CN111549110A - Primer group, kit and detection method for detecting nonspecific amplification in qPCR (quantitative polymerase chain reaction) process - Google Patents

Abstract

The invention provides a primer group, a kit and a detection method for detecting nonspecific amplification in a qPCR (quantitative polymerase chain reaction) process, belonging to the technical field of Real Time PCR detection of nucleic acid, wherein the primer group comprises 4 nonspecific amplification forward primers; the nucleotide sequence of the non-specific amplified forward primer is shown as SEQ ID NO: 3 to SEQ ID NO: and 6. The kit provided by the invention comprises the primer group, and also comprises a universal reverse amplification primer and an LNAFAM hydrolysis probe. The kit for detecting non-specific amplification and the detection method for non-specific amplification provided by the invention can quantitatively detect the non-specific amplification of a PCR reaction system containing polymerase (such as Taq DNA polymerase); quantitatively detecting the influence of the complementary homologous sequence on the detection of real-time PCR non-specific amplification; detecting the influence of the annealing or extension temperature on the detection of real-time PCR non-specific amplification; the effect of annealing or extension time on the detection of real-time PCR non-specific amplification was examined.

Description

Primer group, kit and detection method for detecting nonspecific amplification in qPCR (quantitative polymerase chain reaction) process

The invention has the application number of CN201910219827.4, is named as 'a specific quantitative PCR reaction mixed solution, a miRNA quantitative detection kit and a detection method', and is filed as divisional application of invention patents of 3, month and 22 in 2019.

Technical Field

The invention belongs to the technical field of Real Time PCR detection of nucleic acid, and particularly relates to a primer group, a kit and a detection method for detecting nonspecific amplification in a qPCR process.

Background

PCR is a short term for Polymerase Chain Reaction (Polymerase Chain Reaction), and refers to a method of multiplying the number of DNA molecules by 2 (2 times of the number of DNA molecules)^N) Amplification method, which is one of the most important techniques so far. Because of the great sensitivity and accuracy of PCR, many fields of application, including forensic and clinical diagnostics, where the detection and quantification of specific DNA molecules is required are highly dependent on PCR. Because misleading or erroneous results can lead to serious consequences in these areas, highly accurate analysis, stable, accurate and repeatable results are required.

But is also the very strong sensitivity of PCR, and its non-specific amplification can lead to erroneous results including false positives or false negatives, which can be said to be "millicentimetres poor, spurious". Since the preparation of the PCR reaction solution is carried out at relatively low temperatures (room temperature or on ice), the polymerase (usually Taq DNA polymerase) used in PCR is active at these temperatures, and the primer can form a partially double-stranded structure with a DNA molecule (possibly the other primer) having low complementary homology at these temperatures. When the 3' end of the primer forms a double-stranded structure with others, the primer can be extended to synthesize DNA due to the activity of polymerase at these temperatures. When the newly synthesized DNA has a binding sequence to another primer or the same primer at the other end, the DNA sequence having complete complementarity to the primer at both ends is present, and the DNA can form a true PCR template. That is, at the PCR annealing temperature, the primer can bind to this newly synthesized DNA to form a stable double-stranded structure to initiate DNA synthesis. When the 3' -end between the primers has a complementary sequence, this may result in primer extension using the primer as a template, resulting in primer dimer. It can be seen that the enzymatic activity of the polymerase used in PCR at temperatures below the annealing temperature of PCR can lead to non-specific amplification. The consumption of PCR reaction resources by non-specific amplification is equivalent to the reduction of raw materials which can be amplified originally, namely, the efficiency of specific amplification is reduced, even no specific amplification is generated, the PCR detection sensitivity is reduced or false positive is caused by non-specific amplification signals.

To overcome the above disadvantages of DNA polymerases, researchers have developed hot-start DNA polymerases. This is an important PCR innovation because the use of hot-start DNA polymerase greatly reduces specific amplification, including reducing primer dimer formation, and increases the sensitivity of PCR detection. The enzymatic activity of the hot start polymerase is inhibited below the temperature at which the oligonucleotide primer anneals to the DNA template. This is typically accomplished by inhibiting Taq enzyme activity by binding Taq enzyme with a monoclonal antibody. The inhibitory effect of the monoclonal antibody can be permanently removed by denaturing the monoclonal antibody at 95 ℃. This technological advance allows the preparation of a PCR reaction mixture at room temperature without non-specific amplification of the PCR and without the formation of the original dimers.

However, a recent report demonstrated that 12 of the 17 commonly used hot-start polymerases tested on the market were not completely inhibited before the first 95 ℃ denaturation by gel electrophoresis PCR analysis, resulting in non-specific amplification (Stevens AJ, Appleby S, Kennedy MA. Man. Manual commercial hot-start polymerase to thermal activation. Biotechnology 2016; 61: 293-6). This non-specific amplification activity in Real-Time polymerase chain reaction (Real Time PCR) is probably more extensive than described in the above mentioned literature and is a ubiquitous phenomenon. This is due to the fact that the binding of non-covalent inhibitors of the polymerase, such as monoclonal antibodies or aptamers, is a reversible reaction: a + B Δ C; that is, it is impossible to inhibit the activity of the polymerase 100%, so that the occurrence of non-specific amplification is inevitable.

