Lock nucleic acid probe fluorescence quantitative PCR buffer and application thereof

Abstract

This invention discloses a buffer solution for locked nucleic acid probe-based real-time quantitative PCR (RT-PCR). The buffer solution is composed of dithiothreitol, betaine, tetramethylammonium chloride, ammonium sulfate, tris(hydroxymethyl)aminomethane, magnesium chloride, a Tween-20 / glycerol mixture, and bovine serum albumin. The pH of the buffer solution is 7.0–9.0. The volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:80–120. By adding protective agents and promoters, adjusting the pH value, and adjusting the concentration of corresponding components, this buffer solution effectively protects the enzymes necessary for real-time quantitative PCR detection and improves the stability of modified probes in RTS. It is superior to other solutions for locked nucleic acid modified probes in RTS, improving detection sensitivity, fluorescence intensity, and result reliability. Therefore, this buffer solution has broad application prospects in detections requiring more stringent and precise data.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically relating to a buffer solution for locked nucleic acid probe fluorescence quantitative PCR and its application. Background Technology

[0002] Real-time quantitative PCR (qPCR) is a variant of standard PCR technology, typically used to quantify DNA or RNA in a sample. Using sequence-specific primers, the copy number of a specific DNA or RNA sequence can be determined. Quantification is achieved by measuring the amount of amplified product at each stage of the PCR cycle. If the sample contains an abundant specific sequence (DNA or RNA), amplification is observed in earlier cycles; if the sequence is scarce, amplification is observed in later cycles. Quantification of the amplified product uses fluorescent probes or fluorescent DNA-binding dyes and a real-time PCR instrument, measuring fluorescence during PCR thermal cycling. Currently, qPCR is mainly used in basic research, clinical diagnostics, drug development, food safety, and environmental monitoring.

[0003] Each cycle of real-time quantitative PCR consists of three main steps, and the reaction typically requires 40 cycles:

[0004] (1) High-temperature denaturation: Culturing is used to "melt" single-stranded DNA into single strands and loosen the secondary structure in single-stranded DNA (ssDNA). Usually, the highest temperature that DNA polymerase can withstand (usually 95°C) is used. The higher the GC content of the template, the longer the denaturation time.

[0005] (2) Annealing: During annealing, complementary sequences have the opportunity to hybridize, so an appropriate temperature is used, which is based on the calculated primer melting temperature (Tm) (usually 5°C lower than the primer Tm). If the primer melting temperature is too high, it will greatly increase the reaction temperature of real-time quantitative PCR, resulting in a decrease in fluorescence value and an increase in Ct value.

[0006] (3) Extension: DNA polymerase activity is optimal at 70–72°C, with primer extension rates reaching up to 100 bases per second. When the amplicon in real-time PCR is very small, this step is usually combined with an annealing step.

[0007] However, conventional quantitative PCR suffers from low sensitivity, low reaction efficiency, and poor stability. Current technologies often employ the addition of locked nucleic acid (LNA) probes to the quantitative PCR system to address these issues. The main advantage of LNA-modified probe detection lies in the rigid condensation structure formed by methylene bridges at the 2'-O and 4'-C positions of the LNA through different condensation interactions, increasing the stability of the local structure of the nucleic acid phosphate backbone. As a novel modified nucleic acid, LNA possesses strong hybridization affinity with DNA / RNA, antisense activity, resistance to nucleases, good water solubility, and non-toxicity in vivo. Furthermore, when LNA-modified nucleic acids are used in quantitative PCR (qPCR) probe detection, they can effectively increase the annealing temperature of the detection reaction while simultaneously shortening the required probe length. Therefore, LNA-modified detection probes offer advantages such as higher sensitivity and stronger specificity compared to ordinary probes. However, due to the special chemical structure and properties of LNA probes, an increase in Tm value is inevitable. Therefore, it is necessary to increase the reaction temperature of real-time PCR, but this will lead to a decrease in enzyme activity, resulting in a decrease in fluorescence value and an increase in Ct value. Therefore, it is necessary to develop a specific buffer system to support the stability and activity of enzymes and LNA probes in the reaction system. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a buffer composition.

[0009] A second objective of this invention is to provide the application of the above-described buffer composition in quantitative real-time PCR detection or in the preparation of quantitative real-time PCR detection kits.

