Method for removing primer dimer in ultra-multiplex PCR amplification product purification process, dimer removing liquid and application

Abstract

The invention discloses a method for removing a primer dimer in a purification process of an ultra-multiplex PCR (Polymerase Chain Reaction) amplification product, dimer removing liquid and application, and belongs to the technical field of biology. The dimer removing liquid is used for removing primer dimers in the ultra-multiplex PCR amplification product purification process and comprises the following components in percentage by mass and volume: 4%-6% of PEG (polyethylene glycol) 8000, 0.5%-1.5% of sodium chloride, 0.1%-1.5% of tris (hydroxymethyl) aminomethane hydrochloride and the balance of nuclease-free water. The dimer scavenger disclosed by the invention can be used for efficiently removing primer dimers in a system after super-multiplex PCR amplification, and the yield of target fragments in the amplification system is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for removing primer dimers during the purification of ultra-multiplex PCR amplification products, a dimer scavenging solution, and applications thereof. Technical Background

[0002] As an important clinical diagnostic tool, high-throughput sequencing has attracted the attention of a wide range of scientific researchers and clinicians. With the increasing demand for accurate infection diagnosis, the development of multiplex PCR technology has provided more possibilities for the integration of PCR technology and sequencing technology, enabling more flexible and efficient applications in clinical diagnosis. The emergence of ultra-multiplex PCR technology has further promoted the improvement of the number of PCR amplifications. With the help of high-throughput sequencing technology, the number of amplifications can even be increased by thousands or even tens of thousands of times.

[0003] During this process, the formation of primer dimers becomes a challenge. Especially when the number of amplifications reaches a relatively high level, the removal of small fragment products and primer dimers becomes more complex and difficult, thus affecting the library construction effect. Existing dimer scavenging solutions cannot meet the requirement of efficiently scavenging dimers, and the residual content of dimers in the purified products is high, which is not conducive to subsequent library construction experiments. Therefore, there is an urgent need for an efficient dimer scavenging reagent to improve the yield of target fragments in the amplification system and the amplification efficiency of subsequent experiments. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method for removing primer dimers during the purification of ultra-multiplex PCR amplification products, a dimer scavenging solution, and applications thereof.

[0005] The present invention is implemented as follows. A dimer scavenging solution for removing primer dimers during the purification of ultra-multiplex PCR amplification products, according to mass-volume percentage, comprises the following components: 4%-6% of PEG8000, 0.5%-1.5% of sodium chloride, 0.1%-1.5% of tris(hydroxymethyl)aminomethane hydrochloride, and the balance is nuclease-free water.

[0006] Preferably, according to mass-volume percentage, it comprises the following components: 5% of PEG8000, 1.2% of sodium chloride, 0.16% of tris(hydroxymethyl)aminomethane hydrochloride, and the balance is nuclease-free deionized water.

[0007] A method for removing primer dimers during the purification of ultra-multiplex PCR amplification products comprises the following steps: selecting a suitable sample to extract nucleic acid, performing ultra-multiplex PCR amplification, after one-step purification using nucleic acid purification magnetic beads, adding the dimer scavenging solution according to claim 1 to remove primer dimers.

[0008] Specifically, it comprises the following steps:

[0009] (1) Take out the magnetic beads and the above dimer scavenging solution from the refrigerator in advance and equilibrate at room temperature for more than 30 minutes;

[0010] (2) Vortex to fully suspend the magnetic beads, add 0.9× purified magnetic beads to the ultra-multiplex PCR amplification product, invert or vortex at low speed to mix evenly, and incubate on a PCR instrument at 35 - 39 °C for 3 - 5 min;

[0011] (3) Place the PCR tube on a magnetic rack and let it stand until the solution is clear, then completely remove the supernatant;

[0012] (4) Remove the PCR tube from the magnetic rack, add the dimer scavenging solution to the tube, vortex at low speed or pipette to mix evenly, and incubate on a PCR instrument at 35 - 39 °C for 3 - 5 min;

[0013] (5) Place the PCR tube on a magnetic rack and let it stand until the solution is clear, then completely remove the supernatant;

[0014] (6) Add 180 μL of ethanol with a volume ratio concentration of 80% to each well, place it on a magnetic rack and let it stand until the solution is clear, then discard the supernatant;

