CN115717165A - Method for reducing qPCR (quantitative polymerase chain reaction) nonspecific amplification - Google Patents
Method for reducing qPCR (quantitative polymerase chain reaction) nonspecific amplification Download PDFInfo
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Abstract
The invention provides a method for reducing qPCR (quantitative polymerase chain reaction) nonspecific amplification, belonging to the technical field of biology. According to the invention, the Tween-20 is added into the qPCR full premix system, so that the formation of primer dimer is favorably reduced, the primer consumption and non-specific amplification are reduced, and the loss of functions of the qPCR full premix reagent during long-term storage is further relieved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for reducing qPCR (quantitative polymerase chain reaction) nonspecific amplification.
Background
Polymerase Chain Reaction (PCR) is a molecular biology technology for amplifying and amplifying specific DNA fragments, can be regarded as special DNA replication outside organisms, and has the biggest characteristic that trace DNA can be greatly increased; the quantitative real-time fluorescence PCR (qPCR) is to add a fluorophore in a PCR reaction and analyze the initial amount of a target gene in real time by continuously monitoring the sequence of fluorescent signals and the change of signal intensity. One important point that affects the amplification performance of one-step qPCR reagents is the large amount of primer that is consumed by the large accumulation of primer dimers, resulting in a loss of performance. Primer dimers are recognized by polymerase enzymes from matched primer pairs, resulting in a large amount of non-specific amplification, forming a plurality of primer molecules that hybridize to each other.
The fully premixed reaction solution refers to a one-step fully premixed reaction solution containing a primer probe, wherein the reaction solution contains DNA polymerase, the primer probe required by the reaction and other raw materials required by qPCR amplification reaction, and can be used for immediately carrying out detection reaction after a sample to be detected is added. In the fully premixed reaction solution, all components except the template target exist in the system, so that the primer pairs can be extended and amplified once being overlapped, and particularly, the primer pairs can be overlapped into a dimer after being stored for a long time or being pressurized at high temperature. Since primer dimers are generally short, once formed, these extension products will amplify very efficiently in the subsequent PCR amplification stage, as non-target templates to compete with the target, resulting in loss of reagent function.
Various ways have been reported to reduce the formation of primer dimers, such as blocking Taq enzyme to achieve hot start, optimizing PCR reaction conditions, using modified primers, etc. Stephen G.Will et al (Biology Methods and Protocols,2017, 1-10) report a method of primer covalent modification, such as the exocyclic amine of deoxyadenosine or the alkyl group of cytosine residues at the 3 'end of the primer, which improves the specificity of PCR amplification and increases the yield of specific amplification products at the end of PCR, with the reduction of amplification of non-target products being most pronounced when both primers are modified at their respective 3' ends. This approach actually adds a modifying group to the 3 'terminal base of the primer, increasing steric hindrance when the bases are complementarily paired, thereby reducing the probability of primer dimer formation when the primers are 3' matched. However, the method needs to modify the primer, so that the cost of primer synthesis is greatly increased, and in addition, the modification of the base at the 3' end of the primer not only reduces the probability of overlapping and combining the primer, but also reduces the probability of annealing and matching the primer and the target to a certain extent, and reduces the amplification efficiency of the primer.
U.S. Pat. Nos. 5,607,607 and 6,193,9245A 1 disclose a method for reducing primer dimer by first modifying and blocking the 3 'end of a primer and then excising the non-extendable 3' end of the primer using endonuclease IV to activate the primer after specific primer-template hybridization. Dobosy et al (BMC Biotechnol.11:80, 2011) show an RNase H dependent PCR (rhPCR) method that also uses RNaseH to excise a single RNA base at the 3' end of a primer, thereby activating the primer after specific primer-template hybridization. However, these methods of reducing primer dimer require modification of the primer and additional enzyme for primer activation in the system, which not only greatly increases the cost, but also increases the complexity of the reaction.
Disclosure of Invention
The invention aims to provide a method for reducing the nonspecific amplification of fully premixed qPCR (quantitative polymerase chain reaction), aiming at overcoming the defects in the prior art, and the method can effectively reduce the formation of primer dimers in a fully premixed system by adding Tween-20 into a qPCR fully premixed reaction solution, thereby reducing the nonspecific amplification and improving the amplification activity.
A first aspect of the present application provides a method of reducing qPCR non-specific amplification, the method comprising: (1) Providing a fully premixed reaction solution containing buffer substances, dNTPs, metal salts, DNA polymerase, tween, primers, probes and/or fluorescent dyes;
(2) Adding a nucleic acid template;
(3) The qPCR amplification reaction was performed under appropriate conditions.
