A buffer for supporting multiplex fluorescence quantitative PCR and a preparation method thereof

Abstract

The present invention relates to a buffer solution for supporting multiplex fluorescence quantitative PCR and a preparation method thereof, belonging to the technical field of biochemical reagents. Among them, the buffer solution includes Tris-HCl, (NH4)2SO4, KCl, temperature-controlled magnesium ion nanocapsules, and additives; the additives are polyethylene glycol, betaine, trehalose, tetramethylammonium chloride, and acetylated BSA; the temperature-controlled magnesium ion nanocapsules have a core-shell structure, and the core includes MgCl2 with a concentration of 1 mM - 5 mM in the buffer solution, 0.1 - 0.5 U / μL of pyrophosphatase, 10 - 30 mg / mL of phospholipids, 5 - 15 mg / mL of cholesterol, and an inorganic solution, and the shell includes a thermosensitive polymer with a concentration of 20 - 45 wt% in the buffer solution. The buffer solution of the present invention can dynamically adjust the Mg 2+ concentration and avoid affecting the amplification efficiency due to the difficulty in adjusting the Mg 2+ concentration.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical reagents, and particularly relates to a buffer solution for supporting multiplex fluorescence quantitative PCR, a preparation method thereof, and an application thereof. Background Art

[0002] The basic components of a PCR reaction (including qPCR) include a template, primers, DNA polymerase, Mg 2+ and dNTP. The template is a nucleic acid molecule containing the target region; the primer is an oligonucleotide containing 15 - 30 bases, which can bind to the flanking sequences of the target region 2+ in the template DNA. During the PCR reaction, DNA polymerase catalyzes the extension starting from the 3'-end of the primer; Mg 2+ is a cofactor of DNA polymerase, which helps the binding of dNTP during polymerization; dNTP is composed of four basic nucleotides, dATP, dCTP, dGTP, and dTTP, and is the raw material for the synthesis of new DNA strands. When performing a PCR reaction (including qPCR), a buffer solution is usually added, and the components in the buffer solution can provide a suitable chemical reaction environment for DNA polymerase.

[0003] In dye-based fluorescence quantitative PCR, SYBR Green I is most commonly used as a DNA-binding dye. This dye can produce fluorescence when binding to DNA, but does not produce fluorescence when free. For each newly formed DNA double strand, a certain amount of the dye will bind to it, and the product is quantified based on the accumulation of the signal. Although this method has the advantages of low price and wide applicability, its specificity is poor, it cannot distinguish between amplified products and non-specific amplification, and it cannot even quantify different target products separately. These deficiencies limit the application of dye-based fluorescence quantitative PCR. The fluorescence quantitative PCR of the probe method completely makes up for these deficiencies, and multiplex fluorescence quantitative PCR can even achieve the simultaneous quantification of multiple target products. Due to the preference of multiplex amplification and the strong interference between products, the fluorescence quantitative PCR reagent of the ordinary probe method cannot be used for multiplex fluorescence quantitative PCR, so the optimization of multiplex fluorescence quantitative reagents is very necessary.

[0004] The Chinese patent application with the publication number CN109536587A discloses a high-efficiency multiplex fluorescence quantitative PCR kit and a preparation method thereof. A high-efficiency multiplex fluorescence quantitative PCR reaction solution is composed of a Tris-HCl solution, a dNTPs solution, a KCl solution, an (NH4)2SO4 solution, a MgCl2 solution, a BSA solution, a glycerol solution, and a DTT solution.

[0005] Through the detection of the sensitivity and amplification performance of the reagent, it is found that the high-efficiency multiplex fluorescence quantitative PCR reaction solution has higher sensitivity and amplification performance than traditional reagents. The present invention optimizes the multiplex fluorescence quantitative PCR reaction solution, especially by adding some specific additives, such as a certain proportion of glycerol, BSA, DTT, etc., to improve the sensitivity, specificity and stability of the reaction. However, for some biological samples with complex components in the above reaction solution, for example, fecal samples contain PCR inhibitors such as cholate / deoxycholate, polysaccharides, phytic acid, etc. Residual PCR inhibitors that still exist in the DNA template after extraction, transformation and purification may inhibit the amplification of the target region during the PCR reaction, resulting in a delay in the Ct value of the PCR amplification, or even the inability to detect the target region, thus seriously affecting the detection of the sample.

[0006] The Chinese patent application for invention with the publication number CN117230163A discloses a PCR buffer solution, a kit and their applications. The PCR buffer solution comprises the following components: 1 mM - 8 mM Mg 2+ , 10 mM - 250 mM K + , 5 mM - 80 mM Tris-HCl, 10 mM - 50 mM (NH4)2SO4, glycerol and an additive, and the additive comprises any one or more of betaine, DMSO, 2-pyrrolidone, TMAC, PEG, Tween-20 and BSA.

[0007] Adding an appropriate amount of betaine, DMSO, 2-pyrrolidone, TMAC, PEG, Tween-20 or BSA to the above PCR buffer solution can improve the specificity and sensitivity of amplifying DNA in fecal samples in a common PCR buffer solution. Through the combination method of various additives, it is found that the combination of betaine and most additives has a synergistic effect when amplifying template DNA in fecal samples; and it is found that the combination containing DMSO and 2-pyrrolidone has a synergistic effect on promoting the amplification sensitivity of DNA in fecal samples with one or more of betaine, TMAC, PEG, Tween-20 and BSA, and improves the specificity; further, it is found that adding an appropriate amount of betaine, DMSO, 2-pyrrolidone, TMAC, Tween-20 and BSA to the PCR buffer solution has the best amplification effect. However, the Mg 2+ concentration in the above PCR buffer reaction system is difficult to regulate. When the Mg 2+ concentration is too low, the binding efficiency between the primer and the template will decrease significantly, and the amplification efficiency will also decrease accordingly. When the Mg 2+ concentration is too high, it is easy to cause non-specific amplification or the formation of primer dimers, resulting in false positives. In the multiplex fluorescence quantitative PCR buffer solution, a high concentration of Mg 2+Although it can inhibit partial non-specific amplification, it is easy to mask the target signal, resulting in a delayed Ct value or an abnormal melting curve. Summary of the Invention

[0008] To solve the above technical problem of difficult regulation of Mg 2+ concentration, the present invention provides a buffer solution for supporting multiplex fluorescence quantitative PCR and a preparation method thereof. The buffer solution of the present invention can dynamically adjust the Mg 2+ concentration, avoiding the influence of difficult Mg 2+ concentration regulation on the amplification efficiency.