[0010] A third object of the present invention is to provide a kit comprising the above-described buffer composition.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention provides a buffer composition comprising dithiothreitol (DTT), betaine, tetramethylammonium chloride (TMAC), ammonium sulfate, tris(hydroxymethyl)aminomethane (Tris), magnesium chloride (MgCl2), a Tween-20 / glycerol mixture, and bovine serum albumin (BSA); the pH of the composition is 7.0–9.0; and the volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:80–120.

[0013] Dithiothreitol is a commonly used reducing agent in PCR buffers to maintain enzyme activity. Betaine reduces the likelihood of GC-rich templates forming secondary structures, facilitating PCR amplification and sequencing of these templates. It eliminates the base dependence of denaturation temperature, bringing the Tm values ​​of the two PCR primers closer together, thus lowering the Tm value of DNA. It also improves the stability of DNA polymerase, reduces non-specific amplification, and enhances the specificity of the PCR reaction. Tetramethylammonium chloride is a quaternary ammonium salt that acts as a buffer or stabilizer in PCR buffers. Ammonium sulfate significantly improves the specificity, sensitivity, and accuracy of PCR reactions by improving primer-template binding, enhancing DNA polymerase stability, and optimizing PCR reaction conditions. Tris(hydroxymethyl)aminomethane is a very common component in PCR buffers, used as a buffer to maintain pH stability in the reaction system. Magnesium chloride (MgCl2) serves as a source of magnesium ions in PCR buffers, crucial for DNA polymerase activity; it is an essential component in PCR reactions. Tween-20 / glycerol mixtures can be used as thickeners or stabilizers in PCR reactions. Bovine serum albumin (BSA) is added to PCR reactions to stabilize enzyme activity or reduce nonspecific binding. The pH of the composition can affect enzyme activity, reaction specificity, and DNA stability.

[0014] The addition of protectants and promoters to the above buffer system effectively protects the enzymes essential for real-time quantitative PCR (qPCR) detection, improving detection sensitivity and fluorescence intensity. This formulation, by adjusting the pH and corresponding component concentrations, is superior for detecting locked nucleic acid modified probes in qPCR. The PCR buffer of this invention can improve the amplification efficiency of samples in qPCR, allowing samples of the same concentration to reach the plateau phase earlier; it can improve the sensitivity of modified probes in qPCR, effectively preventing false negatives in low-concentration samples; and it can also improve the stability of modified probes in qPCR, effectively protecting the stability of the enzymes used in the detection reaction, thereby increasing fluorescence value and reducing Ct value.

[0015] Further, the composition comprises 1–5 mM dithiothreitol, 100–500 mM betaine, 5–30 mM tetramethylammonium chloride, 2–10 mM ammonium sulfate, 100–500 mM tris(hydroxymethyl)aminomethane, 1–10 mM magnesium chloride, 1–10% (v / v) Tween-20 / glycerol mixture, and 0.05–1 g / L bovine serum albumin; the pH of the composition is 7.5–9.0; and the volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:80–120.

[0016] Furthermore, the composition comprises 2–4 mM dithiothreitol, 200–400 mM betaine, 10–20 mM tetramethylammonium chloride, 4–8 mM ammonium sulfate, 200–400 mM tris(hydroxymethyl)aminomethane, 3–7 mM magnesium chloride, 3–10% (v / v) Tween-20 / glycerol mixture, and 0.1–1 g / L bovine serum albumin; the pH of the composition is 7.5–9.0; and the volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:80–120.

[0017] Preferably, the composition comprises 3 mM dithiothreitol, 200 mM betaine, 20 mM tetramethylammonium chloride, 8 mM ammonium sulfate, 350 mM tris(hydroxymethyl)aminomethane, 5 mM magnesium chloride, 10% Tween-20 / glycerol mixture, and 1 g / L bovine serum albumin; the pH of the composition is 8; and the volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:100.

[0018] Furthermore, the pH adjuster includes, but is not limited to, hydrochloric acid, sulfuric acid, acetic acid, sodium hydroxide, sodium bicarbonate, phosphate buffer, and borate buffer.

[0019] Furthermore, the composition is prepared by mixing the components in the composition evenly according to the stated proportions and adjusting the pH.

[0020] Furthermore, the composition is prepared by mixing the components in the composition evenly according to the stated proportions, adjusting the pH, and purifying.

[0021] Furthermore, the purification method includes filtration.

[0022] Preferably, the filter membrane used for filtration has a pore size of 0.25 to 0.65 μm.

[0023] Specifically, it includes the following steps:

[0024] A. Weigh or dilute all reagents according to the concentrations described above, dissolve them thoroughly in nuclease-free ultrapure water, and finally bring the volume to the required level.

