WO2021212771A1 - 提升qPCR检测性能的组合物、反应液、用途及方法 - Google Patents
提升qPCR检测性能的组合物、反应液、用途及方法 Download PDFInfo
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- WO2021212771A1 WO2021212771A1 PCT/CN2020/121056 CN2020121056W WO2021212771A1 WO 2021212771 A1 WO2021212771 A1 WO 2021212771A1 CN 2020121056 W CN2020121056 W CN 2020121056W WO 2021212771 A1 WO2021212771 A1 WO 2021212771A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- the present invention belongs to the field of molecular biology detection. Specifically, it relates to a composition, a kit and a method for improving PCR detection performance, and more specifically, it relates to improving the sensitivity, specificity and anti-interference of fluorescent quantitative PCR.
- Quantitative Real-time PCR is a method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in nucleic acid amplification reactions.
- PCR polymerase chain reaction
- Real-time PCR is the real-time detection of the PCR process through fluorescent signals during the PCR amplification process. In the exponential period of PCR amplification, there is a linear relationship between the Ct value of the template and the initial copy number of the template, so it becomes the basis for quantification.
- the fluorescent indicators used 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 double-stranded DNA, such as SYBR Green And EvaGreen etc.
- the technology for improving the detection performance of real-time fluorescent quantitative PCR discussed in the present invention mainly relates to the method of fluorescent probe, especially the improvement of the detection performance of qPCR by the Taqman probe method.
- Chinese patent CN1981055A mentions the application of using a mixture containing polynucleotide polymerase to improve the stability of PCR reaction solution; another example, Chinese patent CN103409540A mentions the use of new dye Gelgreen I with Taq enzyme to improve and optimize qualitative PCR.
- the Chinese patent CN1464070A mentions the use of gold nanoparticles with different particle sizes as the recognition amplifier of the DNA detector to improve the detection sensitivity in DNA detection.
- the present invention provides a composition for improving the detection performance of qPCR, the composition comprising:
- At least one of bovine serum albumin, sorbitol, ammonium sulfate, formamide and tetramethylammonium chloride, and dithiothreitol and betaine At least one of bovine serum albumin, sorbitol, ammonium sulfate, formamide and tetramethylammonium chloride, and dithiothreitol and betaine.
- the composition includes bovine serum albumin, dithiothreitol, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride.
- the composition includes bovine serum albumin, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.
- the composition includes bovine serum albumin, dithiothreitol, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.
- bovine serum albumin has a final concentration of 10-150 ⁇ g/mL, preferably 80-120 ⁇ g/mL in the qPCR reaction solution. For example, 70 ⁇ g/mL, 75 ⁇ g/mL, 80 ⁇ g/mL, 85 ⁇ g/mL, 90 ⁇ g/mL. More preferably, it is a final concentration of 80 ⁇ g/mL.
- dithiothreitol has a final concentration of 1-10 mM, preferably 2-8 mM in the qPCR reaction solution.
- a final concentration of 1-10 mM preferably 2-8 mM in the qPCR reaction solution.
- 2mM, 4mM, 6mM, 8mM More preferably, it is a final concentration of 3 mM.
- sorbitol has a final concentration of 1-10 w/v%, preferably 4-6 w/v% in the qPCR reaction solution. For example, 4w/v%, 5w/v%, 6w/v%. More preferably, it is a final concentration of 4w/v%.
- betaine has a final concentration of 0.5-4 mol/L, preferably 0.6-1 mol/L in the qPCR reaction solution.
- ammonium sulfate has a final concentration of 2-50 mM, preferably 8-15 mM in the qPCR reaction solution.
- the formamide has a final concentration of 0.1-10 v/v% in the qPCR reaction solution, preferably 0.5-5 v/v%.
- a final concentration of 0.1-10 v/v% in the qPCR reaction solution preferably 0.5-5 v/v%.
- tetramethylammonium chloride has a final concentration of 10-100 mM, preferably 20-80 mM in the qPCR reaction solution.
- the present invention provides a qPCR reaction solution, which contains the composition as described above.
- the qPCR reaction solution further includes a sample, for example, a sample with nucleic acid extraction and/or a sample without nucleic acid extraction.
- the qPCR reaction solution also includes primers and probes for qPCR.
- the qPCR reaction solution also includes dNTP, DNA polymerase, and PCR buffer.
