Extraction-free whole blood PCR amplification reaction buffer solution and method thereof

Abstract

The invention provides an extraction-free whole blood PCR amplification reaction buffer solution and a method thereof.The PCR amplification reaction buffer solution comprises a basic buffer solution A and an additive component B. The basic buffer solution A comprises Tris, KCl, MgCl2, dNTP, BSA, glycerin and DMSO; the additive component B comprises nano-silver, and the final concentration of the nano-silver in the amplification reaction buffer solution is 0.5-1.5 mu g / mu L. Nano-silver, spermidine and Ectoine are combined for the first time and matched with a basic buffer solution to be used in the blood direct expansion reaction buffer solution, and experiments prove that the additive combination can remarkably reduce interference generated by a PCR inhibitor in blood, so that the Rn value in PCR amplification is increased, and the Ct value of PCR amplification is reduced.

Description

Technical Field

[0001] The present invention relates to the field of PCR amplification, and particularly to a whole blood PCR amplification reaction buffer without extraction and its method. Background Art

[0002] With the development of molecular biology technology, PCR technology has been widely used in processes such as gene expression, molecular cloning, sequencing, disease detection and treatment, forensic investigation, and agricultural biotechnology. The gene templates for detection may come from human or animal tissues, saliva, feces, sputum, or soil. Due to the complexity of the templates, the extraction of templates often involves many steps and complex operations, and may also introduce new contamination. Therefore, direct amplification PCR, which directly amplifies using the sample as the template, has gradually become the mainstream testing method. Direct amplification PCR does not require DNA purification and can directly add tissue samples, sample lysates, or crude extracts to the PCR system as templates for amplification, providing convenience for experimental operations. The templates for direct amplification PCR are usually complex templates such as tissue samples, and there are often PCR amplification inhibitors. For example, heme and immunoglobulins in blood, urea in urine, and bile salts in feces may all inhibit the activity of Taq enzyme or interfere with the binding of primers to templates, thus limiting the application of direct amplification PCR. Summary of the Invention

[0003] To solve the above problems, the present invention provides a whole blood PCR amplification reaction buffer without extraction. The PCR amplification reaction buffer includes a basic buffer A and an added component B. The basic buffer A includes Tris, KCl, MgCl2, dNTP, BSA, glycerol, and DMSO; the added component B includes nano silver, and the final concentration of nano silver in the amplification reaction buffer is 0.5 - 1.5 μg / μL.

[0004] In one embodiment, the added component B further includes spermidine, and the final concentration of spermidine in the amplification reaction buffer is 0.5 - 1 mM.

[0005] In one embodiment, the final concentration of spermidine in the amplification reaction buffer is 0.5 - 0.7 mM.

[0006] In one embodiment, the added component B further includes ectoine, and the final concentration of ectoine in the amplification reaction buffer is 150 - 250 mM.

[0007] In one embodiment, the added component B further includes spermidine and ectoine. The final concentration of spermidine in the amplification reaction buffer is 0.5 - 1 mM, and the final concentration of ectoine in the amplification reaction buffer is 150 - 250 mM.

[0008] In one embodiment, the basic buffer A includes 100 mM Tris, 40 mM KCl, 3 mM MgCl2, 0.2 mM dNTP, 2 mg / ml BSA, 1% glycerol by volume and 1% DMSO by volume.

[0009] In one embodiment, a whole blood PCR amplification method is provided, in which whole blood is directly used for amplification, and the PCR amplification method uses the above-mentioned PCR amplification reaction buffer.

[0010] The reaction buffer of the present invention mainly relies on the synergistic effect between glycerol, BSA, DMSO, nanosilver, spermidine and ectoine to alleviate the inhibitory effect of whole blood components on PCR reaction. In this way, an efficient and rapid blood direct amplification detection process is successfully realized, which greatly improves the detection efficiency and accuracy. Among them, spermidine can increase the activity of Taq DNA polymerase, so that it can more effectively extend the DNA chain at high temperature. In addition to protecting Taq enzyme, glycerol can also make the conformation of DNA molecules in solution more stretched, and to a certain extent can overcome the adverse effects of complex components in blood on DNA template structure, such as preventing DNA from forming a structure that is not conducive to primer annealing due to certain ions or proteins in blood. BSA not only protects enzymes and nucleic acid templates, but also can bind to inhibitors, thereby reducing their interference with PCR reaction. Nanosilver can significantly improve the specificity of PCR reaction. Ecdoine can maintain the stability of Taq DNA polymerase and improve the efficiency of PCR reaction. DMSO can make DNA more easily unzipped. In some DNA regions with high GC content, DMSO can effectively reduce the stability of its secondary structure, so that primers can smoothly anneal with it. Therefore, the PCR amplification buffer of the present invention can be used for blood direct amplification reaction.

