Buffer solution supporting multiple fluorescent quantitative PCR (Polymerase Chain Reaction) and preparation method thereof

Abstract

The invention relates to a buffer solution supporting multiple fluorescent quantitative PCR and a preparation method thereof, and relates to the technical field of biochemical reagents, the buffer solution comprises Tris-HCl, (NH4) 2SO4, KCl, a temperature control type magnesium ion nanocapsule and an additive; the additive is prepared from polyethylene glycol, betaine, trehalose, tetramethylammonium chloride and acetylated BSA (Bovine Serum Albumin); the temperature control type magnesium ion nanocapsule is of a core-shell structure, the core comprises MgCl2 accounting for 1-5 mM of the concentration of the buffer solution, pyrophosphatase accounting for 0.1-0.5 U / mu L, phospholipid accounting for 10-30 mg / mL, cholesterol accounting for 5-15 mg / mL and an inorganic solution, and the shell comprises a temperature-sensitive polymer accounting for 20-45 wt% of the concentration of the buffer solution. The buffer solution can dynamically adjust the concentration of Mg < 2 + >, and the situation that the amplification efficiency is affected due to the fact that the concentration of Mg < 2 + > is difficult to adjust is avoided.

Description

Technical Field

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

[0002] The basic components of PCR reaction (including qPCR) include 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 sequence of the 2+ domain of the target region in the template DNA. During the PCR reaction, the DNA polymerase catalyzes the extension from the 3' end of the primer; Mg 2+ It 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 chains. When performing PCR reactions (including qPCR), buffer is usually added, and the components in the buffer can provide a suitable chemical reaction environment for DNA polymerase.

[0003] Dye-based fluorescence quantitative PCR most commonly uses SYBR Green I as a DNA binding dye. This dye can produce fluorescence when it binds to DNA, but does not produce fluorescence when it is free. For each DNA double strand formed, a certain amount of dye will bind to it, and the product can be quantified by the accumulation of signals. Although this method has the advantages of low price and wide applicability, its specificity is poor, and it cannot distinguish between amplification products and non-specific amplification, and it cannot quantify different target products separately. These deficiencies limit the application of dye-based fluorescence quantitative PCR. The probe-based fluorescence quantitative PCR completely makes up for these deficiencies, and multiplex fluorescence quantitative PCR can even quantify multiple target products at the same time. Since multiplex amplification has preferences and the products have strong mutual interference, the fluorescence quantitative PCR reagents of the common probe method cannot be used for multiplex fluorescence quantitative PCR, so the optimization of multiplex fluorescence quantitative reagents is very necessary.

[0004] The Chinese invention patent application with publication number CN109536587A discloses an efficient multiplex fluorescence quantitative PCR kit and a preparation method thereof. An efficient multiplex fluorescence quantitative PCR reaction solution comprises Tris-HCl solution, dNTPs solution, KCl solution, (NH 4 ) 2 SO 4 Solution, MgCl 2 The solution is composed of BSA solution, glycerol solution and DTT solution.

[0005] The efficient multiplex fluorescence quantitative PCR reaction solution was tested for reagent sensitivity and amplification performance, and it was found that the reagent has higher sensitivity and amplification performance than traditional reagents. The invention optimizes the multiplex fluorescence quantitative PCR reaction solution, especially by adding some specific additives, such as a certain proportion of glycerol, BSA and DTT, etc., to improve the sensitivity, specificity and stability of the reaction. However, the above-mentioned reaction solution has complex components for some biological samples. For example, fecal samples contain PCR inhibitors such as bile acid / deoxycholic acid, polysaccharides, and phytic acid. The residual PCR inhibitors still present in the DNA template after extraction, conversion, and purification may inhibit the amplification of the target area during the PCR reaction, resulting in a delay in the Ct value of PCR amplification, or even causing the target area to be unable to be detected, thereby seriously affecting the detection of the sample.

[0006] The Chinese invention patent application with publication number CN117230163A discloses a PCR buffer, a kit and an application. The PCR buffer comprises the following components: 1mM-8mMMg 2+ 、10mM-250mMK + ,5mM-80mMTris-HCl,10mM-50mM(NH 4 ) 2 SO 4 , glycerol and additives, wherein the additives include any one or more of betaine, DMSO, 2-pyrrolidone, TMAC, PEG, Tween-20 and BSA.