Life must adapt to a myriad of environments, but genetic material is relatively stable and invariant. How to deal with variable environment with invariable genetic material, life skillfully uses Epigenetics (Epigenetics) as a bridge for communication between heredity and environment to regulate and control gene expression. The most important for epigenetic regulation is microrna (mirna). mirnas are single-stranded RNAs of about 22 nucleotide bases in length, which bind to messenger RNA (mrna) using the base-pairing principle to specifically inhibit translation of the latter. It is now found that there are approximately eight thousand more mirnas in humans. Each miRNA can regulate the expression of hundreds or even thousands of genes. Moreover, mirnas, like hormones, can be secreted from cells into the blood circulation and delivered to other adjacent or distant cells to play a role. Thus, mirnas directly or indirectly regulate almost all genes, regulating various functions of cells. Since mirnas have important gene regulation, their abnormal expression is closely related to various diseases such as cancer. It has now been found that deregulation of mirnas is associated with more than four hundred diseases. When the body is compromised by pathogenic microorganisms or cancer cells, the immune response requires rapid and highly coordinated systemic regulation of many genes to establish an effective defense to identify and eliminate the pathogenic agents. miRNA-mediated gene regulation is faster than other epigenetic mechanisms that require transcription (e.g., methylation). Only miRNA regulatory networks can meet this rapid gene regulation requirement.

Compared with other molecular detection, miRNA detection has undoubtedly great advantages. mirnas are very stable in blood, plasma mirnas are stable under harsh conditions such as freezing and thawing, high temperature storage, acidic conditions and rnase digestion. This makes mirnas attractive as biomarkers and very suitable as biomarker molecules for diseases. However, since miRNA has only twenty bases, it is difficult to detect miRNA, and a technology for accurately detecting miRNA is lacking so far, which greatly limits its clinical application.

The RT-qPCR detection method based on SYBR Green has the defects of high non-specificity and low sensitivity. This is because all kinds of nucleic acids can bind to SYBR Green to produce detectable signals, interfering with the detection of true signals. The method for determining miRNA by Taqman qPCR requires that each miRNA needs a specific reverse transcription primer and a specific probe, and is expensive. Moreover, only 6-8 bases of the reverse transcription primer are miRNA specific. Only hundreds of miRNA specific reverse transcription primers can be designed from six bases, and nearly eight thousand of miRNA are designed for human. Therefore, reverse transcription primers are not specific.

Furthermore, both of the above methods have a common disadvantage in that the presence of the same mature miRNA sequence in genomic DNA and other RNAs such as pri-miRNA, pre-miRNA, and even mRNA can lead to non-specific detection. Whereas PCR typically amplifies millions to tens of millions of times. The difference of milli-centimetres, so called, it is "Qianli". Non-specific amplification will result in non-specific detection. Therefore, the prior art lacks accuracy and sensitivity and is not suitable for high-throughput accurate determination of miRNA. There are reliability problems of the results, for example, the detection results of different laboratories are not comparable, and even the opposite results can be obtained, for example, the reports about miR-195 and let-7a in the blood circulation of breast cancer patients have obvious difference.

Non-specific amplification remains one of the biggest problems for qPCR accurate detection of mirnas.

Disclosure of Invention

In view of the above, the present invention aims to provide a primer set, a kit and a detection method for detecting non-specific amplification in a qPCR process.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a primer group for detecting nonspecific amplification in a qPCR (quantitative polymerase chain reaction) process, which comprises 4 nonspecific amplification forward primers; the nucleotide sequence of the non-specific amplified forward primer is shown as SEQ ID NO: 3 to SEQ ID NO: and 6.

The invention also provides a kit for detecting non-specific amplification in the qPCR process, which comprises the primer group.

Preferably, the kit further comprises a universal reverse amplification primer and an LNAFAM hydrolysis probe.

Preferably, the universal reverse amplification primer has the sequence shown in SEQ ID NO: 2.

Preferably, the LNAFAM hydrolysis probe has the amino acid sequence as set forth in SEQ ID NO: 1.

The invention also provides a method for detecting nonspecific amplification in the qPCR process, which comprises the following steps:

s1) specific reaction: carrying out gradient dilution on the specific template DNA with known concentration to obtain specific template DNA with different concentrations; respectively taking the specific template DNAs with different concentrations as templates, carrying out specific qPCR amplification to obtain specific PCR amplification products, and then making a PCR standard curve according to the amount of the specific template DNAs and the amount of the corresponding amplification products; the specific qPCR amplification system comprises specific template DNA, a specific forward amplification primer, a universal reverse primer, a PCR reaction buffer solution and a hydrolysis probe;

s2) non-specific reaction: adding non-specific template DNA and a non-specifically amplified forward primer in claim 1 into the amplification system in the specific qPCR in the step 1), and then performing non-specific qPCR amplification to obtain a non-specific PCR amplification product; the amplification procedures of the specific qPCR amplification and the non-specific qPCR amplification are consistent;

s3) respectively calculating specific amplification efficiency and non-specific amplification efficiency by using the standard curve in the step S1) according to the non-specific PCR amplification product in the step S2); the non-specific amplification efficiency and the percentage of specific amplification efficiency are quantitative indicators of non-specific amplification.

The invention has the beneficial effects that: the kit for detecting non-specific amplification and the detection method for non-specific amplification provided by the invention can quantitatively detect the non-specific amplification of a PCR reaction system containing polymerase (such as Taq DNA polymerase); quantitatively detecting the influence of the complementary homologous sequence on the detection of real-time PCR non-specific amplification; detecting the influence of the annealing or extension temperature on the detection of real-time PCR non-specific amplification; the effect of annealing or extension time on the detection of real-time PCR non-specific amplification was examined.