[0011] The present invention combines nanosilver, spermidine, and ectoine for the first time, and uses the basic buffer A in the blood direct amplification reaction buffer. Experiments have confirmed that the additive combination can significantly reduce the interference caused by PCR inhibitors in the blood, thereby increasing the Rn value in PCR amplification and reducing the Ct value of PCR amplification. The present invention has successfully developed a PCR reaction buffer that can resist 20% whole blood inhibition, greatly optimizes the amplification effect of blood direct amplification real-time fluorescent PCR, and provides a more efficient and reliable technical solution for the relevant molecular biology detection field. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the accompanying drawings required for use in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments described in the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0013] Figure 1 This is the 20% whole blood PCR amplification result diagram of the PCR buffer containing silver nanoparticles in the present invention;

[0014] Figure 2 This is the comparison diagram of the 20% whole blood PCR amplification effect of the PCR buffer containing silver nanoparticles in the present invention;

[0015] Figure 3 This is the 20% whole blood PCR amplification result diagram of the PCR buffer containing spermidine in the present invention;

[0016] Figure 4 This is the comparison diagram of the 20% whole blood PCR amplification effect of the PCR buffer containing spermidine in the present invention;

[0017] Figure 5 This is the 20% whole blood PCR amplification result diagram of the PCR buffer containing ectoine in the present invention;

[0018] Figure 6 This is the comparison diagram of the 20% whole blood PCR amplification effect of the PCR buffer containing ectoine in the present invention;

[0019] Figure 7 This is the 20% whole blood PCR amplification result diagram of the PCR buffer containing different combinations of added components in the present invention;

[0020] Figure 8 This is the comparison diagram of the 20% whole blood PCR amplification effect of the PCR buffer containing different combinations of added components in the present invention. Detailed implementation manners

[0021] To enable those skilled in the art to better understand the technical solutions in the present application, the following will further illustrate the present invention in combination with embodiments. Obviously, the embodiments are only examples and cannot limit the present invention. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present application. The experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained through commercial channels.

[0022] Example 1. Amplification experiment of a PCR amplification buffer system containing silver nanoparticles on 20% whole blood PCR

[0023] 1) The composition of basic buffer A is 100 mM Tris, 40 mM KCl, 3 mM MgCl2, 0.2 mM dNTP, 2 mg / ml BSA, 1% (v / v) glycerol, and 1% (v / v) DMSO.

[0024] 2) The reaction components and reaction procedures are as follows

[0025] Component Name Reaction Volume (μl) Final Concentration 5X Basic Buffer A 4 1X Taq DNA Polymerase (5U / ul) 0.2 1U Primer mix (10μM) 0.8 0.4μM Probe mix (10μM) 0.4 0.2μM Blood 4 20％ Silver Nanoparticles x 0 - 1.5μg / μL Nuclease-Free Water to 20

[0026] The reaction procedure is: anti-pollution at 50 °C for 2 min, pre-denaturation at 95 °C for 3 - 5 min, denaturation at 95 °C for 20 s, annealing / extension at 55 °C for 20 s (FAM collects fluorescence), 40 cycles.

[0027] 3) Experimental results

[0028]

[0029] Conclusion: Compared with basic buffer A, silver nanoparticles at 0.5 - 1.5 μg / μL have a promoting effect on the qPCR amplification efficiency, with a significant decrease in the Ct value and a significant increase in the Rn value; especially at 1 μg / μL, the Ct value of qPCR amplification decreases by 0.28 and the Rn value increases by 0.29, and the effect of a 20% increase is the most significant. The Rn value is the fluorescence signal value detected in each amplification cycle, and the Ct value refers to the number of cycles when the Rn value reaches the preset fluorescence threshold. The smaller the Ct value, the higher the initial template amount.

[0030] Example 2 Amplification experiment of PCR amplification buffer system containing spermidine for PCR containing 20% whole blood

[0031] 1) The composition of basic buffer A is 100 mM Tris, 40 mM KCl, 3 mM MgCl2, 0.2 mM dNTP, 2 mg / ml BSA, 1% (v / v) glycerol, and 1% (v / v) DMSO.

[0032] 2) The reaction components and reaction procedures are as follows

[0033] Component Name Reaction Volume (μl) Final Concentration 5X Basic Buffer A 4 1X Taq DNA Polymerase (5U / ul) 0.2 1U Primer mix (10μM) 0.8 0.4μM Probe mix (10μM) 0.4 0.2μM Blood 4 20％ Spermidine x 0 - 1mM Nuclease-Free Water to 20

[0034] The reaction procedure is: anti-pollution at 50 °C for 2 min, pre-denaturation at 95 °C for 3 - 5 min, denaturation at 95 °C for 20 s, annealing / extension at 55 °C for 20 s (FAM collects fluorescence), 40 cycles.

[0035] 3) Experimental results

[0036]

[0037] Conclusion: Compared with the basic buffer A, spermidine can improve the qPCR amplification efficiency in the concentration range of 0.5 - 1 mM. Especially at 0.7 mM, the Rn value increased by 0.22, with a 12% increase in the Rn value.

