Reaction system, kit and use for polymerase chain reaction

Abstract

The present invention relates to a PCR reaction system, which comprises reagents necessary for PCR reaction; and a fluorine-containing compound, wherein the fluorine-containing compound is formulated to have a concentration of 4 mM to 24 mM in the reaction system in terms of free fluorine ions and any combination of fluorine ions. The reaction system improves the specificity of detection. The present invention also relates to a kit for PCR and the use of the fluorine-containing compound.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, and in particular to a reaction system for polymerase chain reaction. Background Art

[0002] Nucleic acid detection is at the core of many fields such as drug development, forensics, clinical trials, agricultural products and microbial type identification. It is a common technology for microbial identification, marker search, food and environmental safety assessment, criminal evidence and many other aspects.

[0003] One of the most powerful basic technologies for detecting small amounts of nucleic acids is the method of analyzing amplification products obtained by replicating part or all of a nucleic acid sequence.

[0004] Polymerase Chain Reaction (PCR) is a common technique for simulating in vivo DNA replication in vitro. Although it has only been developed for more than 30 years since it was invented by Mullis in 1983, it has become one of the most powerful technologies in the field of molecular biology. The PCR technology reaction system is simple. It uses sequence-specific primers, thermostable DNA polymerase and thermal cycling to copy or "amplify" specific sequences in DNA or cDNA templates thousands to millions of times. After the traditional PCR reaction is completed, the amplified PCR products are usually analyzed by gel electrophoresis. The analysis of the amplified products requires opening the reaction tube, which is not only time-consuming and troublesome, but also often causes aerosol contamination in the laboratory.

[0005] Real-time fluorescence quantitative PCR (qPCR) can achieve fully closed single-tube amplification by adding fluorescent substances to the reaction system and monitoring the target nucleic acid in real time. It is simple, fast, easy to automate, has good reproducibility and greatly reduces the possibility of contamination.

[0006] Similar to traditional PCR, the entire reaction process of qPCR is also composed of three basic reaction steps: denaturation-annealing-extension. The following is a brief description of the reaction process of qPCR using the TaqMan probe method as an example: ① Template DNA denaturation: After the template or DNA amplified by PCR is heated to the denaturation temperature for a period of time, the hydrogen bonds between the double strands are broken, the DNA double helix structure is opened, and the strands are melted into two DNA single strands to facilitate the next step of binding with the primers; ② Annealing (renaturation) of template DNA and primers: After the DNA is heated to become a single strand, when the temperature drops to a certain level, the primers will pair and bind with the complementary sequence of the template DNA single strand; ③ Primer extension: Under the action of DNA polymerase, the primers on the DNA template use dNTPs as raw materials and synthesize a new chain complementary to the template chain according to the AT, GC base pairing and semi-conservative replication principles. In the qPCR reaction, during the denaturation and annealing process, the probe binds to the template DNA chain before the primer. During the primer extension process, the primer uses the 5'→3' exonuclease activity of Taq DNA polymerase to cut the probe, and the fluorescent probe undergoes energy resonance transfer, thereby generating a fluorescent signal. By repeating the above denaturation-annealing-extension cycle, the template DNA completes exponential amplification and accumulation of fluorescent signals.

[0007] Due to their exponential amplification, PCR and qPCR can theoretically amplify to a detectable level as long as there is one molecule in the reaction tube, which has high sensitivity. However, sensitivity and specificity are two factors that need to be balanced: too high sensitivity may mean the generation of a large number of false positive signals. When the specificity of the test is emphasized, such as when conducting circulating tumor DNA (ctDNA) testing, how to select only extremely rare mutant DNA from a large number of wild-type background DNA is a tricky problem.

