PCR (Polymerase Chain Reaction) premixed solution with high sensitivity and rapid amplification, PCR amplification kit and PCR method

Abstract

The invention provides a PCR (Polymerase Chain Reaction) premixed solution with high sensitivity and rapid amplification, a PCR amplification kit and a PCR method, and relates to the technical field of biology. The PCR premixed solution provided by the invention comprises a PCR enhancer with specific composition, glycerol and TMAC (tetramethylammonium chloride) can reduce non-specific amplification of primers, and the combined use effect of the glycerol and the TMAC is superior to that of any single component; glycine betaine and gelatin have the effect of enhancing sensitivity, and the combined use effect of glycine betaine and gelatin is better; glycerol and betaine can increase the duration of tolerating rapid heating and cooling, have a superposition effect, and do not have an antagonistic effect with other components at the same time; the glycerol and the betaine can obviously improve the stability of the Taq enzyme and obviously increase the temperature tolerance. Therefore, the enhancer provided by the invention can protect the activity of enzyme, promote the amplification efficiency and ensure the stability and accuracy under the condition of rapid amplification.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a PCR premix, PCR amplification kit, and PCR method with high sensitivity and rapid amplification. Background Technology

[0002] Polymerase chain reaction (PCR) is a core technology in modern molecular biology and diagnostics. This temperature-dependent technique amplifies DNA exponentially through repeated cycles of heating and cooling. Traditional thermal cyclers are bulky, and a typical PCR test usually takes 1-2 hours. In point-of-care clinical diagnosis, especially for infectious diseases, reaction time, reaction specificity, and instrument portability are crucial for rapid clinical decision-making at the nursing site. In developing this invention, the inventors discovered at least the following problems with existing technologies: these technologies require amplification equipment, high temperatures and rapid temperature control, and heat-resistant amplification reagents. Currently, the theoretical models for rapid temperature control in instruments are roughly divided into two types: one is the droplet / reagent moving type, where the heating / cooling device and temperature are fixed, and the droplet is moved to achieve rapid temperature control; the other is the droplet / reagent fixed type, where the temperature is adjusted by the instrument's temperature control module to achieve rapid temperature control. However, regardless of the model, reagents that can withstand high temperatures and rapid temperature control are required to perform this operation, making reagent selection particularly important. However, currently, common PCR formulations struggle to maintain superior performance under rapid conditions.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The primary objective of this invention is to provide the application of glycerol and / or betaine as enhancers in improving the high-temperature tolerance and temperature change tolerance of PCR detection reagents.

[0005] The second objective of this invention is to provide a highly sensitive and rapidly amplifying PCR premix to increase the reagent's tolerance to rapid temperature changes, increase the sensitivity of multiplex rapid amplification, and reduce non-specific amplification between primers and probes.

[0006] A third objective of this invention is to provide a PCR amplification kit.

[0007] The fourth objective of this invention is to provide a PCR method.

[0008] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides the application of glycerol and / or betaine as enhancers in improving the high-temperature tolerance and temperature change tolerance of PCR detection reagents.

[0009] As a further technical solution, at least one of glycerol and betaine can increase the duration of tolerance to rapid temperature changes.

[0010] As a further technical solution, at least one of glycerol and betaine can significantly improve the stability of Taq enzyme and significantly increase the tolerance of Taq enzyme to temperature (high temperature or temperature change).

[0011] As a further technical solution, the reinforcing agent also includes at least one of TMAC and gelatin.

[0012] As a further technical solution, the concentration of glycerol is 2 vol%-12 vol%. The concentration of betaine is 50 mM-750 mM; The concentration of TMAC is 20mM-100mM; The concentration of gelatin is 0.01g / 100mL-1g / 100mL.

[0013] Secondly, the present invention provides a PCR premix with high sensitivity and rapid amplification, comprising TMAC, glycerol, betaine and gelatin.