[0007] The above PCR buffer is added with appropriate amounts of betaine, DMSO, 2-pyrrolidone, TMAC, PEG, Tween-20 or BSA to improve the specificity and sensitivity of amplifying DNA in stool samples in ordinary PCR buffer. Through the combination of multiple additives, it is found that the combination of betaine and most additives has a synergistic effect in amplifying template DNA in stool samples; and it is found that the combination of DMSO and 2-pyrrolidone and one or more of betaine, TMAC, PEG, Tween-20 and BSA have the effect of synergistically promoting the sensitivity of DNA amplification in stool samples and improving the specificity; it is further found that the addition of appropriate amounts of betaine, DMSO, 2-pyrrolidone, TMAC, Tween-20 and BSA to the PCR buffer has the best amplification effect. However, Mg in the above PCR buffer reaction system 2+ The concentration is difficult to control. 2+ When the concentration is too low, the efficiency of primer-template binding will decrease significantly, and the amplification efficiency will also decrease. 2+When the concentration is too high, it is easy to cause nonspecific amplification or primer dimer formation, resulting in false positives. In the multiplex fluorescence quantitative PCR buffer, high concentration of Mg 2+ Although it can inhibit some nonspecific amplification, it is easy to mask the target signal, resulting in delayed Ct value or abnormal melting curve. Summary of the invention

[0008] In order to solve the above Mg 2+ The technical problem that the concentration is difficult to control is solved. The present invention provides a buffer supporting multiplex fluorescence quantitative PCR and a preparation method thereof. The buffer of the present invention can dynamically adjust the Mg 2+ concentration, avoid Mg 2+ The concentration is difficult to adjust, which affects the amplification efficiency.

[0009] In a first aspect, the present invention provides a buffer supporting multiplex fluorescence quantitative PCR, the buffer comprising 10mM-20mM Tris-HCl, 15mM-30mM (NH 4 ) 2 SO 4 , 50mM-100mMKCl, 0.5-1.0mg / ml temperature-controlled magnesium ion nanocapsules, additives; The additives include 5-10% (v / v) polyethylene glycol, 0.8M-1.2M betaine, 0.5M-1.0M trehalose, 15mM-50mM tetramethylammonium chloride, and 0.1-0.5mg / mL acetylated BSA at a concentration of 5-10% (v / v) in the buffer; The temperature-controlled magnesium ion nanocapsule is a core-shell structure, wherein the core comprises MgCl 2 , 0.1-0.5U / μL pyrophosphatase, 10-30mg / mL phospholipid, 5-15mg / mL cholesterol, and an inorganic solution, wherein the shell comprises a temperature-sensitive polymer accounting for 20-45wt% of the buffer concentration.

[0010] In the above technical solution, the Tris-HCl, (NH 4 ) 2 SO 4, KCl, as the core component of PCR buffer, mainly plays the role of maintaining pH stability in the reaction system, providing essential ion environment, protecting enzyme activity and enhancing amplification specificity. Betaine is added to the additives mainly to balance the stability of GC / AT base pairs and improve the efficiency of long fragment amplification; acetylated BSA is added mainly to neutralize inhibitors (such as humic acid) and protect enzyme activity; trehalose is added mainly to prevent enzyme denaturation and enhance thermal stability; tetramethylammonium chloride can reduce the difficulty of annealing high GC content templates and reduce non-specific products; polyethylene glycol is added mainly to increase the viscosity of the PCR reaction system, reduce the interaction between primers, and thus reduce the formation of primer dimers. ‌‌‌ When the temperature-controlled magnesium ion nanocapsule is in the low temperature stage, the nanocapsule swells and slowly releases Mg 2+ , reducing the generation of primer dimers and nonspecific amplification. At high temperatures, the nanocapsules shrink and rapidly squeeze to release Mg. 2+ , dynamically adjust Mg 2+ concentration. Among them, MgCl 2 As a magnesium ion source, pyrophosphatase is used as the core of the nanocapsule. Pyrophosphatase can decompose the pyrophosphate produced by the side reaction and prevent Mg from 2+ Chelate with pyrophosphate. The thermosensitive polymer as the shell material has a low critical solution temperature. The polymer chain is easily hydrophilic and swells at low temperatures to form a porous gel structure, encapsulating Mg 2+ Limiting its release, reducing primer dimers and nonspecific binding, the polymer chain is prone to hydrophobic contraction at high temperatures to form a dense structure, squeezing out the internal Mg 2+ The solution meets the high-efficiency extension requirements of DNA polymerase. At the same time, the pyrophosphate produced in the high temperature stage will be hydrolyzed by pyrophosphatase to avoid its reaction with Mg 2+ Combination leads to Mg 2+ Concentration dropped suddenly.