- qPCR reaction solution refers to a mixture capable of detecting nucleic acid using fluorescent quantitative PCR.
- the qPCR reaction solution includes the above-mentioned composition, primers and probes, dNTP, DNA polymerase, and PCR buffer.
- the present invention provides a use of the above composition for improving the detection performance of qPCR.
- the improvement refers to the improvement of sensitivity, specificity, and/or anti-interference performance.
- the term "detection performance” mainly refers to sensitivity, specificity and anti-interference.
- the present invention provides a method for preparing a qPCR reaction solution, the method including the step of mixing a sample with a reaction buffer solution and the above-mentioned composition.
- the reaction buffer includes, for example, dNTP, DNA polymerase, and PCR buffer. Further, primers and probes may be included.
- composition of the present invention can improve the sensitivity, specificity and anti-interference of fluorescent quantitative PCR, and the concentration used in the composition of the present invention can make the sensitivity, specificity and anti-interference of fluorescent quantitative PCR further improved. improve.
- improving detection performance can better provide molecular evidence for disease diagnosis and make adequate preparations for disease prevention and control; timely control of infectious and harmful infectious diseases can be achieved The source of infection, to block the virus pandemic and major outbreak.
- Figure 1 is a graph showing the results of amplification in the absence of the composition of the present invention by using the same set of primer probes for specific amplification of rs7412A in the presence of the APOE gene rs7412A/C template;
- Figure 2 is a graph showing the results of amplification in the presence of the same concentration of APOE gene rs7412A/C template, using the same set of primer probes for specific amplification of rs7412A, in the presence of the composition of the present invention.
- Example 1 HCV detection anti-interference performance of different compositions of the present invention
- a comparative test was performed on low-concentration HCV samples (about 500IU/mL) with interfering factors.
- the interfering factor was hemoglobin at a concentration of 2g/dL.
- the detection protocol was to repeat the sample ten times under each condition for comparative detection. The repeatability and detection rate.
- the sample detection method adopted is the direct amplification method of 10 ⁇ L sample+10 ⁇ L nucleic acid release agent+30 ⁇ L PCR reaction solution without nucleic acid extraction sample.
- Table 1 The influence of different compositions in the present invention on the anti-interference ability
- the low-concentration nucleic acid sample (about 1000 copies/mL) of the 2019 novel coronavirus (hereinafter referred to as the new coronavirus, or 2019-nCoV) with interfering factors was compared and tested.
- the interfering sample was a respiratory oropharyngeal swab with obvious turbid deposits. Samples, the test plan is to repeat the sample ten times under each condition, and compare the repeatability of the test and the detection rate.
- the sample detection method adopts the amplification method of 10 ⁇ L interference sample + 10 ⁇ L nucleic acid release agent + 1 ⁇ L new coronavirus nucleic acid sample + 30 ⁇ L PCR reaction solution to investigate the impact of the new coronavirus nucleic acid amplification effect under different conditions.
- Different combination refers to the combination of different compositions in the PCR reaction solution, as shown in Table 3, where "+” means adding the component, and “-” means not adding the component (after all the components are added to the qPCR reaction solution) It has the following final concentrations: Bovine Serum Albumin 80 ⁇ g/mL, Dithiothreitol 3mM, Sorbitol 4w/v%, Betaine 0.8mol/L, Ammonium Sulfate 10mM, Formamide 3v/v%, Tetramethylammonium Chloride 35mM).
- control 1 no interference sample (replaced with TE), no additive component PCR reaction solution
- control 2 no interference sample (replaced with TE), additive component PCR reaction solution.
- Example 3 The composition of the present invention is used to increase the anti-interference performance of detecting HCV
- the composition of the present invention containing 7 components (bovine serum albumin 80 ⁇ g/mL, dithiothreitol 3MM, sorbitol 4w/v%, betaine 0.8mol/L, ammonium sulfate 10mM, formamide 3v/v %, tetramethylammonium chloride 35mM). It is applied to HCV plasma samples with interfering substances, and compared with HCV plasma samples without interfering substances. At the same time, for comparison, the PCR amplification reagent without the composition of the present invention is used as a control group, and it is also added to the HCV plasma sample with and without interfering substances for comparative detection.
- 7 components bovine serum albumin 80 ⁇ g/mL, dithiothreitol 3MM, sorbitol 4w/v%, betaine 0.8mol/L, ammonium sulfate 10mM, formamide 3v/v %, tetramethylammonium chloride 35mM.