In some embodiments, the tween is any one or more of tween-20, tween-21, tween-40, tween-41, tween-60, tween-61, tween-80, tween-81 and tween-85, preferably the tween is tween-20.
In some embodiments, the volume fraction of tween-20 in the fully premixed reaction solution is 0.01% to 10%, preferably 0.02% to 8%, more preferably 0.03% to 7%, more preferably 0.04% to 6%, most preferably 0.05% to 5%, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, and 10% within the stated range.
In some embodiments, the metal salt is Mg 2+ Salt, K + Salt, mn 2+ Salt, na + Salt and Ca 2+ One or more of a salt; in some embodiments, the concentration of the metal salt in the fully premixed reaction liquid is 20 to 50mmol/L, including 20mmol/L,25mmol/L,30mmol/L,31mmol/L,32mmol/L,33mmol/L,34mmol/L,35mmol/L,36mmol/L,37mmol/L,38mmol/L,39mmol/L,40mmol/L,42mmol/L,45mmol/L,48mmol/L and 50mmol/L in the ranges described.
In some embodiments, the metal salt comprises Mg 2+ Salts and K + Salt; preferably, the metal salt comprises MgCl 2 And KCl, more preferably, the MgCl 2 The concentration in the fully premixed reaction solution is 2-10mmol/L, including 2mmol/L,3mmol/L,4mmol/L,5mmol/L,6mmol/L,7mmol/L,8mmol/L,9mmol/L and 10mmol/L in the range, and the concentration of KCl in the fully premixed reaction solution is 20-50mmol/L, including 20mmol/L,25mmol/L,26mmol/L,27mmol/L,28mmol/L,29mmol/L,30mmol/L,32mmol/L,35mmol/L,38mmol/L,40mmol/L,42mmol/L,45mmol/L,48mmol/L and 50mmol/L in the range.
In some embodiments, the metal salt further comprises KAC, the concentration of KAC in the fully premixed reaction liquid is 60-80mmol/L, including 60mmol/L,61mmol/L,62mmol/L,63mmol/L,64mmol/L,65mmol/L,66mmol/L,67mmol/L,68mmol/L,69mmol/L,70mmol/L,72mmol/L,75mmol/L,78mmol/L,80mmol/L in the range.
In some embodiments, the primers comprise at least one pair of an upstream primer and a downstream primer, the concentration of the upstream primer and the downstream primer in the fully premixed reaction solution is 0.2-0.5. Mu. Mol/L, including 0.2. Mu. Mol/L, 0.3. Mu. Mol/L, 0.4. Mu. Mol/L and 0.5. Mu. Mol/L within the ranges described.
In some embodiments, the probe is a TaqMan probe, the concentration of the TaqMan probe in the fully premixed reaction solution is 0.1-0.5 μmol/L, including 0.1 μmol/L,0.2 μmol/L,0.3 μmol/L,0.4 μmol/L and 0.5 μmol/L within the range. The TaqMan probe comprises a Reporter fluorescent group (Reporter) marked at the 5 'end and a quenching fluorescent group (Quencher) marked at the 3' end; the reporter fluorophore is, for example, FAM, VIC, HEX or TET, and the quencher fluorophore is, for example, BHQ1, TAMRA, MGB.
In some embodiments, the reaction premix further comprises a fluorescent quantitative PCR reference dye.
In some embodiments, the fluorescent dye is SYBRGreen or EvaGreenTM, preferably SYBRGreen.
In some embodiments, the DNA polymerase is a thermostable DNA polymerase, preferably Taq DNA polymerase, the concentration of the Taq DNA polymerase in the whole pre-mix reaction is 0.1-1U/. Mu.L, including 0.1U/. Mu.L, 0.2U/. Mu.L, 0.3U/. Mu.L, 0.4U/. Mu.L, 0.5U/. Mu.L, 0.6U/. Mu.L, 0.7U/. Mu.L, 0.8U/. Mu.L, 0.9U/. Mu.L and 1U/. Mu.L within the ranges described.
In some embodiments, the nucleic acid template is a DNA template.
In some embodiments, the nucleic acid template is an RNA template, and when the nucleic acid template is an RNA, the reaction composition further comprises a reverse transcriptase that is a reverse transcriptase having RNA-guided DNA polymerase activity, such as MMLV, HIV or AMV, preferably MMLV reverse transcriptase, more preferably the concentration of the MMLV reverse transcriptase in the fully premixed reaction is 0.1-1U/. Mu.L, including 0.1U/. Mu.L, 0.2U/. Mu.L, 0.3U/. Mu.L, 0.4U/. Mu.L, 0.5U/. Mu.L, 0.6U/. Mu.L, 0.7U/. Mu.L, 0.8U/. Mu.L, 0.9U/. Mu.L and 1U/. Mu.L within the ranges described.
