Nucleic acid amplification product detection reagent and detection method

Abstract

The present invention discloses a nucleic acid amplification product detection reagent and a detection method, and the detection reagent is prepared by the following method: 1) using cellobiose and 1,3-propanedithiol as carbon sources, and preparing carbon quantum dots by a hydrothermal method; 2) modifying copper ions on the surface of carbon quantum dots to obtain the detection reagent; when there is a byproduct pyrophosphate generated by the nucleic acid amplification reaction, the fluorescence of the carbon quantum dots will be enhanced, so that the solution obtained after the reaction of the detection reagent with the product of nucleic acid amplification is subjected to fluorescence detection, and the qualitative and / or quantitative detection of the nucleic acid amplification product is realized. The present invention provides a nucleic acid amplification product detection reagent and a method, and a reagent that can be used for the detection of pyrophosphate, a byproduct of nucleic acid amplification, is prepared by using copper ions to modify carbon quantum dots, and is successfully applied to the detection of nucleic acid amplification products, and can realize the qualitative and quantitative detection of nucleic acid amplification products, and it has the advantages of being simple and convenient, fast and intuitive, and highly prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a nucleic acid amplification product detection reagent and a detection method. Background Art

[0002] Currently, nucleic acid amplification technology is widely used in food safety testing, environmental monitoring, medical diagnosis, forensic identification and other fields. The earliest and most classic nucleic acid amplification technology is to achieve template denaturation, primer annealing and extension under the action of heat-resistant polymerase, relying on the continuous cycle of temperature to achieve a large amount of target product accumulation. This technology is called polymerase chain reaction (PCR).

[0003] Since its emergence, isothermal amplification technology has been widely used due to its rapid and efficient characteristics, and is considered to be a nucleic acid amplification detection method comparable to PCR. Common isothermal amplification detection technologies include: loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nickase-mediated amplification reaction (NEAR), etc. Since this type of new amplification technology can achieve nucleic acid amplification under relatively low constant temperature conditions (most isothermal amplification technologies require a reaction temperature of around 60°C or 37°C), it gets rid of the precise temperature control equipment required by traditional PCR, greatly reduces costs, and provides great convenience for on-site rapid detection.

[0004] Whether it is PCR or constant temperature amplification technology, the qualitative and quantitative detection of amplification products is of great significance to the development and application of these technologies. The detection of nucleic acid amplification products includes electrophoresis, fluorescence detection, etc. Visual detection is a qualitative detection method of amplification products commonly used in nucleic acid constant temperature amplification. The principle of one of the visualization methods is to detect the byproduct pyrophosphate generated in the nucleic acid amplification reaction. Each time a nucleic acid chain is amplified and extended by one base, a molecule of pyrophosphate ion will be released. The pyrophosphate is decomposed into phosphate ions, and then a phosphoric acid colorimetric reagent is added. The occurrence of a colorimetric reaction indicates that the sample is positive. For example, patent CN 104195040 B discloses a reaction device and method for visual detection of nucleic acid constant temperature amplification, which can judge the detection result by observing the color change produced by the colorimetric reagent reaction with the naked eye, and can realize visual detection, which has the advantages of being simple, convenient, intuitive and fast. However, this type of nucleic acid detection scheme has at least the following shortcomings: 1. This scheme can only realize qualitative detection, not quantitative detection; 2. Its principle is to determine whether the target gene exists by detecting the phosphate produced by the decomposition of pyrophosphate by pyrophosphatase, rather than directly detecting the pyrophosphate by-product of isothermal amplification. Therefore, the activity of pyrophosphatase and the decomposition of pyrophosphate will affect the test results, which can easily cause large errors (especially when the amplification temperature is high).

[0005] The fluorescence detection and analysis method can qualitatively detect the presence of the target through the fluorescence color, and can further realize the quantitative detection of the target through the fluorescence intensity. By using the fluorescence analysis of the nucleic acid amplification product, the fluorescent substance that can produce a fluorescent reaction with the pyrophosphate byproduct of nucleic acid amplification or the phosphate obtained by decomposition of pyrophosphate can realize the qualitative and quantitative detection of the nucleic acid amplification product. Carbon quantum dots (CDs) are a new type of carbon nanomaterial that has emerged in recent years. Its special properties make it possible to use fluorescent substances for biological detection. Similarly, carbon quantum dots have the potential to detect pyrophosphate. For example, patent CN109957399B discloses an iron-doped carbon quantum dot and a preparation method thereof, a peroxide mimetic enzyme composition and its application, and a detection method for p-phenylenediamine, which can use iron-doped carbon quantum dots for the detection of pyrophosphate; patent CN113105892A discloses a copper-doped carbon quantum dot and a preparation method thereof and its application as a probe and a test paper and a method for detecting the pH of a liquid, which can use copper-doped carbon quantum dots for the detection of pyrophosphate. The main principle of using carbon quantum dots to detect pyrophosphate in these patents is that the fluorescence of carbon quantum dots is weakened due to copper / iron doping. When pyrophosphate is present, pyrophosphate competes with carbon quantum dots for copper / iron, which ultimately enhances and restores the fluorescence of carbon quantum dots, thereby achieving pyrophosphate detection through fluorescence measurement. Based on this principle, when metal ions such as copper / iron that affect the fluorescence performance of carbon dots exist in the sample, these metal ions will inevitably affect the detection results.