[0009] In a first aspect, the present invention provides a buffer solution for supporting multiplex fluorescence quantitative PCR, the buffer solution comprising 10 mM - 20 mM Tris-HCl, 15 mM - 30 mM (NH4)2SO4, 50 mM - 100 mM KCl, 0.5 - 1.0 mg / ml temperature-controlled magnesium ion nanocapsules, additives;

[0010] The additives include polyethylene glycol accounting for 5 - 10% (v / v) of the buffer solution concentration, 0.8 M - 1.2 M betaine, 0.5 M - 1.0 M trehalose, 15 mM - 50 mM tetramethylammonium chloride, 0.1 - 0.5 mg / mL acetylated BSA;

[0011] The temperature-controlled magnesium ion nanocapsules have a core-shell structure, the core comprising 1 mM - 5 mM MgCl2 accounting for the buffer solution concentration, 0.1 - 0.5 U / μL pyrophosphatase, 10 - 30 mg / mL phospholipids, 5 - 15 mg / mL cholesterol, an inorganic solution, and the shell comprising 20 - 45 wt% of a temperature-sensitive polymer accounting for the buffer solution concentration.

[0012] In the above technical solution, the Tris-HCl, (NH4)2SO4, and KCl, as the core components of the PCR buffer solution, mainly play the roles of maintaining the pH stability in the reaction system, providing an essential ionic environment, protecting the enzyme activity, and enhancing the amplification specificity. Adding betaine in the additives is mainly to balance the stability of GC / AT base pairs and improve the amplification efficiency of long fragments; adding acetylated BSA is mainly to neutralize inhibitors (such as humic acid) and protect the enzyme activity; adding trehalose is mainly to prevent enzyme denaturation and enhance the thermal stability; adding tetramethylammonium chloride can reduce the annealing difficulty of templates with high GC content and reduce non-specific products; adding polyethylene glycol is mainly to increase the viscosity of the PCR reaction system and reduce the interaction between primers, thereby reducing the formation of primer dimers.

[0013] When the temperature-controlled magnesium ion nanocapsules are at a low temperature stage, the nanocapsules swell and slowly release Mg 2+, reducing the generation of primer dimers and non-specific amplification. During the high-temperature stage, the nanocapsules shrink and rapidly extrude to release Mg 2+ , dynamically regulating the Mg 2+ concentration. Among them, MgCl2 and pyrophosphatase are used as the core of the nanocapsules together as the magnesium ion source. Pyrophosphatase can decompose the pyrophosphate produced by side reactions to prevent Mg 2+ from chelating with pyrophosphate. The temperature-sensitive polymer, as the shell material, has a lower critical solution temperature. The polymer chains are prone to hydrophilic swelling at low temperatures to form a porous gel-like structure, encapsulating Mg 2+ to restrict its release, reducing primer dimers and non-specific binding. The polymer chains are prone to hydrophobic contraction at high temperatures to form a dense structure, extruding the internal Mg 2+ solution to meet the high-efficiency extension requirements of DNA polymerase. At the same time, the pyrophosphate generated during the high-temperature stage will be hydrolyzed by pyrophosphatase to avoid its combination with Mg 2+ resulting in a sudden drop in the Mg 2+ concentration.

[0014] Optionally, the temperature-controlled magnesium ion nanocapsule further includes a cross-linking agent, and the cross-linking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1-2:1. The mass ratio of the cross-linking agent to the temperature-sensitive polymer is 1:15-20.

[0015] In the above technical solution, the cross-linking agent connects the polymer chains in the temperature-sensitive polymer through the carboxyl-amino reaction of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to form a three-dimensional network structure, enhancing the mechanical stability of the shell. At the same time, the cross-linking agent can covalently connect the carboxyl group of the temperature-sensitive polymer and the amino group on the surface of the core to avoid stratification or rupture during centrifugation or temperature cycling.

[0016] Optionally, the additive in the buffer further includes choline hydroxybutyrate with a concentration of 0.1 mM - 0.3 mM in the buffer.

[0017] In the above technical solution, choline hydroxybutyrate is an ionic compound formed by the neutralization reaction of β-hydroxybutyric acid and choline. Choline, as a cation, is combined with the hydroxybutyrate anion through an ionic bond. As an amphoteric ionic compound, choline hydroxybutyrate combines the cationic characteristics of choline and the anionic group of hydroxybutyric acid, has strong water solubility and thermal stability, stabilizes the DNA double strand and polymerase activity by forming a dynamic hydration layer, reduces the influence of Mg 2+ concentration fluctuations. Its amphoteric structure can preferentially bind heavy metal ions (such as Fe 3+ , Cu 2+ ) in the test sample to prevent heavy metal ions from binding to Mg 2+It competes for the active sites of the polymerase, inhibits the formation of primer dimers and template secondary structures, and neutralizes common PCR inhibitors such as phenols and humic acid, thereby improving the detection success rate of complex test samples.

