Composition for improving amplification platform and application thereof

Abstract

The invention provides a composition for improving an amplification platform and application thereof, belongs to the technical field of biology, and particularly relates to an amplification composition containing polyglutamic acid, and the composition can improve the amplification platform of an amplification reaction and improve the target detection rate under the condition of low template input.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition containing polyglutamic acid, which can improve the fluorescence value of an amplification platform of a qPCR reaction and the gray value of a PCR product agarose gel electrophoresis band, and can further increase the amount of a target product in a multiplex amplification reaction. Background Art

[0002] Real-time fluorescence quantitative PCR (Quantitation Real-Time PCR) generally refers to adding a fluorescent group to the reaction system to detect the PCR amplification process through the real-time accumulation of fluorescent signals. This technology was first proposed by Japanese Higuchi in 1992, and the first detection instrument was launched by Applied Biosystems in the United States in 1996, making Real-Time PCR technology available for application and promotion.

[0003] RT-qPCR (Reverse Transcription polymerase chain reaction) is usually divided into two-step and one-step detection. Two-step detection refers to the reverse transcription reaction and quantitative polymerase chain reaction in two tubes, while one-step detection refers to the reverse transcription reaction and quantitative polymerase chain reaction in one tube. One-step RT-qPCR is an important molecular biology tool, widely used in gene expression regulation, disease diagnosis and gene regulation. Compared with the two-step method, the one-step RT-qPCR has fewer experimental errors and fewer pipetting steps because the reverse transcription reaction and quantitative polymerase chain reaction are carried out in one tube. It can reduce the risk of contamination and is suitable for high-throughput amplification and screening. It is fast and highly reproducible. However, compared with the two-step qPCR, the one-step RT-qPCR still has shortcomings. For example, in the RT-qPCR reaction, the amplification platform will gradually decrease with the decrease of template concentration. This may cause the low copy number sample to be unable to be detected during terminal detection, resulting in false negative problems. Therefore, a new method is urgently needed to improve the amplification yield and detection rate of RT-qPCR at low template concentration. Summary of the invention

[0004] The present application can significantly improve the amplification performance, especially the amount of amplified product, by adding polyglutamic acid to the qPCR or PCR amplification system, including increasing the fluorescence platform value of qPCR and the gray value of the agarose gel electrophoresis band of the PCR product, thereby solving the problem of low amplification yield under low template input.

[0005] In a first aspect, the present application provides a composition comprising a buffer substance, a metal salt and polyglutamic acid, wherein the buffer substance comprises at least one of Tris, Hepes, Pipes, Taps or Tricine, and the metal salt comprises at least one of magnesium salt, potassium salt, manganese salt, sodium salt, calcium salt or ammonium salt.

[0006] In some embodiments, the concentration of polyglutamic acid in a single reaction system is in the range of 5-200 μM; preferably 10-150 μM. In some embodiments, the concentration of polyglutamic acid in a single reaction system is 5 μM, 10 μM, 20 μM, 30 μM, 50 μM, 80 μM, 100 μM, 120 μM, 150 μM, 180 μM or 200 μM.

[0007] In some embodiments, the buffer substance is Tris, and the metal salt comprises a magnesium salt and / or a potassium salt. In some embodiments, the composition comprises polyglutamic acid, Tris, MgCl2, KCl and / or KAC.

[0008] In some embodiments, the composition further comprises deoxyribonucleoside triphosphates, and the deoxyribonucleoside triphosphates comprise one or more of dATP, dCTP, dTTP, dGTP, and dUTP. In some embodiments, the deoxyribonucleoside triphosphates are a mixture of dATP, dCTP, dTTP, and dGTP; optionally, the deoxyribonucleoside triphosphates further comprise dUTP. In some embodiments, the composition comprises dATP, dCTP, dTTP, dGTP, polyglutamic acid, Tris, MgCl2, KCl, and / or KAC; optionally, dUTP is further comprised.