[0006] It can be seen that although the prior art discloses a scheme for using carbon quantum dots to realize pyrophosphate detection, there is no disclosed reliable scheme for applying carbon quantum dots to nucleic acid isothermal amplification detection; on the other hand, metal ions such as ferric ions, ferrous ions, and cupric ions are usually inevitably present in nucleic acid samples, and these metal ions will weaken the fluorescence properties of carbon dots. Therefore, when using this type of carbon quantum dot scheme in the prior art for nucleic acid isothermal amplification detection, it is also necessary to solve the problem of metal ion interference in nucleic acid samples. Currently, the existing schemes lack a reliable solution to this problem.

[0007] Therefore, it is necessary to improve the existing technology to provide a more reliable solution. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a nucleic acid amplification product detection reagent and a detection method in view of the deficiencies in the above-mentioned prior art.

[0009] In order to solve the above technical problems, the first aspect of the present invention is to provide a nucleic acid amplification product detection reagent, which is prepared by the following method:

[0010] 1) Using cellobiose and 1,3-propanedithiol as carbon sources, carbon quantum dots were prepared by a hydrothermal method;

[0011] 2) modifying the copper ions on the surface of the carbon quantum dots to obtain the detection reagent;

[0012] The carbon quantum dots prepared in step 1) are EX The carbon quantum dots will emit fluorescence under the irradiation of excitation light, and after the copper ions are modified in step 2), the fluorescence of the carbon quantum dots will be weakened or even quenched; when the by-product pyrophosphate generated by the nucleic acid amplification reaction is present, the fluorescence of the carbon quantum dots will be enhanced, thereby achieving qualitative and / or quantitative detection of the nucleic acid amplification product by performing fluorescence detection on the solution obtained after the detection reagent reacts with the product of nucleic acid amplification.

[0013] Preferably, the step 1) specifically includes:

[0014] 1-1) dissolving 1,3-propanedithiol in anhydrous ethanol and stirring to obtain solution A; dissolving cellobiose in ultrapure water and stirring to obtain solution B;

[0015] 1-2) mixing solution A and solution B, dispersing by ultrasonication, and stirring to obtain a mixture C;

[0016] 1-3) Transferring the mixture C to a hydrothermal reactor and reacting under heating;

[0017] 1-4) After the reaction is completed, the mixture is cooled and centrifuged, the centrifuge liquid is dialyzed, the solution in the dialysis bag is collected, and the solution is freeze-dried to obtain carbon quantum dots.

[0018] Preferably, the step 1) specifically includes:

[0019] 1-1) dissolving 1,3-propanedithiol in anhydrous ethanol and stirring to obtain solution A; dissolving cellobiose in ultrapure water and stirring to obtain solution B;

[0020] 1-2) Mix solution A and solution B, disperse by ultrasonic for 5-30 min, and stir for 2-15 min to obtain mixture C;

[0021] 1-3) Transferring the mixture C to a hydrothermal reactor lined with polytetrafluoroethylene, and reacting at 150-195° C. for 5-18 hours;

[0022] 1-4) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 8000-15000 rpm for 2-15 min. The centrifuged liquid is dialyzed with a dialysis bag with a molecular weight cutoff of 500-1200 Da for 6-20 hours. The solution in the dialysis bag is collected and freeze-dried to obtain carbon quantum dots.

[0023] Preferably, the step 1) specifically includes:

[0024] 1-1) dissolving 1,3-propanedithiol in anhydrous ethanol and stirring to obtain solution A; dissolving cellobiose in ultrapure water and stirring to obtain solution B;

[0025] 1-2) Mix solution A and solution B, disperse by ultrasonic for 5 minutes, and stir for 4 minutes to obtain mixture C;

[0026] 1-3) Transferring the mixture C to a hydrothermal reactor lined with polytetrafluoroethylene and reacting at 185° C. for 12 h;

[0027] 1-4) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 10000 rpm for 5 min. The centrifuged liquid is dialyzed with a dialysis bag with a molecular weight cutoff of 1000 Da for 12 hours. The solution in the dialysis bag is collected and freeze-dried to obtain carbon quantum dots.

[0028] Preferably, the step 1) specifically includes:

[0029] 1-1) dissolving 0.5 g of 1,3-propanedithiol in 50 mL of anhydrous ethanol and stirring to obtain solution A; dissolving 0.64 g of cellobiose in 50 mL of ultrapure water and stirring to obtain solution B;

[0030] 1-2) Mix solution A and solution B, disperse by ultrasonic for 5 minutes, and stir for 4 minutes to obtain mixture C;

[0031] 1-3) Transfer the mixture C to a 200 mL polytetrafluoroethylene-lined hydrothermal reactor and react at 185° C. for 12 h;

[0032] 1-4) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 10000 rpm for 5 min. The centrifuged liquid is dialyzed with a dialysis bag with a molecular weight cutoff of 1000 Da for 12 hours. The solution in the dialysis bag is collected and freeze-dried to obtain carbon quantum dots.

[0033] Preferably, the step 2) is specifically as follows: redissolving the carbon quantum dots obtained in step 1) in ultrapure water to obtain a carbon quantum dot solution, and then adding Cu 2+ The solution is reacted, and after the reaction is completed, centrifugation is performed and the filtrate is freeze-dried to obtain the detection reagent.

[0034] Preferably, the step 2) is specifically as follows: 0.05-0.3 g of the carbon quantum dots prepared by the method of step 1) is redissolved in 50-100 ml of ultrapure water, ultrasonically dispersed for 2-8 min to obtain a carbon quantum dot solution, and then 5-50 ml of Cu is added to the carbon quantum dot solution. 2+ The concentration of Cu is 10 mM 2+ The solution is reacted, and after the reaction is completed, centrifugation is performed and the filtrate is freeze-dried to obtain the detection reagent.

[0035] Preferably, the Cu 2+ The solution is a mixture of one or more of copper sulfate, copper nitrate, copper chloride and copper acetate solutions.

