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.

Claims

1. 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.

2. The composition according to claim 1, 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.

3. The composition according to claim 1, wherein the composition comprises bovine serum albumin, dithiothreitol, sorbitol, ammonium sulfate, formamide, and tetramethylammonium chloride.

4. The composition according to claim 1, wherein the composition comprises bovine serum albumin, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.

5. The composition according to claim 1, wherein the composition comprises bovine serum albumin, dithiothreitol, sorbitol, betaine, ammonium sulfate, formamide, and tetramethylammonium chloride.

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 protein, 10-150 μg/ml; dithiothreitol, 1-10mM; sorbitol, 1-10w/v %; betaine, 0.5-4 mol/L; ammonium sulfate, 2-50 mM; formamide, 0.1-10 v/v %; and tetramethylammonium chloride, 10-100 mM.

7. 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.

8. 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.

9. A qPCR reaction liquid, comprising the composition of claim 1.

10. The qPCR reaction liquid according to claim 9, comprising a sample.

11. The qPCR reaction liquid according to claim 10, wherein the sample comprises a nucleic acid-extracted sample and/or a non-nucleic acid-extracted sample.

12. The qPCR reaction liquid according to claim 9, further comprises a primer and a probe, a dNTP, a DNA polymerase, and a PCR buffer.

13. A method for improving detection performance of qPCR, comprising using the composition of claim 1.

14. The method according to claim 13, wherein the detection performance refers to sensitivity, specificity, and/or interference resistance.

15. A method for improving the detection performance of qPCR, comprising using the qPCR reaction liquid of claim 9.

16. The method according to claim 15, wherein the detection performance refers to sensitivity, specificity, and/or interference resistance.

17. 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.

18. The method according to claim 17, wherein the reaction buffer comprises a dNTP, a DNA polymerase, and a PCR buffer.

19. The method according to claim 17, wherein the reaction buffer further comprises a primer and a probe.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 is a graph showing amplification results in the absence of the composition of the present invention, using the same set of primer and probe for specific amplification of rs7412A in the presence of an APOE gene rs7412A/C template; and

[0030] FIG. 2 is a graph showing amplification results in the presence of the composition of the present invention, using the same set of primer and probe for specific amplification of rs7412A in the presence of an APOE gene rs7412A/C template at the same concentration.

DETAILED DESCRIPTION

[0031] The present invention will be elaborated in detail below in combination with particular embodiments and examples, and advantages and various effects of the present invention will be more clearly presented therefrom. It should be understood by those skilled in the art that these particular embodiments and examples are used to describe the present invention, but not to limit the present invention.

EXAMPLE 1

Interference Resistance Performance of Different Compositions of the Present Invention in HCV Detection

[0032] In a process of clinical application, a substance, such as SDS, bilirubin, and triglyceride, that causes PCR inhibition or interference is often added to a sample. Therefore, the investigation on the interference resistibility of PCR amplification reagents has become one of foci of performance of PCR reagents. In order to investigate the interference resistibility of the composition of the present invention in PCR amplification in a sample with an interferent, the composition of the present invention was applied to an HCV plasma sample with an interferent, and comparative detection was carried out with an HCV plasma sample without an interferent.

[0033] Comparative detection was performed on a low-concentration HCV sample (about 500 IU/mL) with an interference factor which was hemoglobin at a concentration of 2 g/dL. The detection scheme was to repeat detection on the sample ten times under each condition, and compare the detection repeatability and the detection rate. The detection method employed for the sample was a direct amplification method of a sample without nucleic acid extraction of 10 μL sample+10 μL nucleic acid releasing agent+30 μL PCR reaction liquid. Different combinations refer to combinations containing different compositions in a PCR reaction liquid. As shown in Table 1, “+” indicates that the component was added, and “−” indicates that the component was not added (after all ingredients were added to the qPCR reaction liquid, the ingredients had the following final concentrations: 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).