[0025] B. Adjust the pH of the completely dissolved buffer solution using HCl and NaOH;

[0026] C. Filter the pH-adjusted buffer solution using a 0.45 μm filter membrane;

[0027] D. Aliquot the filtered buffer solution into appropriate volumes into storage tubes and store at -20°C.

[0028] Therefore, the present invention also provides the use of the above composition in quantitative real-time PCR detection or in the preparation of quantitative real-time PCR detection kits.

[0029] Furthermore, the quantitative real-time PCR detection system also includes DNA polymerase, dNTPs, primers, and DNA template. Quantitative real-time PCR (qPCR) is a method that uses fluorescent chemicals to measure the total amount of product after each polymerase chain reaction (PCR) cycle during DNA amplification. It is a method for quantitative analysis of specific DNA sequences in the sample using internal or external controls. During qPCR amplification, the PCR progress is detected in real time using fluorescence signals. In the exponential phase of PCR amplification, the Ct value of the template and the initial copy number of the template have a linear relationship, thus serving as the basis for quantification.

[0030] Furthermore, the quantitative real-time PCR detection system also includes a TaqMan probe. A TaqMan probe is an oligonucleotide probe with a fluorescent group (e.g., FAM, TET, VIC, HEX) at its 5' end and a quencher group (e.g., TAMRA, BHQ) at its 3' end. During PCR amplification, a specific fluorescent probe is added along with a pair of primers. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. During PCR amplification, the 5'-3' exonuclease activity of Taq polymerase cleaves and degrades the probe, separating the reporter and quencher fluorescent groups. This allows the fluorescence monitoring system to receive the fluorescent signal; that is, for each DNA strand amplified, one fluorescent molecule is formed, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the PCR product.

[0031] Preferably, the Taqman probe is chemically modified.

[0032] Specifically, the Taqman probe is modified with locked nucleic acid (LNA). Locked nucleic acid (LNA) is a novel and unique bicyclic oligonucleotide derivative. In its structure, the methylene bridges formed at the 2'-O and 4'-C positions of the nucleic acid through different condensation interactions create a rigid condensation structure, increasing the stability of the local structure of the nucleic acid phosphate backbone. LNA probes can improve the thermal stability of the duplex and enhance the specificity of probe-target sequence hybridization, thereby reducing background fluorescence, improving the signal-to-noise ratio, and making gene quantification and allele identification more accurate.

[0033] In the specific application of the example, Taq DNA polymerase, dNTPs, primers, DNA template, probe and the above buffer are mixed and denatured at 95°C; then annealed and extended at 55-65°C for 35-45 cycles, while collecting fluorescence signals to obtain Ct values.

[0034] The present invention also provides a kit comprising the above-described buffer composition.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention provides a buffer for locked nucleic acid probe-based real-time PCR. The buffer composition comprises dithiothreitol, betaine, tetramethylammonium chloride, ammonium sulfate, tris(hydroxymethyl)aminomethane, magnesium chloride, a Tween-20 / glycerol mixture, and bovine serum albumin. The pH of the composition is 7.0–9.0. The volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:80–120. The addition of protectants and promoters to this buffer effectively protects the enzymes essential for real-time quantitative PCR detection, improving sensitivity and fluorescence intensity. This formulation, by adjusting the pH and the concentration of corresponding components, is superior to addressing the detection reaction of locked nucleic acid modified probes in real-time quantitative PCR (manifested as a decrease in Ct value). The PCR buffer of this invention can improve the amplification efficiency of samples in real-time quantitative PCR, allowing samples of the same concentration to reach the plateau phase earlier; it can also improve the sensitivity of modified probes in real-time quantitative PCR, effectively preventing false negatives in low-concentration samples; furthermore, it can improve the stability of modified probes in real-time quantitative PCR, effectively protecting the stability of enzymes used in the detection reaction and improving the reliability of detection results (increasing fluorescence value and decreasing Ct value). Therefore, this buffer has broad application prospects in detections with more stringent and precise data requirements. Attached Figure Description

[0037] Figure 1 This is a graph showing the results at pH=8 in Example 1. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0038] Figure 2 The graph shows the results at pH = 8.5 in Example 1. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0039] Figure 3 This is a graph showing the results at pH=9 in Example 1. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0040] Figure 4 The graph shows the results at pH=7.5 in Example 1. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0041] Figure 5This is a graph showing the results at pH=8 in Example 1. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0042] Figure 6 The results are shown in Comparative Example 1. Note: The substrate in the left figure is cyp2c19*2, and the substrate in the right figure is cyp2c19*3.