[0036] The second aspect of the present invention is to provide a method for detecting nucleic acid amplification products, which method uses the above-mentioned detection reagent to detect the product after nucleic acid amplification of the sample to be tested, so as to perform qualitative and / or quantitative detection of the target in the sample to be tested.

[0037] Preferably, the detection method comprises the following steps:

[0038] Ⅰ. Conduct quality control tests on test reagents:

[0039] Ⅰ-1. Pre-prepare the concentration to C T The detection reagent solution, concentration C p Sodium pyrophosphate solution, nucleic acid amplification reaction solution;

[0040] The detection reagent solution was divided into three parts, each with a volume of V T , take one portion as the test solution for the detection reagent, and take two portions as the quality control solution for the detection reagent;

[0041] Divide the sodium pyrophosphate solution into two equal parts, each with a volume of V p ;

[0042] The nucleic acid amplification reaction solution is divided into two parts, and the sample to be tested is divided into two parts;

[0043] Ⅰ-2. Mix one portion of sodium pyrophosphate solution and one portion of test reagent quality control solution evenly to react. After the reaction is completed, use a wavelength of λ EX The mixed solution 1 obtained after the reaction is irradiated with an excitation light source, and the fluorescence color of the mixed solution 1 is observed;

[0044] When the fluorescent color of the mixed solution 1 matches the fluorescent color that should be exhibited after the detection reagent reacts with pyrophosphate, it is determined that the qualitative detection of the detection reagent is effective;

[0045] Then measure the mixed solution 1 at a wavelength of λ Em The fluorescence intensity at the wavelength λ is determined according to the pre-established characterization test reagent. Em The calculated concentration of pyrophosphate C was calculated from the standard curve f1 of the relationship between the fluorescence intensity at the p ′, compare C p ′ and the actual concentration of pyrophosphate C p The relationship between η and C p ′-C p ) / C p , when η≤ηT When the detection reagent is determined to be effective for quantitative detection, wherein η T is the set threshold, and 0<η T <0.15;

[0046] When it is determined that both the qualitative and quantitative tests of the detection reagent are effective, proceed to the following step II;

[0047] Ⅱ. Detect the nucleic acid amplification products of the samples to be tested:

[0048] Ⅱ-1. A sample to be tested is mixed with a nucleic acid amplification reaction solution and a nucleic acid amplification reaction is performed. After the amplification is completed, a detection reagent test solution is added to the nucleic acid amplification product to react. After the reaction is completed, a mixed solution 2 is obtained. The wavelength is λ EX The excitation light source illuminates the mixed solution 2, and the fluorescence color of the mixed solution 2 is observed. When the fluorescence color of the mixed solution 2 meets the fluorescence color that should be reflected after the detection reagent reacts with the pyrophosphate, it is determined that the target object exists in the sample to be tested, otherwise it is determined that the target object does not exist, thereby achieving qualitative detection of the sample to be tested;

[0049] Ⅱ-2. When it is determined that the target substance exists in the sample to be tested, quantitative detection is performed:

[0050] Measure the mixed liquid 2 at a wavelength of λ EX Under the irradiation of the excitation light source at λ Em The fluorescence intensity at c ;

[0051] Ⅱ-3. Calculate the fluorescence intensity correction value ΔP:

[0052] Take the remaining test reagent quality control solution and measure its EX Under the irradiation of the excitation light source at λ Em The fluorescence intensity at b1 ;

[0053] Then, the detection reagent quality control solution is evenly mixed with a sample to be tested and a nucleic acid amplification reaction solution to obtain a mixed solution 3, and the mixed solution 3 is measured at a wavelength of λ EX Under the irradiation of the excitation light source at λ Em The fluorescence intensity at b2 ; Fluorescence intensity correction ΔP = P b1 -P b2 ;

[0054] Ⅱ-4. Calculate the concentration of the target substance:

[0055] The P obtained in step II-2 c The value of P is corrected to c ',P c '=Pc+ ΔP, and then the fluorescence intensity is calculated as P through the pre-established standard curve f2 that characterizes the relationship between the fluorescence intensity of the solution obtained after the reaction of the detection reagent and the nucleic acid amplification product of the sample to be tested and the concentration of the target in the sample to be tested. c ', and C1' is taken as the target concentration in the sample to be tested.

[0056] The beneficial effects of the present invention are:

[0057] The present invention provides a nucleic acid amplification product detection reagent and a detection method. The present invention uses copper ion-modified carbon quantum dots to prepare a reagent that can be used for nucleic acid amplification byproduct pyrophosphate detection, and is successfully applied to nucleic acid amplification product detection, which can achieve qualitative and quantitative detection of nucleic acid amplification products, and has the advantages of being simple, convenient, rapid, intuitive, and highly ready.

[0058] The present invention can overcome the defect that the conventional visual detection can only be qualitative but not quantitative, and the present invention directly detects the pyrophosphate, which can avoid the defect of increased error that may be caused by the solution of indirectly detecting phosphate to determine the pyrophosphate.