TABLE-US-00001 TABLE 1 Effects of different compositions of the present invention on interference resistibility Ammonium BSA DTT Sorbitol Betaine sulfate Formamide TMAC Combination 1 − + + + + + + Combination 2 + − + + + + + Combination 3 + + − + + + + Combination 4 + + + − + + + Combination 5 + + + + − + + Combination 6 + + + + + − + Combination 7 + + + + + + − Combination 8 + + + + + + + Combination 9 − − − − − − −

TABLE-US-00002 TABLE 2 Detection rates in low-concentration HCV samples with an inhibitor by different compositions Combi- Combi- Combi- Combi- Combi- Combi- Combi- Combi- Combi- Combi- nation nation 1 nation 2 nation 3 nation 4 nation 5 nation 6 nation 7 nation 8 nation 9 Positive 7/10 8/10 6/10 8/10 7/10 6/10 7/10 10/10 0/10 rate

[0034] According to the results in Table 2, the improvement of the detection capability by the compositions of the present invention differed to a certain extent among the low-concentration samples with the inhibitor. For the combination (Combination 8) containing all the ingredients, all of the 10 low-concentration samples were detected. At the same time, for the combination (Combination 9) not containing any component of the present invention, none of the 10 samples was detected. Therefore, the composition of the present invention had obvious advantages regarding the inhibition resistibility in the detection of low-concentration HCV samples. The different compositions of the present invention all had a positive effect on the improvement of the interference resistance.

EXAMPLE 2

Interference Resistance Performance of Different Compositions of the Present Invention for Detection of COVID-19 Viruses

[0035] Comparative detection was performed on a low-concentration COVID-19 virus (hereinafter referred to as novel coronavirus or 2019-nCoV) nucleic acid sample (1000 copies/mL) with an interference factor. The interference sample was a respiratory oropharyngeal swab sample with obvious turbid deposits. The detection scheme was to repeat detection on the sample ten times under each condition, and compare the detection repeatability and the detection rate. The detection method employed for the sample was an amplification method of 10 μL interference sample+10 nucleic acid releasing agent+1 μL novel coronavirus nucleic acid sample+30 μL PCR reaction liquid, to investigate the influence on novel coronavirus nucleic acid amplification effects under different conditions. Different combinations refer to combinations containing different compositions in a PCR reaction liquid. As shown in Table 3, “+” indicates that the component was added, and “−” indicates that the component was not added (after all ingredients were added to the qPCR reaction liquid, the ingredients had the following final concentrations: bovine serum albumin, 80 μg/mL; dithiothreitol, 3 mM; sorbitol, 4 w/v %; betaine, 0.8 mol/L; ammonium sulfate, 10 mM; formamide, 3v/v %; and tetramethylammonium chloride, 35 mM). At the same time, the scheme adopted two control combinations as follows, Control 1: an interference-free sample (replaced with TE), a PCR reaction liquid without additive components; and Control 2: an interference-free sample (replaced with TE), a PCR reaction liquid with additive components.

TABLE-US-00003 TABLE 3 Effects of omission of different components in the present invention on interference resistibility and detection limit Ammonium BSA DTT Sorbitol Betaine sulfate Formamide TMAC Combination 1 / / + + + + + Combination 2 / + + / + + + Combination 3 + + / + / + + Combination 4 / + + / + + + Combination 5 + + + + / + / Combination 6 + + / / + + + Combination 7 + + + + + / / Combination 8 + / + + + / + Combination 9 + + + + + + +

TABLE-US-00004 TABLE 4 Detection rates of low-concentration novel coronavirus samples with an inhibitor by using different combinations Combi- Combi- Combi- Combi- Combi- Combi- Combi- Combi- Combi- Combi- nation nation nation nation nation nation nation nation nation Control Control nation 1 2 3 4 5 6 7 8 9 1 2 Positive 5/10 6/10 6/10 5/10 5/10 6/10 6/10 8/10 10/10 10/10 10/10 rate

[0036] According to the results in Table 4, the improvement of the detection capability by the compositions of the present invention differed to a certain extent among the low concentration samples with the inhibitor. For the combination (Combination 9) containing all the ingredients, all of the 10 low-concentration samples were detected. The different compositions of the present invention all had a positive effect on the improvement of the interference resistance. Compared with the two control tests (Control 1 and Control 2) where both the controls were samples without an inhibition effect, only the pure nucleic acids were amplified and detected, and both were detected positive. With the additive components (Combination 9) adopted in Combination 9 of the present invention, the samples were also all detected positive.