[0018] Optionally, the buffer comprises a stabilizer, wherein the stabilizer is ε-polylysine accounting for 0.1 mM-0.3 mM of the buffer concentration and 0.01-0.05% of sodium tungstosilicate.

[0019] In the above technical solution, ε-polylysine is used as a cationic stabilizer, which is positively charged and partially replaces Mg through electrostatic action. 2+ , directly binds to DNA, reducing the Mg content of the PCR reaction system 2+ The demand for Mg 2+ At low concentrations, ε-polylysine maintains the stability of the DNA template and prevents folding or degradation. 2+ At high concentrations, the flexible long chain structure of ε-polylysine can shield excess Mg 2+ , avoiding the inhibition of polymerase activity, ε-polylysine can also preferentially bind to negatively charged inhibitors (such as heparin and humic acid) in the test sample to prevent them from consuming Mg 2+ .

[0020] C1 in the test sample - 、SO 2- The anions will react with Mg 2+ Form weak binding compounds (such as Mg2Cl) to reduce free Mg 2+ Sodium tungstosilicate acts as an anion stabilizer, and its nanocage structure absorbs these anions through ion exchange, maintaining Mg 2+ Activity. The drastic fluctuation of ionic strength in the buffer will affect the activity of polymerase. Sodium tungstosilicate acts as an "ion buffer" to maintain the electrical neutrality of the reaction system by reversibly adsorbing or releasing anions. In addition, it can also combine with test samples such as Fe 3+ , Cu 2+ to prevent competitive inhibition of Mg 2+ .

[0021] In a second aspect, the present invention provides a method for preparing a buffer supporting multiplex fluorescence quantitative PCR, the preparation method comprising the following steps:

[0022] Preparation of temperature-controlled magnesium ion nanocapsules: Dissolve phospholipids and cholesterol in an inorganic solution, and form a uniform lipid film by rotary evaporation. Hydrate the lipid film with a mixed solution of MgCl2 and pyrophosphatase, shake and centrifuge to obtain unilamellar liposomes. Dissolve the thermosensitive polymer in PBS solution pre-cooled at 3-5°C, and stir magnetically until completely dissolved. Mix the liposomes and the thermosensitive polymer solution at a mass ratio of 1:1.5-2, incubate at 3-5°C for 2-3 h, and centrifuge and purify to obtain temperature-controlled magnesium ion nanocapsules;

[0023] Mixing of each component: Weigh or dilute each component according to specific concentrations and mix them, adjust the pH to 7.5-9.0, and then filter through a 0.22 μM aqueous filter membrane to sterilize and obtain a PCR buffer.

[0024] In the above technical solution, during the preparation process of the temperature-controlled magnesium ion nanocapsules, the surface of the liposomes is negatively charged, and the thermosensitive polymer is adsorbed on the outer layer of the liposomes through electrostatic interaction to form a "liposome-thermosensitive polymer" core-shell structure.

[0025] Optionally, the preparation steps of the temperature-controlled magnesium ion nanocapsules further include mixing the liposomes and the thermosensitive polymer solution at a mass ratio of 1:1.5-2, incubating at 3-5°C for 2-3 h, adding a crosslinking agent and reacting at room temperature for 1-2 h, and centrifuging and purifying to obtain temperature-controlled magnesium ion nanocapsules.

[0026] In the above technical solution, the crosslinking agent connects the polymer chains in the thermosensitive polymer through carboxyl-amino reactions to form a three-dimensional network structure, enhancing the mechanical stability of the outer shell. At the same time, the crosslinking agent can covalently connect the carboxyl groups of the thermosensitive polymer and the amino groups on the surface of the inner core to prevent delamination or rupture during centrifugation or temperature cycling.

[0027] In the third aspect, the present invention provides a kit for PCR reaction, which includes the above-mentioned PCR buffer, and the kit further includes dNTP, DNA polymerase, and TaqMan probe.

[0028] In the fourth aspect, the present invention provides an application of a buffer supporting multiplex fluorescence quantitative PCR, or a buffer prepared by a preparation method of a buffer supporting multiplex fluorescence quantitative PCR, or a kit supporting multiplex fluorescence quantitative PCR in the preparation of DNA amplification products.

[0029] In summary, the present invention includes at least one of the following beneficial technical effects:

[0030] 1. By adding temperature-controlled magnesium ion nanocapsules, the nanocapsules swell at low temperatures and slowly release Mg 2+ , reducing the generation of primer dimers and non-specific amplification. At high temperatures, the nanocapsules shrink and rapidly extrude and release Mg 2+, dynamically adjust Mg 2+ concentration.

[0031] 2. By adding stabilizers, ε-polylysine is used as a cationic stabilizer. Through electrostatic interaction, it partially replaces Mg 2+ , directly binds to DNA, reduces the demand for Mg 2+ in the PCR reaction system, and can also preferentially bind to negatively charged inhibitors (such as heparin and humic acid) in the sample to prevent them from consuming Mg 2+ ; Sodium tungstosilicate is used as an anionic stabilizer. Its nano-cage structure adsorbs anions such as C1 - , SO 2- etc. through ion exchange to maintain the activity of Mg 2+ , and can also maintain the electrical neutrality of the reaction system by reversibly adsorbing or releasing anions. In addition, it can bind metal ions such as Fe 3+ , Cu 2+ etc. in the test sample to prevent their competitive inhibition of Mg 2+ .