[0014] As a further technical solution, the concentration of TMAC (tetramethylammonium chloride) is 20mM-100mM; The concentration of glycerol is 2 vol% - 12 vol%. The concentration of betaine is 50 mM-750 mM; The concentration of gelatin is 0.01g / 100mL-1g / 100mL.

[0015] As a further technical solution, the PCR premix also includes upstream primers, downstream primers, probes, PCR buffer, enzyme catalysts, dNTPs and DNA polymerase, and nonionic surfactants.

[0016] As a further technical solution, the concentration of the upstream primer in the PCR premix is ​​200nM-1800nM; The concentration of the downstream primer is 200 nM-1800 nM; The concentration of the probe is 100 nM-900 nM; The buffer solution comprises Tris-HCl (pH=8.3); the concentration of Tris-HCl is 20mM-100mM; The enzyme catalyst includes MgCl2; the concentration of MgCl2 is 2-8 mM; The nonionic surfactant includes Triton X-100; the concentration of Triton X-100 is 0.005 vol% - 0.05 vol%. dNTP concentrations ranged from 2 mM to 8 mM. The concentration of DNA polymerase was 0.05 U / ul to 0.5 U / ul.

[0017] Thirdly, the present invention provides a PCR amplification kit, the kit comprising the aforementioned PCR premix.

[0018] Fourthly, the present invention provides a PCR method, which uses the aforementioned PCR premix or kit for amplification.

[0019] As a further technical solution, the PCR amplification program includes: pre-denaturation at 95-99℃ for 1-15 seconds; denaturation at 95-99℃ for 0.4-1 seconds; and annealing extension at 60-65℃ for 4-12 seconds. It should be noted that during PCR amplification, the lower limit of the denaturation time is limited by the migration time of the plateau droplets.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The PCR premix provided by this invention includes PCR enhancers. Glycerol reduces non-specific amplification of primers, and TMAC significantly reduces non-specific amplification of primers and probes; the combined effect of these two components is better than either component alone. Betaine and gelatin enhance sensitivity, and their combined effect is even better. Glycerol and betaine increase the duration of tolerance to rapid temperature changes and have an additive effect, without antagonistic effects with other components. Glycerol and betaine significantly improve the stability of Taq enzyme, greatly increasing its temperature tolerance. Therefore, the enhancers provided by this invention can protect enzyme activity, promote amplification efficiency, and ensure stability and accuracy under rapid amplification conditions. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 Digital PCR quantification graphs (top and bottom images show the results of two tests); Figure 2 Melting curve of the amplification product of Scheme 1 in Experiment Example 3; Figure 3 Melting curve of the amplification product in Scheme 2 of Experimental Example 3; Figure 4 Melting curve of the amplification product in Scheme 3 of Experimental Example 3; Figure 5 Melting curve of the amplification product in Scheme 4 of Experimental Example 3; Figure 6 Melting curve of the amplification product in Scheme 5 of Experiment Example 3; Figure 7 Melting curve of the amplification product of scheme 6 in Experiment Example 3. Detailed Implementation

[0023] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] In a first aspect, the present invention provides the application of glycerol and / or betaine as enhancers in improving the high-temperature tolerance and temperature change tolerance of PCR detection reagents.

[0025] The inventors discovered that adding glycerol and betaine to the Taq enzyme amplification system can increase the duration of Taq enzyme tolerance to rapid temperature changes, with a synergistic effect, and without antagonistic effects with other components. Furthermore, adding glycerol and betaine to the Taq enzyme amplification system can significantly improve Taq enzyme stability and greatly increase its temperature tolerance. The enhancer provided by this invention can protect enzyme activity, promote amplification efficiency, and ensure stability and accuracy under rapid amplification conditions.

[0026] In some alternative embodiments, the reinforcing agent further includes at least one of TMAC and gelatin.

[0027] In some alternative embodiments, the concentration of glycerol may be, for example, but not limited to, 2 vol%, 8 vol%, or 12 vol%. The concentration of betaine can be, for example, but not limited to, 50 mM, 300 mM or 750 mM; The concentration of TMAC can be, for example, but not limited to, 20 mM, 50 mM or 100 mM; The concentration of gelatin can be, for example, but not limited to, 0.01 g / 100 mL, 0.1 g / 100 mL or 1 g / 100 mL.