EXAMPLE 3

Use of the Composition of the Present Invention in Improvement of Interference Resistance Performance of HCV Detection

[0037] A composition of the present invention containing 7 components (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) was applied to an HCV plasma sample with an interferent, and comparative detection was carried out with an HCV plasma sample without an interferent. At the same time, for comparison, a PCR amplification reagent without the composition of the present invention was used as a control component, and was also added to HCV plasma samples with an interferent and without an interferent for comparative detection. Direct sample detection was carried out, and the method employed was a method of direct amplification performed on a sample with a total volume of 50 μL in which a sample: a sample release agent: a qPCR reaction liquid=10:10:30 (v/v). As this method employed direct sample amplification without nucleic acid extraction, the interference resistibility of the amplification reagent against the interference and inhibition effects in the sample was investigated in wider dimensions. The preparation method of the interference sample, the experimental comparison scheme, and the PCR amplification procedure used in the experimental process of this experimental scheme are shown in Table 5-7 below:

TABLE-US-00005 TABLE 5 Components and preparation method of interferent samples Sample No. Interferent Concentration Notes Interference Triglyceride 3 g/dL Preparation method of sample 1 each interference sample: Interference Bilirubin 100 mg/dL various interferents:plasma sample 2 sample = 9:1, v/v Interference Hemoglobin 2 g/dL sample 3 Interference IgG 40 g/L sample 4 Control — — Plasma sample sample

TABLE-US-00006 TABLE 6 Experimental comparison scheme for interference resistibility testing Scheme of the Control present invention scheme Forward primer amount 10 pmol  Reverse primer amount 10 pmol  Probe amount 5 pmol Mg.sup.2+ 2 pmol dNTP (U) 2 pmol Enzyme mix 4 μL  Composition  0.125 μL 0 PCR buffer 23.625 μL 23.5 μL Nucleic acid amount 20 μL   Total reaction volume 50 μL  

TABLE-US-00007 TABLE 7 Experiments of testing interference resistibility for direct amplification of HCV samples Step Temperature Time Number of cycles Reverse transcription 50° C. 30 min 1 Pre-denaturation 95° C. 1 min 1 Denaturation 95° C. 15 sec 45 Annealing, extension, and 60° C. 30 sec fluorescence collection

[0038] In order to verify the amplification effect of the composition of the present invention in the interference sample, the composition was added to a PCR amplification reagent without the composition of the present invention, and a comparison test was carried out with a PCR amplification reagent without the composition of the present invention. Comparison of the measured Ct values of the samples was investigated. The Ct value was negatively correlated with the amplification efficiency, i.e., the larger the Ct value, the lower the amplification efficiency.

TABLE-US-00008 TABLE 8 Experiments of testing interference resistibility for direct amplification of HCV samples Interference Interference Interference Interference Control sample 1 sample 2 sample 3 sample 4 sample Comparison results of nucleic acid extraction-free amplification of high- concentration HCV samples Scheme of the 32.42 32.46 32.59 31.76 31.77 30.92 31.83 32.74 29.78 30.08 present invention Control scheme 37.84 38.18 No Ct No Ct No Ct No Ct 36.74 37.12 30.29 30.83 Comparison results of nucleic acid extraction-free amplification of medium/low-concentration HCV samples Scheme of the 35.58 36.37 36.00 36.49 36.29 35.97 36.83 36.34 35.19 35.42 present invention Control scheme No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct 36.43 36.64