[0059] In the method provided by the present invention, the attenuation of the fluorescence intensity of the detection reagent caused by some interfering metal ions present in the nucleic acid sample can be calculated, and the fluorescence measurement result of the solution after the detection reagent reacts with the amplification product can be corrected, so that the error caused by this part of the interfering metal ions to the detection result can be deducted, and the background interference caused by the interfering metal ions in the nucleic acid sample can be basically eliminated, and finally the accuracy of quantitative detection can be improved;

[0060] In the method provided by the present invention, the effectiveness of qualitative detection and quantitative detection of the detection reagent is judged in advance, which can not only ensure the accuracy of the detection result, but also avoid useless detection caused by the failure of the detection reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a transmission electron microscope photograph of CDs prepared in Example 1 of the present invention;

[0062] Figure 2 The fluorescence excitation spectrum and fluorescence emission spectrum of CDs prepared in Example 1 of the present invention;

[0063] Figure 3 The infrared spectrum of CDs prepared in Example 1 of the present invention;

[0064] Figure 4 The relationship between the fluorescence intensity and copper ion concentration of CDs prepared in Example 1 of the present invention;

[0065] Figure 5The relationship between the fluorescence intensity of CDs prepared in Example 1 of the present invention and the mixed concentration of iron and copper ions;

[0066] Figure 6 The relationship between the fluorescence intensity and the pyrophosphate ion concentration of the CDs prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0068] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0069] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0070] The present invention provides a nucleic acid amplification product detection reagent, characterized in that the detection reagent is prepared by the following method:

[0071] 1) Using cellobiose and 1,3-propanedithiol as carbon sources, carbon quantum dots were prepared by a hydrothermal method; specifically:

[0072] 1-1) dissolving 1,3-propanedithiol in anhydrous ethanol and stirring to obtain solution A; dissolving cellobiose in ultrapure water and stirring to obtain solution B;

[0073] 1-2) Mix solution A and solution B, disperse by ultrasonic for 5-30 min, and stir for 2-15 min to obtain mixture C;

[0074] 1-3) Transferring the mixture C to a hydrothermal reactor lined with polytetrafluoroethylene, and reacting at 150-195° C. for 5-18 hours;

[0075] 1-4) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 8000-15000 rpm for 2-15 min. The centrifuged liquid is dialyzed with a dialysis bag with a molecular weight cutoff of 500-1200 Da for 6-20 hours. The solution in the dialysis bag is collected and freeze-dried to obtain carbon quantum dots.

[0076] 2) The copper ions are modified on the surface of the carbon quantum dots to obtain the detection reagent, specifically:

[0077] Take 0.05-0.3g of the carbon quantum dots prepared by the method of step 1) and redissolve them in 50-100ml of ultrapure water, and ultrasonically disperse them for 2-8min to obtain a carbon quantum dot solution. Then, add 5-50ml of Cu 2+ The concentration of Cu is 10 mM 2+ The solution is reacted, and after the reaction is completed, centrifugation is performed and the filtrate is freeze-dried to obtain the detection reagent. 2+ The solution is a mixture of one or more of copper sulfate, copper nitrate, copper chloride and copper acetate solutions. When used, the detection reagent is prepared into a solution of a certain concentration using deionized water.

[0078] The carbon quantum dots prepared in step 1) are EX The carbon quantum dots will emit fluorescence under the irradiation of excitation light, and after the copper ions are modified in step 2), the fluorescence of the carbon quantum dots will be weakened or even quenched; when the by-product pyrophosphate generated by the nucleic acid amplification reaction is present, the fluorescence of the carbon quantum dots will be enhanced, thereby achieving qualitative and / or quantitative detection of the nucleic acid amplification product by performing fluorescence detection on the solution obtained after the detection reagent reacts with the product of nucleic acid amplification.

[0079] Specifically, for positive samples, nucleic acid amplification reaction will be carried out and pyrophosphate will be produced as a byproduct. The pyrophosphate will enhance the fluorescence of the detection reagent and restore the fluorescence. EX Under the irradiation of excitation light, it will emit obvious fluorescence of a specific color, and the fluorescence intensity is positively correlated with the concentration of the target in the sample; for negative samples, no pyrophosphate is produced during the entire reaction process, and the detection reagent has no fluorescence or the fluorescence is very weak, which can be easily distinguished by the naked eye.

[0080] The present invention uses cellobiose and 1,3-propanedithiol as carbon sources and prepares carbon dots with rich surface functional groups such as thiol, hydroxyl, and carbonyl by a bottom-up hydrothermal method. The introduction of rich oxygen-containing functional groups makes the carbon dots have good water solubility. The carbon dots can emit bright blue fluorescence under 355nm wavelength excitation light, and the fluorescence emission peak is at 438nm. With the help of the rich surface photoactive groups such as thiol, hydroxyl, carbonyl, and carboxyl, the carbon dots can bind to a variety of heavy metal ions (copper ions, iron ions, copper ions, mercury ions, nickel ions Ni 2+ The coordination / complexation of these heavy metal ions with the carbon dots will weaken or even quench the fluorescence of the carbon dots.

[0081] Based on the above characteristics of the carbon dots, the present invention further modifies the copper ions on the surface of the carbon dots through the coordination of copper ions and carbon dots to obtain a detection reagent that can be used for nucleic acid amplification product detection: copper ion-modified carbon dots, the detection reagent retains the good water solubility of the carbon dots, and its fluorescence intensity is weakened due to the surface modification of copper ions; when pyrophosphate is present, the pyrophosphate can chelate with the copper ions and combine, and the chelation of pyrophosphate with copper ions is stronger than the coordination of carbon dots with copper ions, so that the pyrophosphate competes for the copper ions on the detection reagent, and finally the fluorescence recovery of the detection reagent is enhanced, and the fluorescence enhancement amount is positively correlated with the concentration of pyrophosphate within a certain range, so the detection of pyrophosphate can be achieved by fluorescence intensity detection through the detection reagent; pyrophosphate is a by-product of the nucleic acid amplification reaction, and finally the qualitative and quantitative detection of the nucleic acid amplification reaction can be achieved through the detection of pyrophosphate.