[0032] 3. By adding choline hydroxybutyrate, through the formation of a dynamic hydration layer, it stabilizes the DNA double strand and polymerase activity, reduces the influence of Mg 2+ concentration fluctuations. Its amphoteric structure can preferentially bind to heavy metal ions (such as Fe 3+ , Cu 2+ ) in the sample to prevent heavy metal ions from competing with Mg 2+ for the polymerase active site. At the same time, it can inhibit the formation of primer dimers and template secondary structures, and can neutralize common PCR inhibitors such as phenols and humic acids, improving the detection success rate of complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the amplification curve of HCV gene amplified using buffer #9;

[0034] Figure 2 is the amplification curve of HBV gene amplified using buffer #9;

[0035] Figure 3 is the amplification curve of HIV-1 gene amplified using buffer #9;

[0036] Figure 4 is the amplification curve of HIV-2 gene amplified using buffer #9;

[0037] Figure 5 is the Mg 2+ release diagram of the temperature-controlled magnesium ion nanocapsule in buffer #9 during the PCR reaction. DETAILED DESCRIPTION OF THE INVENTION

[0038] The following is combined withFigures 1-4 The present invention will be further described in detail with reference to specific embodiments.

[0039] Example 1: This example discloses a buffer solution #1 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0040] A buffer solution #1 for supporting multiplex fluorescence quantitative PCR includes 20 mM Tris-HCl, 15 mM (NH4)2SO4, 100 mM KCl, 0.5 mg / ml temperature-controlled magnesium ion nanocapsules, 5% (v / v) polyethylene glycol, 0.8 M betaine, 1.0 M trehalose, 15 mM tetramethylammonium chloride, 0.1 mg / mL acetylated BSA. The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core includes MgCl2 with a concentration of 1 mM in the buffer solution, 0.1 U / μL pyrophosphatase, 10 mg / mL phospholipids, 5 mg / mL cholesterol, and an inorganic solution. The shell includes a thermosensitive polymer with a concentration of 20 wt% in the buffer solution.

[0041] In this example, the thermosensitive polymer is selected as poly(N-vinylcaprolactam). In other examples, the thermosensitive polymer can also be selected from thermosensitive polymers such as poly(N-isopropylacrylamide) and poly(methyl vinyl ether). The inorganic solvent is selected as chloroform. In other examples, methanol or a mixture of chloroform and methanol can also be used. In this example and other examples, the particle size of the temperature-controlled magnesium ion nanocapsules is 50 - 200 nm.

[0042] A preparation method of a buffer solution #1 for supporting multiplex fluorescence quantitative PCR:

[0043] S1. Preparation of liposomes: Dissolve phospholipids and cholesterol in an inorganic solution, rotate and evaporate to form a uniform lipid film, hydrate the lipid film with a mixed solution of MgCl2 and pyrophosphatase, and oscillate and centrifuge to obtain monolayer liposomes.

[0044] S2. Preparation of temperature-controlled magnesium ion nanocapsules: Dissolve the thermosensitive polymer in PBS solution pre-cooled at 4°C, stir magnetically until completely dissolved, mix the liposomes and the poly(N-vinylcaprolactam) solution according to a mass ratio of 1:2, incubate at 4°C for 2 h, and centrifuge and purify to obtain temperature-controlled magnesium ion nanocapsules.

[0045] S3. Mixing of each component: Weigh or dilute each component according to specific concentrations and mix them, adjust the pH to 8.5, and then sterilize through a 0.22 μM aqueous filter membrane to obtain buffer solution #1 for supporting multiplex fluorescence quantitative PCR.

[0046] Example 2: This example discloses a buffer solution #2 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0047] A buffer solution #2 for supporting multiplex fluorescence quantitative PCR comprises 10 mM Tris-HCl, 30 mM (NH4)2SO4, 50 mM KCl, 1.0 mg / ml temperature-controlled magnesium ion nanocapsules, 10% (v / v) polyethylene glycol, 1.2 M betaine, 0.5 M trehalose, 40 mM tetramethylammonium chloride, 0.5 mg / mL acetylated BSA. The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core comprises MgCl2 at a concentration of 5 mM in the buffer solution, 0.5 U / μL pyrophosphatase, 30 mg / mL phospholipids, 15 mg / mL cholesterol, and methanol. The shell comprises polymethyl vinyl ether at a concentration of 40 wt% in the buffer solution.

[0048] The preparation method of the buffer solution #2 for supporting multiplex fluorescence quantitative PCR is the same as that of Example 1.

[0049] Example 3: This example discloses a buffer solution #3 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0050] A buffer solution #3 for supporting multiplex fluorescence quantitative PCR comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA. The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core comprises MgCl2 at a concentration of 3 mM in the buffer solution, 0.3 U / μL pyrophosphatase, 20 mg / mL phospholipids, 10 mg / mL cholesterol, and a mixed solution of chloroform and methanol with a mass ratio of 3:1. The shell comprises poly-N-isopropylacrylamide at a concentration of 30 wt% in the buffer solution.

[0051] The preparation method of the buffer solution #3 for supporting multiplex fluorescence quantitative PCR is the same as that of Example 1.

[0052] Example 4: This example discloses a buffer solution #4 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0053] A buffer solution #4 for supporting multiplex fluorescence quantitative PCR comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA. The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core comprises MgCl2 with a concentration of 3 mM in the buffer solution, 0.3 U / μL pyrophosphatase, 20 mg / mL phospholipids, 10 mg / mL cholesterol, and a mixed solution of chloroform and methanol with a mass ratio of 3:1. The shell comprises poly-N-isopropylacrylamide with a concentration of 30 wt% in the buffer solution and a cross-linking agent. The cross-linking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0054] A preparation method of a buffer solution #4 for supporting multiplex fluorescence quantitative PCR:

[0055] S1. Preparation of inner core liposomes: Dissolve phospholipids and cholesterol in an inorganic solution, rotate and evaporate to form a uniform lipid film, hydrate the lipid film with a mixed solution of MgCl2 and pyrophosphatase, shake and centrifuge to obtain monolayer liposomes;

[0056] S2. Preparation of the outer shell: Dissolve the thermosensitive polymer in PBS solution pre-cooled at 4°C, and stir magnetically until completely dissolved;

[0057] S3. Combination of the inner core and the outer shell: Mix the liposomes and poly-N-isopropylacrylamide according to a mass ratio of 1:2, incubate at 4°C for 2 h, add the cross-linking agent and react at room temperature for 2 h, and centrifuge and purify to obtain temperature-controlled magnesium ion nanocapsules;

[0058] S4. Mixing of each component: Weigh or dilute each component according to the specific concentration and mix them, adjust the pH to 8.5, and then filter and sterilize through a 0.22 μM aqueous filter membrane to obtain the buffer solution #4 for supporting multiplex fluorescence quantitative PCR.