[0028] Secondly, the present invention provides a PCR premix with high sensitivity and rapid amplification, comprising TMAC, glycerol, betaine and gelatin.

[0029] The PCR premix provided by this invention contains glycerol, which reduces non-specific amplification of primers; and TMAC, which significantly reduces non-specific amplification of primers and probes. The combined effect of these two components is better than either component alone. Betaine and gelatin enhance sensitivity, and their combined effect is even better. Glycerol and betaine increase the duration of tolerance to rapid temperature changes and have an additive effect, without antagonistic effects with other components. Glycerol and betaine significantly improve the stability of Taq enzyme and significantly increase its temperature tolerance. Therefore, the enhancer provided by this invention can protect enzyme activity, promote amplification efficiency, and ensure stability and accuracy under rapid amplification conditions.

[0030] In some alternative implementations, the concentration of TMAC may be, for example, but not limited to, 20 mM, 50 mM or 100 mM; The concentration of glycerol can be, for example, but not limited to, 2 vol%, 8 vol%, or 12 vol%. The concentration of betaine can be, for example, but not limited to, 50 mM, 300 mM or 750 mM; The concentration of gelatin can be, for example, but not limited to, 0.01 g / 100 mL, 0.1 g / 100 mL or 1 g / 100 mL.

[0031] In some optional embodiments, the PCR premix further includes upstream primers, downstream primers, probes, PCR buffer, enzyme catalysts, dNTPs and DNA polymerase, and nonionic surfactants.

[0032] In some optional embodiments, the concentration of the upstream primer in the PCR premix may be, for example, but not limited to, 200 nM, 1000 nM or 1800 nM; The concentration of the downstream primer can be, for example, but not limited to, 200 nM, 1000 nM or 1800 nM; The concentration of the probe can be, for example, but not limited to, 100 nM, 500 nM or 900 nM; The buffer solution comprises Tris-HCl (pH=8.3); the concentration of Tris-HCl may be, for example, but not limited to, 20 mM, 60 mM or 100 mM; The enzyme catalyst includes Mg 2+ For example, MgCl2; the concentration of MgCl2 can be, for example, but not limited to, 2mM, 5mM or 8mM; The nonionic surfactant includes Triton X-100; the volume concentration of Triton X-100 may be, for example, but not limited to, 0.005 vol%, 0.01 vol%, or 0.05 vol%.

[0033] The dNTP concentration can be, for example, but is not limited to, 2 mM, 5 mM or 8 mM.

[0034] The concentration of DNA polymerase can be, for example, but not limited to, 0.05 U / ul, 0.1 U / ul, or 0.5 U / ul.

[0035] In some alternative embodiments, the DNA polymerase is stored in Buffer 1; The PCR enhancer, upstream primer, downstream primer, probe, PCR buffer, MgCl2, Triton X-100, and dNTPs are stored in Buffer 2. Buffer 2 helps to achieve short-term stability of the liquid temperature control during the amplification process.

[0036] Mix Buffer1 and Buffer2 for PCR amplification.

[0037] Thirdly, the present invention provides a PCR amplification kit, the kit comprising the aforementioned PCR premix.

[0038] The PCR amplification kit provided by this invention includes the PCR premix of this invention, and therefore possesses all the technical effects of the PCR premix.

[0039] Fourthly, the present invention provides a PCR method, which uses the aforementioned PCR premix or kit for amplification.

[0040] This method is suitable for amplifying nucleic acids from bacteria, viruses, etc., and has high amplification efficiency and accurate detection.

[0041] In some optional implementations, the PCR amplification program includes: pre-denaturation at 95-99°C for 1-15 seconds; denaturation at 95-99°C for 0.4-15 seconds; and annealing extension at 60-65°C for 4-12 seconds. This allows for the amplification of the target fragment within 9-15 minutes.