[0043] Figure 7 The results are shown in Comparative Example 2. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0044] Figure 8 This is a graph showing the results of Comparative Example 3. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0045] Figure 9 This is a graph showing the results of Comparative Example 4. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0046] Figure 10 This is a graph showing the results of Comparative Example 5. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0047] Figure 11 The results for Comparative Example 6 are shown in the left graph, where the substrate is cyp2c19*2, and the substrate is cyp2c19*3.

[0048] Figure 12 This is a graph showing the results of Comparative Example 7. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0049] Figure 13 This is a graph showing the results of Comparative Example 8. Note: The substrate in the left graph is cyp2c19*2, and the substrate in the right graph is cyp2c19*3.

[0050] Figure 14 The results obtained using the buffer solution of the present invention in Comparative Example 9 are shown in the figure. Note: The reaction substrate shown in the left figure is an equal mixture of wild-type and mutant cyp2c19*2 genes, and the reaction substrate shown in the right figure is an equal mixture of wild-type and mutant cyp2c19*3 genes.

[0051] Figure 15 The results for Comparative Example 9 were obtained using commercially available buffer solutions. Note: The reaction substrate shown in the left figure is a mixture of equal proportions of wild-type and mutant cyp2c19*2 genes, and the reaction substrate shown in the right figure is a mixture of equal proportions of wild-type and mutant cyp2c19*3 genes. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0053] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0054] The dNTPs involved in this invention were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., the Taq enzyme was purchased from Genscript Biotech Co., Ltd., and the probes and primers were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0055] This invention is applied to locked nucleic acid modified probe sets, and the modification content is shown in Table 1 below.

[0056] Table 1. Modification sites and probe sequences of locked nucleic acid probes

[0057]

[0058] Note: In the table, / LNA_C / indicates LNA modification of a C base, / LNA_T / indicates LNA modification of a T base, / LNA_G / indicates LNA modification of a G base, and / LNA_A / indicates LNA modification of an A base. The wild-type probe of cyp2c19*2 has its fluorescent group modified with FAM, the mutant probe with VIC, and the quencher group for both wild-type and mutant probes is BHQ-1; the wild-type probe of cyp2c19*3 has its fluorescent group modified with Cy5, the mutant probe with Texas Red, and the quencher group for both wild-type and mutant probes is BHQ-2.

[0059] The primer sequences used in this invention are shown in Table 2 below.

[0060] Table 2 Primer sequences

[0061]

[0062] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0063] Example 1: Investigating the effect of buffer pH on the detection of Ct value

[0064] I. Experimental Methods

[0065] (1) The qPCR reaction solution contains 1 μL dNTPs, 16.6 μL buffer (pH=8.0), 0.6 μL probe, 0.6 μL primer set, 0.2 μL Taq enzyme and 1 μL DNA substrate; the substrates are the wild-type gene fragment of cyp2c19*2 and the mutant gene fragment of cyp2c19*3 (the probe and primer correspond to Table 1 and Table 2, respectively), and the total volume of each reaction is 20 μL.

[0066] (2) The buffer solution consisted of ① 3 mM dithiothreitol (DTT), ② 200 mM betaine, ③ 20 mM tetramethylammonium chloride (TMAC), ④ 8 mM ammonium sulfate, ⑤ 350 mM tris(hydroxymethyl)aminomethane (Tris), ⑥ 5 mM magnesium chloride (MgCl2); and ⑦ 10% Tween-20 / glycerol mixture (1:100) and ⑧ 1 g / L bovine serum albumin (BSA).

[0067] The specific steps for preparing this buffer solution are as follows:

[0068] A. Weigh or dilute all reagents according to the concentrations described above, dissolve them thoroughly in nuclease-free ultrapure water, and finally bring the volume to the required level.

[0069] B. Adjust the pH of the completely dissolved buffer solution to 7.5, 8.0, 8.5, and 9 using HCl and NaOH, respectively.

[0070] C. Filter the pH-adjusted buffer solution using a 0.45 μm filter membrane;

[0071] D. Aliquot the filtered buffer solution into appropriate volumes into storage tubes and store at -20°C.

[0072] (3) Prepare the reaction system solution as described above and perform the detection reaction using a Thermo Fisher ABI 7500 real-time quantitative PCR instrument. Real-time quantitative PCR instrument detection reaction procedure: 95℃ for 30s; 95℃ for 5s, 65℃ for 30s, cycle 40 times.