[0008] For example, with the rapid development of precision medicine in the past decade, many oncogenic driver genes have been discovered in non-small cell lung cancer (NSCLC), and earth-shaking changes have taken place in the diagnosis and treatment of patients with advanced non-small cell lung cancer. Targeted therapy of the EGFR gene, which is common in patients with non-small cell lung cancer, has become a standard clinical treatment method. The premise of targeted therapy is targeted detection. For the detection of EGFR gene, it is common in clinical practice to detect DNA in the patient's tumor tissue or peripheral blood to observe whether there are mutations to be tested. However, tissue biopsy is somewhat traumatic, and tumor tissue has spatial and temporal heterogeneity, which limits its detection. Studies have found that early screening, medication guidance, prognosis judgment and recurrence monitoring of tumor patients can be achieved by detecting circulating tumor DNA (ctDNA) in the patient's peripheral blood. However, the background of peripheral blood samples is complex, the ctDNA content is scarce, and it is submerged in a large amount of background DNA, which places extremely high requirements on the specificity and sensitivity of the detection method. There are as many as 30 to 40 types of deletion mutations in exon 19 of the EGFR gene. Clinically, a single tube is often used to detect a dozen common mutation types, so extremely specific detection methods and systems are needed.

[0009] At present, it has been reported that adding tetramethylammonium chloride (TMAC) to the reaction system can improve the specificity of the reaction to a certain extent. Nevertheless, research on these additives over the years has shown that they have a synergistic effect on PCR within a certain concentration range and can partially improve the corresponding PCR performance; however, for some complex reaction systems, such as multiplex qPCR reaction systems, due to the presence of a large number of primers and probes in the system, cross reactions are prone to occur, and ordinary PCR additives are difficult to achieve the ideal optimization effect.

[0010] Accordingly, in the field of PCR detection, there is a strong demand for additives that can improve detection specificity (especially in complex reaction systems). Summary of the invention

[0011] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0012] As used hereinafter, the terms "having", "comprising" or "including" are used in a non-exclusive manner. Thus, these terms may refer both to situations in which no further features are present in the entity described in the present context in addition to the features introduced by these terms, and to situations in which one or more further features are present. As an example, the expressions "A has B", "A comprises B" and "A includes B" may refer both to situations in which no further elements are present in A in addition to B (i.e., situations in which it consists solely and exclusively of B), and to situations in which in addition to B, one or more further elements are present in entity A, such as element C, elements C and D or even other elements.

[0013] The term "about" in the context of a particular value or ratio of the present invention means + / - 10% of the stated value or ratio, or in one embodiment, + / - 5% of a given value or ratio.

[0014] As mentioned above, in order to improve the specificity of PCR detection results, attempts have been made to add additives to the reaction system. However, conventional additives have limited applicability, for example, they are not suitable for complex reaction systems involved in multiple PCR reactions. Accordingly, the inventors studied the PCR reaction system and discovered a new additive, thereby completing the present invention.

[0015] Therefore, in a first aspect, the present invention provides a PCR reaction system, comprising:

[0016] Reagents necessary for PCR reactions; and

[0017] Fluorinated compounds,

[0018] The fluorine-containing compound is prepared to have a concentration of about 4 mM to about 24 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

[0019] It should be noted that, for example, when sodium fluoride is used as the fluorine-containing compound, both free F - , there is also a combined form of fluoride ion: HF.

[0020] By adding fluorine-containing compounds to the PCR reaction system, on the one hand, the reaction system of the present invention improves the specificity of the detection results when used for PCR detection; on the other hand, this improvement in specificity has wide applicability, for example, it can be used for multiple PCR detection; on another hand, the reaction system of the present invention can simultaneously reduce the generation of dimers between primers, thereby reducing or avoiding the false positives caused thereby.