Drawings

FIG. 1 is a comparison of the non-specific amplification of the specific quantitative PCR reaction mixture provided by the present invention and three commercially available 2 XqPCRhot-start DNApolymerase MasterMix (CompetitorA, Competitor B, Competitor C);

FIG. 2 shows the effect of the complementary homology between primers and non-specific template on non-specific amplification;

FIG. 3 shows the result of nonspecific amplification of miRNA detected using three nonspecific primers;

FIG. 4 is a graph showing the effect of homology between the 3 'end of the PCR primer and the 3' end of other DNA molecules and internal complementarity on non-specific amplification in PCR; wherein (a) is a partial hot start enzyme; (B) and (C) complete hot start is realized by isolating DNA polymerase with solid paraffin, and the experimental design is as shown in figure 1; (D) influence of partial hot start PCR and complete hot start PCR on PCR non-specific amplification; the numbers on the graph represent the fold and p-value of the relative amplification signal for each PCR experimental group; the graph represents one out of 4 independent experiments;

FIG. 5 is a comparison of the specific quantitative PCR reaction mixture of the present invention and the non-specific amplification results of the 3 'end of the PCR primer and the 3' end and internal complementary homology of other DNAs in a commercially available 2X qPCRhot-start DNApolymerase MasterMix (supplierA);

FIG. 6 is a comparison of PCR extension time and non-specific amplification of the specific quantitative PCR reaction mixture of the present invention and three commercially available 2 XqPCRhot-start DNA polymerase Master Mix (Supplier A, Supplier B, Supplier C); the numbers on the graph represent the fold and p-value of the relative amplification signal at 30 seconds and 18 seconds of PCR extension time for each enzyme, and the graph represents one out of 3 independent experiments;

fig. 7 shows the influence of storage time on the PCR amplification efficiency of the anhydrous miRNA quantitative detection kit provided by the present invention.

Detailed Description

The invention provides a specific quantitative PCR reaction mixed solution, which takes water as a solvent and comprises the following components in concentration: Tris-HCl (70-80) mmol/L, (NH)₄)₂SO₄(15～25)mmol/L，Triton-100(0.08～0.12)％，MgCl₂(2-3) mmol/L, dNTPs (150-250) mu mol/L, trehalose (190-210) mmol/L and hot start Taq DNA polymerase (45000-55000) U/L; the pH value of the Tris-HCl is 8.5-9.0.

In the present invention, the mixture solution for specific quantitative PCR reaction preferably contains water as a solvent, and has the following concentrations: Tris-HCl 75mmol/L, (NH)₄)₂SO₄20mmol/L，Triton-1000.1％，MgCl₂2.5mmol/L, dNTPs 200. mu. mol/L, trehalose 200mmol/L and Taq DNA polymerase 50000U/L; the pH of the Tris-HCl is preferably 8.8.

The specific quantitative PCR reaction mixture is applied to PCR amplification, and the specific quantitative PCR reaction mixture has no limitation on amplified target nucleic acid and can be applied to PCR amplification of all types of DNA or RNA. In the invention, the specific quantitative PCR reaction mixed solution is a solution with 2 times of working concentration; in the specific embodiment of the present invention, the amplification of miRNA is taken as an example. In the invention, the specific quantitative PCR reaction mixed solution can carry out high-specificity PCR amplification, and the non-specific PCR amplification reaction is greatly reduced.

The invention also provides a miRNA quantitative detection kit, which comprises the PCR reaction mixed solution. In the invention, the kit preferably further comprises an LNAFAM hydrolysis probe and a universal reverse amplification primer; the LNAFAM hydrolysis probe has the nucleotide sequence shown in SEQ ID NO: 1; the general reverse amplification primer has the sequence shown in SEQ ID NO: 2. In the invention, the use concentration of the LNAFAM hydrolysis probe is preferably 8-12 mu mol/L, and more preferably 10 mu mol/L; the use concentration of the universal reverse amplification primer is preferably 8-12 mu mol/L, and more preferably 10 mu mol/L.

In the invention, the miRNA quantitative detection kit can also be preferably prepared into an anhydrous miRNA quantitative detection kit; the preparation method of the anhydrous miRNA quantitative detection kit preferably comprises the following steps: and placing the components of the kit in a container, and drying in vacuum to obtain the anhydrous miRNA quantitative detection kit. In the specific implementation process of the invention, the specific quantitative PCR reaction mixed solution, the LNAFAM hydrolysis probe and the universal reverse amplification primer are added into a PCR eight-connected tube, and the anhydrous miRNA quantitative detection kit is obtained by vacuum drying; the vacuum degree of the vacuum drying is preferably-0.08 to-0.1 Mpa, and more preferably-0.09 Mpa; the temperature of the vacuum drying is preferably 20-30 ℃, and more preferably 22-28 ℃. In the invention, the anhydrous miRNA quantitative detection kit is preferably stored in a dark environment; the anhydrous miRNA quantitative detection kit does not need low-temperature storage, is stored for six weeks at room temperature, and keeps the PCR amplification efficiency unchanged.