In some embodiments, the buffer substance comprises one or more of Tris, tris-HCl, tris base or HEPES, preferably Tris, more preferably the concentration of Tris in the fully premixed reaction solution is 50-100mmol/L, including 50mmol/L,55mmol/L,60mmol/L,62mmol/L,65mmol/L,66mmol/L,67mmol/L,68mmol/L,70mmol/L,72mmol/L,75mmol/L,78mmol/L,80mmol/L,82mmol/L,85mmol/L,88mmol/L,90mmol/L,92mmol/L,95mmol/L,98mmol/L and 100mmol/L in the range.
In some embodiments, the concentration of dNTPs in the fully premixed reaction is 0.1 to 1mmol/L, including 0.1mmol/L,0.2mmol/L,0.3mmol/L,0.4mmol/L,0.5mmol/L,0.6mmol/L,0.7mmol/L,0.8mmol/L,0.9mmol/L and 1mmol/L within the stated ranges.
In some embodiments, the qPCR amplification reaction comprises a pre-denaturation and cycling reaction; the temperature of the pre-denaturation is 90-98 ℃, e.g. 92-96 ℃, e.g. 95 ℃ incubation for 20s-60s, e.g. 25s-40s, e.g. 30s; the cycle reaction comprises denaturation at 95 ℃ for 8-12s, annealing at 55-65 ℃ and extension for 10-30s, and 40-50 cycles.
In some embodiments, when the nucleic acid template is RNA, the qPCR amplification reaction is RT (Reverse transcription) -qPCR amplification reaction/Reverse transcription qPCR, the RT-qPCR amplification reaction includes Reverse transcription, pre-denaturation, and cycling reactions; the reverse transcription temperature is 50-60 deg.C, such as 52-58 deg.C, such as 55 deg.C, incubation for 10-20min, such as 12-18min, such as 15min; the temperature of the pre-denaturation is 90-98 ℃, such as 92-96 ℃, such as 95 ℃ incubation for 20s-60s, such as 25s-40s, such as 30s; the cycling reaction comprises denaturation at 95 ℃ for 8-12s, annealing at 55-65 ℃ and extension for 10-30s for 40-50 cycles.
A second aspect of the application provides the use of tween-20 for reducing non-specific amplification of a qPCR system.
In some embodiments, the qPCR system is a fully premixed qPCR system.
In some embodiments, the qPCR system is a fully premixed RT (Reverse transcription) -qPCR system.
In some embodiments, the volume fraction of tween-20 in the qPCR system is 0.01% to 10%, preferably 0.02% to 8%, more preferably 0.03% to 7%, more preferably 0.04% to 6%, most preferably 0.05% to 5%, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, and 10% within the stated range.
In some embodiments, the reducing non-specific amplification of the qPCR system means that there is no non-specific amplification product band in the qPCR amplification product gel electrophoresis image, or the non-specific amplification product band is weak; the size of the non-specific amplification product band is not the same as the designed size of the target amplification product.
In some embodiments, the reduction of non-specific amplification of the qPCR system means that the melting curve of the qPCR amplification product has only one major peak and no bi-or multi-peaks occur, or that the peak of the non-specific amplification product in the bi-or multi-peaks is reduced.
A third aspect of the application provides the use of tween 20 for reducing primer dimers of a qPCR system.
In some embodiments, the qPCR system is a fully premixed qPCR system.
In some embodiments, the qPCR system is a fully premixed RT (Reverse transcription) -qPCR system.
In some embodiments, the volume fraction of tween-20 in the qPCR system is 0.01% to 10%, preferably 0.02% to 8%, more preferably 0.03% to 7%, more preferably 0.04% to 6%, most preferably 0.05% to 5%, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, and 10% within the stated range.
In some embodiments, the reducing primer dimer of the qPCR system means that there is no primer dimer band in the qPCR amplification product gel electrophoresis image, or the primer dimer band is weak; the size of the primer dimer band is not the same as the designed size of the target amplification product.
In some embodiments, reducing primer dimer of the qPCR system means that the melting curve of the qPCR amplification product has only one major peak and no bi-or multi-peaks occur; or to a decrease in the primer dimer peak in said bi-or multi-modal.