[0021] The inventor speculates that the beneficial effect of adding fluorine-containing compounds to the reaction system is derived from the influence of fluorine-containing compounds on the hydrogen bonds formed between AT and GC bases during the amplification process. Specifically, hydrogen bonding is a special intermolecular or intramolecular force, and its formation conditions are as follows: a hydrogen atom is covalently bonded to an atom X with high electronegativity. If it is close to an atom Y with high electronegativity and small radius, such as oxygen (O), fluorine (F), nitrogen (N), etc., then a special intermolecular or intramolecular interaction in the form of XH...Y is generated between X and Y with hydrogen as the medium, which is a hydrogen bond. Its essence is the electrostatic force between the hydrogen nucleus on the strong polar bond (XH) and the atom Y with high electronegativity, lone electron pairs and partial negative charge. The greater the electronegativity of X and Y, the stronger the ability to attract electrons, and the stronger the hydrogen bond. For example: F has the highest electronegativity and the strongest ability to obtain electrons, so FH...F is the strongest hydrogen bond. There are two types of hydrogen bonds formed by AT and GC base pairing: NH...O, NH...N. In the PCR reaction, if F atoms are present, due to the electronegativity F>O>N, they will competitively interfere with the hydrogen bonds formed between AT and GC bases.

[0022] It should be noted that, although fluorine-containing compounds theoretically affect the formation of hydrogen bonds, it is generally not believed that such an effect on hydrogen bonds can be used to improve the specificity of PCR detection results. The reason is that in the PCR reaction, whether it is the binding between primers and templates, between templates and templates, or between primers and primers, they are all hydrogen bonds formed according to AT and GC base pairing. In other words, the effect of fluorine-containing compounds on hydrogen bonds is not selective. It will not only affect the hydrogen bonding between primers and non-target sequences, and between primers, but also affect the normal binding between primers and templates. Considering that both expected and unexpected binding are weakened at the same time, it is generally not believed that such a scheme can have excellent effects in improving specificity.

[0023] The inventors surprisingly found that when fluorine-containing compounds are added to the PCR reaction system at a certain concentration, the specificity of the detection results can be unexpectedly improved; based on further research, the inventors found that the unexpected improvement in specificity is related to the fluorine ions generated by the fluorine-containing compounds being controlled within a certain concentration range.

[0024] In a specific embodiment, the fluorine-containing compound is selected from one or more of the group consisting of: alkali metal fluorides, such as sodium fluoride, potassium fluoride or lithium fluoride, tetrabutylammonium fluoride, hydrofluoric acid, alkali metal salts of fluoroboric acid, such as sodium fluoroborate, potassium fluoroborate or lithium fluoroborate.

[0025] In a specific embodiment, the fluorine-containing compound is formulated to have a concentration of about 4 mM to about 24 mM in terms of free fluoride ions and any combination of fluoride ions in the reaction system, for example, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM or about 24 mM.

[0026] In a preferred embodiment, the fluorine-containing compound is formulated to have a concentration of about 8 mM to about 24 mM in terms of free fluoride ions and any combination of fluoride ions in the reaction system.

[0027] By formulating the fluorine-containing compound to have a concentration of about 8 mM to about 24 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system, the reaction system of the present invention can better improve the reaction specificity and avoid false positive results.

[0028] In a more preferred embodiment, the fluorine-containing compound is formulated to have a concentration of about 8 mM to about 12 mM in terms of free fluoride ions and any combination of fluoride ions in the reaction system.

[0029] By formulating the fluorine-containing compound to have a concentration of about 8 mM to about 12 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system, the reaction system of the present invention improves the specificity of PCR detection results while maintaining a high level of reaction sensitivity.

[0030] In specific embodiments, the alkali metal salt of fluoroboric acid may be added in an amount of about 4 to about 20 mM, for example, about 5 mM, about 10 mM, or about 15 mM.

[0031] In a specific embodiment, the added amount of alkali metal fluoride or hydrofluoric acid can be about 4-about 24 mM, for example: about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM or about 24 mM.

[0032] In a specific embodiment, the reagents necessary for the PCR reaction include DNA polymerase, dNTPs, buffer, divalent cations, a template to be amplified, and primers for amplifying the template.