The invention provides a method for quantitatively detecting miRNA in a sample by utilizing qPCR, which comprises the following steps: 1) mixing the total RNA of the sample, poly A polymerase buffer solution, Escherichia coli poly A polymerase, rATP, MnCl₂Mixing and incubating with RNase-free water to obtain a polyadenylation reaction product; 2) mixing the polyadenylation reaction product obtained in the step 1), an RT buffer solution, dNTP, a reverse transcription primer, a nuclease blocking agent and RNase-free water, and then carrying out reverse transcription to obtain a reverse transcription product; 3) and mixing the reverse transcription product with the specific quantitative PCR reaction mixed solution, and performing qPCR amplification to obtain the quantification of miRNA in the sample.

In the present invention, the total RNA of the sample, poly A polymerase buffer, E.coli poly A polymerase, rATP, MnCl₂And mixing and incubating without RNase water to obtain a polyadenylation reaction product. In the present invention, the reaction system is preferably 20. mu.L; the invention is used for the total RNA, the poly A polymerase buffer solution, the rATP and the MnCl of the sample₂The volume ratio and the amount of RNase-free water are not particularly limited, and may be determined by referring to a polyadenylation reaction system which is conventional in the art. In the specific implementation process of the invention, the volume of the total RNA of the sample is 1 mu L, and the total amount is 30 ng-1 mu g; the poly A polymerase buffer solution is 5X poly A polymerase buffer solution, and the volume of the poly A polymerase buffer solution is 4 mu L; the volume of the escherichia coli poly A polymerase is 1 mu L; the concentration of the rATP is 10mmol/L, and the volume of the rATP is 1 mu L; the MnCl₂The concentration of (1) is 25mmol/L, and the MnCl is₂The volume of (D) is 1. mu.L; the volume of RNase-free water was 12. mu.L. In the invention, the temperature of the mixed incubation is preferably 36-38 ℃, and more preferably 37 ℃; the mixing incubation time is preferably 25-35 min, and more preferably 30 min. The present invention preferably performs termination of the reaction after the mixed incubation; the termination temperature is preferably 95 ℃, and the termination time is 5 min; after the termination reaction is completed, it is preferable to store the obtained polyadenylation reaction product at 0 ℃, and it is more preferable to store the obtained polyadenylation reaction productThe material was placed on ice.

After the polyadenylation reaction product is obtained, mixing the polyadenylation reaction product, an RT buffer solution, dNTP, a reverse transcription primer, a nuclease blocking agent and RNase-free water, and carrying out reverse transcription to obtain a reverse transcription product; the volume and the amount of each component of the reverse transcription system are not required to be referred to in the invention in any way according to the volume and the amount of each component of the reverse transcription system which are conventional in the field. In the specific implementation process of the invention, the reverse transcription system comprises the following components in 20 μ L:

polyadenylation reaction product, 4 μ L;

10 × RT buffer, 2 μ L;

100mM dNTP mix, 0.8. mu.L;

10 mu mol/L reverse transcription primer, 1 mu L;

nuclease blocker NEB, 1 μ L;

RNase-free water, 11.2. mu.L.

In the invention, the reverse transcription temperature is preferably 40-44 ℃, and more preferably 42 ℃; the reverse transcription time is preferably 50-70 min, and more preferably 60 min. In the invention, the reverse transcription system is preferably incubated at 55 ℃ for 5min before the reverse transcription; so as to be beneficial to the uniform mixing of all components in a reverse transcription system. The present invention preferably performs termination of reverse transcription after the reverse transcription; the temperature of the termination is preferably 95 ℃ and the time of the termination is 5 min.

After the reverse transcription is finished, mixing the obtained reverse transcription product with the PCR reaction mixed solution, and then carrying out qPCR amplification to obtain the quantification of miRNA in the sample. In the present invention, the amplification system of qPCR amplification comprises the following composition in concentration, in 10 μ L:

. In the present invention, the amplification procedure of qPCR amplification preferably comprises the steps of: 60 s-10 min at 95 ℃; 95 ℃, 5-30 s, 65 ℃, 18-120 s and 40 cycles.

In the invention, the specific primers comprise a specific upstream primer and a specific downstream primer; the nucleotide sequence of the specific primer meets the following requirements: 3' and 5 ' ends of the same primer do not have more than 3 homologous bases, 3' ends of any two different primers do not have more than 3 complementary bases, and 3' ends between one primer and 5 ' ends of the other primer do not have more than 3 homologous bases; the 3' end of the primer does not have 3 or more complementary bases with the 3' end of the DNA molecule other than the specific template in the amplification system, and should not have 3 or more homologous bases with the 5 ' end of the other DNA molecule.

In the invention, a qPCR amplification product is obtained after the qPCR amplification is finished; the present invention utilizes analytical methods conventional in the art to analyze the amplification product to obtain the quantification of miRNA in a sample.

According to the method for quantitatively detecting miRNA in the sample by utilizing qPCR, the specificity of PCR amplification can be greatly improved by adopting the specific quantitative PCR reaction mixed solution, and meanwhile, the specific requirements of primer design are further limited in the detection method, and non-specific amplification can also be reduced; furthermore, the detection method limits the extension time to 18-30 s, and can reduce non-specific amplification by properly shortening the extension time; the detection method of the invention can avoid non-specific amplification in PCR.

The invention provides a kit for detecting nonspecific amplification in a qPCR (quantitative polymerase chain reaction) process, which comprises 4 nonspecific amplification forward primers; the nucleotide sequence of the non-specifically amplified forward primer is shown as SEQ ID NO: 3 to SEQ ID NO: and 6.

