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 PDF

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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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composition
present
detection
qpcr
amplification
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PCT/CN2020/121056
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French (fr)
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戴立忠
纪博知
吴康
刘佳
邓中平
缪为民
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圣湘生物科技股份有限公司
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Priority to EP20931793.2A priority Critical patent/EP4006167B1/en
Priority to BR112022021338A priority patent/BR112022021338A2/pt
Publication of WO2021212771A1 publication Critical patent/WO2021212771A1/zh
Priority to US17/936,128 priority patent/US20230193369A1/en
Priority to ZA2022/12648A priority patent/ZA202212648B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

一种用于提升荧光定量PCR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。还涉及含有所述组合物的qPCR反应液，提及其配置方法。所述组合物可以提高荧光定量PCR的灵敏度、特异性以及抗干扰性。

Description

提升qPCR检测性能的组合物、反应液、用途及方法 技术领域
本发明属于分子生物学检测领域，具体地，涉及提升PCR检测性能的组合物、试剂盒及方法，更具体地，涉及提升荧光定量PCR的灵敏度、特异性以及抗干扰性。
背景技术
实时荧光定量PCR(Quantitative Real-time PCR)是一种在核酸扩增反应中，以荧光化学物质测每次聚合酶链式反应(PCR)循环后产物总量的方法。通过内参或者外参法对待测样品中的特定DNA序列进行定量分析的方法。Real-time PCR是在PCR扩增过程中，通过荧光信号，对PCR进程进行实时检测。由于在PCR扩增的指数时期，模板的Ct值和该模板的起始拷贝数存在线性关系，所以成为定量的依据。用于qPCR检测的荧光指示剂主要分为两大类：一类是荧光探针，如Taqman探针，分子信标探针；另一类是能与双链DNA结合的荧光染料，如SYBR Green和EvaGreen等。本发明讨论的提升实时荧光定量PCR检测性能的技术主要涉及荧光探针的方法，尤其是Taqman探针法的qPCR检测性能的提升。
影响qPCR检测性能的因素非常多，迄今已有许多研究专注于改善qPCR的检测效果。例如，中国专利CN1981055A中提到利用含有多核苷酸聚合酶的混合物提升PCR反应液的稳定性的应用；又例如，中国专利CN103409540A中提到利用新型染料Gelgreen I搭配Taq酶进行提升和优化定性PCR的扩增效率，再例如，中国专利CN1464070A中提到利用不同粒径大小的纳米金颗粒作为DNA检测器的识别放大器，用于提升DNA检测中的检测灵敏度。
然而，在qPCR检测领域，目前尚缺乏一种简单、便捷的方案，能够同时 提升qPCR检测性能的多个方面，包括但不局限于针对DNA/RNA的扩增检测灵敏度、抗干扰能力以及检测特异性等。
发明内容
有鉴于此，第一方面，本发明提供了一种用于提升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。
