to be tested in the first round of reaction as the target to be tested (the template molecule with known sequence);

[0187] 1.2. Principle of data analysis

[0188] A. Count the number of bright spots (fluorescent signals appear as bright spots or bright spots on the image) in each picture, for example, a fixed size image area of 1024*1024 or 512*512 can be selected for statistics;

[0189] B. Analyze the scatter plot of the point number changing with time, and analyze and calculate the quenching time by origin software fitting.

[0190] 1.3. Test process

[0191] Assuming the base to be tested is C_Atto532 (dCTP with Atto532), test by selecting a single target of G for the first round of target sequence: add C_Atto532 base according to the usual single molecule sequencing process, ensure that the laser intensity is constant, select a fixed area, collect a photo every 100 ms until all the fluorescent signals are completely quenched;

[0192] 2. Long sequencing verification

[0193] The additives obtained through the quenching curve screening are verified for their effect on sequencing quality through two-channel sequencing (two-color sequencing). The test process is as follows: base polymerization / elongation reaction — collect photos under fluorescent detection reagent — cut and process, and repeat the cycle for 60 cycles of target sequencing or 80 cycles of biological sample sequencing.

[0194] 3. Specific test

[0195] Substrate (modified nucleotide / reformed nucleotide, also known as reversible terminator, a nucleotide with a fluorescent molecule that can inhibit the binding of other nucleotides to the next position of the template to be tested): 50-150 nM, for example 125 nM C_Atto647N (dCTP with Atto647N), 50-150 nM, for example 125 nM T_Atto647N (dTTP with Atto647N), 100-200, for example 200 nM A_Atto647N (dATP with Atto647N), 50-200 nM, for example 75 nM G_Atto647N (dGTP with Atto647N), 1000 nM T_Atto532 (dTTP with Atto532), and 1000 nM C_Atto532 (dCTP with Atto532), AT mixture mix (A_Atto647N and T_Atto532), CG mixture mix (G_Atto647N and C_Atto532) 1 mL, specifically prepared as shown in the table below, and the specific test volume is adjusted according to the experimental situation.

[0196]

[0197] The fluorescent detection solution used contains the components in the following two tables:

[0198]

[0199]

[0200] The single target sequence of the first round of reaction C base (the binding position of the first round of reaction base is underlined in the table) is selected, and the target sequence is provided with 3'-fam. The specific sequence can be referred to the following table. The single target sequence, target sequence, target, etc. referred to in this paper refer to the template; the C target in the following table refers to the sequence of the template preset for the first round of reaction C base (C nucleotide analogues are added).

[0201]

[0202]

[0203] The target sequence is connected to the designated surface / chip by hybridization with the probe on the surface, and C_Atto647N and C_Atto532 are added for detection, including base extension reaction, and after the above-mentioned fluorescent detection reagent is introduced into the designated surface, the surface is imaged, wherein the laser exposure setting of C_Atto647N is 100ms or 500ms, and the laser is 60mW. The exposure setting of C_Atto532 is 100ms, and the laser is 30mW; in this way, different exposure times are selected according to different test bases to continuously collect photos, until the number of bright spots is almost quenched, and finally the fitting analysis is carried out by origin software;

[0204] The results are shown in Figure 16 The quenching curve of Atto532 under different concentrations of BME is shown in the figure. As can be seen from the curve, the quenching curves of 1mM BME, 10mM BME and 25mM BME have no obvious difference from those without BME, but the starting point number of 50mM BME is significantly lower than that of other experimental groups. Under the same concentration and the same condition, the hybridization density cannot have a one-fold difference. Combined with the structure of the base linker and the characteristics of BME, it can be inferred that BME has a cleavage effect on the disulfide bond of the base linker, thereby causing the phenomenon of lower starting point number. Combined with Figure 17 It can be seen that the quenching time of Atto532 under different concentrations of BME has no obvious change.