[0082] When preparing the detection reagent, the fluorescence intensity of the obtained detection reagent can be controlled by controlling the ratio of carbon dots and copper ions, that is, the greater the ratio of copper ions, the weaker the fluorescence. In a preferred embodiment, by controlling the ratio of copper ions, the detection reagent still has a certain fluorescence intensity, and a small amount of copper ions can still be bound to its surface, but compared with the carbon dots without copper ions, the fluorescence intensity of the two is significantly different, ensuring that they can be distinguished by naked eye observation.

[0083] In the following embodiments, the detection of nucleic acid isothermal amplification products is taken as an example for the convenience of description, but it should be understood that the detection reagent and detection method of the present invention can also be applied to the detection of non-isothermal amplification products.

[0084] The amplification reaction solution in the present invention is a conventional product and is selected according to the specific detection object. The amplification reaction solution generally includes primers, dNTPs, Tris-HCl buffer, Mg 2+ , amplification template, polymerase, etc.

[0085] The above is the general concept of the present invention. The detection reagent, the structure of the isothermal amplification detection device and the specific detection method of the present invention are further described below in combination with specific embodiments.

[0086] Example 1

[0087] A nucleic acid amplification product detection reagent is prepared by the following method:

[0088] 1. Using cellobiose and 1,3-propanedithiol as carbon sources, carbon quantum dots were prepared by hydrothermal method; specifically:

[0089] 1-1. Dissolve 0.5 g of 1,3-propanedithiol in 50 mL of anhydrous ethanol and stir to obtain solution A; dissolve 0.64 g of cellobiose in 50 mL of ultrapure water and stir to obtain solution B;

[0090] 1-2. Mix solution A and solution B, disperse by ultrasonic for 5 minutes, and stir for 4 minutes to obtain mixture C;

[0091] 1-3. Transfer the mixture C to a 200 mL polytetrafluoroethylene-lined hydrothermal reactor and react at 185° C. for 12 h;

[0092] 1-4. After the reaction was completed, the mixture was cooled to room temperature and centrifuged at 10000 rpm for 5 min. The centrifuged liquid was dialyzed with a dialysis bag with a molecular weight cutoff of 1000 Da for 12 hours. The solution in the dialysis bag was collected and freeze-dried to obtain carbon quantum dots (CDs).

[0093] 2. The copper ions are modified on the surface of carbon quantum dots to obtain the detection reagent, specifically:

[0094] Take 0.1g of the carbon quantum dots prepared by the method in step 1 and dissolve it in 80ml of ultrapure water, and ultrasonically disperse it for 5min to obtain a carbon quantum dot solution. Then, add 20ml of Cu 2+ The concentration of Cu is 10 mM 2+ The solution was reacted, and after the reaction was completed, centrifugation was performed, and the filtrate was freeze-dried to obtain the detection reagent: copper ion modified carbon dots (Cu-CDs), which were stored at -10°C. 2+ The solution is a copper acetate solution. When used, the detection reagent is prepared into a detection reagent of a certain concentration (such as 0.1-2.0 mg / mL) using deionized water and stored at 0-2°C for use.

[0095] 1. Perform the following performance tests on the carbon quantum dots CDs obtained in step 1:

[0096] Reference Figure 1 , is the transmission electron microscope photo of CDs, it can be seen that the particle size is relatively uniform, about 4-8nm;

[0097] Reference Figure 2 , which are the fluorescence excitation spectrum (Ex) and fluorescence emission spectrum (Em) of CDs. It can be seen that its excitation peak is at 355nm and the emission peak is at 438nm. It can emit bright blue fluorescence under the excitation light of 355nm.

[0098] Reference Figure 3 , which is the infrared spectrum of CDs, indicating that the carbon quantum dots CDs have functional groups such as thiol (-SH), hydroxyl (OH), carbonyl (C=O), and carboxyl (COOH).

[0099] Reference Figure 4 , which is the fluorescence intensity change result after a series of copper ion solutions are added to the CDs solution, where the CDs solution concentration is 0.2 mg / mL, the copper ion solution is a copper acetate aqueous solution with concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mM, and the concentration of 0 represents a pure deionized water solution. It can be seen that with the increase of copper ion concentration, the fluorescence intensity of CDs gradually decreases.

[0100] Reference Figure 5 , is the fluorescence intensity change result after a series of concentrations of iron ion and copper ion mixed solution are added to the CDs solution (fluorescence intensity at 438nm under 355nm excitation light), wherein the concentration of carbon quantum dot solution is 0.2mg / mL, and the concentration of mixed solution is: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10mM aqueous solution of iron acetate and copper acetate, and the concentration ratio of iron acetate to copper acetate is 1:1, and the concentration of 0 is represented by pure deionized water solution. It can be seen that with the increase of the concentration of the mixed metal ion solution, the fluorescence intensity of CDs gradually decreases. With the total concentration of iron acetate and copper acetate as the X-axis and the fluorescence intensity of CDs as the Y-axis, curve fitting is performed, and the standard curve obtained is: y=-0.248x+3019, R 2 =0.998; using this curve, the total concentration of iron ions and copper ions (y) can be calculated by the fluorescence intensity (x) of CDs.

[0101] 2. Detect the fluorescence enhancement performance of the copper ion modified carbon dots (Cu-CDs) obtained in step 2 by pyrophosphate ions (PPi):

[0102] Reference Figure 6 , which is the fluorescence intensity change result after a series of concentrations of pyrophosphate ion solution are added to the Cu-CDs solution (fluorescence intensity at 438nm under 355nm excitation light), wherein the concentration of Cu-CDs solution is 0.2mg / mL, the pyrophosphate (PPi) ion solution is a sodium pyrophosphate solution with concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0mM, and the concentration of 0 represents a deionized water solution. It can be seen that with the increase of the concentration of the mixed pyrophosphate solution, the fluorescence intensity of Cu-CDs gradually increases. With the concentration of sodium pyrophosphate as the X-axis and the fluorescence intensity of Cu-CDs as the Y-axis, curve fitting is performed, and the obtained standard curve (i.e., f1) is: y=5.502x+481, R 2=0.997; The concentration of sodium pyrophosphate (y) can be calculated by using the curve through the fluorescence intensity (x) of the Cu-CDs. Therefore, the concentration of pyrophosphate can be detected by Cu-CDs, and finally the detection of nucleic acid amplification products can be realized.