In the invention, the kit for detecting non-specific amplification in the qPCR process preferably further comprises a universal reverse primer URP and an LNAFAM hydrolysis probe.

The invention provides a method for detecting nonspecific amplification in a qPCR (quantitative polymerase chain reaction) process, which comprises the following steps of:

s1) specific reaction: carrying out gradient dilution on the specific template DNA with known concentration to obtain specific template DNA with different concentrations; respectively taking the specific template DNAs with different concentrations as templates, carrying out specific qPCR amplification to obtain specific PCR amplification products, and then making a PCR standard curve according to the amount of the specific template DNAs and the amount of the corresponding amplification products; the specific qPCR amplification system comprises specific template DNA, a specific forward amplification primer, a universal reverse primer, a PCR reaction buffer solution and a hydrolysis probe;

s2) non-specific reaction: adding non-specific template DNA and the non-specific amplified forward primer into the amplification system in the specific qPCR in the step 1), and then performing non-specific qPCR amplification to obtain a non-specific PCR amplification product; the amplification procedures of the specific qPCR amplification and the non-specific qPCR amplification are consistent;

s3) respectively calculating specific amplification efficiency and non-specific amplification efficiency by using the standard curve in the step S1) according to the non-specific PCR amplification product in the step S2); the non-specific amplification efficiency and the percentage of specific amplification efficiency are quantitative indicators of non-specific amplification.

In the present invention, the gradient dilution in step S1) is preferably a 10-fold gradient dilution; the gradient dilution is preferably provided with 5-7 gradients, and more preferably 6 gradients.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The specific quantitative PCR reaction mixture provided in this example: water is used as a solvent, and the water-soluble organic solvent comprises the following components in concentration: Tris-HCl 75mmol/L, (NH)₄)₂SO₄20mmol/L，Triton-1000.1％，MgCl₂(2-3) mmol/L, dNTPs200 mu mol/L, trehalose 200mmol/L and Taq DNA polymerase 50000) U/L; the pH value of the Tris-HCl is 8.8.

The specific quantitative PCR reaction mixture was compared with the non-specific amplification of three commercially available 2 XqPCRhot-startDNApolymerase MasterMix (CompetitorA, Competitor B, Competitor C). The foregoing references to completer a, completer B, completer C (SupplierA,SupplierB, Supplier C) are each

Universal Probe qPCRMastermix (New England Biolabs (NEB), Ipshow, MA, USA), TaqMan FastAdvanced Master Mix (Thermo Fisher scientific, Waltham, MA, USA) and

Multiplex Master Mix(Thermo FisherScientific,Waltham，MA，USA)

experimental design reference (Stevens AJ, Appleby S, Kennedy MA. Manual commercial hot-start polymers detection activity reagent to thermal activity reagent 2016; 61: 293-6), differs from literature in that the amplification signal is detected using Taqman probes.

The materials used for the experiments were as follows:

1)LNARTP(0.1μmol/L)

2)P36F1(NSP1)10μmol/L

3)miR0601P7925(NSP2)10μmol/L

4) CompetitorA 2 XqPCR Hot-start DNApolymerase MasterMix

5) CompetitorB 2 XqPCR Hot-start DNApolymerase MasterMix

6) Competitor C2 XqPCR Hot-start DNApolymerase MasterMix

7) The specific quantitative PCR reaction mixture (Universal and specific quantification of miRNA)2 XqPCR MasterMix, referred to as USmiR for short

8)LNAFAM(10μmol/L)

9)URP(10μmol/L)

10)RoxII

11)Nuclease-free Water

The specific reaction system is as follows:

the system using the PCR reaction solution of CometithorA:

the system using the PCR reaction solution of company Competor B:

the system using the PCR reaction solution of company Competor C:

the system using the mixture solution for specific quantitative PCR reaction provided in this example:

the amplification procedure of the above reaction system was as follows:

the experimental results are shown in FIG. 1, wherein the left side of FIG. 1 shows the results of amplification using Non-Specific Primer-1(NSP1) as a Primer; FIG. 1 shows the right result of amplification using Non-Specific Primer (NSP2) as a Primer.

The 3 'ends of Non-Specific Primer-1(NSP1) and Non-Specific Primer (NSP2) share some complementary homology with the 3' end of a synthetic DNA fragment having a probe and reverse Primer binding site (see Table 3 for Non-Specific template, this sequence is amplified with Non-Specific Primer sequence to form the sequence of interest amplified in this example). The complementary homology of NSP1 is higher than that of NSP 2. The numbers on the graph represent the fold and p-value of the relative amplification signal for each enzyme versus the US-miR amplification signal. The graph represents one out of 3 independent experiments.

Since non-specific amplification is mainly determined by complementary homology between the polymerase amplification system and the primer/non-specific template. The above results have demonstrated that different "hot start" polymerase amplification systems have significantly different effects on the efficiency of non-specific amplification. The nonspecific amplification of the amplification by using the specific quantitative PCR reaction mixed liquid system provided by the embodiment is obviously reduced.

Example 2

Two pairs of non-specific primers NSP1 and NSP1a were detected using the specific quantitative PCR reaction mixture described in example 1; results of non-specific amplification of NSP3 and NSP3 a.