[0059] Example 5: This example discloses a buffer solution #5 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0060] A buffer solution #5 for supporting multiplex fluorescence quantitative PCR comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA, and 0.2 mM choline hydroxybutyrate. The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core comprises MgCl2 at a concentration of 3 mM in the buffer solution, 0.3 U / μL pyrophosphatase, 20 mg / mL phospholipid, 10 mg / mL cholesterol, and a mixed solution of chloroform and methanol with a mass ratio of 3:1. The shell comprises poly(N-isopropylacrylamide) at a concentration of 30 wt% in the buffer solution.

[0061] The preparation method of the buffer solution #5 for supporting multiplex fluorescence quantitative PCR is the same as that of Example 1.

[0062] Example 6: This example discloses a buffer solution #6 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0063] A buffer solution #6 for supporting multiplex fluorescence quantitative PCR comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA, and 0.2 mM choline hydroxybutyrate. The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core comprises MgCl2 at a concentration of 3 mM in the buffer solution, 0.3 U / μL pyrophosphatase, 20 mg / mL phospholipid, 10 mg / mL cholesterol, and a mixed solution of chloroform and methanol with a mass ratio of 3:1. The shell comprises poly(N-isopropylacrylamide) at a concentration of 30 wt% in the buffer solution and a cross-linking agent. The cross-linking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0064] The preparation method of the buffer solution #6 for supporting multiplex fluorescence quantitative PCR is the same as that of Example 4.

[0065] Example 7: This example discloses a buffer solution #7 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0066] A buffer solution #5 for supporting multiplex fluorescence quantitative PCR comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA, 0.2 mM ε-polylysine, 0.03% sodium tungstosilicate. The temperature-controlled magnesium ion nanocapsules are of a core-shell structure. The core comprises MgCl2 at a concentration of 3 mM in the buffer solution, 0.3 U / μL pyrophosphatase, 20 mg / mL phospholipid, 10 mg / mL cholesterol, and a mixed solution of chloroform and methanol with a mass ratio of 3:1. The shell comprises poly-N-isopropylacrylamide at a concentration of 30 wt% in the buffer solution.

[0067] The preparation method of buffer solution #7 for supporting multiplex fluorescence quantitative PCR is the same as that in Example 1.

[0068] Example 8: This example discloses a buffer solution #8 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0069] A buffer solution #8 for supporting multiplex fluorescence quantitative PCR comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA, 0.2 mM ε-polylysine, 0.03% sodium tungstosilicate. The temperature-controlled magnesium ion nanocapsules are of a core-shell structure. The core comprises MgCl2 at a concentration of 3 mM in the buffer solution, 0.3 U / μL pyrophosphatase, 20 mg / mL phospholipid, 10 mg / mL cholesterol, and a mixed solution of chloroform and methanol with a mass ratio of 3:1. The shell comprises poly-N-isopropylacrylamide at a concentration of 30 wt% in the buffer solution and a cross-linking agent. The cross-linking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1.5:1. The mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0070] The preparation method of buffer solution #8 for supporting multiplex fluorescence quantitative PCR is the same as that in Example 4.

[0071] Example 9: This example discloses a buffer solution #9 for supporting multiplex fluorescence quantitative PCR and its preparation method.

[0072] A buffer solution #6 for supporting multiplex fluorescence quantitative PCR comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA, 0.2 mM choline hydroxybutyrate, 0.2 mM ε-polylysine, 0.03% sodium tungstosilicate. The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core comprises MgCl2 at a concentration of 3 mM in the buffer solution, 0.3 U / μL pyrophosphatase, 20 mg / mL phospholipid, 10 mg / mL cholesterol, and a mixed solution of chloroform and methanol with a mass ratio of 3:1. The shell comprises poly(N-isopropylacrylamide) at a concentration of 30 wt% in the buffer solution and a cross-linking agent. The cross-linking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1.5:1. The mass ratio of the cross-linking agent to the temperature-sensitive polymer is 1:18.

[0073] The preparation method of buffer solution #9 for supporting multiplex fluorescence quantitative PCR is the same as that in Example 4.

[0074] Comparative Example 1: This comparative example provides a comparative buffer solution D1 for supporting multiplex fluorescence quantitative PCR. The buffer solution comprises 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 3 mM MgCl2, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA, 0.2 mM choline hydroxybutyrate, 0.2 mM ε-polylysine, 0.03% sodium tungstosilicate.

[0075] The preparation method of the comparative buffer solution D1 for supporting multiplex fluorescence quantitative PCR is as follows:

[0076] Mixing of each component: Weigh or dilute the above components according to specific concentrations and mix them, adjust the pH to 8.5, and then sterilize through a 0.22 μM aqueous filter membrane to obtain the buffer solution D1 for supporting multiplex fluorescence quantitative PCR.