[0033] As those skilled in the art will appreciate, DNA polymerase is used to polymerize new DNA chains, and a commonly used DNA polymerase is Taq polymerase.

[0034] "dNTPs" can be used interchangeably with "deoxynucleotide triphosphates" or "nucleotides containing triphosphate groups". DNA polymerase uses dNTPs to synthesize new DNA chains. dNTPs generally include dATP, dGTP, dTTP, and dCTP.

[0035] "Buffer" is intended to provide a suitable chemical environment for DNA polymerase. An exemplary buffer may be a pH 8.3-9.0 buffer, such as Tris buffer. In some embodiments, the pH of the buffer may be 8.5, 8.8.

[0036] An exemplary "divalent cation" may be Mg 2+ 、Zn 2+ , Mn 2+ .

[0037] The "template to be amplified" includes the target region to be amplified.

[0038] The primers used to amplify the template are complementary to the 3' end of each of the sense strand and the antisense strand of the DNA target, respectively.

[0039] In one variation of the present invention, the concentration of the fluorine-containing compound in the reaction system is about 4 mM to about 24 mM as alkali metal fluoride.

[0040] It should be noted that, for example, when sodium fluoroborate is used as the fluorine-containing compound, it will be partially hydrolyzed into NaF in the reaction system, and the concentration of sodium fluoroborate can be characterized by the amount of NaF.

[0041] In a specific embodiment, the concentration of the fluorine-containing compound of the present invention in the reaction system as alkali metal fluoride is about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM or about 24 mM.

[0042] In a preferred embodiment, the concentration of the fluorine-containing compound of the present invention in the reaction system is about 8 mM to about 24 mM as alkali metal fluoride.

[0043] In a more preferred embodiment, the concentration of the fluorine-containing compound of the present invention in the reaction system is about 8 mM to about 12 mM as alkali metal fluoride.

[0044] In a second aspect, the present invention provides a kit for PCR, comprising:

[0045] DNA polymerase, dNTPs, buffer, divalent cations, and fluorine-containing compounds; and

[0046] Optionally, primers for amplifying the template,

[0047] The fluorine-containing compound is prepared to have a concentration of about 4 mM to about 24 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

[0048] In a specific embodiment, each component in the kit of the present invention is packaged separately or in a mixed form with one or more other components. For example, the fluorine-containing compound can be combined with a buffer, a divalent cation and dNTPs to form a PCR mix.

[0049] In some embodiments, the components of the kit of the present invention are present in concentrated form. For example, the buffer may be present in a concentrated (eg, 10×) form.

[0050] It is understood that, depending on the needs, the kit of the present application may further include other components. For example, in the case of reverse transcription PCR, it may further include reverse transcriptase, RNase inhibitor, etc. For another example, in the case of a positive control, it may further include a positive control. For another example, in the case of qPCR based on TaqMAN fluorescent labeled probes, it may further include a labeled TaqMAN probe, etc.

[0051] In some embodiments, the reaction system or kit of the present invention does not include a modified oligonucleotide, such as a 3'-modified oligonucleotide, especially an oligonucleotide with a 3'-silyl ether modification.

[0052] In some embodiments, the reaction system or kit of the present invention does not include an oligonucleotide with a reversibly modified 3' end.

[0053] In some embodiments, the reaction system or kit of the present invention does not include such an oligonucleotide, the 3' end of which needs to be regenerated to a hydroxyl group during and / or before amplification.

[0054] In a third aspect, the present invention provides use of an additive for improving the specificity of PCR, wherein the additive is a fluorine-containing compound, and wherein the fluorine-containing compound is formulated to have a concentration of about 4 mM to about 24 mM in terms of free fluoride ions and any combination of fluoride ions in the reaction system.

[0055] In some embodiments, the fluorine-containing compound of the present invention is used in multiplex PCR detection with high specificity requirements.