[0009] In some embodiments, the composition further comprises a DNA polymerase, and the DNA polymerase comprises a DNA-dependent DNA polymerase and / or an RNA-dependent DNA polymerase. In some embodiments, the DNA-dependent DNA polymerase comprises one or more of Taq DNA polymerase, Pfu DNA polymerase, and Bst DNA polymerase. In some embodiments, the RNA-dependent DNA polymerase comprises one or more of MMLV reverse transcriptase, AMV reverse transcriptase, and HIV reverse transcriptase.

[0010] In some embodiments, the composition comprises Taq DNA polymerase, dATP, dCTP, dTTP, dGTP, polyglutamate, Tris, MgCl2, KCl and / or KAC; optionally, the composition further comprises MMLV or AMV reverse transcriptase.

[0011] In some embodiments, the composition further comprises a primer and a probe; preferably, the probe is a TaqMan probe. In some embodiments, the composition further comprises a primer and a fluorescent dye; preferably, the fluorescent dye comprises one of SYBR Green I, LC Green PLUS or EvaGreen; more preferably, the fluorescent dye is SYBR Green I.

[0012] In some embodiments, the composition comprises a primer, a probe or a fluorescent dye, Taq DNA polymerase, dATP, dCTP, dTTP, dGTP, polyglutamate, Tris, MgCl2, KCl and / or KAC; optionally, the composition further comprises MMLV or AMV reverse transcriptase.

[0013] In some embodiments, the composition further comprises uracil-DNA glycosylase.

[0014] In some embodiments, the composition further comprises a DNA and / or RNA template.

[0015] The second aspect of the present application provides a kit, which comprises the composition described in the first aspect of the present application.

[0016] The third aspect of the present application provides a method for increasing the yield of DNA or RNA amplification products, which comprises the step of using the composition described in the first aspect of the present application to perform an amplification reaction on a DNA or RNA template. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A :The effect of adding polyglutamic acid to H-01 gene system on RT-qPCR in buffer system 1 environment;

[0018] Figure 1B :The effect of adding polyglutamic acid on RT-qPCR in M-51 gene system under buffer system 1 environment;

[0019] Figure 2A : The effect of adding polyglutamic acid on RT-qPCR in the H-01 gene system under buffer system 2;

[0020] Figure 2B :The effect of adding polyglutamic acid on RT-qPCR in M-51 gene system under buffer system 2 environment;

[0021] Figure 3A :The effect of different polyglutamic acid concentrations on RT-qPCR reaction in H-01 gene system;

[0022] Figure 3B :The effect of different polyglutamic acid concentrations on RT-qPCR reaction in M-51 gene system;

[0023] Figure 4A : Experimental results under FAM fluorescence channel in the first set of multiplex amplification system;

[0024] Figure 4B :The experimental results of the first group of multiplex amplification systems under ROX fluorescence channel;

[0025] Figure 4C : Experimental results of the first set of multiplex amplification system under VIC fluorescence channel;

[0026] Figure 5A : Experimental results under FAM fluorescence channel in the second set of multiplex amplification system;

[0027] Figure 5B : Experimental results of the second set of multiplex amplification system under ROX fluorescence channel;

[0028] Figure 5C : Experimental results of the second set of multiplex amplification system under the VIC fluorescence channel;

[0029] Figure 6 :Agarose gel electrophoresis of RT-PCR multiple products;

[0030] Fig. 7A :The effect of adding polyglutamic acid in H-01 system on qPCR;

[0031] Figure 7B :The effect of adding polyglutamic acid on qPCR in M-51 system. DETAILED DESCRIPTION

[0032] The technical solution of the present application is further explained below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are merely simple examples of the present application and do not represent or limit the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

[0033] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0034] Example 1: Material preparation

[0035] 1. Sample preparation

[0036] This application uses the Nanjing Novozyme Biotechnology Co., Ltd. The Cell / Tissue Total RNA Isolation Kit (Vazyme, RC101) was used to extract RNA from Hela cells and mouse hybridoma cells. The concentrations of the extracted RNA were 2459 ng / μl and 1890 ng / μl, respectively. After detection by qPCR reagents without reverse transcriptase, there was no DNA contamination and subsequent testing was possible.

[0037] In the present application, HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme R312) of Nanjing Novazon Biotech Co., Ltd. was used to reverse transcribe Hela cell RNA and mouse hybridoma cell RNA to obtain Hela cDNA and mouse hybridoma cell cDNA.

