US-12618105-B2 - Composition, reaction liquid and method for improving QPCR test performance, and use thereof

Abstract

A composition for improving the detection performance of fluorescent quantitative PCR. The composition comprises bovine serum albumin, sorbitol, ammonium sulfate, formamide, tetramethylammonium chloride, and at least one of dithiothreitol and betaine. The present invention further relates to a qPCR reaction liquid containing the composition and a preparation method therefor. The composition can improve the sensitivity, specificity, and interference resistance of real-time fluorescent quantitative PCR.

Inventors

• Lizhong Dai
• BOZHI JI
• Kang Wu
• Jia Liu
• ZHONGPING DENG
• Weimin MIAO

Assignees

• SANSURE BIOTECH INC.

Dates

Publication Date

20260505

Application Date

20220928

Priority Date

20200423

Claims (15)

1. 1 . A composition for improving qPCR detection performance, consisting of the following components: bovine serum albumin, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride, and at least one of dithiothreitol and betaine; wherein components in the composition are formulated to have the following final concentrations after being added to a qPCR reaction liquid: bovine serum protein, 10-150 μg/ml; dithiothreitol, 1-10 mM; sorbitol, 1-10 w/v %, betaine, 0.5-4 mol/L; ammonium sulfate, 2-50 mM; formamide, 0.1-10 v/v %; and tetramethylammonium chloride, 10-100 mM.
2. 2 . The composition according to claim 1 , wherein the composition comprises bovine serum albumin, dithiothreitol, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride.
3. 3 . The composition according to claim 1 , wherein the composition comprises bovine serum albumin, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.
4. 4 . The composition according to claim 1 , wherein the composition comprises bovine serum albumin, dithiothreitol, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.
5. 5 . The composition according to claim 1 , wherein the components in the composition are formulated to have the following final concentrations after being added to the qPCR reaction liquid: bovine serum protein, 80-120 μg/ml; dithiothreitol, 2-8 mM; sorbitol, 4-6 w/v %; betaine, 0.6-1 mol/L; ammonium sulfate, 8-15 mM; formamide, 0.5-5 v/v %; and tetramethylammonium chloride, 20-80 mM.
6. 6 . The composition according to claim 1 , wherein the components in the composition are formulated to have the following final concentrations after being added to the qPCR reaction liquid: bovine serum albumin, 80 μg/mL; dithiothreitol, 3 mM; sorbitol, 4 w/v %; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3 v/v %; and tetramethylammonium chloride, 35 mM.
7. 7 . A qPCR reaction liquid, comprising the composition of claim 1 .
8. 8 . The qPCR reaction liquid according to claim 7 , comprising a sample.
9. 9 . The qPCR reaction liquid according to claim 8 , wherein the sample comprises a nucleic acid-extracted sample and/or a non-nucleic acid-extracted sample.
10. 10 . The qPCR reaction liquid according to claim 7 , further comprises a primer and a probe, a dNTP, a DNA polymerase, and a PCR buffer.
11. 11 . A method for improving detection performance of qPCR, comprising adding the composition of claim 1 to a qPCR reaction system.
12. 12 . A method for improving the detection performance of qPCR, comprising adding the qPCR reaction liquid of claim 7 to a qPCR reaction system.
13. 13 . A method for preparing a qPCR reaction liquid, wherein the method comprising a step of mixing a sample with a reaction buffer and the composition of claim 1 .
14. 14 . The method according to claim 13 , wherein the reaction buffer comprises a dNTP, a DNA polymerase, and a PCR buffer.