[0205] Figure 18 The quenching of Atto532 under different concentrations of DTT is shown in the figure. As can be seen from the quenching curve, compared with the control group without DTT, the quenching curve of 1mM and 10mM DTT decreases more slowly, but DTT has similar functions to BME. High concentration of 50mM DTT has a significant cleavage effect on the disulfide bond, and the starting point number is significantly lower than that of other test groups. For the quenching of 1mM and 10mM DDT, the quenching time is counted, as shown in Figure 19The figure shows that as the concentration of DTT increases, the quenching time increases significantly. The quenching time is doubled under 10 mM DTT treatment, that is, DTT has a significant effect on increasing the quenching time of Atto532, but it is difficult to apply due to the excision effect on the base linker. For the AA test case, we tried the combination of AA and MV, Figure 20 Under the same proportion condition (AA: MV = 1: 1), as the concentration of AA and MV increases, the quenching time is relatively improved compared with the control group without adding. The quenching time of Atto532 is extended to 5s under 20mM (AA: MV = 1: 1). In addition, we conducted a gradient experiment on the proportion of AA and MV, compared with the control group of 20mM AA, combined with Figure 21 and 22 Two bar charts show that under the condition of 20mM AA, the quenching time of Atto532 is shortened by adding low concentration of MV. As the concentration of MV increases, the quenching time can reach 10-15s, but it is still significantly lower than the control group of 20mM AA, that is, for Atto532, adding MV on the basis of AA is not conducive to prolonging its quenching time.

[0206] Figure 23 and 24 The test results of the combination of AA and MV are shown. The read length distribution Figure 23 shows that after adding 20mM AA and 20mM MV (AA: MV = 1: 1), the main peak of the read length moves to the left, the sequencing read length becomes shorter, and the single molecule fluorescence brightness is only about 300, which is significantly lower than the control group, resulting in a high deletion rate (the proportion of bases that cannot be identified). For example, Figure 25 The mapping results show that the mapped rate is low, and the overall sequencing quality is poor. Therefore, adding AA alone does not significantly affect the quality of single-color sequencing, but adding MV in combination significantly increases the deletion rate, moves the read length to the left (indicating that the read length becomes shorter), and the sequencing quality is poor, which cannot be applied.

[0207] Example 32

[0208] After introducing various antioxidants, the effect of the antioxidant DABCO in the original formula is unknown, and the effect of sodium iodide as a catalyst for the GOD oxygen removal system is also unknown. Therefore, we tried to remove DABCO and sodium iodide in the original formula for comparison test.

[0209]

[0210]

[0211] The results are shown in the following table, and there is no difference in each index before and after DABCO and NaI removal.

[0212] Experimental conditions Signal intensity_Atto532 Signal intensity_Atto647N Imaging quality score_Atto532 Imaging quality score_Atto647N Error rate Control group 172 346 2.95 2.15 6.50％ Experimental group 177 341 3.07 2.11 6.39％

[0213] Example 34

[0214] Formulation 1: 100 mM glucose, 10 U / mL glucose oxidase, 30 mM ascorbic acid, 1 mM gallic acid, water-soluble vitamin E (Trolox), 0.50 mM p-benzoquinone (BQ), 0.0001 wt% cyanuric acid, 5 μΜ 5'-adenylic acid, 5 μΜ guanosine-5'-monophosphate, 200 mM Tris, and 30 v / v% acetonitrile, 15 v / v% liquid paraffin, pH 7.5.

[0215] Formulation 2: 120 mM glucose, 12 U / mL glucose oxidase, 5 mM ascorbic acid, 15 mM gallic acid ethyl ester, 6 mM water-soluble vitamin E (Trolox), 0.0001 wt% cyanuric acid, 10 μΜ 5'-adenylic acid, 10 μΜ guanosine-5'-monophosphate, 200 mM Tris, 20 mM NaCl, and 20 v / v% acetonitrile, 10 v / v% liquid paraffin, pH 7.5. And, when formulating the reagent, water-soluble vitamin E was dissolved with acetonitrile, and other components were added after being left in air for more than 10 h to complete the formulation.

[0216] Formulation 3: 150 mM glucose, 15 U / mL glucose oxidase, 10 mM ascorbic acid, 10 mM gallic acid propyl ester, 5 mM hydroquinone, 12 mM water-soluble vitamin E (Trolox), 0.0005 wt% cyanuric acid, 2 μΜ 5'-adenylic acid, 2 μΜ guanosine-5'-monophosphate, 250 mM Tris, and 20 v / v% acetonitrile, 10 v / v% liquid paraffin, pH 8.0.

[0217] Referring to any of the above examples, under the same other conditions, the three formulations were tested, and the results of sequencing the same sample using the three formulations were all good in quality, which was reflected in the similar read length, throughput, and error rate.