A fourth aspect of the present application provides a qPCR fully premixed reaction composition comprising buffer substances, dNTPs, metal salts, DNA polymerase, tween-20 in a volume fraction of 0.01% to 10%, upstream and downstream primers and Taqman probes; optionally, the qPCR full premix reaction composition further comprises a reverse transcriptase.
In some embodiments, the buffer substance comprises 50-100mmol/L Tris, the concentration of dNTPs is 0.1-1mmol/L, and the metal salt comprises 2-10mmol/L MgCl 2 And 20-50mmol/L KCl, the metal salt further comprises 60-80mmol/L KAC, the DNA polymerase is 0.1-1U/μ L Taq DNA polymerase, the concentration of the upstream and downstream primers is 0.2-0.5 μmol/L, the concentration of the TaqMan probe is 0.1-0.5 μmol/L, the volume fraction of the Tween-20 is 0.02-8%, and the reverse transcriptase is 0.1-1U/μ L MMLV reverse transcriptase.
In some embodiments, the buffer substance comprises 50-70mmol/L Tris, the concentration of dNTPs is 0.1-0.5mmol/L, and the metal salt comprises 2-6mmol/L MgCl 2 And 20-30mmol/L KCl, the metal salt further comprises 60-70mmol/L KAC, the DNA polymerase is 0.2-0.5U/μ L Taq DNA polymerase, the concentration of the upstream and downstream primers is 0.2-0.3 μmol/L, the concentration of the TaqMan probe is 0.1-0.2 μmol/L, the volume fraction of Tween-20 is 0.04-6%, and the reverse transcriptase is 0.5-0.7U/μ L MMLV reverse transcriptase.
In some embodiments, the reaction composition comprises 67mmol/L Tris, 0.4mmol/L dNTPs, 5mmol/L MgCl 2 27mmol/L KCl and 67mmol/LKAC, 0.3U/mu L DNA polymerase, 0.05% -5% volume fraction Tween-20, 0.3 mu mol/L upstream and downstream primers and 0.2 mu mol/L Taqman probe; optionally, the reaction composition further comprises 0.7U/μ L of MMLV reverse transcriptase.
A fifth aspect of the present application provides a qPCR fully premixed reaction composition comprising buffer substances, dNTPs, metal salts, DNA polymerase, tween-20 in a volume fraction of 0.01% to 10%, upstream and downstream primers and SYBR Green fluorochrome; optionally, the qPCR fully premixed reaction composition further comprises a reverse transcriptase.
In some embodiments, the buffer substance comprises 50-100mmol/L Tris, the concentration of dNTPs is 0.1-1mmol/L, and the metal salt comprises 2-10mmol/L MgCl 2 And 20-50mmol/L KCl, the metal salt further comprises 60-80mmol/L KAC, the DNA polymerase is TaqD of 0.1-1U/. Mu.LNA polymerase, the concentration of the upstream primer and the downstream primer is 0.2-0.5 mu mol/L, the volume fraction of the Tween-20 is 0.02-8%, and the reverse transcriptase is 0.1-1U/mu L MMLV reverse transcriptase.
In some embodiments, the buffer substance comprises 50-70mmol/L Tris, the concentration of dNTPs is 0.1-0.5mmol/L, and the metal salt comprises 2-6mmol/L MgCl 2 And 20-30mmol/L KCl, the metal salt further comprises 60-70mmol/L KAC, the DNA polymerase is 0.2-0.5U/μ L Taq DNA polymerase, the concentration of the upstream and downstream primers is 0.2-0.3 μmol/L, the volume fraction of Tween-20 is 0.04-6%, and the reverse transcriptase is 0.5-0.7U/μ L MMLV reverse transcriptase.
In some embodiments, the reaction composition comprises 67mmol/L Tris, 0.4mmol/L dNTPs, 5mmol/L MgCl 2 27mmol/L KCl and 67mmol/L KAC, 0.3U/L DNA polymerase, 0.05% -5% volume fraction Tween-20, 0.3 μmol/L upstream and downstream primers; optionally, the reaction composition further comprises 0.7U/μ L of MMLV reverse transcriptase.
A sixth aspect of the present application provides a qPCR amplification kit comprising a qPCR whole premix reaction composition according to the fourth aspect and/or the fifth aspect.
The concentration of tween 20 in the present application refers to volume fraction, for example, tween-20 with the final concentration of 0.1% in the fully premixed reaction solution refers to 0.015 μ L of tween-20 in 15 μ L of the fully premixed reaction system.