15. 15 . The method according to claim 13 , wherein the reaction buffer further comprises a primer and a probe.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Application No. PCT/CN2020/121056, filed on Oct. 15, 2020, which claims priority to Chinese Patent Application No. 202010326989.0, filed on Apr. 23, 2020. The disclosures of the aforementioned applications are hereby incorporated by reference in their entireties. TECHNICAL FIELD The present invention belongs to the field of molecular biology detection, in particular to a composition, a kit, and a method for improving PCR detection performance, and more particularly to the improvement of sensitivity, specificity, and interference resistance of fluorescent quantitative PCR. BACKGROUND Real-time fluorescent quantitative PCR (quantitative real-time PCR) is a method for measuring the total amount of a product after each polymerase chain reaction (PCR) cycle with a fluorescent chemical in a nucleic acid amplification reaction. It is a method for quantitative analysis of a specific DNA sequence in a sample under test by an internal reference or external reference method. Real-time PCR is real-time detection of a PCR process by fluorescence signals during PCR amplification. The Ct value of a template has a linear relationship with an initial copy number of the template during an exponential period of the PCR amplification, which thus becomes a basis for quantification. Fluorescent indicators for qPCR detection are mainly divided into two categories: one is fluorescent probes, such as Taqman probes and molecular beacon probes; the other is fluorescent dyes that can bind to a double-stranded DNA, such as SYBR Green and EvaGreen. Technologies for improving the detection performance of real-time fluorescent quantitative PCR discussed herein mainly involve improvement of the qPCR detection performance of a fluorescent probe method, especially a Taqman probe method. There are many factors affecting the qPCR detection performance, and many studies have so far focused on the improvement of qPCR detection effects. For example, Chinese Patent CN1981055A mentioned the application of a mixture containing a polynucleotide polymerase to improve the stability of a PCR reaction liquid; for another example, Chinese Patent CN103409540A mentioned the use of a novel dye Gelgreen I combined with a Taq enzyme to improve and optimize the amplification efficiency of qualitative PCR; for still another example, Chinese Patent CN1464070A mentioned the use of gold nanoparticles with different particle sizes as an identification amplifier of a DNA detector to improve the detection sensitivity in DNA detection. However, in the field of qPCR detection, there is currently a lack of a simple and convenient solution that can simultaneously improve various aspects of qPCR detection performance, including but not limited to detection sensitivity, interference resistibility, detection specificity, and the like, of DNA/RNA amplification. SUMMARY In view of this, in a first aspect, the present invention provides a composition for improving qPCR detection performance, the composition comprising: bovine serum albumin, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride, and at least one of dithiothreitol and betaine. In a particular embodiment, the composition comprises bovine serum albumin, dithiothreitol, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride. In a particular embodiment, the composition comprises bovine serum albumin, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride. In a particular embodiment, the composition comprises bovine serum albumin, dithiothreitol, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride. In the present invention, the bovine serum albumin has a final concentration of 10-150 μg/mL, preferably 80-120 μg/mL in the qPCR reaction liquid, for example, 70 μg/mL, 75 μg/mL, 80 μg/mL, 85 μg/mL, or 90 μg/mL, more preferably, a final concentration of 80 μg/mL. In the present invention, the dithiothreitol has a final concentration of 1-10 mM, preferably 2-8 mM in the qPCR reaction liquid, for example, 2 mM, 4 mM, 6 mM, or 8 mM, more preferably, a final concentration of 3 mM. In the present invention, the sorbitol has a final concentration of 1-10 w/v %, preferably 4-6 w/v % in the qPCR reaction liquid, for example, 4 w/v %, 5 w/v %, or 6 w/v %, more preferably, a final concentration of 4 w/v %. In the present invention, the betaine has a final concentration of 0.5-4 mol/L, preferably 0.6-1 mol/L in the qPCR reaction liquid, for example, 0.6 mol/L, 0.7 mol/L, 0.8 mol/L, or 0.9 mol/L, more preferably, a final concentration of 0.8 mol/L. In the present invention, the ammonium sulfate has a final concentration of 2-50 mM, preferably 8-15 mM in the qPCR reaction liquid, for example, 8 mM, 9 mM, 10 mM, 11 mM, or 12 mM, more preferably, a final concentration of 10 mM. In the present invention, the formamide has a final concentration of 0.1-10 v/v %,