[0042] In some alternative implementations, the PCR amplification program includes: pre-denaturation at 97°C for 15 seconds; denaturation at 97°C for 15 seconds; and annealing extension at 60°C for 12 seconds.

[0043] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0044] Example 1 A PCR premix comprising: DNA polymerase (final concentration 0.05 U / ul, Nanjing Novizan Biotechnology Co., Ltd., Taq Pro HS DNA Polymerase), reverse transcriptase (final concentration 1 U / ul, Suzhou Nearshore Protein Technology Co., Ltd., NovoScript®Ⅲ Reverse Transcriptase), UDG enzyme (final concentration 0.001 U / ul, Shanghai Yisheng Biotechnology Co., Ltd., Uracillary DNA Glycosylase (UDG), Glycerol-Free), and RNase inhibitor (final concentration 0.5 U / ul, Shanghai Yisheng Biotechnology Co., Ltd., Murine RNase inhibitor (40 U / μL). The following reagents were used: Glycerol-free, forward and reverse primers (final concentration 200 nM), probe (final concentration 100 nM), KCl (final concentration 20 mM), MgCl2 (final concentration 2 mM), Tris-HCl (final concentration 20 mM, pH=8.3), Triton X-100 (final concentration 0.005 vol%), dNTP (final concentration 2 mM), TMAC (final concentration 20 mM), glycerol (final concentration 2 vol%), betaine (final concentration 50 mM), and gelatin (final concentration 0.01 g / 100 mL). TMAC, glycerol, betaine, and gelatin were used as enhancers.

[0045] Example 2 A PCR premix comprises: DNA polymerase (final concentration 0.1 U / ul, manufacturer and model same as in Example 1), reverse transcriptase (final concentration 3 U / ul, manufacturer and model same as in Example 1), UDG enzyme (final concentration 0.005 U / ul, manufacturer and model same as in Example 1), RNase inhibitor (final concentration 1 U / ul, manufacturer and model same as in Example 1), upstream and downstream primers (final concentration 1000 nM), probe (final concentration 500 nM), KCl (final concentration 80 mM), MgCl2 (final concentration 4 mM), Tris-HCl (final concentration 50 mM, pH=8.3), Triton X-100 (final concentration 0.01 vol %), dNTP (final concentration 5 mM), TMAC (final concentration 50 mM), glycerol (final concentration 8 vol %), betaine (final concentration 300 mM), and gelatin (final concentration 0.1 g / 100 mL). TMAC, glycerin, betaine, and gelatin are used as reinforcing agents.

[0046] Example 3 A PCR premix comprises: DNA polymerase (final concentration 0.5 U / ul, manufacturer and model same as in Example 1), reverse transcriptase (final concentration 6 U / ul, manufacturer and model same as in Example 1), UDG enzyme (final concentration 0.01 U / ul, manufacturer and model same as in Example 1), RNase inhibitor (final concentration 2 U / ul, manufacturer and model same as in Example 1), upstream and downstream primers (final concentration 1800 nM), probe (final concentration 900 nM), KCl (final concentration 130 mM), MgCl2 (final concentration 8 mM), Tris-HCl (final concentration 100 mM, pH=8.3), Triton X-100 (final concentration 0.05 vol %), dNTP (final concentration 8 mM), TMAC (final concentration 100 mM), glycerol (final concentration 12 vol %), betaine (final concentration 750 mM), and gelatin (final concentration 1 g / 100 mL). TMAC, glycerin, betaine, and gelatin are used as reinforcing agents.

[0047] Experimental Example 1 Primer and probe sequences and equipment: The primer and probe reference patent CN 112458212A.

[0048] Upstream primer: CTTCTAACCGAGGTHGAAACG (SEQ ID NO.1); Downstream primer: GAGGTGACAGGATYGGTCTTGT (SEQ ID NO.2); The probe is: CCMTCAGGCCCCCTCAAAGCCGA (SEQ ID NO.3).