[0011] Optionally, the temperature-controlled magnesium ion nanocapsules also include a crosslinker, which is a mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1-2:1, and the mass ratio of the crosslinker to the temperature-sensitive polymer is 1:15-20.

[0012] In the above technical scheme, the cross-linking agent connects the polymer chains in the thermosensitive polymer to each other through the carboxyl-amino reaction of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to form a three-dimensional network structure, thereby enhancing the mechanical stability of the shell. At the same time, the cross-linking agent can covalently connect the carboxyl group of the thermosensitive polymer and the amino group on the surface of the inner core to avoid stratification or rupture during centrifugation or temperature cycling.

[0013] Optionally, the additive in the buffer further comprises choline hydroxybutyrate at a concentration of 0.1 mM-0.3 mM in the buffer.

[0014] In the above technical solution, choline hydroxybutyrate is an ionic compound formed by neutralization reaction of β-hydroxybutyric acid and choline, and choline as a cation is bonded to the hydroxybutyrate anion through an ionic bond. Choline hydroxybutyrate is a zwitterionic compound that combines the cationic properties of choline and the anionic group of hydroxybutyric acid, has strong water solubility and thermal stability, stabilizes DNA double-strands and polymerase activity by forming a dynamic hydration layer, and reduces Mg 2+ The amphoteric structure can preferentially bind to heavy metal ions (such as Fe 3+ , Cu 2+ ), to prevent heavy metal ions from reacting with 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.

[0015] 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.

[0016] 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+ .

[0017] C1 in the test sample - 、SO 2- The anions will react with Mg 2+ Formation of weakly bound compounds (such as Mg 2 Cl), reducing 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+ .

[0018] 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: Preparation of temperature-controlled magnesium ion nanocapsules: Phospholipids and cholesterol were dissolved in an inorganic solution and rotary evaporated to form a uniform lipid film. 2 and pyrophosphatase mixed solution to hydrate the lipid film, shake and centrifuge to obtain single-layer liposomes, dissolve the thermosensitive polymer in a PBS solution precooled at 3-5°C, 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 2-3h, and purify by centrifugation to obtain temperature-controlled magnesium ion nanocapsules; Mixing of components: Weigh or dilute the components according to specific concentrations and mix them, adjust the pH to 7.5-9.0, and then sterilize through a 0.22 μM water filter to obtain a PCR buffer.

[0019] In the above technical solution, during the preparation of the temperature-controlled magnesium ion nanocapsules, the surface of the liposome is negatively charged, and the thermosensitive polymer is adsorbed on the outer layer of the liposome by electrostatic action to form a "liposome-thermosensitive polymer" core-shell structure.

[0020] Optionally, the preparation step of the temperature-controlled magnesium ion nanocapsules further includes mixing the liposomes and the thermosensitive polymer solution in a mass ratio of 1:1.5-2, incubating at 3-5°C for 2-3h, adding a cross-linking agent to react at room temperature for 1-2h, and centrifuging and purifying to obtain the temperature-controlled magnesium ion nanocapsules.

[0021] In the above technical scheme, the cross-linking agent connects the polymer chains in the thermosensitive polymer to each other through the carboxyl-amino reaction to form a three-dimensional network structure, thereby enhancing the mechanical stability of the shell. At the same time, the cross-linking agent can covalently connect the carboxyl group of the thermosensitive polymer and the amino group on the surface of the inner core to prevent stratification or rupture during centrifugation or temperature cycling.

[0022] In a third aspect, the present invention provides a kit for PCR reaction, the kit comprising the above-mentioned PCR buffer, the kit further comprising dNTP, DNA polymerase and TaqMan probe.

[0023] In a fourth aspect, the present invention provides a buffer supporting multiplex fluorescence quantitative PCR, or a buffer prepared by a method for preparing a buffer supporting multiplex fluorescence quantitative PCR, or an application of a kit supporting multiplex fluorescence quantitative PCR in the preparation of DNA amplification products.

[0024] In summary, the present invention includes at least one of the following beneficial technical effects: 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 nonspecific amplification, the nanocapsules shrink during the high temperature stage, and the rapid extrusion releases Mg 2+ , dynamically adjust Mg 2+ concentration.

[0025] 2. By adding stabilizers, ε-polylysine is used as a cationic stabilizer to partially replace Mg through electrostatic action. 2+ , directly binds to DNA, reducing the Mg content of the PCR reaction system 2+ It can also preferentially bind 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 anion stabilizer, and its nanocage structure adsorbs C1 through ion exchange - 、SO 2- Isoanions, maintaining Mg 2+ activity, and can also 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+ .