- sample release agent 10:10:30 (v/v) for direct sample amplification with a total volume of 50 ⁇ L.
- This method uses direct sample amplification.
- the increased method of nucleic acid-free extraction is because the anti-interference ability of the amplification reagents on the interference and inhibition effects in the sample is investigated in a larger dimension.
- the preparation method of the interference sample of the experimental program, the experimental comparison program, and the PCR amplification program used in the experimental process are shown in Table 5-7 as follows:
- the composition was added to the PCR amplification reagent without the composition of the present invention, and a comparative test was performed with the PCR amplification reagent without the composition of the present invention, and the results of each sample were examined. Comparing the detection of Ct value, the Ct value is negatively correlated with the amplification efficiency, that is, the larger the Ct value, the lower the amplification efficiency.
- the composition of the present invention has a significant improvement in anti-interference ability against common interference substances in serum samples. Regardless of whether it is a low-concentration or a medium-to-high concentration of HCV samples, there is no significant difference in amplification efficiency between interference samples with interference factors and control samples without interference factors. However, compared to the control amplification reagent without the composition of the present invention, the amplification efficiency is significantly reduced in samples with interference factors, in high-concentration HCV samples, and in samples containing triglycerides and IgG (amplification).
- the increased Ct value is negatively correlated with the amplification efficiency, that is, the larger the Ct value, the lower the amplification efficiency, and No Ct means no amplification).
- the control reagent cannot achieve the effect of amplification detection.
- samples with lower and medium concentrations of HCV all interfering samples cannot be amplified.
- This control experiment shows that the composition of the present invention has a significant improvement in the anti-interference ability of qPCR amplification, especially sample lysis amplification technology that does not require nucleic acid extraction and purification.
- Example 4 The composition of the present invention is used to increase the sensitivity of detecting HCV
- composition of the invention containing all 7 ingredients (all the ingredients have the following final concentrations after being added to the qPCR reaction solution: bovine serum albumin 80 ⁇ g/mL, dithiothreitol 3mM, sorbitol 4w/v%, sugar beet Base 0.8mol/L, ammonium sulfate 10mM, formamide 3v/v%, tetramethylammonium chloride 35mM).
- bovine serum albumin 80 ⁇ g/mL
- dithiothreitol 3mM dithiothreitol 3mM
- sorbitol 4w/v% sugar beet Base 0.8mol/L
- ammonium sulfate 10mM formamide 3v/v%
- tetramethylammonium chloride 35mM tetramethylammonium chloride
- the method of comparison is to use the clinically diagnosed positive hepatitis C virus (HCV) sample to gradually dilute it, diluted 10 times (1:9, v/v), diluted 100 times (1:99, v/v), diluted 1000 Times (1:999, v/v), the comparison test was carried out with 10 ⁇ L nucleic acid release agent + 10 ⁇ L HCV sample + 30 ⁇ L PCR reaction solution.
- Real-time qPCR Real-time qPCR
- the comparison result in Table 10 shows that for the composition of the present invention in the PCR amplification system of the present invention, there is a significant improvement in the sensitivity of RT-PCR.
- the detection ability of the kit has a negative correlation with the Cycle threshold (Ct) value, and the smaller the Ct value at the same concentration, the higher the detection ability, the larger the Ct value, the lower the detection ability, and No Ct means No amplification.
- the composition of the present invention has a significant improvement in the detection ability of nucleic acids, and the detection ability of low-concentration nucleic acids has a significant improvement effect compared to the control results of the composition of the present invention. For the detection ability of low-concentration samples in the clinic, there is Significant lifting effect.
- Using the PCR additive component of the present invention can significantly improve the sensitivity and anti-interference ability of detecting hepatitis C virus (HCV), and can achieve timely and rapid diagnosis of diseases.
- HCV hepatitis C virus
- Table 10 The influence of the composition of the present invention on the Ct value of nucleic acid amplification.
- Example 5 The composition of the present invention is used to increase the anti-interference performance of detecting new crowns
- composition of the present invention contains all 7 ingredients (all the ingredients are added to the qPCR reaction solution and have the following final concentration of bovine serum albumin 80 ⁇ g/mL, dithiothreitol 3MM, sorbitol 4w/v%, sugar beet Base 0.8mol/L, ammonium sulfate 10mM, formamide 3v/v%, tetramethylammonium chloride 35mM).