[0049] Using the aforementioned primers and probes (the probes were modified with FAM fluorescent groups and BHQ1 quenching groups) as upstream and downstream primers and probes for the PCR premixes in Examples 1-3, nucleic acid amplification and fluorescence detection were performed on the extracted nucleic acid samples of influenza A virus. Extraction was carried out according to the instructions of the nucleic acid extraction and purification reagent (Antu Bioengineering Co., Ltd., Henan Zhengzhou Medical Device Registration No. 20180037) to obtain the corresponding samples. Subsequently, amplification experiments were performed on a microfluidic platform for verification. The platform instruments and amplification process were based on patent CN119186662 A.

[0050] Temperature tolerance verification: For the PCR premixes of Examples 1-3, the composition of their enhancers was adjusted, and the adjusted PCR premixes were tested for temperature tolerance and tolerance to rapid temperature rise and fall.

[0051] 1. Temperature Tolerance Test: Temperature tolerance tests were conducted on the protocols containing different enhancer components. Each protocol underwent pre-denaturation at 95℃ / 97℃ / 99℃ and followed by conventional PCR testing. PCR tests were performed on influenza A virus nucleic acid sample 1 (5000 copies / mL) and influenza A virus nucleic acid sample 2 (1000 copies / mL). The test procedures were as follows: reverse transcription 50℃ for 2 min, pre-denaturation 95℃ / 97℃ / 99℃ for 15 s; denaturation 95℃ / 97℃ / 99℃ (pre-denaturation and denaturation temperatures were consistent) for 15 s; annealing extension 60℃ for 12 s. The CT value data at the highest tolerance temperature were obtained after testing, and the data are as follows.

[0052] Results regarding the adjustment of the reinforcing agent in Example 1:

[0053] Regarding the results after adjusting the reinforcing agent in Example 2:

[0054] Regarding the results after adjusting the reinforcing agent in Example 3:

[0055] Results: When glycerol and betaine are used alone or in combination, their CT values ​​do not change much when the maximum temperature increases. Glycerol, betaine and their combination can significantly improve the stability of Taq enzyme and significantly increase its temperature tolerance. The combined temperature tolerance of glycerol and betaine can reach 99℃, and the CT value does not decline.

[0056] Experimental Example 2 The primer and probe sequences and equipment are the same as in Experiment 1.

[0057] Tolerance to rapid temperature changes: Tests were conducted on a microfluidic platform using two influenza A virus nucleic acid samples: Sample 1 (5000 copies / ml) and Sample 2 (1000 copies / ml). The amplification program was set to a final result of 15 minutes (reverse transcription 50℃ for 2 minutes, pre-denaturation 99℃ for 15 seconds; denaturation 99℃ for 0.4 seconds; annealing extension 60℃ for 6 seconds). The CT values ​​at the highest tolerance duration are given below: Results regarding the adjustment of the reinforcing agent in Example 1:

[0058] Regarding the results after adjusting the reinforcing agent in Example 2:

[0059] Regarding the results after adjusting the reinforcing agent in Example 3:

[0060] Results: Both glycerol and betaine can increase the duration of tolerance to rapid temperature changes, and they have a synergistic effect.

[0061] Experimental Example 3 The primer and probe sequences and equipment are the same as in Experiment 1.

[0062] Sensitivity and specificity tests were performed using Example 2 as an example.

[0063] Sensitivity test: The sensitivity test consists of two steps: target digital PCR determination and sensitivity assessment.

[0064] Target digital PCR determination steps: 1. Preparation of amplification system: The test was performed according to the BIO-RAD One-Step RT-ddPCR Advanced Kit for Probes.

[0065] Based on the reagent readings, the copy number for each system was statistically calculated to confirm the copy number of the measured sample concentration: in conclusion: Figure 1 This is digital PCR settling data, with the settling sample concentration being 1.2*10^5 Copy / ml.