[0026] 3. By adding choline hydroxybutyrate, a dynamic hydration layer is formed to stabilize the DNA double strand and polymerase activity, reducing Mg 2+ The amphoteric structure can preferentially bind heavy metal ions (such as Fe 3+ , Cu 2+ ), to prevent heavy metal ions from reacting with Mg 2+ It competes for the active sites of the polymerase and can inhibit the formation of primer dimers and template secondary structures. It can also neutralize common PCR inhibitors such as phenols and humic acid, thereby improving the detection success rate of complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the amplification curve of HCV gene amplified using buffer #9; Figure 2 is the amplification curve of HBV gene amplified using buffer #9; Figure 3 is the amplification curve of HIV-1 gene amplified using buffer #9; Figure 4 is the amplification curve of HIV-2 gene amplified using buffer #9; Figure 5 The temperature-controlled magnesium nanocapsules in buffer #9 are Mg-free as the PCR reaction proceeds. 2+ Release diagram. DETAILED DESCRIPTION

[0028] The following combination Figure 1-Figure 4 The present invention is further described in detail with reference to the accompanying drawings and specific embodiments.

[0029] Example 1: This example discloses a buffer #1 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0030] A buffer #1 supporting multiplex fluorescence quantitative PCR includes 20 mM Tris-HCl, 15 mM (NH 4 ) 2 SO 4 , 100mM KCl, 0.5mg / ml temperature-controlled magnesium ion nanocapsules, 5% (v / v) polyethylene glycol, 0.8M betaine, 1.0M trehalose, 15mM tetramethylammonium chloride, 0.1mg / mL acetylated BSA, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 1mM of the buffer concentration 2 , 0.1 U / μL pyrophosphatase, 10 mg / mL phospholipid, 5 mg / mL cholesterol, and an inorganic solution, wherein the shell comprises a temperature-sensitive polymer accounting for 20 wt % of the buffer concentration.

[0031] In this embodiment, the temperature-sensitive polymer is poly-N-vinyl caprolactam. In other embodiments, the temperature-sensitive polymer can also be poly-N-isopropylacrylamide, polymethyl vinyl ether and other temperature-sensitive polymers. Chloroform is selected as the inorganic solvent. In other embodiments, methanol or a mixture of chloroform and methanol can also be selected. The particle size of the temperature-controlled magnesium ion nanocapsules described in this embodiment and other embodiments is 50-200nm.

[0032] A method for preparing a buffer #1 supporting multiplex fluorescence quantitative PCR: S1. Preparation of liposomes: Dissolve phospholipids and cholesterol in inorganic solution, and form a uniform lipid film by rotary evaporation. 2 and pyrophosphatase mixture to hydrate the lipid film, and then centrifuge to obtain unilamellar liposomes; S2. Preparation of temperature-controlled magnesium ion nanocapsules: The thermosensitive polymer was dissolved in a PBS solution precooled at 4°C, and magnetically stirred until completely dissolved. The liposomes were mixed with the poly (N-vinyl caprolactam) solution at a mass ratio of 1:2, and the mixture was incubated at 4°C for 2 h. The temperature-controlled magnesium ion nanocapsules were purified by centrifugation. S3. Mixing of components: Weigh or dilute the components according to specific concentrations and mix them, adjust the pH to 8.5, and then sterilize through a 0.22 μM water filter to obtain buffer #1 that supports multiplex fluorescence quantitative PCR.

[0033] Example 2: This example discloses a buffer #2 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0034] A buffer #2 supporting multiplex fluorescence quantitative PCR includes 10 mM Tris-HCl, 30 mM (NH 4 ) 2 SO 4 , 50mM KCl, 1.0mg / ml temperature-controlled magnesium ion nanocapsules 10% (v / v) polyethylene glycol, 1.2M betaine, 0.5M trehalose, 40mM tetramethylammonium chloride, 0.5mg / mL acetylated BSA, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 5mM of the buffer concentration 2 , 0.5 U / μL pyrophosphatase, 30 mg / mL phospholipid, 15 mg / mL cholesterol, methanol, the shell comprising polymethyl vinyl ether accounting for 40 wt % of the buffer concentration.

[0035] The preparation method of buffer #2 supporting multiplex fluorescence quantitative PCR is the same as that in Example 1.