[0073] II. Experimental Results

[0074] The results are as follows: Figure 1-4 As shown in Table 3, when the pH of the buffer solution is 8, the Ct values ​​of both genes are significantly lower than those when the pH is 8.5, 9, and 7.5. Therefore, it can be seen that the pH of the buffer solution has a significant impact on the Ct value, and the optimal solution is when the pH of the buffer solution is 8.

[0075] Table 3. Statistical results of Ct values ​​for buffer solutions at different pH values.

[0076]

[0077] Example 2

[0078] The qPCR reaction system and detection reaction procedure are the same as in Example 1. In Example 2, the buffer system is changed to consist of 1 mM dithiothreitol, 100 mM betaine, 30 mM tetramethylammonium chloride, 10 mM ammonium sulfate, 450 mM tris(hydroxymethyl)aminomethane, 2 mM magnesium chloride, 8% (v / v) Tween-20 / glycerol mixture, and 0.1 g / L bovine serum albumin.

[0079] Example 3

[0080] The qPCR reaction system and detection reaction procedure are the same as in Example 1. In Example 3, the buffer system is changed to consist of 5 mM dithiothreitol, 450 mM betaine, 10 mM tetramethylammonium chloride, 5 mM ammonium sulfate, 150 mM tris(hydroxymethyl)aminomethane, 8 mM magnesium chloride, 3% (v / v) Tween-20 / glycerol mixture, and 0.5 g / L bovine serum albumin.

[0081] The results of Examples 2 and 3 show that when the concentrations of the components in the buffer solution are adjusted, the Ct values ​​obtained by detection using the method of Example 1 are still in the range of 24 to 30.

[0082] Comparative Example 1

[0083] The qPCR reaction system and detection reaction procedure are the same as in Example 1. In Comparative Example 1, only ①3mM dithiothreitol (DTT) was removed from the buffer, and the pH of the buffer was set to 8.

[0084] Comparative Example 2

[0085] The qPCR reaction system and detection reaction procedure are the same as in Example 1. In Comparative Example 2, only ②200mM betaine was removed from the buffer, and the pH of the buffer was set to 8.

[0086] Comparative Example 3

[0087] The qPCR reaction system and detection reaction procedure are the same as in Example 1. In Comparative Example 3, only 20 mM tetramethylammonium chloride (TMAC) was removed from the buffer, and the pH of the buffer was set to 8.

[0088] Comparative Example 4

[0089] The qPCR reaction system and detection reaction procedure are the same as in Example 1. In Comparative Example 4, only the ④8mM ammonium sulfate in the buffer was removed, and the pH of the buffer was set to 8.

[0090] Comparative Example 5

[0091] The qPCR reaction system and detection reaction procedure are the same as in Example 1. Comparative Example 5 only removes 350mM tris(hydroxymethyl)aminomethane (Tris) from the buffer solution and sets the pH of the buffer solution to 8.

[0092] Comparative Example 6

[0093] The qPCR reaction system and detection reaction procedure are the same as in Example 1. Comparative Example 6 only removes 5mM magnesium chloride (MgCl2) from the buffer and sets the pH of the buffer to 8.

[0094] Comparative Example 7

[0095] The qPCR reaction system and detection reaction procedure are the same as in Example 1. Comparative Example 7 only removes the ⑦10% Tween-20 / glycerol mixture (1:100) from the buffer, and the pH of the buffer is set to 8.

[0096] Comparative Example 8

[0097] The qPCR reaction system and detection reaction procedure are the same as in Example 1. Comparative Example 8 only removes 1 g / L bovine serum albumin (BSA) from the buffer and sets the pH of the buffer to 8.

[0098] The results are as follows: Figure 5-13 As shown in Table 4, the Ct values ​​of both genes in Example 1 were significantly lower than those in Comparative Examples 1-8, thus the effect was significantly better than that in Comparative Examples 1-8. Therefore, Example 1 is the optimal solution. Thus, each component in the buffer composition provided by this invention is essential.

[0099] Table 4. Statistical results of Ct values ​​for Example 1 and the comparative example.