[0039] The above experiments showed that in the direct amplification test of the nucleic acid extraction-free sample, the composition of the present invention had a significantly improved interference resistibility against common interferents in serum samples. No matter whether the sample was a high-concentration or a medium/low-concentration HCV sample, there was no significant difference in the amplification efficiency between the interference sample with an interference factor and the control sample without the interference factor. However, compared with the control amplification reagent without the composition of the present invention, in the sample with the interference factor added, in the high-concentration HCV sample, for the sample containing the triglyceride and the IgG, the amplification efficiency was significantly decreased (the amplification Ct value was negatively correlated with the amplification efficiency, i.e., the larger the Ct value, the lower the amplification efficiency. No Ct means no amplification.). For the interference samples containing the bilirubin and the hemoglobin, the control reagent could not achieve the effect of an amplification test. For the low/medium-concentration HCV samples with lower concentrations, none of the interference samples could be amplified. This control experiment showed that the composition of the present invention had a significantly improved interference resistibility for qPCR amplification, especially in a sample lysis amplification technology that does not require nucleic acid extraction or purification.

EXAMPLE 4

Use of the Composition of the Present Invention in Improvement of Sensitivity of HCV Detection

[0040] In order to evaluate the nucleic acid detection performance of the composition of the present invention containing all 7 ingredients (all the ingredients had the following final concentrations after being added to a 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) in emergency situations, a nucleic acid amplification reagent with the composition of the present invention was comparatively analyzed with a commercial reagent without the composition of the present invention. The comparison method adopted was gradual gradient dilution of a clinically diagnosed positive hepatitis C virus (HCV) sample, i.e., 10-fold dilution (1:9, v/v), 100-fold dilution (1:99, v/v), and 1000-fold dilution (1:999, v/v). A comparative test was carried out by using 10 μL nucleic acid releasing agent+10 μL HCV sample+30 μL PCR reaction liquid. The real-time fluorescent quantitative PCR (Real-time qPCR) amplification test procedure was as shown in Table 9:

TABLE-US-00009 TABLE 9 Amplification test procedure for HCV samples employed in the present invention Step Temperature Time Number of cycles Reverse transcription 50° C. 30 min 1 Pre-denaturation 95° C. 1 min 1 Denaturation 95° C. 15 sec 45 Annealing, extension, and 60° C. 30 sec fluorescence collection

[0041] The comparison results in Table 10 showed that for the composition of the present invention in the PCR amplification system of the present invention, the sensitivity aspect of the RT-PCR was significantly improved. The detection capability of the kit was negatively correlated with the cycle threathold (Ct) value. That is, the smaller the Ct value at the same concentration, the higher the detection capability; the larger the Ct value, the lower the detection capability. No Ct means no amplification. The composition of the present invention had a significant improvement in the detection capability for nucleic acids. The composition had a significantly improved detection capability for low-concentration nucleic acids as compared with the results of the control groups for the composition of the present invention, and had a significantly improved detection capability for clinically low-concentration samples. The use of the PCR additive components in the present invention could significantly improve the sensitivity and interference resistance of hepatitis C virus (HCV) detection, and could achieve timely and rapid diagnosis of diseases.

TABLE-US-00010 TABLE 10 Effects of the composition of the present invention on Ct values of nucleic acid amplification PCR reaction liquid with the composition of Commercial PCR the present invention reaction liquid HCV sample 26.64 28.09 HCV sample diluted 10x 29.89 32.21 HCV sample diluted 100x 33.11 35.83 HCV sample diluted 1000x 36.32 No Ct

EXAMPLE 5

Use of the Composition of the Present Invention in Improvement of Interference Resistance Performance of Novel Coronavirus Detection

[0042] In order to prove the application of the composition of the present invention containing all 7 ingredients (after all the ingredients were added to a qPCR reaction liquid, the ingredients had the following final concentrations: 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) in the novel coronavirus detection, and the sampling method of the novel coronavirus is using an oropharyngeal swab/nasopharyngeal swab and storing the sample in a preservation solution for detection. For comparative verification to verify the interference resistibility of the composition of the present invention in the detection of novel coronavirus nucleic acids, the comparison scheme was to add 1 μL of a novel coronavirus nucleic acid at a concentration of about 1000 copies/mL into PCR Mastermix prepared, and at the same time add 10 μL of a sample with an inhibition effect and 10 μL of a nucleic acid releasing agent, to construct a reaction system with a total volume of 50 so as to verify the interference resistibility of the novel coronavirus detection system in the amplification system. For comparative verification, both a control group and a blank group were used for the verification, specifically as shown in Table 11. Amplification tests were performed using the amplification procedure in Table 7 for all conditions.