[0036] Example 3: This example discloses a buffer #3 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0037] A buffer #3 supporting multiplex fluorescence quantitative PCR includes 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 3mM of the buffer concentration 2, 0.3U / μL pyrophosphatase, 20mg / mL phospholipid, 10mg / mL cholesterol, and a mixture of chloroform and methanol in a mass ratio of 3:1, wherein the shell includes poly N-isopropylacrylamide accounting for 30wt% of the buffer concentration.

[0038] The preparation method of buffer #3 supporting multiplex fluorescence quantitative PCR is the same as that in Example 1.

[0039] Example 4: This example discloses a buffer #4 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0040] A buffer #4 supporting multiplex fluorescence quantitative PCR includes 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 3mM of the buffer concentration 2 , 0.3U / μL pyrophosphatase, 20mg / mL phospholipids, 10mg / mL cholesterol, and a mixture of chloroform and methanol in a mass ratio of 3:1, the shell comprising poly N-isopropylacrylamide accounting for 30wt% of the buffer concentration, and a cross-linking agent, the cross-linking agent is a mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0041] A method for preparing buffer #4 supporting multiplex fluorescence quantitative PCR: S1. Preparation of core liposomes: Dissolve phospholipids and cholesterol in inorganic solution, and form a uniform lipid film by rotary evaporation. 2 and pyrophosphatase mixture to hydrate the lipid film, and centrifuge to obtain unilamellar liposomes; S2. Preparation of the shell: dissolve the thermosensitive polymer in a PBS solution precooled at 4°C and stir magnetically until completely dissolved; S3, combining the inner core with the outer shell: liposomes were mixed with poly (N-isopropylacrylamide) at a mass ratio of 1:2, incubated at 4°C for 2 h, a cross-linking agent was added and reacted at room temperature for 2 h, and purified by centrifugation to obtain temperature-controlled magnesium ion nanocapsules; S4. Mixing of components: Weigh or dilute the components according to specific concentrations and mix them, adjust the pH to 8.5, and then sterilize through a 0.22 μM water filter to obtain buffer #4 that supports multiplex fluorescence quantitative PCR.

[0042] Example 5: This example discloses a buffer #5 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0043] A buffer #5 supporting multiplex fluorescence quantitative PCR includes 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, 0.2mM choline hydroxybutyrate, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 3mM of the buffer concentration 2 , 0.3U / μL pyrophosphatase, 20mg / mL phospholipid, 10mg / mL cholesterol, and a mixture of chloroform and methanol in a mass ratio of 3:1, wherein the shell includes poly N-isopropylacrylamide accounting for 30wt% of the buffer concentration.

[0044] The preparation method of buffer #5 supporting multiplex fluorescence quantitative PCR is the same as that in Example 1.

[0045] Example 6: This example discloses a buffer #6 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0046] A buffer #6 supporting multiplex fluorescence quantitative PCR includes 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, 0.2mM choline hydroxybutyrate, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 3mM of the buffer concentration 2 , 0.3U / μL pyrophosphatase, 20mg / mL phospholipids, 10mg / mL cholesterol, and a mixture of chloroform and methanol in a mass ratio of 3:1, the shell comprising poly N-isopropylacrylamide accounting for 30wt% of the buffer concentration, and a cross-linking agent, the cross-linking agent is a mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0047] The preparation method of buffer #6 supporting multiplex fluorescence quantitative PCR is the same as that of Example 4.

[0048] Example 7: This example discloses a buffer #7 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0049] A buffer #5 supporting multiplex fluorescence quantitative PCR includes 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, 0.2mM ε-polylysine, 0.03% sodium tungstosilicate, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 3mM of the buffer concentration 2 , 0.3U / μL pyrophosphatase, 20mg / mL phospholipid, 10mg / mL cholesterol, and a mixture of chloroform and methanol in a mass ratio of 3:1, wherein the shell includes poly N-isopropylacrylamide accounting for 30wt% of the buffer concentration.

[0050] The preparation method of buffer #7 supporting multiplex fluorescence quantitative PCR is the same as that in Example 1.

[0051] Example 8: This example discloses a buffer #8 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0052] A buffer #8 supporting multiplex fluorescence quantitative PCR includes 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, 0.2mM ε-polylysine, 0.03% sodium tungstosilicate, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 3mM of the buffer concentration 2, 0.3U / μL pyrophosphatase, 20mg / mL phospholipids, 10mg / mL cholesterol, and a mixture of chloroform and methanol in a mass ratio of 3:1, the shell comprising poly N-isopropylacrylamide accounting for 30wt% of the buffer concentration, and a cross-linking agent, the cross-linking agent is a mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0053] The preparation method of buffer #8 supporting multiplex fluorescence quantitative PCR is the same as that of Example 4.