在一个具体的实施方案中，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。
在一个具体的实施方案中，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。
在一个具体的实施方案中，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。
在本发明中，牛血清蛋白在qPCR反应液中具有10-150μg/mL、优选80-120μg/mL的终浓度。例如，70μg/mL、75μg/mL、80μg/mL、85μg/mL、90μg/mL。更优选为80μg/mL的终浓度。
在本发明中，二硫苏糖醇在qPCR反应液中具有1-10mM，优选2-8mM的终浓度。例如，2mM、4mM、6mM、8mM。更优选为3mM的终浓度。
在本发明中，山梨醇在qPCR反应液中具有1-10w/v％，优选4-6w/v％的终浓度。例如，4w/v％、5w/v％、6w/v％。更优选为4w/v％的终浓度。
在本发明中，甜菜碱在qPCR反应液中具有0.5-4mol/L，优选0.6-1mol/L的终浓度。例如，0.6mol/L、0.7mol/L、0.8mol/L、0.9mol/L。更优选为0.8mol/L的终浓度。
在本发明中，硫酸铵在qPCR反应液中具有2-50mM，优选8-15mM的终浓度。例如，8mM、9mM、10mM、11mM、12mM。更优选为10mM的终浓度。
在本发明中，甲酰胺在qPCR反应液中具有0.1-10v/v％，优选0.5-5v/v％的 终浓度。例如1v/v％、2v/v％、3v/v％、4v/v％、5v/v％。更优选为3v/v％的终浓度。
在本发明中，四甲基氯化铵在qPCR反应液中具有10-100mM，优选20-80mM的终浓度。例如，20mM、40mM、60mM、80mM。更优选为35mM的终浓度
第二方面，本发明提供一种qPCR反应液，其含有如上所述的组合物。
进一步地，所述qPCR反应液还包括样本，例如，经核酸提取的样本和/或未经核酸提取的样本。
进一步地，所述qPCR反应液还包括用于qPCR的引物和探针。
更进一步地，所述qPCR反应液还包括dNTP、DNA聚合酶、PCR缓冲液。
在本发明的定义中，术语“qPCR反应液”是指能够使用荧光定量PCR检测核酸的混合物。
在一个具体的实施方案中，qPCR反应液包括如上所述组合物、引物和探针、dNTP、DNA聚合酶、PCR缓冲液。
第三方面，本发明提供一种上述组合物在改善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。
在本发明的定义中，术语“检测性能”主要是指灵敏度、特异性和抗干扰性。
第四方面，本发明提供一种用于配制qPCR反应液的方法，所述方法包括将样本与反应缓冲液和上述组合物混合的步骤。
所述反应缓冲液包括例如dNTP、DNA聚合酶、PCR缓冲液。进一步地，可以包括引物和探针。
使用本发明的组合物，可以提高荧光定量PCR的灵敏度、特异性以及抗干扰性，并且本发明的组合物所使用的浓度，能够使得荧光定量PCR的灵敏度、特异性以及抗干扰性得到进一步地提高。特别地，在复杂的临床检验情况下，提升检测性能能够更好的为疾病诊断提供分子证据，为疾病的防控做充分的准备；对传染性和危害性大的传染病可以做到及时控制传染源，阻断病毒大流行和大爆发。
附图说明
图1为APOE基因rs7412A/C模板存在的情况下，利用同一套针对rs7412A特异性扩增的引物探针，在无本发明组合物的情况下的扩增结果图；
图2为在同样浓度的APOE基因rs7412A/C模板存在的情况下，利用同一套针对rs7412A特异性扩增的引物探针，在有本发明组合物的情况下的扩增结果图。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
实施例1、本发明不同组合物的HCV检测抗干扰性能
在临床应用过程中，样本中经常会加入带有PCR抑制或者干扰的物质，如SDS，胆红素，甘油三酯等，因此考察PCR扩增试剂的抗干扰的能力成为了PCR试剂性能的重点内容之一。为了考察本发明中的组合物在带有干扰物质的样本中进行PCR扩增的抗干扰能力验证。将本发明的组合物应用到带有干扰物质的HCV血浆样本当中，与无干扰物质的HCV血浆样本进行对比检测。