[0038] The reverse transcriptase in the present application is from HiScript II Reverse Transcriptase (Vazyme, R201) of Novazon Biotech Co., Ltd., and the Taq DNA Polymerase in the examples is from Champagne Taq DNA Polymerase (Vazyme, P122) of Nanjing Novazon Biotech Co., Ltd.

[0039] 2. Primer design

[0040] qPCR primers and probes were designed based on the human FMR1 (XM_001185076.2), MYC (XM_002467.6), B2M (XM_004048.4), EGFR (XM_054357418.1), GAPDH (XM_001256799.3) gene sequences and mouse Ncor2 (XM_030254302.1) and Chd7 (XM_006538005.5) gene sequences. The specific sequences are shown in Table 1.

[0041] Table 1: qPCR primer and probe sequences for human and mouse targets in Example 1

[0042]

[0043]

[0044]

[0045] Among them, the H series sequence name is human gene target, and the M series sequence name is mouse gene target. When the test system in the embodiment is the M system, the added template is hybridoma cell RNA; when the test system in the embodiment is the H system, the added template is Hela cell RNA.

[0046] Example 2: Effect of polyglutamic acid in one-step RT-qPCR

[0047] The RT-qPCR reaction system was prepared according to Table 2. The system without polyglutamic acid was used as the control group. The effect of polyglutamic acid in different buffer components on RT-qPCR amplification was tested in the H-01 and M-51 systems. Two template concentration gradients were tested, and two replicates were performed for each concentration.

[0048] Table 2: RT-qPCR reaction system

[0049] Components Volume (μL) <![CDATA[ddH2O]]> To 20μL 5×RT-qPCR buffer system 4 Upstream primer (10 μM) 0.4 Downstream primer (10 μM) 0.4 Probe (10 μM) 0.2 Taq DNA Polymerase (5U / μL) 1 M-MLV reverse transcriptase (10U / μL) 1 Polyglutamic acid (1mM) 1 / 0 RNA template 100pg / 10pg

[0050] The "5×RT-qPCR buffer system" is divided into two groups. The component concentrations of "5×RT-qPCR buffer system 1" are: 250mmol / L Tris, 200mmol / L KAC, 50mmol / L KCl, 2mmol / L dNTPs, 25mmol / L MgCl2; the component concentrations of "5×RT-qPCR buffer system 2" are: 250mmol / L Tris, 300mmol / L KCl, 10mM NH4Cl, 1.5mmol / LdNTPs, 25mmol / L MgCl2, which are used to study whether different buffer components affect the role of polyglutamic acid in RT-qPCR.

[0051] The reaction was carried out according to the conditions in Table 3.

[0052] Table 3: RT-qPCR reaction procedure

[0053]

[0054] Result analysis:

[0055] The experimental results are shown in Figure 1A , Figure 1B , Figure 2A and Figure 2B In the figure, 1-1 represents no polyglutamic acid added at an input amount of 100 pg; 1-2 represents no polyglutamic acid added at an input amount of 10 pg; 2-1 represents polyglutamic acid added at an input amount of 100 pg; 2-2 represents polyglutamic acid added at an input amount of 10 pg. Figure 1A To investigate the effect of adding polyglutamic acid to the H-01 gene system on RT-qPCR in the buffer system 1 environment, Figure 1BTo investigate the effect of adding polyglutamic acid to the M-51 gene system on RT-qPCR in buffer system 1, Figure 2A To investigate the effect of adding polyglutamic acid to the H-01 gene system on RT-qPCR in buffer system 2, Figure 2B The effect of adding polyglutamic acid to the M-51 gene system on RT-qPCR in buffer system 2. From the experimental results, it can be seen that no matter what buffer system is added with 50 μM polyglutamic acid, the fluorescence platform of the amplification system without adding polyglutamic acid is significantly improved. Figure 1A In the experiment, compared with the reaction system without polyglutamic acid, the fluorescence platform values ​​after adding polyglutamic acid at 100pg and 10pg template concentrations increased by 60% and 100%, respectively; Figure 1B After adding polyglutamic acid, the fluorescence platform values ​​at 100pg and 10pg template concentrations increased by 30% and 170%, respectively; Figure 2A In the experiment, after adding polyglutamic acid, the fluorescence platform values ​​at 100pg and 10pg template concentrations increased by 85% and 95%, respectively; Figure 2B After adding polyglutamic acid, the fluorescence platform values ​​at 100pg and 10pg template concentrations increased by 80% and 81% respectively. It can be seen that polyglutamic acid can increase the fluorescence value of the amplification platform, and this effect is not limited to the specific buffer environment, template input amount and amplification gene system.