Interpretation of terms
Tween-20: polysorbate 20, having a trunk structure as follows:
real-time fluorescent quantitative PCR: also known as RT (Real-Time) -qPCR, may be abbreviated as qPCR. The method is characterized in that a fluorescent group is added in a PCR reaction, and the initial amount of a target gene is analyzed in real time by continuously monitoring the sequence of fluorescent signals and the change of signal strength.
Fully premixing reaction liquid: the method refers to a one-step fully premixed reaction solution containing a primer probe/dye, wherein the reaction solution containing DNA polymerase, a primer probe required by reaction and other raw materials required by PCR amplification reaction can be used for carrying out detection reaction immediately after a sample to be detected is added.
Non-specific amplification: the PCR reaction process is mainly based on target-specific amplification and simultaneously has non-specific amplification, the presence of which can not only interfere with the sensitivity of specific amplification by consuming raw materials in the reaction system but also can cause false positive results due to non-specific amplification, such as amplification of non-target products caused by primer-primer interactions with templates and non-specific binding of primers to templates.
Primer dimer: a pair of primers or the 3 'terminal parts of the primers are subjected to base complementary binding, and a small molecular weight double-stranded DNA fragment is formed by extending from the 3' terminal under the action of polymerase.
Drawings
FIG. 1A: effect of storage at-20 ℃ and 37 ℃ for 7 days on the fully premixed RT-qPCR (0.1% and 0% tween-20 added, respectively) amplification curve for the H-09 gene;
FIG. 1B: -effect of storage at 20 ℃ and 37 ℃ for 7 days on the fully premixed RT-qPCR (0.1% and 0% tween-20 added, respectively) amplification curve of the M-51 gene;
FIG. 2A: when the sample input amount is 1ng, adding Tween-20 with different concentrations to influence the full-premix RT-qPCR amplification curve of the M-52 gene;
FIG. 2B: when the sample input amount is 100pg, adding Tween-20 with different concentrations to influence the full-premix RT-qPCR amplification curve of the M-52 gene;
FIG. 2C: when the sample input amount is 10pg, adding Tween-20 with different concentrations to influence a full-premix RT-qPCR amplification curve of the M-52 gene;
FIG. 3A: when the sample input amount is 1ng, adding Tween-20 with different concentrations to influence the full-premix RT-qPCR amplification curve of the M-54 gene;
FIG. 3B: when the sample input amount is 100pg, adding Tween-20 with different concentrations to influence the full-premix RT-qPCR amplification curve of the M-54 gene;
FIG. 3C: when the sample input amount is 10pg, adding Tween-20 with different concentrations to influence the full-premix RT-qPCR amplification curve of the M-54 gene;
FIG. 4A: when the sample input amount is 1ng, adding tween-20 with different concentrations to influence a full-premix RT-qPCR amplification curve of the M-5 gene;
FIG. 4B: when the sample input amount is 100pg, adding Tween-20 with different concentrations to influence a full-premix RT-qPCR amplification curve of the M-5 gene;
FIG. 4C: when the sample input amount is 10pg, adding Tween-20 with different concentrations to influence the full-premix RT-qPCR amplification curve of the M-5 gene;
FIG. 5 is a schematic view of: gel electrophoresis picture of the full premix RT-qPCR amplification product of M-52, M-54 and M-5 genes;
FIG. 6A: a melting curve diagram of a full-premixed RT-qPCR amplification product of the H-01 gene;
FIG. 6B: a melting curve diagram of a fully premixed RT-qPCR amplification product of the H-07 gene;
FIG. 6C: melting curve diagram of the fully premixed RT-qPCR amplification product of the H-19 gene;
FIG. 6D: melting curve diagram of the fully premixed RT-qPCR amplification product of the H-31 gene.
Detailed Description
The technical scheme of the invention is further explained by the specific embodiment in combination with the attached drawings. However, the following examples are merely illustrative of the present invention and do not represent or limit the scope of the present invention. The protection scope of the invention is subject to the claims. In the following examples, reagents and consumables used are purchased from suppliers common in the art, and experimental methods and technical means used are conventional in the art, unless otherwise specified.
Taq DNA Polymerase (Taq DNA Polymerase) in the examples was from Champagne Taq DNA Polymerase, product number P122, from Novokea Biotech, nanjing Transcriptase, M-MLV (H-) Reverse Transcriptase, product number R021, from Novokea Biotech, product number, tween-20 was from sigma (Lot # WXBD1913V, CASNO. 9005-64-5).