[0056] In a fourth aspect, the present invention also provides a reaction system for nucleic acid (in vitro) amplification, a kit for nucleic acid (in vitro) amplification, and the use of an additive in improving the specificity of nucleic acid (in vitro) amplification. Specifically, the reaction system and the kit contain a fluorine-containing compound as defined in the present invention; and the additive is a fluorine-containing compound as defined in the present invention. It is understood that the reaction system and the kit also include reagents necessary for nucleic acid amplification.

[0057] By adding the fluorinated compound of the present invention to the reaction system of nucleic acid amplification, the specificity of the reaction is improved. For example, when used in the reaction system of LAMP, the fluorinated compound of the present invention improves the specificity of the reaction. For another example, when used in the reaction system of NASBA, the fluorinated compound of the present invention improves the specificity of the reaction. For another example, when used in the reaction system of TMA, the fluorinated compound of the present invention improves the specificity of the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The qPCR amplification results (FAM channel) of reaction system ① in Example 1 are shown;

[0059] Figure 2 The qPCR amplification results (FAM channel) of reaction system ② in Example 1 are shown;

[0060] Figure 3 The qPCR amplification results (FAM channel) of reaction system ③ in Example 1 are shown;

[0061] Figure 4 The qPCR amplification results (FAM channel) of reaction system ④ in Example 1 are shown;

[0062] Figure 5 The qPCR amplification results of the no-template control group in Example 2 are shown (ROX channel);

[0063] Figure 6 The qPCR amplification results of the positive template group in Example 2 are shown (ROX channel);

[0064] Figure 7 The amplification results of the no-template control and the negative control of the four reaction systems in Example 3 are shown (ROX channel); and

[0065] Figure 8 The amplification results of the X3 template in the four reaction systems in Example 3 are shown (ROX channel). DETAILED DESCRIPTION

[0066] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0067] In some embodiments, there is no limitation on the concentration / amount of the fluorine-containing compound in the reaction system, the kit or the use of the present invention.

[0068] Example 1 The additive of the present invention is used to detect multiple deletion mutations in exon 19 of the EGFR gene

[0069] The best reaction system was screened by adding different concentrations of sodium fluoroborate (NaBF4) to the EGFR gene exon 19 multiple deletion mutation detection system. The fixed components of the system are shown in Table 1:

[0070] Table 1

[0071]

[0072] * Primer and probe combination: Commercially available primer and probe combination targeting 21 EGFR gene exon 19 deletion mutations (including D1, D2 and D3 mutations below).

[0073] Based on the above fixed components, different concentrations of sodium fluoroborate (NaBF4) were added as shown in Table 2 to prepare 4 reaction systems.

[0074] Table 2

[0075]

[0076]

[0077] Prepare 4 groups of PCR reaction systems according to the above table, each containing a fixed component and a variable component. The prepared PCR reaction system was divided into 8 PCR tubes, and each group of systems was divided into 8 tubes; finally, equal amounts of templates were added to the 8 tubes in each group: 2 tubes were NTC (no template control), 3 tubes were Neg (negative control, no mutant type), and the remaining 3 tubes were 3 types of test templates containing different 19del mutation types (named D1, D2, and D3, respectively, all of which were mixed templates containing mutant and wild type).

[0078] The prepared PCR reaction system was placed on a StepOne Plus fluorescent PCR instrument for PCR amplification. The program used was: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 56°C annealing and extension for 30 seconds, and collecting fluorescent signals, for a total of 45 cycles; 10°C to terminate the reaction.

[0079] Result analysis:

[0080] Figure 1-Figure 4 Table 3 below shows the qPCR amplification results of the four reaction systems.

[0081] Table 3

[0082]

[0083] As can be seen from Table 3, in the Neg reaction, a weak false positive fluorescence signal appeared in the FAM channel in system ① (NaBF4 = 0mM), and the false positive fluorescence signal in Neg was eliminated by adding NaBF4 in the three reaction systems of system ② (NaBF4 = 5mM), system ③ (NaBF4 = 10mM), and system ④ (NaBF4 = 20mM). In other words, the false positive results caused by the mismatch between the primer and the template can be eliminated by adding fluorine-containing compounds.