[0218] Example 36

[0219] The (original) base recipe disclosed in US 7,282,337 or US 7,666,593: 134 μΐ HEPES / NaCl, 24 μΐ 100 mM aqueous Vitamin E Trolox (buffered with 2-(N-morpholino)ethanesulfonic acid, MES, pH 6.1), 10 μΐ triethylenediamine DABCO (buffered with MES, pH 6.1), 8 μΐ 2 M glucose, 20 μΐ NaI (50 mM, buffered with water), 4 μΐ glucose oxidase, and 30% acetonitrile.

[0220] The new base recipe: does not contain 10 μΐ triethylenediamine DABCO (buffered with MES, pH 6.1), optionally contains or does not contain NaI, and the rest is the same as the above base recipe.

[0221] With reference to any of the above embodiments, the two recipes were tested under the same other conditions, and the results obtained from sequencing the same sample using the two recipes were similar to those disclosed in the patent literature, both showing that single molecule sequencing could be achieved in principle, but with shorter read length and higher error rate.

[0222] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "exemplary embodiment", "example", "certain examples", "specific examples" or "embodiments" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the exemplary description of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0223] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application. SEQUENCE LISTING <110> Shenzhen Zymabio Technology Co., Ltd. <120> Fluorescent detection reagent and preparation method and application thereof <130> PI2021001 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 78 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(73) <223> Target sequence <400> 1 ctagactgga cttgacgact tgcacgtagc tagctgggtg cggaagagaa agaataccat 60 gcagaaggag gcaaagta 78 <210> 2 <211> 77 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(77) <223> Target sequence <400> 2 gcagctaatg catgcgcatc tagagtcgta cgctgggtgc ggaagagaaa gaataccatg 60 cagaaggagg caaagta 77 <210> 3 <211> 78 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(73) <223> Target sequence <400> 3 tgacgtacct agactgagtc acgttcgacg atgctgggtg cggaagagaa agaataccat 60 gcagaaggag gcaaagta 78 <210> 4 <211> 78 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(73) <223> Target sequence <400> 4 tcatctatta tgccatctcc cttgtggaat cagctgggtg cggaagagaa agaataccat 60 gcagaaggag gcaaagta 78

Claims

1. A method of incorporating tagged nucleotides, characterized in that, The method comprises the following steps: (a) providing a hybridization complex, the hybridization complex being a hybrid of a primer and a template molecule, the primer being configured to hybridize to the 3' end of the template molecule, the template molecule being a single-stranded nucleic acid molecule; (b) placing a polymerase, a nucleotide analogue and the hybridization complex under conditions suitable for a polymerization reaction, to obtain an extension product by binding the nucleotide analogue to the hybridization complex, the nucleotide analogue comprising a linked sugar unit, a base, a cleavable blocking group and a fluorescent label; (c) replacing the solution system of (b) with a fluorescence detection reagent, the fluorescence detection reagent comprising an enzymatic oxygen scavenging system and a plurality of reducing agents and not comprising triethylenediamine, the fluorescence detection reagent comprising a combination of 6mM-12mM of a water-soluble vitamin E or a derivative thereof and 0.36mM-0.96mM of a quinone compound, and in addition to the 6mM-12mM of the water-soluble vitamin E or the derivative thereof in the aforementioned combination, the plurality of reducing agents in the fluorescence detection reagent further comprises at least one of 1mM-200mM of ascorbic acid, 1mM-20mM of gallic acid or an analogue or derivative of gallic acid and 0.0001%-0.001% of cyanuric acid by mass; (d) irradiating at least a portion of the hybridization complex in the presence of the fluorescence detection reagent and collecting at least a portion of a signal from the fluorescent label; (e) replacing the fluorescence detection reagent with a cleavage reagent for cleaving the cleavable blocking group and the fluorescent label on the extension product.

2. The method of claim 1, wherein, The polymerization reaction in (b) comprises two of the four nucleotide analogues with different fluorescent labels, wherein the four nucleotide analogues comprise dATP, dUTP or dTTP, dCTP and dGTP, two of the four nucleotide analogues with fluorescent label X and the other two with fluorescent label Y, the fluorescent label X and the fluorescent label Y being two fluorescent labels with different emission wavelengths.