[0103] Example 2

[0104] A method for detecting a nucleic acid amplification product, wherein the method uses the detection reagent of Example 1 to detect the product after nucleic acid amplification of a sample to be tested, so as to perform qualitative and / or quantitative detection of a target in the sample to be tested, and the detection method comprises the following steps:

[0105] Ⅰ. Conduct quality control tests on test reagents:

[0106] Ⅰ-1. Pre-prepare the concentration to C T The detection reagent solution, concentration C p Sodium pyrophosphate solution, nucleic acid amplification reaction solution;

[0107] The detection reagent solution was divided into three parts, each with a volume of V T , take one portion as the test solution for the detection reagent, and take two portions as the quality control solution for the detection reagent;

[0108] Divide the sodium pyrophosphate solution into two equal parts, each with a volume of V p ;

[0109] The nucleic acid amplification reaction solution is divided into two parts, and the sample to be tested is divided into two parts;

[0110] Ⅰ-2. Mix one portion of sodium pyrophosphate solution and one portion of test reagent quality control solution evenly to react. After the reaction is completed, use a wavelength of λ EX The mixed solution 1 obtained after the reaction is irradiated with an excitation light source, and the fluorescence color of the mixed solution 1 is observed;

[0111] When the fluorescent color of the mixed solution 1 matches the fluorescent color that should be exhibited after the detection reagent reacts with pyrophosphate, it is determined that the qualitative detection of the detection reagent is effective;

[0112] Then measure the mixed solution 1 at a wavelength of λ Em The fluorescence intensity at the wavelength λ is determined according to the pre-established characterization test reagent. Em The calculated concentration of pyrophosphate C was calculated from the standard curve f1 of the relationship between the fluorescence intensity at the p ′, compare C p ′ and the actual concentration of pyrophosphate C p The relationship between η and C p ′-C p ) / C p , when η≤η TWhen the detection reagent is determined to be effective for quantitative detection, wherein η T is the set threshold, and 0<η T <0.15; η T That is, it represents the allowable difference threshold. The smaller η is, the closer the detection reagent is to the standard, and the higher the accuracy level of the detection reagent is.

[0113] In this embodiment, the excitation wavelength λ EX =355nm, emission wavelength λ Em =438nm; f1 is obtained through Example 1, specifically: 5.502x+481, R 2 =0.997; η T =0.05. In this embodiment, when the detection reagent reacts with a pyrophosphate solution of known concentration, the detection reagent is considered to be effective for quantitative detection only when the concentration value of pyrophosphate calculated by the standard curve f1 and its actual value are within the allowable error range, that is, the detection reagent is considered to meet the requirements (not deteriorated or the concentration meets the required range, etc.), and the difference with the standard is within the allowable range. This result is used as a quality control indicator to ensure the accuracy of the quantitative detection result.

[0114] When it is determined that both the qualitative and quantitative tests of the detection reagent are effective, proceed to the following step II;

[0115] Ⅱ. Detect the nucleic acid amplification products of the samples to be tested:

[0116] II-1. A sample to be tested is mixed with a nucleic acid amplification reaction solution and a nucleic acid amplification reaction is performed. After the amplification is completed, a detection reagent test solution is added to the product after the nucleic acid amplification for reaction. After the reaction is completed, a mixed solution 2 is obtained. An excitation light source with a wavelength of 355 nm is used to irradiate the mixed solution 2, and the fluorescence color of the mixed solution 2 is observed. When the fluorescence color of the mixed solution 2 is consistent with the fluorescence color that should be reflected after the reaction of the detection reagent and pyrophosphate, it is determined that the target object exists in the sample to be tested, otherwise it is determined that the target object does not exist, thereby achieving qualitative detection of the sample to be tested;

[0117] Ⅱ-2. When it is determined that the target substance exists in the sample to be tested, quantitative detection is performed:

[0118] Measure the fluorescence intensity of the mixed solution 2 at 438 nm under the irradiation of the excitation light source with a wavelength of 355 nm, and record it as P c ;

[0119] Ⅱ-3. Calculate the fluorescence intensity correction value ΔP:

[0120] Take the remaining test reagent quality control solution and measure its fluorescence intensity at 438nm under the excitation light source with a wavelength of 355nm, which is recorded as P b1 ;

[0121] Then, the detection reagent quality control solution is evenly mixed with a sample to be tested and a nucleic acid amplification reaction solution to obtain a mixed solution 3, and the mixed solution 3 is measured at a wavelength of λ EX Under the irradiation of the excitation light source at λ Em The fluorescence intensity at b2 ; Fluorescence intensity correction ΔP = P b1 -P b2 ;

[0122] In this embodiment, the purpose of calculating the fluorescence intensity correction amount ΔP is to eliminate background interference caused by some metal ions in the nucleic acid sample that can weaken the fluorescence of the detection reagent;