[0077] Comparative Example 2: This comparative example provides a buffer D2 for supporting multiplex fluorescence quantitative PCR, including 15 mM Tris-HCl, 20 mM (NH4)2SO4, 80 mM KCl, 0.8 mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0 M betaine, 0.7 M trehalose, 30 mM tetramethylammonium chloride, 0.3 mg / mL acetylated BSA, 0.2 mM choline hydroxybutyrate, 0.2 mM ε-polylysine, 0.03% sodium tungstosilicate. The temperature-controlled magnesium ion nanocapsules are of a shell-less structure, including MgCl2 at a concentration of 3 mM in the buffer, 0.3 U / μL pyrophosphatase, 30 wt% poly-N-isopropylacrylamide, 20 mg / mL phospholipids, 10 mg / mL cholesterol, a mixed solution of chloroform and methanol with a mass ratio of 3:1, and a cross-linking agent. The cross-linking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0078] The preparation method of the buffer D2 for supporting multiplex fluorescence quantitative PCR is as follows:

[0079] S1. Preparation of temperature-controlled magnesium ion nanocapsules: Dissolve phospholipids and cholesterol in an inorganic solution, rotate and evaporate to form a uniform lipid film, hydrate the lipid film with a mixed solution of MgCl2 and pyrophosphatase, dissolve the thermosensitive polymer in PBS solution pre-cooled at 4°C, stir magnetically until completely dissolved, dissolve the lipid film in the poly-N-isopropylacrylamide solution at a mass ratio of 1:2, incubate at 4°C for 2 h, add the cross-linking agent and react at room temperature for 2 h, and centrifuge and purify to obtain shell-less temperature-controlled magnesium ion nanocapsules;

[0080] S2. Mixing of each component: Weigh or dilute each component according to the specific concentration and mix them, adjust the pH to 8.5, and then filter and sterilize through a 0.22 μM aqueous filter membrane to obtain the buffer D2 for supporting multiplex fluorescence quantitative PCR.

[0081] Comparative Example 3: This comparative example provides a buffer D3 for supporting multiplex fluorescence quantitative PCR, which is the same as that in Example 9, except that silica is selected instead of poly-N-isopropylacrylamide.

[0082] Comparative Example 4: This comparative example provides a buffer D4 for supporting multiplex fluorescence quantitative PCR, which is the same as that in Example 9, except that the temperature-controlled magnesium ion nanocapsules lack pyrophosphatase.

[0083] Comparative Example 5: This comparative example provides a buffer D5 for supporting multiplex fluorescence quantitative PCR, which is the same as that in Example 9, except that DNA polymerase is selected instead of pyrophosphatase.

[0084] Comparative Example 6: This comparative example provides a buffer D6 for supporting multiplex fluorescence quantitative PCR, which is the same as that in Example 9, except that phenolic resin is selected to replace the mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

[0085] Comparative Example 7: The buffer in this comparative example is PCR buffer D7 purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0086] The buffers #1-#9 in Examples 1-9 of the present application and the comparative buffers D1-D7 in Comparative Examples 1-7 were respectively applied to the kit. The kit also included detection primer pairs, dNTP (Tiangen Biochemical Technology (Beijing) Co., Ltd.), Taq DNA polymerase (Tiangen Biochemical Technology (Beijing) Co., Ltd.), TaqMan probe, and M-MLV reverse transcriptase (Shanghai Zeye Biotechnology Co., Ltd.). HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene were respectively selected as target genes. The sources of the target genes are shown in Table 1, and the target genes of the present application are the products after nucleic acid extraction by the magnetic bead method. The monitoring regions of the target genes are shown in Table 2. The nucleotide sequences of the detection primer pairs and detection probes for each target gene are shown in Table 3. The quadruple PCR system configurations including the buffers #1-#9 of the present application and the comparative buffers D1-D7 are shown in Table 4. In addition to configuring the reaction systems of the experimental wells, positive control wells and negative control wells also need to be set. The template of the positive control well is 10 3 copies / μL of the plasmid containing the transformed target region, and the template of the negative control well is ultrapure water. After completing the PCR system configuration, qPCR reactions were carried out on the SLAN fully automatic medical PCR analysis system, and the program settings are shown in Table 5.

[0087] Table 1

[0088]

[0089] Table 2

[0090]

[0091] Table 3

[0092]

[0093] Table 4

[0094]

[0095] Table 5

[0096]

[0097] After the PCR reaction is completed, set the baseline and threshold line, export the Ct values of each reaction well, and analyze them in combination with the amplification curve. If the positive control well has an obvious exponential amplification curve and the Ct value of the positive control is between 25 and 32, while the negative control well does not show a curve (no amplification), it indicates that this experiment is valid. The Ct values of the DNA detected using the reaction systems containing buffer #1 - #9 and the comparative buffers D1 - D7 are shown in Tables 6 - 9.

[0098] Table 6

[0099]

[0100] Table 7

[0101]

[0102] Table 8

[0103]

[0104] Table 9

[0105]

[0106] As can be seen from Table 6, based on the data of Examples 1 - 3, when detecting four target regions in the target gene with negative detection, the quadruple PCR amplification system containing the buffer of the present application does not cause non-specific amplification. Through further optimization of the buffer formulation, especially from the data of Example 3, it can be seen that the average Ct values of the HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene detected by the reaction system containing buffer #3 are 29.78, 29.63, 30.92, and 29.92 respectively. Compared with the average Ct values of the four target genes detected by the reaction systems containing buffer #1 and #2, the Ct values of the four target genes are all advanced, indicating that the detection sensitivity has been improved.