3. The method of claim 1, wherein, The polymerization reaction in (b) comprises four nucleotide analogues, wherein the four nucleotide analogues comprise dATP, dUTP or dTTP, dCTP and dGTP, the four nucleotide analogues respectively with four fluorescent labels with different emission wavelengths.

4. The method of claim 1, wherein, The blocking group and the fluorescent label on the nucleotide analogue are located on the same side of the base.

5. The method of claim 4, wherein, The 3' position of the sugar unit on the nucleotide analogue is -OH.

6. The method of claim 1, wherein, The blocking group and the fluorescent label on the nucleotide analogue are located on different sides of the base.

7. The method of claim 6, wherein, The blocking group on the nucleotide analogue is located at the 3' position of the sugar unit.

8. The method of claim 1, wherein, Further comprising (f) performing (b)-(e) at least once.

9. The method of claim 1, wherein, The hybridization complex is attached to a substrate surface, (b) comprises irradiating the substrate surface with light of a specific wavelength to excite the fluorescent label on the substrate surface to emit fluorescence, and collecting at least a portion of the fluorescence from the fluorescent label.

10. The method of claim 9, wherein, The fluorescent label comprises at least one of ROX, ATTO532 and Alexafluor532, and at least one of CY5, IF700 and ATTO647N.

11. The method of claim 10, wherein, The fluorescent label comprises ATTO532 and ATTO647N.

12. The method according to any one of claims 9-11, characterized in that, The enzymatic oxygen removal system is selected from combination I, combination II or combination III, combination I comprises glucose and glucose oxidase, combination II comprises glucose, glucose oxidase and catalase, and combination III comprises protocatechuic acid and protocatechuate 3,4-dioxygenase.

13. The method of claim 12, wherein, The fluorescent detection reagent comprises combination I, ascorbic acid and gallic acid or an analogue or derivative of gallic acid.

14. The method according to claim 13, characterized in that The fluorescent detection reagent comprises 50mM-300mM glucose, 2U / mL-20U / mL glucose oxidase, 1mM-200mM ascorbic acid and 1mM-20mM gallic acid or an analogue or derivative of gallic acid.

15. The method of claim 14, wherein, The fluorescent detection reagent comprises 80mM-150mM glucose, 8U / mL-12U / mL glucose oxidase, 1mM-50mM ascorbic acid and 1mM-10mM gallic acid or an analogue or derivative of gallic acid.

16. The method of claim 13, wherein, The fluorescent detection reagent comprises a combination of 6mM-12mM water-soluble vitamin E and 0.36mM-0.96mM p-benzoquinone.

17. The method according to any one of claims 13-16, characterized by, The fluorescent detection reagent further comprises 0.0001%-0.001% cyanuric acid by mass percentage.

18. The method of claim 17, wherein, The mass percentage of cyanuric acid is 0.0003%-0.0009%.

19. The method according to any of claims 13-16 and 18, characterized by, The fluorescent detection reagent further comprises at least one of adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate and thymidine monophosphate.

20. The method of claim 17, wherein, The fluorescent detection reagent further comprises at least one of adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate and thymidine monophosphate.

21. The method of claim 19, wherein, The fluorescent detection reagent further comprises 1mM-50μM 5'-adenylic acid and 1μM-50μM guanosine-5'-monophosphate.

22. The method of claim 20, wherein, The fluorescent detection reagent further comprises 1mM-50μM 5'-adenylic acid and 1μM-50μM guanosine-5'-monophosphate.

23. The method of claim 21 or 22, wherein, The fluorescent detection reagent further comprises 4mM-12μM 5'-adenylic acid and 4μM-12μM guanosine-5'-monophosphate.

24. The method of claim 17, wherein, The fluorescent detection reagent adopts a Tris buffer system, and the pH of the fluorescent detection reagent is 6.5-8.

5.

25. The method of any one of claims 13-16, 18, and 20-22, wherein, The fluorescent detection reagent adopts a Tris buffer system, and the pH of the fluorescent detection reagent is 6.5-8.

5.

26. The method of claim 19, wherein, The fluorescent detection reagent adopts a Tris buffer system, and the pH of the fluorescent detection reagent is 6.5-8.

5.