The experimental materials were as follows:

1)ArtDNA4(Internal Complementary-2)

2)P36F1(NSP1)10μmol/L

3)P36F1a(NSP1a)10μmol/L

4)miR0601P7933(NSP3)10μmol/L

5)miR0601P7933a(NSP3a)10μmol/L

6) the specific quantitative PCR reaction mixture (Universal and specific quantification of miRNA)2X qPCR MasterMix, abbreviated as USmiR 2X qPCRMix, provided in example 1

7)LNAFAM(10μmol/L)

8)URP(10μmol/L)

9)RoxII

10)Nuclease-free Water。

The specific amplification system is as follows:

amplification procedure of the above reaction system:

the result of nonspecific detection of amplification is shown in FIG. 2, and the primer with a high complementary homology sequence at the 3 'end is several hundred times higher than the primer with a low complementary homology sequence at the 3' end, indicating that the influence of the complementary homology between the 3 'end of the primer and the 3' end of other DNA fragments on nonspecific amplification is significantly influenced.

The experimental results of the left and right panels of fig. 1 are similar to the experiment of fig. 2: the complementary homology between NSP1 and the 3' end of the non-specific template was higher than that of NSP2, and as a result, the non-specific amplification of NSP1 was about eighty thousand times higher than that of NSP 2. Therefore, complementary homology of the 3 'end of the primer to the 3' end of other DNA fragments, which might otherwise lead to severe non-specific amplification, should be avoided or minimized in designing the primer. In addition, when there are other non-template DNA molecules in the non-specific template, the preferred primers can significantly reduce non-specific amplification.

Example 3

Investigation of whether residual reverse transcription primer in RT cDNA could be used as template for non-specific amplification

Examples the non-specific template used in the experiment shown in FIG. 1 is a universal reverse transcription primer, and thus the universal reverse transcription primer remaining in the cDNA solution after RT may act as a non-specific template resulting in non-specific amplification. To confirm this possibility, Phi29DNApolymerase was used to degrade the single stranded residual universal reverse transcription primer, since Phi29DNApolymerase had 3 '-5' exonuclease activity.

Experimental Material

1)LNARTP(0.1μmol/L)

2)10x Phi29 buffer

3)Phi29 DNAPolymerase

4)dNTPs(10mmol/L)

5)DTT(0.1mol/L)

6)Trehalose(1mol/L)

7)P36F1(NSP1)10μmol/L

8)miR0601P7925(NSP2)10μmol/L

9)miR0601P7933(NSP3)10μmol/L

10)(Universal and Specific Quantification ofmiRNA，USQmiR)2XqPCRMasterMix

11)LNAFAM(10μmol/L)

12)URP(10μmol/L)

13)ROXⅡ

14)Nuclease-free Water。

2xPhi29 mix system:

note: preparing on ice, adding into equal volume of RT product, blowing, beating, and mixing at 50 deg.C for 60min, 65 deg.C for 10 min.

Then carrying out qPCR reaction;

the qPCR reaction system was as follows:

the reaction procedure for qPCR was as follows:

as shown in FIG. 3, it was confirmed that the non-specific amplification was significantly reduced by about one hundred fold by treating cDNA with Phi29DNApolymerase (FIG. 3, left), but the Phi29DNApolymerase treatment did not affect the specific amplification (FIG. 3, right). This experiment demonstrates that when miRNA is reverse transcribed into cDNA using polyA/universal reverse transcription primer methods, the residual reverse transcription primer may act as a non-specific template leading to non-specific amplification.

Example 4

Investigation of the influence of 3 'terminal of PCR primer and 3' terminal of other DNA and internal complementary homology on non-specific amplification of PCR

Experimental Material

1)LNARTP(0.1μmol/L)

2)P36F1(NSP1)10μmol/L

3)MiR0601P7925(NSP2)10μmol/L

4) CompetitorA 2 XqPCR Hot-start DNApolymerase Master Mix

5) CompetitorB 2 XqPCR Hot-start DNApolymerase Master Mix

6) Competitor C2 XqPCR Hot-start DNApolymerase Master Mix

7) Specific quantitative PCR reaction mix (Universal and specific quantification of miRNA)2 XqPCR MasterMix as provided in example 1

8)LNAFAM(10μmol/L)

9)URP(10μmol/L)

10)RoxII

11)Nuclease-free Water

12) Compettor D company Paraffin (50-52 ℃ C.)

13) Compettor D company Paraffin (58-60 ℃ C.)

The amplification system is as follows:

the procedure for the amplification was as follows:

the results are shown in figure 4 of the drawings,

the 3 'end of the PCR primers and the 3' end of other DNA and internal complementary homology have significantly different effects on PCR non-specific amplification. The amount of non-specific amplification was 202 times greater than the amount of internal complementary homology when the complementary homology of the other DNA to the 3 'end of the PCR primer was at the 3' end of the DNA (FIG. 4-A). Because of the commonly used hot start DNA polymerases in the market, the enzyme activity is not completely inhibited before the first 95 ℃ denaturation and has a certain polymerase activity, the enzymes do not have complete hot start function, but have partial hot start function, namely, the DNA polymerases have partial polymerases capable of DNA synthesis in the process of preparing PCR and before the first 95 ℃ denaturation of the PCR. By isolating the PCR primer from other components such as DNA polymerase with paraffin wax (the specific method is to add 7.5. mu.l of other components except the forward primer, add 2.5. mu.l of melted paraffin wax, and add 2.5. mu.l of primer after paraffin wax is solidified), the polymerase can not extend the primer, i.e., can not synthesize DNA, until the temperature reaches a certain temperature during the preparation of PCR and at the start of PCR, and complete hot start is achieved (FIG. 4-B, 4-C). In the case of complete hot start, when the other DNAs have complementary homology to the 3 'end of the PCR primer at the 3' end of the DNA, the amount of non-specific amplification is 37 times and 33 times as large as the amount of internal complementary homology (FIG. 4-B, using paraffin wax having a melting point of 50-52 ℃ C.) and (FIG. 4-C, using paraffin wax having a melting point of 58-60 ℃ C.). This indicates that the nonspecific amplification is inversely related to the temperature in the temperature range in which the DNA polymerase has an enzymatic activity, i.e., the lower the temperature, the more severe the nonspecific amplification, whereas the higher the temperature, the less nonspecific amplification. This is in accordance with the thermodynamics of DNA renaturation. It is noteworthy that in the case where the DNA synthesis is allowed only at temperatures as high as the renaturation temperature, there is still non-specific amplification.