[0100]

[0101] Comparative Example 9

[0102] The qPCR reaction solution contained 1 μL dNTPs, 13 μL of the buffer solution from Example 1 (pH = 8.0) or commercial general-purpose buffer (purchased from Baori Biotechnology (Beijing) Co., Ltd.), 0.6 μL probe set, 0.6 μL primer set, 0.2 μL Taq enzyme, and 1 μL DNA substrate; the substrates were a stoichiometric mixture of wild-type and mutant cyp2c19*2 genes; and a stoichiometric mixture of wild-type and mutant cyp2c19*3 genes (probes and primers correspond to Tables 1 and 2, respectively), with a total reaction volume of 20 μL for each reaction. The reaction system solutions were prepared as described above, and the reactions were performed using a Thermo Fisher ABI 7500 real-time quantitative PCR instrument, with the reaction procedure as shown in Example 1.

[0103] The results are as follows: Figure 14-15As shown in Table 5, when using locked nucleic acid modified probes and commercial ordinary buffer for real-time PCR, the reaction Ct values ​​all increased to varying degrees. Therefore, from the perspective of Ct value analysis, the reaction results using the buffer of the present invention are significantly better than those using commercial ordinary buffer.

[0104] Secondly, when using locked nucleic acid modified probes with commercially available general-purpose buffers for quantitative PCR, the fluorescence values ​​of the overall reaction curves showed a significant decrease. Therefore, from the perspective of fluorescence values, the reaction results using the buffers of this invention are significantly better than those using commercially available general-purpose buffers.

[0105] Finally, by Figure 14 and 15 As can be seen from the comparison, when using the locked nucleic acid modified probe with the buffer described in this invention for real-time PCR, the specificity is good, and no non-specific amplification occurs. The curve shape is also "S"-shaped, indicating a good overall reaction process. When using the locked nucleic acid modified probe with commercial ordinary buffer for real-time PCR, significant non-specific amplification occurs, indicating a decrease in specificity to varying degrees. The curve shape is also abnormal, with some sections being linear, indicating a poor overall reaction process. Therefore, in terms of reaction specificity, sensitivity, and overall amplification, the reaction results using the buffer of this invention are significantly better than those using commercial ordinary buffer.

[0106] Table 5. Statistical analysis of the results of the buffer solution of this invention and commercially available buffer solutions.

[0107]

[0108] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A buffer solution composition, characterized in that, The composition comprises dithiothreitol, betaine, tetramethylammonium chloride, ammonium sulfate, tris(hydroxymethyl)aminomethane, magnesium chloride, a Tween-20 / glycerol mixture, and bovine serum albumin; the pH of the composition is 7.0–9.0; the volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:80–120.

2. The buffer composition according to claim 1, characterized in that, The composition comprises 1–5 mM dithiothreitol, 100–500 mM betaine, 5–30 mM tetramethylammonium chloride, 2–10 mM ammonium sulfate, 100–500 mM tris(hydroxymethyl)aminomethane, 1–10 mM magnesium chloride, 1–10% (v / v) Tween-20 / glycerol mixture, and 0.05–1 g / L bovine serum albumin; the pH of the composition is 7.5–9.0; and the volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:80–120.

3. The buffer composition according to claim 2, characterized in that, The composition comprises 3 mM dithiothreitol, 200 mM betaine, 20 mM tetramethylammonium chloride, 8 mM ammonium sulfate, 350 mM tris(hydroxymethyl)aminomethane, 5 mM magnesium chloride, 10% Tween-20 / glycerol mixture, and 1 g / L bovine serum albumin; the pH of the composition is 8; the volume ratio of Tween-20 to glycerol in the Tween-20 / glycerol mixture is 1:

100.

4. The buffer composition according to any one of claims 1 to 3, characterized in that, The pH adjusters include hydrochloric acid, sulfuric acid, acetic acid, sodium hydroxide, sodium bicarbonate, phosphate buffer, and borate buffer.

5. The buffer composition according to any one of claims 1 to 3, characterized in that, The preparation method involves mixing the components in the composition evenly according to the stated proportions and adjusting the pH.

6. The use of the buffer composition according to any one of claims 1 to 3 in quantitative real-time PCR detection or in the preparation of quantitative real-time PCR detection kits.

7. The application according to claim 6, characterized in that, The system for quantitative real-time PCR detection also includes DNA polymerase, dNTPs, primers, and DNA template.

8. The application according to claim 7, characterized in that, The system for quantitative real-time PCR detection also includes Taqman probes.

9. The application according to claim 8, characterized in that, The Taqman probe is modified with locked nucleic acid.

10. A reagent kit, characterized in that, The kit comprises any one of the buffer compositions described in claims 1 to 3.

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