[0015] (7) Repeat the above operation to aspirate and remove all the remaining ethanol with a volume ratio concentration of 80%;

[0016] (8) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 1 - 2 min to dry the magnetic beads and completely volatilize the remaining ethanol;

[0017] (9) Add nuclease-free water, remove the PCR tube from the magnetic rack, invert or vortex at low speed to mix evenly, and let it stand at room temperature for 2 - 5 min;

[0018] (10) Centrifuge briefly, place the PCR tube on a magnetic rack and let it stand for 2 - 5 min until the solution is clear;

[0019] (11) Use a pipette to aspirate the supernatant and transfer it to a new PCR tube. The supernatant in the tube is the PCR purified product after removing primer dimers.

[0020] Further, the sample is a metagenomic sample.

[0021] Further, the sample is an infected metagenomic sample.

[0022] Application of the above dimer scavenging solution in removing primer dimers from the purification of ultra-multiplex PCR amplification products.

[0023] Advantages and technical effects of the present invention: The dimer scavenging solution of the present invention can efficiently remove primer dimers in the system after ultra-multiplex PCR amplification, and improve the yield of target fragments in the amplification system. Description of the Drawings

[0024] Figure 1 This is the Qsep result diagram of the purified products after multiplex PCR amplification of the control group and the experimental group in the embodiments of the present invention (1 is the primer residue fragment of 30-50 bp, 2 is the dimer fragment of 100-150 bp, and 3 is the target fragment of 250-400 bp). Specific Embodiments

[0025] The following will describe the implementation schemes of the present invention in detail in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through market purchase.

[0026] Definition of some terms. Unless otherwise defined below, the meanings of all technical terms and scientific terms used in the specific embodiments of the present invention are intended to be the same as those commonly understood by those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are still provided to better explain the present invention.

[0027] As used in the present invention, the terms "comprising", "including", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If in the following a group is defined as comprising at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting only of these embodiments.

[0028] In addition, the terms first, second, third, (1), (2), (3), and the like in the specification and claims are used to distinguish similar elements and are not necessarily used to describe the order or time sequence. It should be understood that such terms can be interchanged under appropriate circumstances, and the embodiments described in the present invention can be implemented in an order different from that described or illustrated in the present invention.

[0029] The present invention will be described below in conjunction with specific embodiments.

[0030] Example 1

[0031] Accurately weigh 94.9 g of magnesium chloride hexahydrate powder and 0.8 g of tris(hydroxymethyl)aminomethane hydrochloride powder using an electronic balance. Add the weighed powders to a clean beaker, add 80 ml of deionized water, stir until the powders are completely dissolved, cool to room temperature, and then transfer the liquid to a 100-ml clean volumetric flask. Slowly add deionized water to make the volume up to 100 mL. A mixed solution of magnesium chloride with a mass-volume percentage of 44.5% and tris(hydroxymethyl)aminomethane hydrochloride with a mass-volume percentage of 0.8% is obtained. After filtering and sterilizing the solution with a 0.22 μM filter membrane, it can be used for subsequent experiments.

[0032] Example 2

[0033] Accurately weigh 4 g of PEG8000 powder, 0.5 g of sodium chloride powder, and 0.1 g of tris(hydroxymethyl)aminomethane hydrochloride powder using an electronic balance. Add the weighed powders to a clean beaker, add 80 ml of deionized water, stir until the powders are completely dissolved, cool to room temperature, and then transfer the liquid to a 100-ml clean volumetric flask. Slowly add deionized water to make the volume up to 100 mL. A mixed solution of PEG8000 with a mass-volume percentage of 4%, sodium chloride with a mass-volume percentage of 0.5%, and tris(hydroxymethyl)aminomethane hydrochloride with a mass-volume percentage of 0.1% is obtained. After filtering and sterilizing the solution with a 0.22 μM filter membrane, it can be used for subsequent experiments.