TABLE-US-00011 TABLE 11 Comparison of the interference resistibility of the scheme of the present invention in 2019-nCoV nucleic acid detection systems Scheme of the Control Blank present invention scheme scheme Forward primer amount 10 pmol  Reverse primer amount 10 pmol  Probe amount 5 pmol Mg.sup.2+ 2 pmol dNTP (T) 2 pmol Enzyme mix 4 μL  Composition of the present 0.15 μL   / / invention Nucleic acid 1 μL  PCR buffer 23.65 μL   23.5 μL   23.5 μL   Nucleic acid releasing agent 10 μL 10 μL 10 μL Sample with an inhibition 10 μL 10 μL / effect Sample without an / / 10 μL inhibition effect Total reaction volume 50 μL  

TABLE-US-00012 TABLE 12 Improvement of interference resistibility for 2019-nCoV nucleic acid detection by the scheme of the present invention Scheme of the Control Blank Combination present invention scheme scheme Positive rate 10/10 2/10 10/10

[0043] This experimental scheme aimed to verify the effect of the composition of the present invention on the interference resistibility of the 2019-nCoV detection system. It can be seen from the comparison results in Table 12 that in the nucleic acid detection reagent with the composition of the present invention added thereto, in the presence of a sample with an obvious inhibition effect, 2019-nCoV nucleic acids at a concentration of about 1000 copies/mL were all detected. Under the same condition, in the control group without the composition of the present invention added, in the presence of a sample with an inhibition effect, the positive detection rate of 2019-nCoV nucleic acids at the same concentration was much lower, only 2/10. In contrast, in the absence of the composition of the present invention, in the presence of a sample without an inhibition effect, all the 2019-nCoV nucleic acids at the same concentration were detected positive. Therefore, it was found that the sample with a strong inhibition effect had a very obvious effect on the amplification system, and could strongly inhibit the amplification effect of the commercial control system, but the composition of the present invention could significantly improve the interference resistibility of the detection reagent components.

EXAMPLE 6

Use of the Composition of the Present Invention in Improvement of Sensitivity of Novel Coronavirus Detection

[0044] In order to evaluate the nucleic acid detection performance of the composition of the present invention containing all 7 ingredients (after all the ingredients were added to a qPCR reaction liquid, the ingredients had the following final concentrations: 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) in emergency situations, a nucleic acid amplification reagent with the composition of the present invention was comparatively analyzed with a commercial reagent without the composition of the present invention. The comparison method adopted was gradual gradient dilution of a clinically diagnosed positive novel coronavirus (2019-nCoV) nucleic acid sample, i.e., 10-fold dilution (1:9, v/v), 100-fold dilution (1:99, v/v), 1000-fold dilution (1:999, v/v), and 10000-fold dilution (1:9999, v/v). The comparative test was carried out by using 45 μL PCR reaction liquid+5 μL nucleic acid sample. The real-time fluorescent quantitative PCR (Real-time qPCR) amplification test procedure was as shown in Table 13:

TABLE-US-00013 TABLE 13 Amplification test procedure for 2019-nCoV nucleic acid employed in the present invention Step Temperature Time Number of cycles Reverse transcription 50° C. 30 min 1 Pre-denaturation 95° C. 1 min 1 Denaturation 95° C. 15 sec 45 Annealing, extension, and 60° C. 30 sec fluorescence collection

[0045] The comparison results in Table 14 showed that for the composition of the present invention in the PCR amplification system of the present invention, the sensitivity of RT-PCR was significantly improved. The detection capability of the kit was negatively correlated with the cycle threathold (Ct) value. That is, the smaller the Ct value at the same concentration, the higher the detection capability; the larger the Ct value, the lower the detection capability. No Ct means no amplification. The composition of the present invention had a significant improvement in the detection capability for nucleic acids. The composition had a significantly improved detection capability for low-concentration nucleic acids as compared with the results of the control groups for the composition of the present invention, and had a significantly improved detection capability for clinically low-concentration samples.