对带有干扰因素的低浓度HCV样本(约为500IU/mL)进行对比检测，干扰因素为浓度在2g/dL的血红蛋白，检测的方案为在每种条件下对样本进行十次重复，对比检测的重复性和检出率。采用的样本检测方式的10μL样本+10μL核酸释放剂+30μL PCR反应液的免核酸提取的样本直接扩增的方式。不同的组合是指在PCR反应液含有不同组合物的组合，如表1，其中“+”表示加入该组分，“-”表示未加入该组分(所有成分被添加至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)。
表1本发明中不同组合物对抗干扰能力的影响
Figure PCTCN2020121056-appb-000001
表2不同组合物对有抑制物的低浓度HCV样本的检出率
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
根据表2中的结果，本发明组合物对于低浓度下有抑制物的样本的检测能力的提升存在一定的差异，在含有全部成分的组合中(组合8)，对于10个低浓度的样本全部检出。同时对于未含有本发明中任何组分的组合(组合9)，10个样本中全部未检出，因此对于低浓度下的HCV样本检测中的抗抑制能力，本发明的组合物有明显的优势。本发明的不同组合物均能对抗干扰性的提升具有积极的作用。
实施例2、本发明不同组合物的2019新型冠状病毒检测抗干扰性能
对带有干扰因素的低浓度2019新型冠状病毒(下称新冠，或2019-nCoV)核酸样本(约为1000拷贝/mL)进行对比检测，干扰样本为带有明显浑浊沉淀的呼吸道口咽拭子样本，检测的方案为在每种条件下对样本进行十次重复，对比检测的重复性和检出率。采用的样本检测方式的10μL干扰样本+10μL核酸释放剂+1μL新冠病毒核酸样本+30μL PCR反应液的扩增的方式，考察在不同条 件下对新冠病毒核酸扩增效果的影响。不同的组合是指在PCR反应液含有不同组合物的组合，如表3，其中“+”表示加入该组分，“-”表示未加入该组分(所有成分被添加至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)。同时该方案采用了两组对照组合，对照1：无干扰样本(用TE替代)，无添加剂组分PCR反应液；对照2：无干扰样本(用TE替代)，带添加剂组分PCR反应液。
表3本发明中不同组分缺失对干扰能力和检出限的影响
  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 + + + + + + +
表4不同组合对有抑制物的低浓度新冠样本的检出率
Figure PCTCN2020121056-appb-000002
根据表4中的结果，本发明组合物对于低浓度下有抑制物的样本的检测能力的提升存在一定的差异，在含有全部成分的组合中(组合9)，对于10个低 浓度的样本全部检出。本发明的不同组合物均能对抗干扰性的提升具有积极的作用。相比较两组对照试验而言(对照1和对照2)，两组对照均为未带有抑制效果的样本，仅对纯核酸进行扩增检测，全部检测为阳性。本发明的组合9采纳的添加剂组分(组合9)，也全部检测为阳性。
实施例3、本发明组合物用于增加检测HCV的抗干扰性能
将本发明的含有7种组分的组合物(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)。应用到带有干扰物质的HCV血浆样本当中，与无干扰物质的HCV血浆样本进行对比检测。同时为了对比，将无本发明组合物的PCR扩增试剂作为对照组分，也同样加入到带有干扰物质和无干扰物质的HCV血浆样本进行对比检测。进行直接的样本检测，采取的方法是样本：样本释放剂：qPCR反应液＝10:10:30(v/v)进行总体积为50μL的样本直接扩增方式，该方式因为采用了样本直接扩增的免核酸提取方式，因为更大维度的考察了扩增试剂对样本中干扰和抑制效果的抗干扰能力。该实验方案的干扰样本的制备方法、试验对比方案以及实验过程中使用的PCR扩增程序分别如下表5-7所示：
表5干扰物质样本的组分和配置方式
Figure PCTCN2020121056-appb-000003
Figure PCTCN2020121056-appb-000004
表6用于抗干扰能力检测的实验对比方案
Figure PCTCN2020121056-appb-000005
表7用于HCV样本直接扩增的抗干扰能力的检测实验
Figure PCTCN2020121056-appb-000006
为了验证本发明中组合物在干扰样本的扩增效果，往无本发明组合物的PCR扩增试剂中加入组合物，与无本发明组合物的PCR扩增试剂进行对比测 试，考察各样本的检测Ct值的对比，其Ct值与扩增效率呈负相关，即Ct值数值越大，则扩增效率越低。