[0123] Generally, it is inevitable that there will be a certain amount of metal ions such as ferric ions, ferrous ions, and copper ions in nucleic acid samples. These ions can bind to the surface of the detection reagent through coordination / complexation to weaken its fluorescence intensity (while other metal ions present in nucleic acid samples, such as sodium ions, potassium ions, etc., will not affect the fluorescence of the detection reagent). Although the detection reagent has been modified with copper ions, resulting in reduced fluorescence, since the copper ions that react with carbon quantum dots during the preparation process are not excessive, there will still be a small amount of active functional groups (thiol, hydroxyl or carbonyl) that are not bound to metal ions on the detection reagent, so the detection reagent can also bind a small amount of metal ions (ferric ions, ferrous ions, copper ions), so its fluorescence intensity will continue to decline to a certain extent; when the fluorescence of the detection reagent is restored by pyrophosphate, these ferric ions, ferrous ions, and copper ions present in the nucleic acid sample will consume a certain amount of pyrophosphate, so that the pyrophosphate actually produced during the isothermal amplification process is larger than the measured amount, causing errors in nucleic acid quantification;

[0124] In this embodiment, the reduction of fluorescence intensity ΔP caused by these interfering metal ions is calculated, and P b1 Corrected to P b1 ', so that the error caused by this part of interfering metal ions on the detection result can be deducted, thereby basically eliminating the background interference caused by these metal ions in the nucleic acid sample.

[0125] Ⅱ-4. Calculate the concentration of the target substance:

[0126] The P obtained in step II-2 c The value of P is corrected to c ',P c '=P c+ ΔP, and then the fluorescence intensity is calculated as P through the pre-established standard curve f2 that characterizes the relationship between the fluorescence intensity of the solution obtained after the reaction of the detection reagent and the nucleic acid amplification product of the sample to be tested and the concentration of the target in the sample to be tested. c', and C1' is taken as the target concentration in the sample to be tested.

[0127] The construction method of the standard curve f2 is similar to that of f1. Different target nucleic acids have different standard curves f2. No specific examples are given in this embodiment, and only a brief description of the construction method is given. The method for obtaining f2 is: a series of concentrations of target nucleic acids are prepared in advance, and then mixed with the amplification reaction solution, and amplified at a constant temperature in the amplification system, and then the amplification product is mixed with a certain concentration of a detection reagent, and the fluorescence intensity of the detection mixture at 438nm under 355nm excitation light; finally, the target nucleic acid concentration is used as the X-axis and the fluorescence intensity of the detection reagent is used as the Y-axis. The curve is fitted to obtain the standard curve f2 that characterizes the relationship between the fluorescence intensity of the detection reagent and the target nucleic acid concentration.

[0128] Several specific examples are provided below.

[0129] In one example, the method of Example 2 is used to detect Salmonella, and the amplification reaction solution includes: primers, dNTPs, Tris-HCl buffer, Mg 2+ NH 4+ , template, etc., the constant temperature amplification temperature is 63°C, and the amplification time is 65min. Positive samples emit bright blue fluorescence under the 355nm excitation light source, and no obvious fluorescence is observed in negative samples.

[0130] In one example, the method of Example 2 is used to detect Vibrio parahaemolyticus, and the amplification reaction solution includes: primers F3, B3, LF, BIP, LB for Vibrio parahaemolyticus; dNTP; Betaine, Bst polymerase; DTT, template, etc.; the constant temperature amplification temperature is 63°C, and the amplification time is 65 minutes. The positive sample emits bright yellow-green fluorescence under the irradiation of the 355nm excitation light source, and no obvious fluorescence is observed in the negative sample.

[0131] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A nucleic acid amplification product detection reagent, characterized in that: The detection reagent is prepared by the following method: 1) Using cellobiose and 1,3-propanedithiol as carbon sources, carbon quantum dots were prepared by hydrothermal method; 2) Modifying the surface of carbon quantum dots with copper ions to obtain the detection reagent; The carbon quantum dots prepared in step 1) are EX The carbon quantum dots will emit fluorescence under the irradiation of the excitation light, and after the copper ions are modified in step 2), the fluorescence of the carbon quantum dots will be weakened or even quenched; when the byproduct pyrophosphate generated by the nucleic acid amplification reaction is present, the fluorescence of the carbon quantum dots will be enhanced, so that the qualitative and / or quantitative detection of the nucleic acid amplification product can be achieved by performing fluorescence detection on the solution obtained after the detection reagent reacts with the nucleic acid amplification product; Step 1) specifically includes: 1-1) Dissolve 1,3-propanedithiol in anhydrous ethanol and stir to obtain solution A; dissolve cellobiose in ultrapure water and stir to obtain solution B; 1-2) Mix solution A and solution B, disperse by ultrasonic for 5-30 minutes, and stir for 2-15 minutes to obtain mixture C; 1-3) Transfer the mixture C to a hydrothermal reactor lined with polytetrafluoroethylene and react at 150-195°C for 5-18h; 1-4) After the reaction is completed, the mixture is cooled to room temperature, centrifuged at 8000-15000 rpm for 2-15 min, and the centrifuged liquid is dialyzed with a dialysis bag with a molecular weight cutoff of 500-1200 Da for 6-20 hours. The solution in the dialysis bag is collected and freeze-dried to obtain carbon quantum dots; Step 2) is as follows: 0.05-0.3 g of the carbon quantum dots prepared by the method of step 1) is redissolved in 50-100 ml of ultrapure water, and ultrasonically dispersed for 2-8 min to obtain a carbon quantum dot solution, and then 5-50 ml of Cu is added to the carbon quantum dot solution. 2+ The concentration of Cu is 10 mM 2+ The solution is reacted, and after the reaction is completed, centrifugation is performed and the filtrate is freeze-dried to obtain the detection reagent.