Example 1
1. Sample preparation
Using Nanjing NuoZan Biotechnology Ltd
And (2) extracting RNA from the Cell/Tissue Total RNA Isolation Kit (Vazyme, RC 101), extracting Hela Cell Total RNA with the concentration of 2578 ng/mu l, extracting hybridoma Cell Total RNA with the concentration of 2356 ng/mu l, detecting by a qPCR reagent without reverse transcriptase, and carrying out subsequent tests without DNA pollution.
2. Primer design
qPCR primer probes were designed based on the human (DCT, XM _ 017020401.3) gene sequence and the mouse (Ncor 2, XM _ 030254302.1) gene sequence, and the specific sequences are shown in Table 1.
Table 1: human and murine target qPCR primer Probe sequences in example 1 (orientation: 5 '-3')
Wherein, the name of the H series sequence is a human gene target, and the name of the M series sequence is a mouse gene target.
3. Preparation and amplification of fully premixed reaction solution
RT-qPCR full premix amplification reaction solution is prepared according to the following table 2, wherein the 5 x amplification system is: tris (250 mmol/L), KAC (250 mmol/L), KCl (100 mmol/L), dNTPs (1.5 mmol/L), mgCl 2 (20mmol/L)。
Table 2: RT-qPCR full premix system
Two sets of RT-qPCR fully premixed reaction solutions of H-09 gene were prepared according to Table 2, wherein Tween-20 was not added to the first set, tween-20 was added to the second set until the final concentration of the reaction solution was 0.1% (volume fraction, the same applies hereinafter), and two sets were prepared, the first set was stored at-20 ℃ for 7 days as a control group, and the other set was stored at 37 ℃ for 7 days (simulated long-term storage conditions) as an experimental group.
Respectively adding human-derived and murine RNA samples into the H-09 gene fully premixed reaction liquid treated at the temperature of-20 ℃ and 37 ℃, wherein the input amount of the human-derived RNA sample is 100pg (5 mu L), the input amount of the murine RNA sample is 100pg (5 mu L), carrying out an RT-qPCR amplification experiment on a human or murine target according to a reaction program shown in a table 3, and an amplification curve is shown in a figure 1A.
Two groups of RT-qPCR fully premixed reaction liquid of the M-51 gene are prepared according to the table 2, the subsequent treatment method is the same as the RT-qPCR fully premixed reaction liquid of the H-09 gene, and the amplification curve is shown in a figure 1B.
Table 3: RT-qPCR reaction procedure
4. Analysis of results
The RT-qPCR amplification curve of the H-09 gene is shown in FIG. 1A, and the RT-qPCR amplification curve of the M-51 gene is shown in FIG. 1B. It can be seen that the amplification performance of the fully premixed reagent is significantly affected by the treatment at 37 ℃, and after tween-20 with the final concentration of 0.1% is added into the fully premixed reagent, the platform retention of the amplification curve is significantly improved, and the amplification efficiency is significantly improved.
Example 2
1. Sample preparation was consistent with example 1.
2. Primer design
Based on the mouse (nco 2, XM _030254302.1/Zbtb20, NM _001393397.1/Chd7, XM _ 006538005.5) gene sequences, qPCR primer probes were designed, and the specific sequences are shown in table 4.
Table 4: qPCR primer probe sequence for murine target in example 2 (orientation: 5 '-3')
Wherein, the M series sequence name is mouse gene target.
3. Preparation and amplification of fully premixed reaction solution
RT-qPCR full premix amplification reaction solution was prepared according to Table 5, wherein the 5 × amplification system was: tris (250 mmol/L), KAC (250 mmol/L), KCl (100 mmol/L), dNTPs (1.5 mmol/L), mgCl 2 (20mmol/L)。
Table 5: RT-qPCR full premix system
Preparing 6 groups of RT-qPCR full-premix reaction liquid of M-52 genes according to the table 5, wherein each group is prepared into two parts, and Tween-20 is not added in the first group to be used as a control group; adding tween-20 into the second group to the sixth group respectively until the final concentration of the reaction solution is 5%, 1%, 0.5%, 0.1% and 0.05%, and taking the reaction solution as an experimental group; the control and experimental groups were stored at 37 ℃ for 7 days.
The murine RNA samples were added to the fully premixed reaction solutions of the M-52 gene of the control group and the experimental group after treatment, respectively, at the amounts of 1ng (5 μ L), 100pg (5 μ L) and 10pg (5 μ L), and the RT-qPCR amplification experiments for murine targets were performed according to the reaction procedures in table 3, and the amplification curves are shown in fig. 2A (1 ng), fig. 2B (100 pg) and fig. 2C (10 pg).