Example 5

The specific quantitative PCR reaction mixture provided in example 1 was compared with a commercially available 2 XqPCRhot-start DNApolymerase MasterMix (supplierA) for non-specific amplification of the 3 'end of the PCR primers and the 3' end and internal complementary homology of other DNAs.

Experimental Material

Template:

1)LNARTP(0.1μmol/L)

2)ArtDNA3(10pmol/L)

3)ArtDNA4(10pmol/L)

primer:

4)P36F1(NSP1)10μmol/L

5)URP(10μmol/L)

enzymes and others:

6) CompetitorA 2 XqPCR Hot-start DNApolymerase Master Mix

7) Specific quantitative PCR reaction mixture in example 1

8)LNAFAM(10uM)

9)ROXⅡ

10)Nuclease-free water

qPCR reaction system 1:

qPCR reaction system 2:

the amplification procedure was as follows:

the experimental results are shown in FIG. 5, wherein in FIG. 5, the numbers on the graph indicate the fold and p-value of the relative amplification signals of the respective enzymes and the amplification signal of the mixture solution for specific quantitative PCR reaction provided by the present invention; the graph represents one out of 3 independent experiments. As is clear from FIG. 5, in the system to which the mixture solution for specific quantitative PCR reaction of the present invention was added, no non-specific amplification was detected, regardless of whether the complementary homologous sequences of the non-specific template and the primer were present at the 3' -end of the non-specific template or inside the non-specific template.

Example 6

Comparison of PCR extension time for specific quantitative PCR reaction mixture USQ-miR2X qPCR Master Mix in example 1 with three commercially available 2X qPCR Hot-start DNA polymerase Master Mix (Supplier A, Supplier B, Supplier C) and non-specific amplification.

Experimental design reference (Stevens AJ, Appleby S, Kennedy MA. Manual commercial hot-start polymers detection activity reagent to thermal activity reagent 2016; 61: 293-6), differs from literature in that the amplification signal is detected using Taqman probes.

The experimental materials and amplification system were the same as those described in example 1; the amplification procedure changed the extension times to 18s and 30 s.

The results are shown in FIG. 6, and the numbers in FIG. 6 represent the fold and p-value of the relative amplification signals at 30s and 18s PCR extension times for the respective enzymes; the graph represents one out of 3 independent experiments.

When the extension time is short (18-30 s), the reduction of non-specific amplification can be more than twenty thousand times, but when the extension time is long (60s), the ratio of the relative amount of non-specific amplification is reduced to more than hundreds of times to one thousand times. This may be the reason why the consumption of PCR resources tends to saturate, but the relative amount of non-specific amplification is far from saturating. According to the USQ-miR-qPCR amplification reaction system, when the extension time is relatively short (18-30 s), the relative amount of non-specific amplification is not changed, but after the extension time is increased to 60s, the relative amount of non-specific amplification is increased by about 22 times. Therefore, the extension time of the USQ-miR qPCR amplification reaction system is preferably within 18-30 s

TABLE 1 comparison of non-specific amplification (ratio of relative amounts) of the commercially available major qPCR amplification reaction System with the qPCR amplification reaction System provided by the present invention (USQ-miR2X qPCR MasterMix)

Example 7

Preparation of anhydrous miRNA quantitative detection kit

Adding the specific quantitative PCR reaction mixed solution (2X USQ-miR qPCR) in the embodiment 1, a forward primer with a final concentration of 0.2 mu mol/L, a universal reverse primer URP with a final concentration of 0.2 mu mol/L and an LNAFAM hydrolysis probe with a final concentration of 0.1 mu mol/L into the trace PCR amplification eight-row calandria according to the amount of 20 mu L of the total volume of PCR, and after the mixture is prepared by vacuum drying at room temperature (the vacuum degree is-0.09 Mpa and the time is 3-8 h), placing the mixture in a dark place at room temperature for later use.

Detection of storage period of anhydrous miRNA quantitative detection kit

The template was ArtDNA4 at a final concentration of 10nmol/L and the forward primer was ArtRNA 4. The PCR reaction solution except the template was vacuum-dried overnight, and 20. mu.l/well was added after diluting the template at one week, two weeks, four weeks and six weeks after drying, respectively. The upper 4 wells (A-D) of each octant tube were the ArtDNA4 primer (positive) and the lower 4 wells (E-H) were the NSP2 (negative) results. The results are shown in fig. 7, and the amplification efficiency of the anhydrous miRNA quantitative detection kit remains unchanged within 6 weeks of storage at room temperature.