TABLE-US-00014 TABLE 14 Effects of the composition of the present invention on Ct values of nucleic acid amplification PCR reaction liquid with the composition of Commercial PCR the present invention reaction liquid Nucleic acid diluted 10x 28.64 29.89 Nucleic acid diluted 100x 31.89 33.32 Nucleic acid diluted 1000x 35.07 37.03 Nucleic acid diluted 10000x 38.34 No Ct

[0046] By improving the sensitivity and interference resistance in the detection of the novel coronavirus 2019-nCoV, infection sources can be controlled in a timely manner, and virus pandemics and outbreaks can be blocked.

EXAMPLE 7

Use of the Composition of the Present Invention in Improvement of Detection Specificity

[0047] In order to evaluate the effect of the composition of the present invention containing 7 components (after all the ingredients were added to a qPCR reaction liquid, the ingredients had the following final concentrations: 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) for detection specificity, the composition of the present invention was added to a PCR amplification system, and a comparative study was carried out with a PCR amplification system without the composition of the present invention. In order to highlight the effect of the composition, the same primer and probe sequence and concentration and the same nucleic acid concentration were used for amplification tests. In terms of verifying the detection specificity, the composition of the present invention was applied to analysis of human genome polymorphism (human APOE gene r57412) for specificity comparison of amplification tests. The experimental design was as follows:

TABLE-US-00015 TABLE 15 Comparison test of specific amplification based on polymorphism analysis of human APOE gene rs7412 Amplification scheme of Control the present invention scheme Forward primer 5′-GCGGACATGGAGGACGTG-3′ sequence (SEQ ID NO: 1) Reverse primer 5′-GCTGCCCATCTCCTCCATC-3′ sequence (SEQ ID NO: 2) Taqman probe 5′-CTTCTGCAGGTCATCGGCATCGC-3′ sequence (SEQ ID NO: 3) Forward primer 10 pmol amount Reverse primer 10 pmol amount Probe amount  5 pmol Mg.sup.2+  2 pmol dNTP (T)  2 pmol Enzyme mix  4 μL Composition of the  0.15 μL  0 present invention PCR buffer 33.65 μL 33.5 μL Nucleic acid amount  5 μL Total reaction 50 μL volume

TABLE-US-00016 TABLE 16 qPCR amplification test procedure for human gene APOE polymorphism analysis Step Temperature Time Number of cycles UNG enzyme 50° C. 5 min 1 Thermal activation 95° C. 1 min 1 Denaturation 95° C. 15 sec 45 Annealing, extension, and 60° C. 30 sec fluorescence collection

[0048] Human genome polymorphism analysis has a very high requirement for amplification specificity. It can be seen from the results of FIG. 1 of the present experimental design that in the conventional qPCR reaction system without the composition of the present invention, the probe for detecting the APOE gene rs7412A had an obvious non-specific amplification curve in the presence of an APOE gene rs7412C template. Although the non-specific amplification curve had a low curve fluorescence intensity due to a weak binding force, it still had an adverse effect on result determination. After the addition of the composition of the present invention, the non-specific amplification of the detection system was obviously improved. The presence of the APOE gene rs7412C template had no effect on the detection at all, and there was no amplification signal at all. This control test showed that even in the case of a very small amount, the composition of the present invention had an obvious inhibition capability against non-specific amplification of qPCR, and significantly improved the qPCR specificity.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0049] The contents of the electronic sequence listing (CU707SequenceListing.xml; Size: 4,076 bytes; and Date of Creation: Sep. 27, 2022) is herein incorporated by reference in its entirety.