[0054] Example 9: This example discloses a buffer #9 supporting multiplex fluorescence quantitative PCR and a preparation method thereof.

[0055] A buffer #6 supporting multiplex fluorescence quantitative PCR includes 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, 0.2mM choline hydroxybutyrate, 0.2mM ε-polylysine, 0.03% sodium tungstosilicate, the temperature-controlled magnesium ion nanocapsules are core-shell structures, the core includes MgCl accounting for 3mM of the buffer concentration 2 , 0.3U / μL pyrophosphatase, 20mg / mL phospholipids, 10mg / mL cholesterol, and a mixture of chloroform and methanol in a mass ratio of 3:1, the shell comprising poly N-isopropylacrylamide accounting for 30wt% of the buffer concentration, and a cross-linking agent, the cross-linking agent is a mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the thermosensitive polymer is 1:18.

[0056] The preparation method of buffer #9 supporting multiplex fluorescence quantitative PCR is the same as that of Example 4.

[0057] Comparative Example 1: This comparative example provides a buffer D1 for supporting multiplex fluorescence quantitative PCR, the buffer comprising 15 mM Tris-HCl, 20 mM (NH 4 ) 2 SO 4 , 80 mM KCl, 3 mM MgCl 2, 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, and 0.03% sodium tungstosilicate.

[0058] The preparation method of buffer D1 supporting multiplex fluorescence quantitative PCR is as follows: Mixing of components: 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 water filter to obtain a buffer D1 that supports multiplex fluorescence quantitative PCR.

[0059] Comparative Example 2: This comparative example provides a buffer D2 for supporting multiplex fluorescence quantitative PCR, including 15mM Tris-HCl, 20mM (NH 4 ) 2 SO 4 , 80mM KCl, 0.8mg / ml temperature-controlled magnesium ion nanocapsules, 8% (v / v) polyethylene glycol, 1.0M betaine, 0.7M trehalose, 30mM tetramethylammonium chloride, 0.3mg / mL acetylated BSA, 0.2mM choline hydroxybutyrate, 0.2mM ε-polylysine, 0.03% sodium tungstosilicate, the temperature-controlled magnesium ion nanocapsules are shell-free structures, including MgCl accounting for 3mM of the buffer concentration 2 , 0.3U / μL pyrophosphatase, 30wt% poly (N-isopropylacrylamide), 20mg / mL phospholipids, 10mg / mL cholesterol, a mixture of chloroform and methanol in a mass ratio of 3:1, and a cross-linking agent, wherein the cross-linking agent is a mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1.5:1, and the mass ratio of the cross-linking agent to the temperature-sensitive polymer is 1:18.

[0060] The preparation method of buffer D2 supporting multiplex fluorescence quantitative PCR is as follows: S1. Preparation of temperature-controlled magnesium ion nanocapsules: Phospholipids and cholesterol were dissolved in an inorganic solution and rotary evaporated to form a uniform lipid film. 2 and pyrophosphatase mixture to hydrate the lipid film, dissolve the thermosensitive polymer in a PBS solution precooled at 4°C, stir magnetically until completely dissolved, dissolve the lipid film in a poly (N-isopropylacrylamide) solution at a mass ratio of 1:2, incubate at 4°C for 2h, add a crosslinker and react at room temperature for 2h, and purify by centrifugation to obtain a temperature-controlled magnesium ion nanocapsule with a shell-free structure; S2. Mixing of components: Weigh or dilute the components according to specific concentrations and mix them, adjust the pH to 8.5, and then sterilize through a 0.22 μM water filter to obtain a buffer D2 that supports multiplex fluorescence quantitative PCR.

[0061] Comparative Example 3: This comparative example provides a buffer D3 for supporting multiplex fluorescence quantitative PCR. The buffer D3 is the same as Example 9, except that silicon dioxide is used instead of poly (N-isopropylacrylamide).

[0062] Comparative Example 4: This comparative example provides a buffer D4 for supporting multiplex fluorescence quantitative PCR. The buffer D4 is the same as Example 9, except that the temperature-controlled magnesium ion nanocapsules lack pyrophosphatase.

[0063] Comparative Example 5: This comparative example provides a comparison of buffer D5 supporting multiplex fluorescence quantitative PCR. It is the same as Example 9, except that DNA polymerase is used instead of pyrophosphatase.