表8用于HCV样本直接扩增的抗干扰能力的测试实验
Figure PCTCN2020121056-appb-000007
通过上述实验表明，在免核酸提取的样本直扩扩增试验当中，本发明中的组合物对于血清样本中的针对常见干扰物质的抗干扰能力有着明显的提升。无论是低浓度还是中高浓度的HCV样本，添加干扰因素的干扰样本与无干扰因素的对照样本，在扩增效率上无明显区别。但是相对于无本发明组合物的对照扩增试剂，在添加了干扰因素的样本中，在高浓度HCV样本中，对于含有甘油三酯和IgG的样本中，扩增效率有明显的下降(扩增的Ct值与扩增效率呈负相关，即Ct值数值越大，代表扩增效率越低，No Ct则代表无扩增)。对于含有胆红素和血红蛋白的干扰样本中，对照试剂则无法达到扩增检测的效果。对于浓度更低的中低浓度HCV样本中，则所有的干扰样本均无法扩增。此对照试验表明了，本发明的组合物对于qPCR扩增尤其是无需核酸提取纯化的样本 裂解扩增技术中的抗干扰的能力，有着明显的提升。
实施例4、本发明组合物用于增加检测HCV的灵敏度
为了测评该发明含有全部7种成分的组合物(所有成分被添加至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)。对应急情况中的核酸检测性能，将带有本发明组合物的核酸扩增试剂与无本发明组合物的商用试剂进行对比分析。对比的方法为用临床诊断为阳性的丙肝病毒(HCV)样本逐级的梯度稀释，稀释10倍(1:9,v/v)，稀释100倍(1:99,v/v)，稀释1000倍(1:999,v/v)，以10μL核酸释放剂+10μLHCV样本+30μL PCR反应液的方式进行对比测试。实时荧光定量PCR(Real-time qPCR)扩增检测程序如表9：
表9本发明采用的对HCV样本的扩增检测程序。
Figure PCTCN2020121056-appb-000008
表10的对比结果表明，对于本发明中的PCR扩增体系中的本发明组合物，在RT-PCR的灵敏度方面有这显著的提升。试剂盒的检测能力与Cycle threathold(Ct)值呈负相关的关系，及同等浓度下Ct值越小，则表示检测能力越高，Ct值越大，则表示检测能力越低，No Ct则表示无扩增。本发明组合物对于核酸检测能力有明显的提升，对于低浓度核酸的检出能力，相对于本发明组合物的对照组结果，有明显的提升效果，对于临床中低浓度样本的检测能力，有显著的提升作用。用本发明中的PCR添加剂组分可显著提升检测丙肝病毒(HCV)的灵敏度和抗干扰性，可以做到疾病的及时快速诊断。
表10本发明组合物对核酸扩增Ct值的影响。
Figure PCTCN2020121056-appb-000009
实施例5、本发明组合物用于增加检测新冠的抗干扰性能
为了证明本发明中含有全部7种成分的组合物(所有成分被添加至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)。在新冠病毒检测中的应用，由于新冠病毒采样方式为口咽拭子/鼻咽拭子并且用保存液保存起来用于检测。为了验证本发明组合物在新冠病毒核酸检测的抗干扰能力的对比验证，对比方案为在配制的PCR Mastermix中间加入1μL的浓度约为1000拷贝/mL的新冠病毒核酸，同时分别加入带有抑制效果的样本10μL和核酸释放剂10μL，构建成总体积为50μL的反应体系，验证扩增体系中新冠病毒检测体系的抗干扰能力。为了对比验证，同时采用对照组和空白组验证，具体如表11。所有条件均采用表7的扩增程序进行扩增检测。
表11本发明方案在2019-nCoV核酸检测体系中的抗干扰能力对比
Figure PCTCN2020121056-appb-000010
Figure PCTCN2020121056-appb-000011
表12本发明方案对2019-nCoV核酸检测的抗干扰能力提升
Figure PCTCN2020121056-appb-000012
本实验方案为验证本发明组合物对于2019-nCoV检测体系的抗干扰能力的影响。从表12的对比结果可以看出来，在加有本发明组合物的核酸检测试剂当中，对于带有明显抑制效应的样本存在的条件下，对浓度约为1000拷贝/mL的2019-nCoV核酸均能全部检出。同等条件下，在未加入本发明组合物的对照组中，对于带有抑制效果的样本存在的条件下，对同等浓度的2019-nCoV核酸的检测阳性率就低很多，只有2/10的检出。相比较而言，如果在没有本发明的组合物中，对于没有抑制效果的样本存在下，同等浓度的2019-nCoV核酸全部检测出阳性。因此发现有强烈抑制效果的样本对于扩增体系的作用非常明显，能够很强烈的抑制商用对照体系的扩增效果，但本发明的组合物对于检测试剂组分的抗干扰能力有明显的提升。