[0056] Example 3: Effect of polyglutamic acid concentration in the amplification system on RT-qPCR reaction

[0057] The RT-qPCR reaction system was prepared according to the method in Table 2, wherein the concentration of polyglutamic acid added was 200 μM, 600 μM, 1 mM, and 2 mM, and the amount added was 1 μl. Therefore, the concentration of polyglutamic acid in a single reaction system was 10 μM, 30 μM, 50 mM, and 100 mM. This example was used to verify the effect of different concentrations of polyglutamic acid in the reaction system on amplification. The test system was the H-01 and M-51 systems, the RNA template input amount was 10 pg, and each reaction was repeated twice.

[0058] Result analysis:

[0059] The experimental results are shown in Figure 3A-3B In the figure, 1 represents the control without adding polyglutamic acid; 2 represents the addition of 10 μM polyglutamic acid; 3 represents the addition of 20 μM polyglutamic acid; 4 represents the addition of 50 μM polyglutamic acid; and 5 represents the addition of 100 μM polyglutamic acid. Figure 3A The effect of different polyglutamic acid concentrations on RT-qPCR reaction in the H-01 gene system. Figure 3B The effect of different polyglutamic acid concentrations on RT-qPCR reaction in the M-51 gene system. Figure 3A-3BIt can be seen that compared with the control group without polyglutamic acid, adding different concentrations of polyglutamic acid to the reaction system improves the reaction amplification platform. Figure 3A Compared with the control group, adding 10μM polyglutamic acid to the H-01 gene system can increase the amplification fluorescence platform by 3%, adding 30μM polyglutamic acid can increase the amplification platform by 40%, adding 50μM polyglutamic acid can increase the amplification platform by 54%, and adding 100μM polyglutamic acid can increase the amplification platform by 45%. Figure 3B In the experiment, compared with the control group, adding 10μM, 30μM, 50μM and 100μM polyglutamic acid to the M-51 gene system increased the amplification fluorescence platform by 38.9%, 145%, 158% and 150%, respectively.

[0060] Example 4: Effect of polyglutamic acid on one-step multiplex RT-qPCR reaction

[0061] H-02, H-03 and H-04 were used as the first multiplex system, and the RT-qPCR reaction system was prepared according to Table 2, in which the primers and probes of the three systems of H-02, H-03 and H-04 were added. M-51, M-52 and M-53 were used as the second multiplex system, and the RT-qPCR reaction system was prepared according to Table 2, in which the primers and probes of the three systems of M-51, M-52 and M-53 were added. The reaction was carried out according to the procedure in Table 3 to detect the effect of polyglutamic acid in the one-step multiplex RT-qPCR reaction system under two template input amounts.

[0062] Result analysis:

[0063] The experimental results are shown in Figure 4A-4C and Figure 5A-5C In the figure, 1-1 represents no polyglutamic acid added at an input amount of 100 pg; 1-2 represents no polyglutamic acid added at an input amount of 10 pg; 2-1 represents polyglutamic acid added at an input amount of 100 pg; 2-2 represents polyglutamic acid added at an input amount of 10 pg. Figure 4A This is the experimental result of the first group of multiplex amplification system under FAM fluorescence channel. Figure 4B This is the experimental result of the first group of multiplex amplification system under ROX fluorescence channel. Figure 4C This is the experimental result of the first group of multiplex amplification system under the VIC fluorescence channel. Figure 5A This is the experimental result of the second set of multiplex amplification system under the FAM fluorescence channel. Figure 5B This is the experimental result of the second set of multiplex amplification system under the ROX fluorescence channel. Figure 5C The experimental results of the second group of multiplex amplification system under the VIC fluorescence channel. From the experimental results, it can be seen that adding 50μM polyglutamic acid to the multiplex amplification system can also increase the fluorescence value of the multiplex amplification platform. Figure 4AIn the multiplex amplification system, compared with the one without polyglutamic acid, at the template concentrations of 100pg and 10pg, the addition of polyglutamic acid to the system increased the fluorescence value of the amplification platform by 31% and 37%, respectively; Figure 4B In the experiment, the fluorescence value of the amplification platform can be increased by 14% and 79% respectively; Figure 4C In the experiment, the fluorescence value of the amplification platform can be increased by 14% and 84% respectively. Figure 5A Compared with the multiplex amplification system without polyglutamic acid, the addition of polyglutamic acid to the system at 100pg and 10pg template concentrations increased the fluorescence value of the amplification platform by 44% and 189%, respectively; Figure 5B In the experiment, the fluorescence value of the amplification platform can be increased by 20% and 14% respectively; Figure 5C In the amplification system, the fluorescence value of the amplification platform can be increased by 86% and 412% respectively. It can be seen that adding polyglutamic acid to the amplification system can not only increase the fluorescence value of the amplification platform in the single-plex system, but also significantly increase the fluorescence value of the amplification platform in the multiplex one-step RT-qPCR system.

[0064] Example 5: Effect of polyglutamic acid on the amount of multiplex RT-PCR products

[0065] H-05, H-06, H-07, H-08 and H-09 were used as the third group of multiplex systems. The RT-PCR system was prepared according to Table 4. The reaction was carried out according to the procedure in Table 5. After the reaction, the products were subjected to agarose gel electrophoresis to detect the effect of polyglutamic acid on the product amount in the one-step multiplex RT-PCR reaction system with an input amount of 100 pg Hela RNA. Each reaction was repeated twice.

[0066] Table 4: Multiplex RT-PCR reaction system

[0067]

[0068] The component concentrations of the "5×RT-PCR buffer system" are: 250mmol / L Tris, 25mmol / L (NH4)2SO4, 250mmol / L KCl, 2mmol / L dNTPs, and 15mmol / LMgCl2.

[0069] Table 5 Multiplex RT-PCR reaction procedure

[0070]

[0071]

[0072] Result analysis:

[0073] Figure 6The figure is an agarose gel electrophoresis diagram of RT-PCR multiple products, wherein lane 1 represents the multiple amplification products (two replicates) without adding polyglutamic acid to the amplification system, and lane 2 represents the multiple amplification products (two replicates) with 50 μM polyglutamic acid added to the amplification system. Figure 6 It can be seen from the figure that the amount of target product increased significantly after adding polyglutamic acid to the system. The gray value of the target fragment of 497 bp was statistically analyzed, and the yield of the target fragment increased by about 11 times after adding polyglutamic acid to the multiple PCR amplification system. Figure 6 The gray values ​​of the target fragments of 310 bp, 279 bp, 247 bp and 214 bp in lanes 1 and 2 of Figure 2 show that the yield of the target fragments increased by about 5 times, 1.5 times, 0.8 times and 0.5 times, respectively. It can be seen that adding polyglutamic acid to the RT-PCR amplification system can effectively increase the amount of the final amplification product.

[0074] Example 6: Effect of polyglutamic acid on qPCR

[0075] Using the Hela cDNA and mouse hybridoma cDNA in Example 1 as templates, the qPCR reaction system was prepared according to Table 6, and the system without polyglutamic acid was used as the control group. The effect of polyglutamic acid on qPCR was detected in the H-01 and M-51 systems, wherein the Hela cDNA template diluted 100 times and 1000 times was added to the H-01 system, and the mouse hybridoma cell cDNA template diluted 10 times and 100 times was added to the M-51 system, and two replicates were made for each concentration. The configured reaction system was amplified according to the program in Table 7.