27. The method according to any one of claims 13-16, 18, 20-22, 24 and 26, characterized in that, The fluorescent detection reagent further comprises acetonitrile, and the volume percentage of the acetonitrile is 10%-40%.

28. The method of claim 17, wherein, The fluorescent detection reagent further comprises acetonitrile, and the volume percentage of the acetonitrile is 10%-40%.

29. The method of claim 19, wherein, The fluorescent detection reagent further comprises acetonitrile, and the volume percentage of the acetonitrile is 10%-40%.

30. The method of claim 27, wherein, The fluorescent detection reagent further comprises acetonitrile, and the volume percentage of the acetonitrile is 18%-35%.

31. The method of claim 28 or 29, wherein, The fluorescent detection reagent further comprises acetonitrile, and the volume percentage of the acetonitrile is 18% to 35%.

32. The method of claim 27, wherein, The volume percentage of the acetonitrile is 28% to 35%.

33. The method of any one of claims 13-16, 18, 20-22, 24, 26, and 28-30, wherein, The fluorescent detection reagent comprises the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 1 mM to 15 mM gallic acid or gallic acid ethyl ester or gallic acid propyl ester, 6 mM to 15 mM water-soluble vitamin E, 0.36 mM to 0.96 mM p-benzoquinone, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 µM 5'-adenylic acid, 4 µM to 12 µM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

34. The method of claim 33, wherein, The fluorescent detection reagent comprises the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 10 mM to 15 mM gallic acid ethyl ester, 6 mM to 12 mM water-soluble vitamin E, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 µM 5'-adenylic acid, 4 µM to 12 µM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

35. The method of claim 33, wherein, The fluorescent detection reagent comprises the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 10 mM to 15 mM gallic acid propyl ester, 3 to 7 mM hydroquinone, 6 mM to 12 mM water-soluble vitamin E, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 µM 5'-adenylic acid, 4 µM to 12 µM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

36. The method of any one of claims 9-11, 13-16, 18, 20-22, 24, 26, 28-30, 32, 34, and 35, wherein, The irradiation time is 10-100 milliseconds.

37. The method of claim 36, wherein, The irradiation time is 50-100 milliseconds.

38. The method of any one of claims 11, 13-16, 18, 20-22, 24, 26, 28-30, 32, 34, and 35, wherein, In (d), the light intensity for irradiating ATTO532 is 30-40 milliwatts, and the light intensity for irradiating ATTO647N is 60-80 milliwatts.

39. The method of any one of claims 11, 13-16, 18, 20-22, 24, 26, 28-30, 32, 34, and 35, wherein, The duration of irradiating ATTO532 or ATTO647N in one field of view is 50-500 milliseconds.

40. The method of claim 39, wherein, The duration of irradiating ATTO532 or ATTO647N in one field of view is 50-100 milliseconds.

41. A fluorescent detection reagent, characterized in that, The fluorescent detection reagent comprises an enzymatic oxygen removal system and a plurality of reducing agents, and does not comprise triethylenediamine, the fluorescent detection reagent comprises a combination of 6mM-12mM water-soluble vitamin E or a derivative thereof and 0.36mM-0.96mM quinone, and in addition to 6mM-12mM water-soluble vitamin E or a derivative thereof in the foregoing combination, the plurality of reducing agents of the fluorescent detection reagent further comprises at least one of 1mM-200mM ascorbic acid, 1mM-20mM gallic acid or an analogue or derivative of gallic acid, and 0.0001%-0.001% by mass of cyanuric acid.

42. The agent of claim 41, wherein The enzymatic oxygen removal system is selected from combination I, combination II or combination III, combination I comprises glucose and glucose oxidase, combination II comprises glucose, glucose oxidase and catalase, and combination III comprises protocatechuic acid and protocatechuate 3,4-dioxygenase.

43. The agent of claim 42, wherein The fluorescent detection reagent comprises combination I, ascorbic acid and gallic acid or an analogue or derivative of gallic acid.

44. The agent of claim 43, wherein The fluorescent detection reagent comprises 50mM-300mM glucose, 2U / mL-20U / mL glucose oxidase, 1mM-200mM ascorbic acid and 1mM-20mM gallic acid or an analogue or derivative of gallic acid.