Example 8

The specific quantitative PCR reaction mixture in the example 1 is applied to the detection of miRNA of clinical samples

The amplification system and procedure were the same as in the qPCR reaction system 2 of example 5:

TABLE 2 specific amplification detection of plasma and tissue miRNA (Cq values + -SD)

NSP1 and NSP 2: non-specific primers (artificial sequences, see table 1), SP1, SP2 and SP3: specific primers correspond to SP1: hsa-miR-548a-5p, SP2: hsa-miR-93-5p and SP3: hsa-miR-92a-3p respectively. NTC No template control. SP1, SP2, SP3 indicate the synthesis of cDNA with the corresponding miRNA specific reverse transcription primers (see table 3). "No" means no amplification signal.

As can be seen from table 2, since the sequences of the two non-specific primers (NSP1 and NSP2) are artificial and do not exist in nature, no signal should be detected, and indeed all non-specific primers (artificial sequences) do not have PCR amplification signals. Furthermore, although three specific primers can detect mirnas naturally occurring in the human body, amplification signals can be detected only after their corresponding mirnas are reverse transcribed.

Example 9

Kit for detecting non-specific amplification in qPCR process

The kit comprises the following components: all components required for qPCR, i.e.the template (Table 3: non-specific template LNARTP), forward and reverse primers and LNAFAM probe and the specific quantitative PCR reaction Mix from example 1 (2 XqPCR Master Mix) were included as controls. Probes, reverse transcription primers, PCR forward and reverse primers and other oligonucleotides were synthesized by the firm of Competition Biotechnology (Shanghai). 2X PCR master DNA polymerase mix NEB, Thermo Fisher scientific (Waltham, MA, USA), TAKARA. Chemical reagents were purchased from Sigma and paraffin wax from shanghai point model factory.

The detection method comprises the following steps:

the experimental setup included a standard curve to quantify template amount and as a positive control, and detection of nonspecific amplification included four primers with two pairs of nonspecific forward primers, four experimental groups, with at least three replicates per group of experiments. PCR reaction mixtures were prepared using a 2xDNA polymerase cocktail and contained 0.2. mu. mol/L (final concentration) of the forward primer, Universal Reverse Primer (URP) and 0.2. mu. mol/L (final concentration) of the LNAFAM probe, according to the manufacturer's instructions. PCR template usage in 10 μ LPCR reaction volume: the concentration of the synthesized DNA template is 0.1-10 mu mol/L, 0.08 mu L is taken as the template, and 0.08 mu L is taken after the RT product is diluted by 10 times.

The PCR cycle parameters were:

95 ℃ for 10min (reaction System to which the mixture of specific quantitative PCR reaction of the present invention in example 1 was added)

Or 95 ℃ for 1min (adding other reaction systems of commercially available PCR reaction mixed liquid);

then 95 ℃ for 30s, 65 ℃ for 1min, 40 cycles.

TABLE 3 summary of the names and sequences of the oligonucleotides used in the above examples of the invention

Oligonucleotides include the template required for qPCR (non-specific template), forward and reverse primers and hydrolysis probes. Wherein the forward primer is non-specific and the underlined bases are complementary homologous sequences of the forward primer to the 3' end of the non-specific template LNARTP. The nonspecific forward primers 1 and 2 are a pair, and 3 and 4 are a pair. The second primer of each pair is obtained by removing several bases from the complementary homologous sequence at the 3' end of the first primer.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims (6)

1. A primer group for detecting nonspecific amplification in a qPCR process is characterized by comprising 4 nonspecific amplification forward primers; the nucleotide sequence of the non-specific amplified forward primer is shown as SEQ ID NO: 3 to SEQ ID NO: and 6.

2. A kit for detecting non-specific amplification in a qPCR process comprising the primer set of claim 1.

3. The kit of claim 2, further comprising a universal reverse amplification primer and an LNAFAM hydrolysis probe.

4. The kit of claim 3, wherein the universal reverse amplification primer has the sequence as set forth in SEQ ID NO: 2.

5. The kit of claim 3, wherein said LNAFAM hydrolysis probe has the sequence set forth in SEQ ID NO: 1.

6. A method of detecting non-specific amplification in a qPCR process comprising the steps of:

s1) specific reaction: carrying out gradient dilution on the specific template DNA with known concentration to obtain specific template DNA with different concentrations; respectively taking the specific template DNAs with different concentrations as templates, carrying out specific qPCR amplification to obtain specific PCR amplification products, and then making a PCR standard curve according to the amount of the specific template DNAs and the amount of the corresponding amplification products; the specific qPCR amplification system comprises specific template DNA, a specific forward amplification primer, a universal reverse primer, a PCR reaction buffer solution and a hydrolysis probe;

s2) non-specific reaction: adding non-specific template DNA and a non-specifically amplified forward primer in claim 1 into the amplification system in the specific qPCR in the step 1), and then performing non-specific qPCR amplification to obtain a non-specific PCR amplification product; the amplification procedures of the specific qPCR amplification and the non-specific qPCR amplification are consistent;

s3) respectively calculating specific amplification efficiency and non-specific amplification efficiency by using the standard curve in the step S1) according to the non-specific PCR amplification product in the step S2); the non-specific amplification efficiency and the percentage of specific amplification efficiency are quantitative indicators of non-specific amplification.

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