[0107] By comparing Example 3 with Example 4, it can be known that the temperature-controlled magnesium ion nanocapsule in Example 4 added a cross-linking agent. The average Ct values of the HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene detected by the reaction system containing buffer #4 are all advanced compared with the average Ct values of the four target genes detected by the reaction system containing buffer #3. This is because the cross-linking agent connects the polymer chains in the temperature-sensitive polymer through the carboxyl-amino reaction of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to form a three-dimensional network structure, enhancing the mechanical stability of the outer shell. At the same time, the cross-linking agent can covalently connect the carboxyl group of the temperature-sensitive polymer and the amino group on the surface of the inner core to prevent stratification or rupture during centrifugation or temperature cycling.

[0108] The buffer #5 in Example 5 was added with choline hydroxybutyrate. The average Ct values of HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene detected in the reaction system containing buffer #5 were all earlier than the average Ct values of the four target genes detected in the reaction system containing buffer #3. And the buffer #6 in Example 6 was added with both choline hydroxybutyrate and cross-linking agent. The average Ct values of the four target genes detected in the reaction system containing buffer #6 were earlier than the average Ct values of the four target genes detected in the reaction system containing buffer #5. The reason is that choline hydroxybutyrate, as an amphoteric ionic compound, combines the cationic characteristics of choline and the anionic group of hydroxybutyric acid, has strong water solubility and thermal stability, stabilizes the DNA double strand and polymerase activity by forming a dynamic hydration layer, and reduces the influence of Mg 2+ concentration fluctuations. Its amphoteric structure can preferentially bind heavy metal ions (such as Fe 3+ , Cu 2+ ) in the sample, prevent heavy metal ions from competing with Mg 2+ for the polymerase active site, and at the same time can inhibit the formation of primer dimers and template secondary structures, and can neutralize common PCR inhibitors such as phenols and humic acids, improving the detection success rate of complex samples.

[0109] Compared with Example 3, the buffer in Example 7 was added with the positive stabilizer ε-polylysine and the negative stabilizer sodium tungstosilicate. The average Ct values of HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene detected in the reaction system containing buffer #7 were earlier than the average Ct values of the target genes detected in the reaction system containing buffer #3. Compared with Example 7, the buffer #8 in Example 8 was added with the positive stabilizer ε-polylysine, the negative stabilizer sodium tungstosilicate and cross-linking agent. The average Ct values of the four target genes detected in the reaction system containing buffer #8 were earlier than the average Ct values of the four target genes detected in the reaction system containing buffer #7. This is because ε-polylysine, as a cationic stabilizer, is positively charged and partially replaces Mg 2+ through electrostatic interaction, directly binds to DNA, reduces the demand of the PCR reaction system for Mg 2+ . When the concentration of Mg 2+ is low, ε-polylysine maintains the stability of the DNA template, preventing folding or degradation. When the concentration of Mg 2+ is high, the flexible long-chain structure of ε-polylysine can shield excessive Mg 2+ , avoiding polymerase activity inhibition. ε-polylysine can also preferentially bind negatively charged inhibitors (such as heparin and humic acid) in the sample, preventing them from consuming Mg 2+ . Anions such as C1 - , SO 2- in the detection sample will combine with Mg 2+Form weak binding compounds (such as Mg2Cl) to reduce free Mg 2+ Sodium tungstosilicate acts as an anion stabilizer, and its nanocage structure absorbs these anions through ion exchange, maintaining Mg 2+ Activity. The drastic fluctuation of ionic strength in the buffer will affect the activity of polymerase. Sodium tungstosilicate acts as an "ion buffer" to maintain the electrical neutrality of the reaction system by reversibly adsorbing or releasing anions. In addition, it can also combine with test samples such as Fe 3+ , Cu 2+ to prevent competitive inhibition of Mg 2+ .

[0110] like Figures 1-4 As shown in Tables 6 to 9, the buffer #9 in Example 9 simultaneously added positive stabilizer ε-polylysine, negative stabilizer sodium tungstosilicate, cross-linking agent, and choline hydroxybutyrate. The Ct value averages of the four target genes detected by the reaction system containing buffer #9 were significantly earlier than the Ct value averages of the four target genes detected by the reaction system containing buffers #1 to #8, indicating that the kit containing buffer #9 has the highest detection sensitivity and no nonspecific amplification. Figure 5 As can be seen from the figure, as the PCR reaction proceeds, the Mg in the temperature-controlled magnesium ion nanocapsules 2+ The cumulative release amount and release rate will change according to the temperature changes in different steps. In the reverse transcription stage, the temperature is 50℃, and the Mg in the temperature-controlled magnesium ion nanocapsules 2+ The release rate is slow, Mg 2+ The cumulative release amount increased slowly. As the temperature rose to 95°C during the pre-denaturation stage, the Mg in the temperature-controlled magnesium nanocapsules 2+ The release rate increased rapidly and then stabilized. 2+ The cumulative release amount increased significantly until the Mg in the controlled magnesium nanocapsules 2+ All released completely, Mg 2+ The concentration remains constant in the reaction system.

[0111] Compared with Example 9, in Comparative Examples 1-7, the comparison buffer D1 in Comparative Example 1 does not contain temperature-controlled magnesium ion nanocapsules, but is replaced by MgCl2 solution; the preparation steps of the temperature-controlled magnesium ion nanocapsules in Comparative Example 2 are different, resulting in the structure of the temperature-controlled magnesium ion nanocapsules not being a core-shell structure; in Comparative Example 3, silica is used instead of poly (N-isopropylacrylamide); the temperature-controlled magnesium ion nanocapsules in Comparative Example 4 lack pyrophosphatase; in Comparative Example 5, DNA polymerase is used instead of pyrophosphatase; in Comparative Example 6, phenolic resin is used instead of the cross-linking agent of the present application; and in Comparative Example 7, a buffer purchased on the market is used instead of the buffer of the present application.