实施例6、本发明组合物用于增加检测新冠的灵敏度
为了测评该发明含有全部7种成分的组合物(所有成分被添加至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)。对应急情况中的核酸检测性能，将带有本发明组合物的核酸扩增试剂与无本发明组合物的商用试剂进行对比分析。对比的方法为用临床诊断为阳性的新型冠状病毒(2019-nCoV)核酸样本逐级的梯度稀释，稀释10倍(1:9,v/v)，稀释100倍(1:99,v/v)，稀释1000倍(1:999,v/v)，稀释10000倍(1:9999,v/v)，以45μL PCR反应液+5μL核酸样本的方式进行对比测试。实时荧光定量PCR(Real-time qPCR)扩增检测程序如表13：
表13本发明采用的对2019-nCoV核酸的扩增检测程序。
Figure PCTCN2020121056-appb-000013
表14的对比结果表明，对于本发明中的PCR扩增体系中的本发明组合物，在RT-PCR的灵敏度方面有这显著的提升。试剂盒的检测能力与Cycle threathold(Ct)值呈负相关的关系，及同等浓度下Ct值越小，则表示检测能力越高，Ct值越大，则表示检测能力越低，No Ct则表示无扩增。本发明组合物对于核酸检测能力有明显的提升，对于低浓度核酸的检出能力，相对于本发明组合物的对照组结果，有明显的提升效果，对于临床中低浓度样本的检测能力，有显著的提升作用。
表14本发明组合物对核酸扩增Ct值的影响。
Figure PCTCN2020121056-appb-000014
Figure PCTCN2020121056-appb-000015
提升检测新型冠状病毒2019-nCoV的灵敏度和抗干扰性，可以做到及时控制传染源，阻断病毒大流行和大爆发。
实施例7、本发明组合物用于增加检测特异性
为了测评本发明中含有7种组分的组合物(所有成分被添加至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)。用于检测特异性的作用，将本发明组合物添加到PCR扩增体系当中，与没有本发明组合物的PCR扩增体系进行对比研究。为了突出组合物的作用，使用同样引物探针序列及浓度以及同等核酸浓度进行扩增检测。本发明的组合物在验证检测特异性方面，应用于人基因组多态性分析(人APOE基因rs7412)进行扩增检测的特异性对比。该实验设计如下：
表15基于人APOE基因rs7412多态性分析的特异性扩增对比试验
Figure PCTCN2020121056-appb-000016
Figure PCTCN2020121056-appb-000017
表16检测人基因APOE多态性分析的qPCR扩增程序
Figure PCTCN2020121056-appb-000018
人基因组多态性分析对于扩增的特异性要求非常高。通过本实验设计结果图1结果可以看出，在不含本发明组合物的常规qPCR反应体系中，检测APOE基因rs7412A的探针在APOE基因rs7412C模板存在的情况下，存在明显的非特异扩增曲线，其非特异扩增曲线由于其结合力较弱，曲线荧光强度较低，但是依然会对结果判断产生不利的影响。加入本发明组合物后，明显改善了检测体系的非特异性扩增，APOE基因rs7412C模板的存在对于检测完全没有影响，完全没有扩增信号。此对照试验表明了，即使在用量非常小的情况下，本发明的组合物对于qPCR非特异性扩增有明显的抑制能力，显著改善qPCR的特异性。

Claims (10)

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. qPCR反应液，其含有如权利要求1～6中任一项所述的组合物。
  8. 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。
  9. 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。
  10. 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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