- the new coronavirus sampling method is an oropharyngeal swab/nasopharyngeal swab and it is stored in a preservation solution for testing.
- the comparison scheme is to add 1 ⁇ L of the new coronavirus nucleic acid with a concentration of about 1000 copies/mL in the prepared PCR Mastermix, and at the same time add the inhibitory effect.
- 10 ⁇ L of the sample and 10 ⁇ L of the nucleic acid release agent were constructed into a reaction system with a total volume of 50 ⁇ L to verify the anti-interference ability of the new coronavirus detection system in the amplification system.
- both the control group and the blank group are used for verification, as shown in Table 11. All conditions used the amplification procedure in Table 7 for amplification detection.
- This experimental scheme is to verify the influence of the composition of the present invention on the anti-interference ability of the 2019-nCoV detection system. It can be seen from the comparison results in Table 12 that among the nucleic acid detection reagents added with the composition of the present invention, for the presence of a sample with a significant inhibitory effect, the concentration of 2019-nCoV nucleic acid with a concentration of about 1000 copies/mL is uniform. All can be detected. Under the same conditions, in the control group that did not add the composition of the present invention, the positive rate of detection of the same concentration of 2019-nCoV nucleic acid was much lower in the presence of a sample with inhibitory effect, only 2/10 of the detection rate. out.
- Example 6 The composition of the present invention is used to increase the sensitivity of detecting new crowns
- composition of the invention containing all 7 components (all components are added to the qPCR reaction solution and have the following final concentrations: bovine serum albumin 80 ⁇ g/mL, dithiothreitol 3MM, sorbitol 4w/v%, sugar beet Base 0.8mol/L, ammonium sulfate 10mM, formamide 3v/v%, tetramethylammonium chloride 35mM).
- bovine serum albumin 80 ⁇ g/mL dithiothreitol 3MM
- sorbitol 4w/v% sugar beet Base 0.8mol/L
- ammonium sulfate 10mM formamide 3v/v%
- tetramethylammonium chloride 35mM tetramethylammonium chloride
- the method of comparison is to use the new coronavirus (2019-nCoV) nucleic acid sample that is clinically diagnosed to be positive. ), diluted 1000 times (1:999, v/v), diluted 10000 times (1:9999, v/v), and carried out a comparative test with 45 ⁇ L PCR reaction solution + 5 ⁇ L nucleic acid sample.
- Real-time qPCR Real-time qPCR
- the comparison result in Table 14 shows that for the composition of the present invention in the PCR amplification system of the present invention, there is a significant improvement in the sensitivity of RT-PCR.
- the detection ability of the kit has a negative correlation with the Cycle threshold (Ct) value, and the smaller the Ct value at the same concentration, the higher the detection ability, the larger the Ct value, the lower the detection ability, and No Ct means No amplification.
- the composition of the present invention has a significant improvement in the detection ability of nucleic acids, and the detection ability of low-concentration nucleic acids has a significant improvement effect compared to the control results of the composition of the present invention. For the detection ability of low-concentration samples in the clinic, there is Significant lifting effect.
- Table 14 The influence of the composition of the present invention on the Ct value of nucleic acid amplification.
- Example 7 The composition of the present invention is used to increase the detection specificity
- composition of the present invention containing 7 components (all components have the following final concentration after being added to the qPCR reaction solution: bovine serum albumin 80 ⁇ g/mL, dithiothreitol 3MM, sorbitol 4w/v%, Betaine 0.8mol/L, ammonium sulfate 10mM, formamide 3v/v%, tetramethylammonium chloride 35mM).
- the composition of the present invention is added to the PCR amplification system, and the comparison study is carried out with the PCR amplification system without the composition of the present invention.