45. The agent of claim 44, wherein The fluorescent detection reagent comprises 80mM-150mM glucose, 8U / mL-12U / mL glucose oxidase, 1mM-200mM ascorbic acid and 1mM-10mM gallic acid or an analogue or derivative of gallic acid.

46. The agent of claim 42, wherein The fluorescent detection reagent comprises a combination of 6mM-12mM water-soluble vitamin E and 0.36mM-0.96mM p-benzoquinone.

47. The agent of any one of claims 43-46, wherein, The fluorescent detection reagent further comprises 0.0001%-0.001% by mass of cyanuric acid.

48. The agent of claim 47, wherein The mass percentage of the cyanuric acid is 0.0003%-0.0009%.

49. The agent of any of claims 43-46, 48, wherein, The fluorescent detection reagent further comprises at least one of adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate and thymidine monophosphate.

50. The agent of claim 49, wherein The fluorescent detection reagent further comprises 1mM-50μM 5'-adenylic acid and 1μM-50μM guanosine-5'-monophosphate.

51. The agent of claim 50, wherein The fluorescent detection reagent further comprises 4mM-12μM 5'-adenylic acid and 4μM-12μM guanosine-5'-monophosphate.

52. The agent of any one of claims 43-46, 48, 50, and 51, wherein, The fluorescent detection reagent adopts a Tris buffer system, and the pH of the fluorescent detection reagent is 6.5-8.

5.

53. The agent of any one of claims 43-46, 48, 50, and 51, wherein, The fluorescent detection reagent further comprises acetonitrile, and the volume percentage of the acetonitrile is 10%-40%.

54. The agent of claim 49, wherein The fluorescent detection reagent further comprises acetonitrile, and the volume percentage of the acetonitrile is 10%-40%.

55. The agent of claim 53, wherein The volume percentage of the acetonitrile is 18%-35%.

56. The agent of claim 55, wherein The volume percentage of the acetonitrile is 28%-35%.

57. The agent of any one of claims 43-46, 48, 50, 54-56, wherein, The fluorescent detection reagent comprises the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 1 mM to 15 mM gallic acid or gallic acid ethyl ester or gallic acid propyl ester, 6 mM to 15 mM water-soluble vitamin E, 0.36 mM to 0.96 mM p-benzoquinone, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 mM 5'-adenylic acid, 4 mM to 12 mM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

58. The agent of claim 57, wherein The fluorescent detection reagent comprises the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 10 mM to 15 mM gallic acid ethyl ester, 6 mM to 12 mM water-soluble vitamin E, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 mM 5'-adenylic acid, 4 mM to 12 mM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

59. The agent of claim 58, wherein The fluorescent detection reagent comprises the following components: 80 mM to 150 mM glucose, 8 U / mL to 12 U / mL glucose oxidase, 1 mM to 200 mM ascorbic acid, 10 mM to 15 mM gallic acid propyl ester, 3 to 7 mM hydroquinone, 6 mM to 12 mM water-soluble vitamin E, 0.0003 wt% to 0.0009 wt% cyanuric acid, 4 mM to 12 mM 5'-adenylic acid, 4 mM to 12 mM guanosine-5'-monophosphate, 100 mM to 300 mM tris-hydroxymethyl aminomethane, and 18 v / v% to 35 v / v% acetonitrile.

60. The agent of any one of claims 41-46, 48, 50, 54-56, 58, and 59, wherein, The fluorescent detection reagent further comprises liquid paraffin and / or silicone oil.

61. The agent of claim 60, wherein The fluorescent detection reagent comprises 8 to 20 v / v% of liquid paraffin and / or silicone oil.

62. A method of making the agent of any one of claims 41-61, wherein, Liquid paraffin and / or silicone oil is added before the fluorescent detection reagent is packaged.

63. The method of claim 62, wherein, 8 to 20 v / v% of liquid paraffin and / or silicone oil is added.

64. Use of the reagent of any one of claims 41-61 in detecting a fluorescent signal.

65. A kit comprising, The fluorescent detection reagent and the nucleotide analogue in the method of any one of claims 1-40.

66. A mixture system, characterized in that, It comprises a hybridization complex placed in a solution, the hybridization complex being a hybrid of a primer and a template molecule, the solution being the reagent of any one of claims 41-61.

67. The mixture system of claim 66, wherein, The hybridization complex is immobilized on the surface of a solid phase substrate.

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