The amplification products of the M-52 gene fully premixed reaction solution with the final concentration of 0.05% and 0.1% of Tween-20 in the control group and the experimental group were selected for 2% agarose gel electrophoresis detection, and the electrophoresis gel images are shown in FIG. 5.
RT-qPCR fully premixed reaction liquid of 6 groups of M-54 genes is prepared according to the table 5, the subsequent treatment and experimental method are the same as the RT-qPCR fully premixed reaction liquid of the M-52 gene, the obtained amplification curve is shown in figures 3A-C, and the electrophoresis gel diagram is shown in figure 5.
RT-qPCR fully premixed reaction liquid of 6 groups of M-5 genes is prepared according to the table 5, the subsequent treatment and experiment methods are the same as the RT-qPCR fully premixed reaction liquid of the M-52 gene, the obtained amplification curves are shown in figures 4A-C, and the electrophoresis gel diagram is shown in figure 5.
4. Analysis of results
FIGS. 2-4 show the RT-qPCR amplification curves after adding different final concentrations of Tween-20 to the fully premixed reaction solutions of M-52, M-54 and M-5 genes, respectively, and storing at 37 ℃ for 7 days. The result shows that compared with the control group, the addition of 0.05-5% of tween-20 can improve the platform retention of the fully premixed reaction solution after being stored for 7 days at 37 ℃, wherein the effect of tween-20 with final concentration of 0.05% and 0.1% is optimal, which indicates that tween-20 is beneficial to improving the amplification performance of the fully premixed reaction solution.
As shown in fig. 5, compared with the fully premixed qPCR amplification reaction without tween-20, the amplification product of the reaction with tween-20 added to a final concentration of 0.05% to 0.1% was single, indicating that adding tween-20 to the fully premixed qPCR reaction solution significantly reduced the formation of primer dimer, promoted the specific amplification of more primers for the target, and alleviated the problem of primer consumption due to long-term storage.
Example 3
1. Sample preparation was consistent with example 1.
2. Primer design
qPCR primer probes were designed based on the human (FMR 1, NM-001185076.2/MYC, NM-002467.6/B2M, NM-004048.4/GUSB, XM-047420289.1) gene sequences, and the specific sequences are shown in Table 6.
Table 6: human target qPCR primer Probe sequence in example 3 (orientation: 5 '-3')
Wherein, the name of the H series sequence is human gene target.
3. Preparation and amplification of fully premixed reaction solution
RT-qPCR full premix amplification reaction solution was prepared according to table 7, wherein the 5 × amplification system was: tris (250 mmol/L), KAC (250 mmol/L), KCl (100 mmol/L), dNTPs (1.5 mmol/L), mgCl 2 (20mmol/L)。
Table 7: RT-qPCR full premix system
| Components | Amount of addition |
| 5X amplification System | 4μL |
| Upstream primer (10. Mu. Mol/L) | 0.4μL |
| Downstream primer (10. Mu. Mol/L) | 0.4μL |
| SYBRGreen(20×) | 0.3μL |
| Taq DNA Polymerase(5U/μL) | 1μL |
| Reverse Transcriptase(10U/μL) | 1μL |
| Tween-20/without Tween-20 | 0.1％/0％ |
| ddH 2 O | To15μL |
Preparing two groups of RT-qPCR full-premix reaction liquid of H-01 genes according to the table 7, wherein the two groups are respectively prepared into two parts, wherein Tween-20 is not added in the first group and is used as a control group; adding Tween-20 to the final concentration of 0.1% as experimental group; the control and experimental groups were stored at 37 ℃ for 7 days.
Human source RNA samples with the sample input amount of 100pg (5 μ L) were added to the H-01 gene total premixed reaction solutions of the control group and the experimental group after treatment, and human source target dye method RT-qPCR detection was performed according to the reaction procedure of Table 8, and the obtained melting curve was as shown in FIG. 6A.
The preparation of RT-qPCR fully premixed reaction liquid of H-07, H-19 and H-31 genes and the subsequent reaction method are the same as those of the H-01 gene, and the obtained melting curves are respectively shown in FIG. 6B, FIG. 6C and FIG. 6D.
Table 8: dye method RT-qPCR reaction procedure
4. Analysis of results
As shown in fig. 6A-D, tween-20 with a final concentration of 0.1% was added to the fully premixed qPCR amplification system for different genes, and after treatment at 37 ℃, the primer dimer peak of non-target amplification in the amplification product was significantly reduced, and it was found that adding tween-20 with a final concentration of 0.1% to the fully premixed reaction solution could mitigate the formation of primer dimer in the reaction system, protect the primer probe from being consumed, and further reduce non-specific amplification.