[0034] Example 3

[0035] Accurately weigh 5 g of PEG8000 powder, 1 g of sodium chloride powder, and 1 g of tris(hydroxymethyl)aminomethane hydrochloride powder using an electronic balance. Add the weighed powders to a clean beaker, add 80 ml of deionized water, stir until the powders are completely dissolved, cool to room temperature, and then transfer the liquid to a 100-ml clean volumetric flask. Slowly add deionized water to make the volume up to 100 mL. A mixed solution of PEG8000 with a mass-volume percentage of 5%, sodium chloride with a mass-volume percentage of 1%, and tris(hydroxymethyl)aminomethane hydrochloride with a mass-volume percentage of 1% is obtained. After filtering and sterilizing the solution with a 0.22 μM filter membrane, it can be used for subsequent experiments.

[0036] Example 4

[0037] Accurately weigh 6 g of PEG8000 powder, 1.5 g of sodium chloride powder, and 1.5 g of tris(hydroxymethyl)aminomethane hydrochloride powder using an electronic balance. Add the weighed powders to a clean beaker, add 80 ml of deionized water, stir until the powders are completely dissolved, cool to room temperature, and then transfer the liquid to a 100-ml clean volumetric flask. Slowly add deionized water to make the volume up to 100 mL. A mixed solution of PEG8000 with a mass-volume percentage of 6%, sodium chloride with a mass-volume percentage of 1.5%, and tris(hydroxymethyl)aminomethane hydrochloride with a mass-volume percentage of 1.5% is obtained. After filtering and sterilizing the solution with a 0.22 μM filter membrane, it can be used for subsequent experiments.

[0038] Example 5

[0039] Accurately weigh 5 g of PEG8000 powder, 1.2 g of sodium chloride powder and 0.16 g of tris(hydroxymethyl)aminomethane hydrochloride powder using an electronic balance. Add the weighed powders into a clean beaker, add 80 ml of deionized water, stir until the powders are completely dissolved. After cooling to room temperature, transfer the liquid to a clean 100-ml volumetric flask, slowly add deionized water, and make up the volume to 100 mL. A mixed solution with a mass-volume percentage of 5% PEG8000, 1.2% sodium chloride, and 0.16% tris(hydroxymethyl)aminomethane hydrochloride is obtained. After filtering and sterilizing the solution with a 0.22 μM filter membrane, it can be used for subsequent experiments.

[0040] Example 6. Comparison of dimer scavenging solutions

[0041] Set up an experiment with a positive reference as the sample to compare the dimer scavenging ability of the currently used dimer scavenging solution and the dimer scavenging solution of the present invention in the ultra-multiplex PCR amplification products. Perform five groups of ultra-multiplex PCR amplifications on the reference nucleic acid and purify the amplification products, which are the control group and experimental groups 1, 2, 3, and 4. The amplification products of the five groups are all purified according to the following purification steps.

[0042] 1) Take out the magnetic beads and dimer scavenging solution in advance and equilibrate at room temperature for more than 30 minutes;

[0043] 2) Vortex to fully suspend the magnetic beads, add 0.9× purified magnetic beads to the amplification products, invert or vortex at low speed to mix evenly, and incubate at 35 - 39 °C on a PCR instrument for 3 - 5 min;

[0044] 3) Place the PCR tube on a magnetic rack, let it stand until the solution is clear, and then completely remove the supernatant;

[0045] 4) Take the PCR tube off the magnetic rack, add the dimer scavenging solution into the tube, vortex at low speed to mix evenly or pipette to mix evenly, and incubate at 35 - 39 °C on a PCR instrument for 3 - 5 min;

[0046] 5) Place the PCR tube on a magnetic rack, let it stand until the solution is clear, and then completely remove the supernatant;

[0047] 6) Add 180 μL of 80% ethanol to each well, place it on a magnetic rack, let it stand until the solution is clear, and then completely remove the supernatant;

[0048] 7) Repeat the above operation to suck out the remaining 80% ethanol;

[0049] 8) Keep the PCR tube on the magnetic rack, let it stand at room temperature for 1 - 2 min to dry the magnetic beads and completely volatilize the remaining ethanol;

[0050] 9) Add nuclease-free water, remove the PCR tube from the magnetic rack, invert or vortex it gently at low speed, and let it stand at room temperature for 2 - 5 min;

[0051] 10) Centrifuge briefly, place the PCR tube on the magnetic rack, and let it stand for 2 - 5 min until the solution becomes clear.

[0052] 11) Use a pipette to aspirate the supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared PCR purified product.