2. The nucleic acid amplification product detection reagent according to claim 1, characterized in that: The step 1) specifically includes: 1-1) Dissolve 1,3-propanedithiol in anhydrous ethanol and stir to obtain solution A; dissolve cellobiose in ultrapure water and stir to obtain solution B; 1-2) Mix solution A and solution B, disperse by ultrasonic for 5 minutes, and stir for 4 minutes to obtain mixture C; 1-3) Transfer the mixture C to a hydrothermal reactor lined with polytetrafluoroethylene and react at 185°C for 12 hours; 1-4) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 10,000 rpm for 5 minutes. The centrifuged liquid is dialyzed with a dialysis bag with a molecular weight cutoff of 1,000 Da for 12 hours. The solution in the dialysis bag is collected and freeze-dried to obtain carbon quantum dots.

3. The nucleic acid amplification product detection reagent according to claim 2, characterized in that: The step 1) specifically includes: 1-1) Dissolve 0.5 g of 1,3-propanedithiol in 50 mL of anhydrous ethanol and stir to obtain solution A; dissolve 0.64 g of cellobiose in 50 mL of ultrapure water and stir to obtain solution B; 1-2) Mix solution A and solution B, disperse by ultrasonic for 5 minutes, and stir for 4 minutes to obtain mixture C; 1-3) Transfer the mixture C to a 200 mL polytetrafluoroethylene-lined hydrothermal reactor and react at 185°C for 12 hours; 1-4) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 10,000 rpm for 5 minutes. The centrifuged liquid is dialyzed with a dialysis bag with a molecular weight cutoff of 1,000 Da for 12 hours. The solution in the dialysis bag is collected and freeze-dried to obtain carbon quantum dots.

4. The nucleic acid amplification product detection reagent according to claim 1, characterized in that: The Cu 2+ The solution is a mixture of one or more of copper sulfate, copper nitrate, copper chloride and copper acetate solutions.

5. The nucleic acid amplification product detection reagent according to claim 1, characterized in that: The method for qualitatively and / or quantitatively detecting a target in a product of nucleic acid amplification of a sample to be tested by a detection reagent comprises the following steps: Ⅰ. Conduct quality control tests on test reagents: Ⅰ-1. Pre-prepare the concentration to C T The detection reagent solution, concentration C p Sodium pyrophosphate solution, nucleic acid amplification reaction solution; The detection reagent solution was divided into three parts, each with a volume of V T , take one portion as the test solution for the detection reagent, and take two portions as the quality control solution for the detection reagent; Divide the sodium pyrophosphate solution into two equal parts, each with a volume of V p ; The nucleic acid amplification reaction solution is divided into two parts, and the sample to be tested is divided into two parts; Ⅰ-2. Mix one portion of sodium pyrophosphate solution and one portion of test reagent quality control solution evenly to react. After the reaction is completed, use a wavelength of λ EX The mixed solution 1 obtained after the reaction is irradiated with an excitation light source, and the fluorescence color of the mixed solution 1 is observed; When the fluorescent color of the mixed solution 1 matches the fluorescent color that should be exhibited after the detection reagent reacts with pyrophosphate, it is determined that the qualitative detection of the detection reagent is effective; Then measure the mixed solution 1 at a wavelength of λ Em The fluorescence intensity at the wavelength λ is determined according to the pre-established characterization test reagent. Em Standard curve of the relationship between fluorescence intensity and pyrophosphate concentration f 1 Calculate the concentration of pyrophosphate C p ´, compare C p ´ and the actual concentration of pyrophosphate C p The relationship between η and C p ´-C p ) / C p , when η≤η T When the detection reagent is determined to be effective for quantitative detection, wherein η T is the set threshold, and 0<η T <0.15; When it is determined that both the qualitative and quantitative tests of the detection reagent are effective, proceed to the following step II; Ⅱ. Detect the nucleic acid amplification products of the samples to be tested: Ⅱ-1. A sample to be tested is mixed with a nucleic acid amplification reaction solution and a nucleic acid amplification reaction is performed. After the amplification is completed, a detection reagent test solution is added to the nucleic acid amplification product to react. After the reaction is completed, a mixed solution 2 is obtained. The wavelength is λ EX The excitation light source illuminates the mixed solution 2, and the fluorescence color of the mixed solution 2 is observed. When the fluorescence color of the mixed solution 2 meets the fluorescence color that should be reflected after the detection reagent reacts with the pyrophosphate, it is determined that the target object exists in the sample to be tested, otherwise it is determined that the target object does not exist, thereby achieving qualitative detection of the sample to be tested; Ⅱ-2. When it is determined that the target substance exists in the sample to be tested, quantitative detection is performed: Measure the mixed liquid 2 at a wavelength of λ EX Under the irradiation of the excitation light source at λ Em The fluorescence intensity at c ; Ⅱ-3. Calculate the fluorescence intensity correction value ΔP: Take the remaining test reagent quality control solution and measure its EX Under the irradiation of the excitation light source at λ Em The fluorescence intensity at b1 ; Then, the detection reagent quality control solution is evenly mixed with a sample to be tested and a nucleic acid amplification reaction solution to obtain a mixed solution 3, and the mixed solution 3 is measured at a wavelength of λ EX Under the irradiation of the excitation light source at λ Em The fluorescence intensity at b2 ; Fluorescence intensity correction ΔP=P b1 -P b2 ; Ⅱ-4. Calculate the concentration of the target substance: The P obtained in step II-2 c The value of P is corrected to c ',P c =P c+ ΔP, and then a standard curve is established in advance to characterize the relationship between the fluorescence intensity of the solution obtained after the reaction of the detection reagent and the nucleic acid amplification product of the sample to be tested and the concentration of the target in the sample to be tested. f 2 Calculate the fluorescence intensity as P c ', and C1' is taken as the target concentration in the sample to be tested.

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