Claims (21)
1. A method of reducing qPCR non-specific amplification, the method comprising: (1) Providing a fully premixed reaction solution containing buffer substances, dNTPs, metal salts, DNA polymerase, tween, primers, probes and/or fluorescent dyes; (2) adding a nucleic acid template; (3) carrying out qPCR amplification reaction under appropriate conditions.
2. The method according to claim 1, wherein the tween is tween-20, and the volume fraction of the tween-20 in the fully premixed reaction solution is 0.01% to 10%, preferably 0.02% to 8%, more preferably 0.03% to 7%, more preferably 0.04% to 6%, and most preferably 0.05% to 5%.
3. The method according to claim 1, wherein the primers comprise at least one pair of forward and backward primers, preferably 0.2-0.5 μmol/L.
4. The method of claim 1, the metal salt comprising Mg 2+ Salt, K + Salt, mn 2+ Salt, na + Salt and Ca2 + One or more of a salt; preferably, the metal salt comprises Mg 2+ Salts and K + Salt; preferably, the metal salt comprises MgCl 2 And KCl; preferably, the metal salt comprises 2-10mmol/L MgCl 2 And 20-50mmol/L KCl; more preferably, the metal salt further comprises KAC, preferably 60-80mmol/L KAC.
5. The method of claim 1, said probe is a TaqMan probe, preferably a TaqMan probe of 0.1-0.5 μmol/L; the fluorescent dye is a SYBR Green fluorescent dye.
6. The method according to claim 1, wherein the DNA polymerase is a thermostable DNA polymerase, preferably Taq DNA polymerase, preferably 0.1-1U/. Mu.L Taq DNA polymerase.
7. The method of claim 1, wherein the buffer substance comprises Tris, preferably 50-100mmol/L Tris.
8. The method of claim 1, wherein the nucleic acid template is a DNA template.
9. The method of claim 7, wherein the qPCR amplification reaction comprises a pre-denaturation at 92-96 ℃ for 25s-40s and a cycling reaction comprising a denaturation at 95 ℃ for 8-12s, annealing at 55-65 ℃ and extension for 10-30s for 40-50 cycles.
10. The method of claim 1, wherein the nucleic acid template is an RNA template.
11. The method of claim 10, the reaction premix further comprising a reverse transcriptase, preferably MMLV reverse transcriptase.
12. The method of claim 11, wherein the qPCR amplification reaction is reverse transcription qPCR comprising reverse transcription at 52-58 ℃ for 12-18min, pre-denaturation comprising pre-denaturation at 92-96 ℃ for 25s-40s, and cycling reaction comprising denaturation at 95 ℃ for 8-12s, annealing at 55-65 ℃ and extension for 10-30s for 40-50 cycles.
13. Use of tween-20 for reducing non-specific amplification of a qPCR system.
14. The use according to claim 13, the qPCR system is a fully premixed qPCR system or a fully premixed reverse transcription qPCR system.
15. Use according to claim 13, wherein the volume fraction of tween-20 is 0.01% to 10%, preferably 0.02% to 8%, more preferably 0.03% to 7%, more preferably 0.04% to 6%, most preferably 0.05% to 5%.
16. Use of tween 20 for reducing primer dimers of a qPCR system.
17. The use according to claim 16, the qPCR system being a fully premixed qPCR system or a fully premixed reverse transcription qPCR system.
18. Use according to claim 16, wherein tween-20 is in a volume fraction of 0.01% to 10%, preferably 0.02% to 8%, more preferably 0.03% to 7%, more preferably 0.04% to 6%, most preferably 0.05% to 5%.
19. A qPCR full-premix reaction composition comprises buffer substances, dNTPs, metal salts, DNA polymerase, 0.01-10% of Tween-20 by volume fraction, upstream and downstream primers and a Taqman probe; optionally, the qPCR fully premixed reaction composition further comprises a reverse transcriptase.
20. A qPCR full premix reaction composition, the reaction composition includes buffer substance, dNTPs, metal salt, DNA polymerase, 0.01% -10% volume fraction Tween-20, upstream and downstream primers and SYBR Green fluorescent dye; optionally, the qPCR fully premixed reaction composition further comprises a reverse transcriptase.
21. A qPCR amplification kit comprising a qPCR whole premix reaction composition according to any of claims 19-20.
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