[0076] Table 5 qPCR reaction system

[0077] Components Volume (μL) <![CDATA[ddH20]]> To 20μL 5×cDNA Buffer System 4 Upstream primer (10 μmol / L) 0.4 Downstream primer (10 μmol / L) 0.4 Probe (10 μmol / L) 0.2 Taq DNA Polymerase (5U / μL) 1 Polyglutamic acid (1mM) 1 cDNA template 2

[0078] Among them, the component concentrations of the "5×cDNA buffer system" are 250mmol / L Tris, 200mmol / L KAC, 50mmol / LKCl, 2mmol / L dNTPs, and 25mmol / L MgCl2.

[0079] Table 6 qPCR reaction procedure

[0080]

[0081] Result analysis:

[0082] The experimental results are shown in Fig. 7A and 7BIn the figure, 1-1 represents no addition of polyglutamic acid at high input; 1-2 represents no addition of polyglutamic acid at low input; 2-1 represents addition of polyglutamic acid at high input; 2-2 represents addition of polyglutamic acid at low input. Fig. 7A This represents the effect of adding polyglutamic acid to the H-01 system on qPCR. Figure 7B This represents the effect of adding polyglutamic acid to the M-51 system on qPCR. Figure 7A-7B As can be seen from the figure, polyglutamic acid can effectively increase the fluorescence value of the amplification platform in the qPCR reaction. Fig. 7A In the experiment, compared with the control group, the fluorescence platform values ​​after adding polyglutamic acid at two concentration gradients increased by 60% and 58%, respectively; Figure 7B In the experiment, the fluorescence platform value of the amplification system with polyglutamic acid added increased by 56% and 315% at two concentrations, respectively, indicating that polyglutamic acid can also increase the fluorescence value of the amplification platform in the qPCR amplification system.

Claims

1. A composition comprising a buffer substance, a metal salt and polyglutamic acid, wherein the buffer substance comprises at least one of Tris, Hepes, Pipes, Taps or Tricine, and the metal salt comprises at least one of a magnesium salt, a potassium salt, a manganese salt, a sodium salt, a calcium salt or an ammonium salt.

2. The composition according to claim 1, wherein the concentration of the polyglutamic acid in a single reaction system is in the range of 5-200 μM; preferably 10-150 μM.

3. The composition of claim 1, wherein the buffer substance is Tris, and the metal salt comprises a magnesium salt and / or a potassium salt; preferably, the composition comprises polyglutamic acid, Tris, MgCl2, KCl and / or KAC.

4. compositions as claimed in claim 1, it also comprises deoxyribonucleoside triphosphate, and described deoxyribonucleoside triphosphate comprises one or more among dATP, dCTP, dTTP, dGTP, dUTP; Preferably, described deoxyribonucleoside triphosphate is the mixture of dATP, dCTP, dTTP, dGTP, optionally, also comprises dUTP.

5. The composition of claim 1, further comprising a DNA polymerase, wherein the DNA polymerase comprises a DNA-dependent DNA polymerase and / or an RNA-dependent DNA polymerase; preferably, the DNA-dependent DNA polymerase comprises one or more of Taq DNA polymerase, Pfu DNA polymerase, and Bst DNA polymerase, and the RNA-dependent DNA polymerase comprises one or more of MMLV reverse transcriptase, AMV reverse transcriptase, and HIV reverse transcriptase.

6. The composition of claim 1, comprising Taq DNA polymerase, dATP, dCTP, dTTP, dGTP, polyglutamic acid, Tris, MgCl2, KCl and / or KAC; optionally, the composition further comprises MMLV or AMV reverse transcriptase.

7. The composition of claim 1, further comprising a primer and a probe; preferably, the probe is a TaqMan probe.

8. The composition according to claim 1, further comprising a primer and a fluorescent dye; preferably, the fluorescent dye comprises one of SYBR Green I, LC Green PLUS or EvaGreen; more preferably, the fluorescent dye is SYBR Green I.

9. The composition of claim 1, further comprising a DNA and / or RNA template.

10. A kit comprising the composition according to any one of claims 1 to 9.

11. A method for increasing the yield of DNA or RNA amplification products, comprising the step of using the composition according to any one of claims 1 to 9 to perform an amplification reaction on a DNA or RNA template.

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