[0038] Example 3: Amplification experiment of PCR amplification buffer system containing ectoine on PCR with 20% whole blood

[0039] 1) The composition of the basic buffer A is Tris 100 mM, KCl 40 mM, MgCl2 3 mM, dNTP 0.2 mM, BSA 2 mg / ml, glycerol 1% (v / v), DMSO 1% (v / v).

[0040] 2) The reaction components and reaction procedures are as follows

[0041]

[0042]

[0043] The reaction procedure is: anti - pollution at 50°C for 2 min, pre - denaturation at 95°C for 3 - 5 min, denaturation at 95°C for 20 s, annealing / extension at 55°C for 20 s (FAM collects fluorescence), 40 cycles.

[0044] 3) Experimental results

[0045]

[0046] Conclusion: Compared with the basic buffer A, ectoine at 150 - 250 mM has a slight promoting effect on PCR containing blood. The maximum Ct value can be reduced by 0.7, and the Rn value can be increased by 13%.

[0047] Example 4: Amplification experiment of combined PCR amplification buffer system on PCR with 20% whole blood

[0048] 1) The composition of the basic buffer A is Tris 100 mM, KCl 40 mM, MgCl2 3 mM, dNTP 0.2 mM, BSA 2 mg / ml, glycerol 1% (v / v), DMSO 1% (v / v).

[0049] 2) The reaction components and reaction procedures are as follows

[0050] Component Name Reaction Volume (μl) Final Concentration 5X Basic Buffer A 4 1X Taq DNA Polymerase (5U / ul) 0.2 1U Primer mix (10μM) 0.8 0.4μM Probe mix (10μM) 0.4 0.2μM Blood 4 20％ Spermidine x 0.7mM Silver Nanoparticles x 1μg / μL Ectoine x 200mM Nuclease-Free Water to 20

[0051] The reaction procedure is: anti - pollution at 50°C for 2 min, pre - denaturation at 95°C for 3 - 5 min, denaturation at 95°C for 20 s, annealing / extension at 55°C for 20 s (FAM collects fluorescence), 40 cycles.

[0052] 3) Experimental results

[0053]

[0054] Conclusion: Compared with the basic buffer A, the pairwise combinations of silver nanoparticles + spermidine, silver nanoparticles + ectoine, and spermidine + ectoine can improve the amplification speed. However, the combination of the three (basic Buffer + silver nanoparticles + spermidine + ectoine) is the optimal combination. The combined addition can further enhance the amplification efficiency of direct blood amplification PCR, with the Ct value decreasing by 0.23 and the Rn value increasing by 0.62, and the Rn value increasing by more than 40%.

[0055] The present invention has achieved an innovative breakthrough in the application of PCR reaction buffer additives, and for the first time, silver nanoparticles, spermidine, and ectoine are added in combination. Verified by experiments, this combination can significantly reduce the interference caused by PCR inhibitors in blood, and has a significant synergistic effect on the Rn value in real-time fluorescence PCR experiments. The present invention has successfully developed a PCR reaction buffer that can resist the inhibitory effect of 20% whole blood, greatly optimizing the amplification effect of direct blood amplification real-time fluorescence PCR, and providing a more efficient and reliable technical solution for related molecular biology detection fields.

[0056] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims.

[0057] Those skilled in the art will also recognize, or be able to confirm using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also included in the appended claims.

Claims

1. A whole blood PCR amplification reaction buffer without extraction, characterized in that, The PCR amplification reaction buffer includes a basic buffer A and an additive component B. The basic buffer A includes Tris, KCl, MgCl2, dNTP, BSA, glycerol, and DMSO. The additive component B includes silver nanoparticles, and the final concentration of the silver nanoparticles in the amplification reaction buffer is 0.5 - 1.5 μg / μL.

2. The PCR amplification reaction buffer according to claim 1, wherein The additive component B further includes spermidine, and the final concentration of the spermidine in the amplification reaction buffer is 0.5 - 1 mM.

3. The PCR amplification reaction buffer according to claim 2, wherein, The final concentration of the spermidine in the amplification reaction buffer is 0.5 - 0.7 mM.

4. The PCR amplification reaction buffer according to claim 1, wherein The additive component B further includes ectoine, and the final concentration of the ectoine in the amplification reaction buffer is 150 - 250 mM.

5. The PCR amplification reaction buffer according to claim 1, wherein The additive component B further includes spermidine and ectoine. The final concentration of the spermidine in the amplification reaction buffer is 0.5 - 1 mM, and the final concentration of the ectoine in the amplification reaction buffer is 150 - 250 mM.

6. The PCR amplification reaction buffer according to claim 1, characterized in that, The basic buffer A includes 100 mM Tris, 40 mM KCl, 3 mM MgCl2, 0.2 mM dNTP, 2 mg / ml BSA, 1% (v / v) glycerol, and 1% (v / v) DMSO.

7. A method for whole blood PCR amplification, characterized in that, In the PCR amplification method, whole blood is directly used for amplification, and the PCR amplification method uses the PCR amplification reaction buffer according to any one of claims 1 - 6.

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