[0066] Sensitivity test protocol: 1. Formulas containing different enhancer components were prepared (the enhancer was adjusted based on Example 2), and nucleic acid samples with high values ​​of influenza A were tested (the sample concentration after the set value was 1.2*10^5 copies / ml, and the subsequent concentrations were converted after gradient dilution, and the amplification efficiency of the tested samples was between 90% and 110%, which met the requirements).

[0067] 2. The amplification program used a rapid PCR instrument with reverse transcription at 50℃ for 2 min; pre-denaturation at 99℃ for 15 s; denaturation at 99℃ for 0.4 s; and annealing extension at 60℃ for 6 s. Sensitivity test data are shown in the table below:

[0068] Conclusion: Both betaine and gelatin enhance sensitivity, and the combined effect is better than the amplification effect of either ingredient alone, exhibiting a mixed gain effect, and there is no antagonistic effect between their components.

[0069] Nonspecific amplification: Results analysis: such as Figures 2-7As shown, the composition of the enhancer was adjusted based on the qPCR amplification system of Example 2. The sample was prepared according to the sample addition method of HiScriptⅡ One Step qRT-PCR SYBR Green Kit and amplified (Agilent Mx3005P). The sample amplified was a nucleic acid sample of influenza A virus with a concentration of 5000 copies / ml.

[0070]

[0071] The results above show that there are significant differences in the primer dimer peaks of non-target amplification in the amplification products. Glycerol reduces the primer dimer peaks of non-target amplification, especially after the addition of TMAC, which has a significant effect, protecting the primer probes from being consumed and thus reducing non-specific amplification.

[0072] Conclusion: Glycerol can reduce non-specific amplification of primers, and TMAC can significantly reduce non-specific amplification of primers and probes. The combined effect is better than either component alone.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of glycerol and / or betaine as enhancer to improve high temperature tolerance and temperature change tolerance of PCR detection reagent.

2. Use according to claim 1, characterized in that, The enhancer further comprises at least one of TMAC and gelatin.

3. Use according to claim 1 or 2, characterized in that, The concentration of glycerol is 2vol%-12vol%; The concentration of betaine is 50mM-750mM; The concentration of TMAC is 20mM-100mM; The concentration of gelatin is 0.01g / 100mL-1g / 100mL.

4. A PCR premix solution with high sensitivity and rapid amplification, characterized in that, The enhancer comprises TMAC, glycerol, betaine and gelatin.

5. The PCR master mix of claim 4, wherein, The concentration of TMAC is 20mM-100mM; The concentration of glycerol is 2vol%-12vol%; The concentration of betaine is 50mM-750mM; The concentration of gelatin is 0.01g / 100mL-1g / 100mL.

6. The PCR master mix of claim 4, wherein, The PCR premix further comprises upstream primer, downstream primer, probe, PCR buffer, enzymatic agent, dNTP and DNA polymerase, non-ionic surfactant.

7. The PCR master mix of claim 6, wherein, The concentration of upstream primer in the PCR premix is 200nM-1800nM; The concentration of downstream primer is 200nM-1800nM; The concentration of probe is 100nM-900nM; The buffer comprises Tris-HCL; the concentration of Tris-HCL is 20mM-100mM; The enzymatic agent comprises MgCl2; the concentration of MgCl2 is 2-8mM; The non-ionic surfactant comprises TritonX-100; the concentration of TritonX-100 is 0.005vol%-0.05vol%; The concentration of dNTP is 2mM-8mM; The concentration of DNA polymerase is 0.05U / ul-0.5U / ul.

8. A PCR amplification kit, characterized by, The kit comprises the PCR premix of any one of claims 4-7.

9. A PCR method characterized in that, The amplification is performed by using the PCR premix of any one of claims 4-7 or the kit of claim 8.

10. The PCR method according to claim 9, characterized by, The PCR amplification procedure comprises: pre-denaturation 95-99℃, 1-15s; denaturation 95-99℃, 0.4-1s; annealing extension 60-65℃, 4-12s.

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