[0053] The dimer removal solution used in the control group is the existing dimer removal solution prepared in Example 1, and its components are 44.5% magnesium chloride and 0.8% tris(hydroxymethyl)aminomethane hydrochloride; the first experimental group uses the dimer removal solution of the present invention prepared in Example 2, and its components are 4% PEG8000, 0.5% sodium chloride, and 0.1% tris(hydroxymethyl)aminomethane hydrochloride according to mass - volume percentage; the second experimental group uses the dimer removal solution of the present invention prepared in Example 3, and its components are 5% PEG8000, 1% sodium chloride, and 1% tris(hydroxymethyl)aminomethane hydrochloride according to mass - volume percentage; the third experimental group uses the dimer removal solution of the present invention prepared in Example 4, and its components are 6% PEG8000, 1.5% sodium chloride, and 1.5% tris(hydroxymethyl)aminomethane hydrochloride according to mass - volume percentage; the fourth experimental group uses the dimer removal solution of the present invention prepared in Example 5, and its components are 5% PEG8000, 1.2% sodium chloride, and 0.16% tris(hydroxymethyl)aminomethane hydrochloride according to mass - volume percentage. The purified products are subjected to Qubit quantification and Qseq experiments. The experimental results are shown in Table 1 and Figure 1 。

[0054] Table 1 Nucleic acid concentration and Qsep peak area of amplified products after purification

[0055]

[0056] The four experimental groups use the dimer removal solution of the present invention to purify dimers from multiplex amplification products. After purification, the nucleic acid concentration decreases compared with the control group, the proportion of primer residual fragments and dimer fragments in the system decreases, and the proportion of target fragments increases. It shows that the dimer removal solution of the present invention can effectively remove primer residues and dimer fragments in the amplification system, and the removal effect is better than the currently used removal solution.

[0057] Example 7. Performance verification of dimer removal solution

[0058] In this embodiment, the dimer removal solution is used in the construction of ultra-multiplex PCR combined with targeted sequencing libraries, and the applications of the existing dimer removal solution and the dimer removal solution of the present invention in tNGS are compared. The nucleic acid of the positive reference is subjected to two sets of ultra-multiplex PCR amplification. After one-step purification using nucleic acid purification magnetic beads, a dimer removal solution is added to remove primer dimers; the obtained product is then subjected to library PCR amplification. After one-step purification using nucleic acid purification magnetic beads, a dimer removal solution is added to remove primer dimers, and a sample sequencing library is obtained. The dimer removal solution in the control group is the existing dimer removal solution, and its components are magnesium chloride at 44.5% by mass / volume and tris(hydroxymethyl)aminomethane hydrochloride solution at 0.8%; the dimer removal solution in the experimental group is the dimer removal solution of the present invention, and its components are PEG8000 at 5% by mass / volume, sodium chloride at 1.2%, and tris(hydroxymethyl)aminomethane hydrochloride at 0.16%. The products after the two amplifications and purifications are both subjected to Qubit quantification. After the library quality control is qualified, the BGISEQ-200 sequencer is used for on-machine sequencing, and bioinformatics analysis is performed after the data is downloaded.

[0059] The experimental results are as follows:

[0060] Table 2 Nucleic acid concentration after purification of amplification products

[0061]

[0062] Table 3 Detection control of positive reference

[0063]

[0064] During the construction of the ultra-multiplex PCR combined with targeted sequencing library, compared with the control group, when the dimer removal solution of the present invention is used for the purification of amplification products, the concentration after purification of the PCR1 product decreases, the amplification efficiency of PCR2 increases, the library concentration increases, and the overall data detected by on-machine sequencing of the library is better than that of the control group.