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Abstract
Description
组合 | 组合1 | 组合2 | 组合3 | 组合4 | 组合5 | 组合6 | 组合7 | 组合8 | 组合9 |
阳性率 | 7/10 | 8/10 | 6/10 | 8/10 | 7/10 | 6/10 | 7/10 | 10/10 | 0/10 |
BSA | DTT | 山梨醇 | 甜菜碱 | 硫酸铵 | 甲酰胺 | TMAC | |
组合1 | / | / | + | + | + | + | + |
组合2 | / | + | + | / | + | + | + |
组合3 | + | + | / | + | / | + | + |
组合4 | / | + | + | / | + | + | + |
组合5 | + | + | + | + | / | + | / |
组合6 | + | + | / | / | + | + | + |
组合7 | + | + | + | + | + | / | / |
组合8 | + | / | + | + | + | / | + |
组合9 | + | + | + | + | + | + | + |
Claims (10)
- 一种用于提升qPCR检测性能的组合物,所述组合物包括:牛血清蛋白、山梨醇、硫酸铵、甲酰胺和四甲基氯化铵,以及二硫苏糖醇和甜菜碱中的至少一种;其中,所述组合物中各组分的浓度被配制为使得在被添加至qPCR反应液中后具有以下终浓度:牛血清蛋白10-150μg/mL、二硫苏糖醇1-10mM、山梨醇1-10w/v%、甜菜碱0.5-4mol/L、硫酸铵2-50mM、甲酰胺0.1-10v/v%,和四甲基氯化铵10-100mM。
- 根据权利要求1所述的组合物,所述组合物包括牛血清蛋白、二硫苏糖醇、山梨醇、硫酸铵、甲酰胺和四甲基氯化铵。
- 根据权利要求1所述的组合物,所述组合物包括牛血清蛋白、山梨醇、甜菜碱、硫酸铵、甲酰胺和四甲基氯化铵。
- 根据权利要求1所述的组合物,所述组合物包括牛血清蛋白、二硫苏糖醇、山梨醇、甜菜碱、硫酸铵、甲酰胺和四甲基氯化铵。
- 根据权利要求4所述的组合物,其中,所述组合物中各组分的浓度被配制为使得在被添加至qPCR反应液中后具有以下终浓度:牛血清蛋白80-120μg/mL、二硫苏糖醇2-8mM、山梨醇4-6w/v%、甜菜碱0.6-1mol/L、硫酸铵8-15mM、甲酰胺0.5-5v/v%、四甲基氯化铵20-80mM。
- 根据权利要求4所述的组合物,其中,所述组合物中各组分的浓度被配制为使得在被添加至qPCR反应液中后具有以下终浓度:牛血清蛋白80μg/mL、二硫苏糖醇3mM、山梨醇4w/v%、甜菜碱0.8mol/L、硫酸铵10mM、甲酰胺3v/v%、四甲基氯化铵35mM。
- qPCR反应液,其含有如权利要求1~6中任一项所述的组合物。
- 根据权利要求7所述的qPCR反应液,其进一步包括样本,例如,经核酸提取的样本和/或未经核酸提取的样本。
- 权利要求1~6中任一项所述组合物在改善qPCR检测性能中的用途,有 利地,所述改善是指灵敏度、特异性,和/或抗干扰性的性能的提升。
- 一种用于配制qPCR反应液的方法,所述方法包括将样本与反应缓冲液和权利要求1~6中任一项所述的组合物混合的步骤。
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US17/936,128 US20230193369A1 (en) | 2020-04-23 | 2022-09-28 | Composition, reaction liquid and method for improving qpcr test performance, and use thereof |
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CN111218502B (zh) * | 2020-04-23 | 2020-07-21 | 圣湘生物科技股份有限公司 | 提升qPCR检测性能的组合物、反应液、用途及方法 |
CN114480589B (zh) * | 2021-12-09 | 2023-05-02 | 四川省医学科学院·四川省人民医院 | 一种pcr反应体系稳定剂及其应用 |
CN116179656B (zh) * | 2022-12-09 | 2024-04-09 | 南京诺唯赞生物科技股份有限公司 | 一种qPCR扩增反应液及用途 |
CN118222764A (zh) * | 2024-04-22 | 2024-06-21 | 北京森康生物技术开发有限公司 | 一种抗多种消毒剂干扰的非洲猪瘟病毒荧光pcr扩增反应试剂、试剂盒及检测方法 |
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ZA202212648B (en) | 2023-03-29 |
EP4006167B1 (en) | 2023-11-29 |
CN111218502A (zh) | 2020-06-02 |
CN111218502B (zh) | 2020-07-21 |
EP4006167A1 (en) | 2022-06-01 |
EP4006167A4 (en) | 2022-11-30 |
BR112022021338A2 (pt) | 2022-12-13 |
US20230193369A1 (en) | 2023-06-22 |
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