[0112] The average Ct values of the four target genes detected in the reaction system containing buffers D1 - D7 were significantly delayed compared to the average Ct values of the four target genes detected in the reaction system containing buffer #9, and non-specific amplification occurred in all cases. It can be seen from this that the materials lacking or replaced in the buffer cannot play a role in the buffer, but instead reduce the function of the buffer. Therefore, each component cannot be randomly replaced by other materials.

[0113] The above are all preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby. Therefore, all equivalent changes made according to the structure, shape, and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A buffer solution for supporting multiplex fluorescence quantitative PCR, characterized in that, The buffer solution comprises 10 mM - 20 mM Tris-HCl, 15 mM - 30 mM (NH4)2SO4, 50 mM - 100 mM KCl, 0.5 - 1.0 mg / ml temperature-controlled magnesium ion nanocapsules, and additives; The additives comprise polyethylene glycol at a concentration of 5 - 10% (v / v) of the buffer solution, 0.8 M - 1.2 M betaine, 0.5 M - 1.0 M trehalose, 15 mM - 40 mM tetramethylammonium chloride, and 0.1 - 0.5 mg / mL acetylated BSA; The temperature-controlled magnesium ion nanocapsules have a core-shell structure. The core comprises 1 mM - 5 mM MgCl2 at a concentration of the buffer solution, 0.1 - 0.5 U / μL pyrophosphatase, 10 - 30 mg / mL phospholipids, 5 - 15 mg / mL cholesterol, chloroform or methanol or a mixed solution of chloroform and methanol with a mass ratio of 3:

1. The shell comprises a thermosensitive polymer at a concentration of 20 - 40 wt% of the buffer solution, and the thermosensitive polymer is any one of poly(N-vinylcaprolactam), poly(N-isopropylacrylamide), and poly(methyl vinyl ether); The preparation steps of the temperature-controlled magnesium ion nanocapsules are as follows: Dissolve phospholipids and cholesterol in chloroform or methanol or a mixed solution of chloroform and methanol with a mass ratio of 3:1, and rotary evaporate to form a uniform lipid film. Hydrate the lipid film with a mixed solution of MgCl2 and pyrophosphatase, shake and centrifuge to obtain monolayer liposomes. Dissolve the thermosensitive polymer in PBS solution pre-cooled at 3 - 5°C, and stir magnetically until completely dissolved. Mix the liposomes and the thermosensitive polymer solution at a mass ratio of 1:2, incubate at 3 - 5°C for 2 - 3 h, and centrifuge and purify to obtain the temperature-controlled magnesium ion nanocapsules.

2. A buffer for supporting multiplex fluorescence quantitative PCR according to claim 1, characterized in that, The temperature-controlled magnesium ion nanocapsules further comprise a cross-linking agent, and the cross-linking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1 - 2:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:15 - 20.

3. A buffer for supporting multiplex fluorescence quantitative PCR according to any one of claims 1 or 2, characterized in that, The additives in the buffer solution further comprise choline hydroxybutyrate at a concentration of 0.1 mM - 0.3 mM of the buffer solution.

4. A buffer solution for supporting multiplex fluorescence quantitative PCR according to claim 3, characterized in that, The buffer solution comprises a stabilizer, and the stabilizer is ε-polylysine at a concentration of 0.1 mM - 0.3 mM of the buffer solution and sodium tungstosilicate at 0.01 - 0.05%.

5. A method for preparing a buffer solution for supporting multiplex fluorescence quantitative PCR according to any one of claims 1-4, characterized in that, The preparation method comprises the following steps: Preparation of temperature-controlled magnesium ion nanocapsules: Dissolve phospholipids and cholesterol in chloroform or methanol or a mixed solution of chloroform and methanol with a mass ratio of 3:1, and rotary evaporate to form a uniform lipid film. Hydrate the lipid film with a mixed solution of MgCl2 and pyrophosphatase, shake and centrifuge to obtain monolayer liposomes. Dissolve the thermosensitive polymer in PBS solution pre-cooled at 3 - 5°C, and stir magnetically until completely dissolved. Mix the liposomes and the thermosensitive polymer solution at a mass ratio of 1:2, incubate at 3 - 5°C for 2 - 3 h, and centrifuge and purify to obtain the temperature-controlled magnesium ion nanocapsules; Mixing of each component: Weigh or dilute each component according to specific concentrations and mix them, adjust the pH to 7.5 - 9.0, and then filter through a 0.22 μM aqueous filter membrane to sterilize, obtaining the PCR buffer.

6. The preparation method of a buffer solution for supporting multiplex fluorescence quantitative PCR according to claim 5, characterized in that, The preparation steps of the temperature-controlled magnesium ion nanocapsule further include mixing the liposome and the thermosensitive polymer solution at a mass ratio of 1:2, incubating at 3 - 5 °C for 2 - 3 h, adding a cross-linking agent and reacting at room temperature for 1 - 2 h, and centrifuging and purifying to obtain the temperature-controlled magnesium ion nanocapsule.

7. A kit for supporting multiplex fluorescence quantitative PCR, characterized in that, The kit includes the buffer for supporting multiplex fluorescence quantitative PCR described in any one of claims 1 - 4, or the buffer prepared by the preparation method of the buffer for supporting multiplex fluorescence quantitative PCR described in any one of claims 5 - 6. The kit further includes dNTP, DNA polymerase, and TaqMan probe.

8. Use of the buffer for supporting multiplex fluorescence quantitative PCR described in any one of claims 1 - 4, or the buffer prepared by the preparation method of the buffer for supporting multiplex fluorescence quantitative PCR described in any one of claims 5 - 6, or the kit for supporting multiplex fluorescence quantitative PCR described in claim 7 in the preparation of DNA amplification products.

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