[0064] Comparative Example 6: This comparative example provides a comparison buffer D6 supporting multiplex fluorescence quantitative PCR, which is the same as Example 9, except that phenolic resin is used instead of the mixture of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide.

[0065] Comparative Example 7: The buffer solution of this comparative example is PCR buffer solution D7 purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0066] The buffers #1-#9 of Examples 1-9 of the present application and the comparison buffers D1-D7 of Comparative Examples 1-7 are respectively applied to the kit, which also includes a detection primer pair, dNTP (Tiangen Biochemical Technology (Beijing) Co., Ltd.), TaqDNA 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 are selected as target genes, respectively. The sources of the target genes are shown in Table 1, and the target genes of the present application are products after nucleic acid extraction by magnetic beads. The monitoring area of ​​the target gene is shown in Table 2. The nucleotide sequences of each target gene detection primer pair and detection probe are shown in Table 3. The quadruple PCR system configuration including buffers #1-#9 of the present application and comparison buffers D1-D7 is shown in Table 4. In addition to configuring the reaction system of the experimental wells, positive control wells and negative control wells need to be set, and the template of the positive control well is 10 3 The template of the negative control well was ultrapure water. After completing the PCR system configuration, the qPCR reaction was performed on the SLAN fully automatic medical PCR analysis system. The program settings are shown in Table 5.

[0067] Table 1

[0068] Table 2

[0069] Table 3

[0070] Table 4

[0071] Table 5

[0072] After the PCR reaction is completed, the baseline and threshold lines are set, and the Ct values ​​of each reaction well are derived and analyzed 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, and the negative control well does not have a line (no amplification), it indicates that this experiment is effective. The Ct values ​​of DNA detected using the reaction system containing buffers #1-#9 and comparison buffers D1-D7 are shown in Tables 6-9.

[0073] Table 6

[0074] Table 7

[0075] Table 8

[0076] Table 9

[0077] It can be seen from Table 6 that, through the data of Examples 1-3, when detecting the four target regions in the negative target gene, the quadruple PCR amplification system containing the buffer of the present application will not cause nonspecific amplification. Through further optimization of the buffer formula, especially the data of Example 3, it can be seen that the reaction system containing buffer #3 detects the HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene with an average Ct value of 29.78, 29.63, 30.92, and 29.92, respectively. Compared with the average Ct value of the four target genes detected by the reaction system containing buffers #1 and #2, the Ct values ​​of the four target genes are all advanced, indicating that the sensitivity of the detection has been improved.

[0078] By comparing Example 3 with Example 4, it can be seen that the temperature-controlled magnesium ion nanocapsules in Example 4 are added with a cross-linker, and the Ct value averages of the HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene detected by the reaction system containing buffer #4 are all earlier than the Ct value averages of the four target genes detected by the reaction system containing buffer #3. This is because the cross-linker connects the polymer chains in the thermosensitive polymer to each other through the carboxyl-amino reaction of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to form a three-dimensional network structure, thereby enhancing the mechanical stability of the shell. At the same time, the cross-linker can covalently connect the carboxyl group of the thermosensitive polymer and the amino group on the surface of the inner core to prevent stratification or rupture during centrifugation or temperature cycling.

[0079] Buffer #5 in Example 5 added choline hydroxybutyrate, and the mean Ct values ​​of HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene detected by the reaction system containing buffer #5 were all earlier than the mean Ct values ​​of the four target genes detected by the reaction system containing buffer #3. Buffer #6 in Example 6 added choline hydroxybutyrate and a cross-linking agent at the same time, and the mean Ct values ​​of the four target genes detected by the reaction system containing buffer #6 were all earlier than the mean Ct values ​​of the four target genes detected by the reaction system containing buffer #5. The reason is that choline hydroxybutyrate, as a zwitterionic compound, combines the cationic properties 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 Mg 2+ The amphoteric structure can preferentially bind heavy metal ions (such as Fe 3+ , Cu 2+ ), to prevent heavy metal ions from reacting with Mg 2+ It competes for the active sites of the polymerase and can inhibit the formation of primer dimers and template secondary structures. It can also neutralize common PCR inhibitors such as phenols and humic acid, thereby improving the detection success rate of complex samples.