[0065] Example 8. Verification of clinical samples

[0066] In this embodiment, the dimer scavenging solution of the present invention is used in the construction of a super-multiplex PCR combined targeted sequencing library to verify the effect of the dimer scavenging solution of the present invention on the detection of clinical samples during tNGS. Twenty clinical respiratory samples positive for pathogens detected by mNGS were selected, and a full-process experiment of nucleic acid extraction, tNGS library construction, and sequencing was performed on the samples. The nucleic acid was amplified by super-multiplex PCR. After the amplification products were purified in one step using nucleic acid purification magnetic beads, a dimer scavenging solution was added to remove primer dimers; the resulting products were then subjected to library PCR amplification. After the amplification products were purified in one step using nucleic acid purification magnetic beads, a dimer scavenging solution was added to remove primer dimers, and a sample sequencing library was obtained. The dimer scavenging solution used in this embodiment is the dimer scavenging solution of the present invention, and its components are 5% PEG8000, 1.2% sodium chloride, and 0.16% tris(hydroxymethyl)aminomethane hydrochloride by mass / volume percentage. After the library quality control was qualified, sequencing was performed on a BGISEQ-200 sequencer. After the data was downloaded, bioinformatics analysis was performed. By comparing the pathogens detected by mNGS and tNGS in clinical samples, it can be seen that the pathogens detected by tNGS using the dimer scavenging solution of the present invention are consistent with those detected by mNGS. The experimental results are as follows:

[0067] Table 4 Comparison of pathogen detection in clinical samples

[0068]

[0069]

[0070]

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements 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. A dimer removal solution, which is used to remove primer dimers during the purification process of ultra-multiplex PCR amplification products, and is characterized in that, By mass-volume percentage, it comprises the following components: 4%-6% of PEG8000, 0.5%-1.5% of sodium chloride, 0.1%-1.5% of tris(hydroxymethyl)aminomethane hydrochloride, and the balance is nuclease-free water.

2. The dimer scavenging solution according to claim 1, wherein By mass-volume percentage, it comprises the following components: 5% of PEG8000, 1.2% of sodium chloride, 0.16% of tris(hydroxymethyl)aminomethane hydrochloride, and the balance is nuclease-free deionized water.

3. A method for removing primer dimers during the purification of ultra-multiplex PCR amplification products, characterized in that, It comprises the following steps: Select a suitable sample to extract nucleic acid, perform ultra-multiplex PCR amplification, after one-step purification using nucleic acid purification magnetic beads, add the dimer removal solution described in Claim 1 to remove primer dimers.

4. The method for removing primer dimers during the purification process of ultra-multiplex PCR amplification products according to claim 3, characterized in that, It comprises the following steps: (1) Take out the magnetic beads and the dimer removal solution described in Claim 1 from the refrigerator in advance and equilibrate at room temperature for more than 30 minutes; (2) Vortex to fully suspend the magnetic beads, add 0.9× purification magnetic beads to the ultra-multiplex PCR amplification product, invert or vortex at low speed to mix evenly, and incubate at 35-39°C on a PCR instrument for 3-5 min; (3) Place the PCR tube on a magnetic rack and let it stand until the solution is clear, then completely remove the supernatant; (4) Take the PCR tube off the magnetic rack, add the dimer removal solution to the tube, vortex at low speed or pipette to mix evenly, and incubate at 35-39°C on a PCR instrument for 3-5 min; (5) Place the PCR tube on a magnetic rack and let it stand until the solution is clear, then completely remove the supernatant; (6) Add 180 μL of ethanol with a volume ratio concentration of 80% to each well, place it on a magnetic rack and let it stand until the solution is clear, then discard the supernatant; (7) Repeat the above operation to suck out all the remaining ethanol with a volume ratio concentration of 80%; (8) Keep the PCR tube on the magnetic rack and let it stand at room temperature for 1-2 min to dry the magnetic beads and completely volatilize the remaining ethanol; (9) Add nuclease-free water, take the PCR tube off the magnetic rack, invert or vortex at low speed to mix evenly, and let it stand at room temperature for 2-5 min; (10) Centrifuge instantaneously, place the PCR tube on a magnetic rack and let it stand for 2-5 min until the solution is clear; (11) Use a pipette to aspirate the supernatant and transfer it to a new PCR tube. The supernatant in the tube is the PCR purified product after removing primer dimers.

5. The method for removing primer dimers during the purification process of ultra-multiplex PCR amplification products according to claim 3 or 4, characterized in that, The sample is a metagenomic sample.

6. The method for removing primer dimers during the purification process of ultra-multiplex PCR amplification products according to claim 5, wherein, The sample is an infected metagenomic sample.

7. Use of the dimer removal solution described in Claim 1 in removing primer dimers in the purification of ultra-multiplex PCR amplification products.

Images