[0080] Compared with Example 3, the buffer in Example 7 adds a positive stabilizer ε-polylysine and a negative stabilizer sodium tungstosilicate. The average Ct values ​​of the HCV gene, HBV gene, HIV-1 gene, and HIV-2 gene detected by the reaction system containing buffer #7 are earlier than the average Ct values ​​of the target genes detected by the reaction system containing buffer #3. Compared with Example 7, the buffer #8 in Example 8 adds a positive stabilizer ε-polylysine, a negative stabilizer sodium tungstosilicate, and a cross-linking agent. The average Ct values ​​of the four target genes detected by the reaction system containing buffer #8 are earlier than the average Ct values ​​of the four target genes detected by the reaction system containing buffer #7. This is because ε-polylysine, as a cationic stabilizer, 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 inhibition of polymerase activity, ε-polylysine can also preferentially bind to negatively charged inhibitors in the sample (such as heparin, humic acid), preventing them from consuming Mg 2+ . Detection of C1 in samples - 、SO 2- The anions will react with Mg 2+ Formation of weakly bound compounds (such as Mg 2 Cl), reducing 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+ .

[0081] like Figure 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.

[0082] Comparative Examples 1-7 Compared with Example 9, the comparative buffer D1 in Comparative Example 1 does not contain temperature-controlled magnesium ion nanocapsules, but uses MgCl 2 solution instead; 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; in Comparative Example 7, a buffer purchased on the market is used instead of the buffer of the present application.

[0083] The Ct value averages of the four target genes detected by the reaction system containing buffer D1-D7 were significantly delayed compared to the Ct value averages of the four target genes detected by the reaction system containing buffer #9, and non-specific amplification occurred in both. It can be seen that the missing or replaced materials in the buffer cannot play a role in the buffer, but will reduce the effect of the buffer, so each component cannot be arbitrarily replaced by other materials.

[0084] The above are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A buffer supporting multiplex fluorescence quantitative PCR, characterized in that: The buffer comprises 10mM-20mM Tris-HCl, 15mM-30mM (NH4)2SO4, 50mM-100mM KCl, 0.5-1.0mg / ml temperature-controlled magnesium ion nanocapsules, and additives; The additives include 5-10% (v / v) polyethylene glycol, 0.8M-1.2M betaine, 0.5M-1.0M trehalose, 15mM-50mM tetramethylammonium chloride, and 0.1-0.5mg / mL acetylated BSA at a concentration of 5-10% (v / v) in the buffer; The temperature-controlled magnesium ion nanocapsule has a core-shell structure, wherein the core comprises MgCl2 accounting for 1 mM-5 mM of the buffer concentration, 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 comprises a temperature-sensitive polymer accounting for 20-45 wt% of the buffer concentration.

2. A buffer supporting multiplex fluorescence quantitative PCR according to claim 1, characterized in that: The temperature-controlled magnesium ion nanocapsules also include a crosslinking agent, which is a mixed solution of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1-2:1, and the mass ratio of the crosslinking agent to the temperature-sensitive polymer is 1:15-20.

3. A buffer supporting multiplex fluorescence quantitative PCR according to any one of claims 1 or 2, characterized in that: The additive in the buffer further includes choline hydroxybutyrate in a concentration of 0.1 mM to 0.3 mM in the buffer.

4. A buffer supporting multiplex fluorescence quantitative PCR according to claim 3, characterized in that: 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.

5. A method for preparing a buffer supporting multiplex fluorescence quantitative PCR according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Preparation of temperature-controlled magnesium ion nanocapsules: phospholipids and cholesterol are dissolved in an inorganic solution, and a uniform lipid film is formed by rotary evaporation. The lipid film is hydrated with a mixture of MgCl2 and pyrophosphatase, and the mixture is shaken and centrifuged to obtain a single-layer liposome. The thermosensitive polymer is dissolved in a PBS solution precooled at 3-5°C, and magnetic stirring is performed until it is completely dissolved. The liposome and the thermosensitive polymer solution are mixed at a mass ratio of 1:1.5-2, and the mixture is incubated at 3-5°C for 2-3 hours. The mixture is purified by centrifugation to obtain the temperature-controlled magnesium ion nanocapsules. Mixing of components: Weigh or dilute the components according to specific concentrations and mix them, adjust the pH to 7.5-9.0, and then sterilize through a 0.22 μM water filter to obtain a PCR buffer.

6. The method for preparing a buffer supporting multiplex fluorescence quantitative PCR according to claim 5, characterized in that: The preparation step of the temperature-controlled magnesium ion nanocapsules also includes mixing the liposomes and the thermosensitive polymer solution in a mass ratio of 1:1.5-2, incubating at 3-5°C for 2-3h, adding a crosslinking agent to react at room temperature for 1-2h, and centrifuging and purifying to obtain the temperature-controlled magnesium ion nanocapsules.

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

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

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