[0084] On the other hand, as the concentration of NaBF4 increases, the positive fluorescence signal of the FAM channel is inhibited to a certain extent when detecting the D1 / D2 / D3 test templates, which is partially reflected in Figure 2-Figure 4 The fluorescence signal values ​​decrease in sequence on the vertical axis.

[0085] Compared with system ① (NaBF4 = 0mM), system ② (NaBF4 = 5mM) improved the specificity of the system, that is, eliminated false positives. At the same time, when detecting D1 / D2 / D3 test templates, there was no obvious difference in the Ct value, that is, the sensitivity of the system was not reduced.

[0086] In summary, the specificity of the system can be improved by adding fluorine-containing compounds to the EGFR gene exon 19 multiple deletion mutation detection system; at the same time, the optimal sensitivity of the system was further maintained when 5mM NaBF4 was added.

[0087] Example 2 The additive of the present invention is used for multiple detection of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, Mycoplasma pneumoniae, rhinovirus and new coronavirus

[0088] The best reaction system was screened by adding different concentrations of sodium fluoride (NaF) to multiple systems of adenovirus (AdV), influenza B virus, respiratory syncytial virus, influenza A virus, Mycoplasma pneumoniae, rhinovirus and new coronavirus. The fixed components of the system are shown in Table 4:

[0089] Table 4

[0090]

[0091]

[0092] Based on the above fixed components, different concentrations of NaF were added as shown in Table 5 to prepare 4 groups of reaction systems.

[0093] Table 5

[0094]

[0095] Prepare 4 PCR reaction systems according to the above table, each containing a fixed component and a variable component. Divide the prepared PCR reaction systems into 8-tube PCR strips, with 4 tubes for each system; finally, add equal amounts of templates to the 4 tubes of each reaction system: 2 tubes for NTC (no template control group) and 2 tubes for adenovirus positive template (positive template group).

[0096] The prepared PCR reaction system was placed in a fluorescent quantitative PCR instrument (Shanghai Hongshi, -96P) for PCR amplification. The program used was: 50°C, reverse transcription for 10 minutes; 95°C pre-denaturation for 3 minutes; 95°C denaturation for 10 seconds, 60°C annealing and extension for 15 seconds, for a total of 10 cycles, without light collection; 95°C denaturation for 10 seconds, 56°C annealing and extension for 15 seconds, for a total of 40 cycles, with light collection; 94°C denaturation for 10 seconds, 50°C annealing for 120 seconds, 50-90°C melting curve analysis, and the heating rate was 0.05°C per second.

[0097] Result analysis:

[0098] Figure 5 , Figure 6 The qPCR amplification results of the no-template control group and the positive template group are shown respectively. Table 6 below compares the qPCR amplification results of the four reaction systems.

[0099] Table 6

[0100]

[0101] As can be seen from Table 6, in the NTC of system ① (NaF = 0 mM), the ROX channel showed a relatively obvious false positive fluorescence signal, in the NTC of system ② (NaF = 5 mM), the ROX channel showed only a weak false positive fluorescence signal, and in the two reaction systems of system ③ (NaF = 10 mM) and system ④ (NaF = 20 mM), the false positive fluorescence signal in the NTC was eliminated by adding different concentrations of NaF. In other words, by adding fluorine-containing compounds, the false positive results caused by primer dimers can be weakened or even eliminated.

[0102] On the other hand, as the concentration of NaF increases, the positive fluorescence signal of the ROX channel is inhibited to a certain extent when detecting the adenovirus template to be tested, which is partially reflected in Figure 6The fluorescence signal values ​​decrease from system ② to system ④.

[0103] Compared with system ② (NaF = 5mM), system ③ (NaF = 10mM) further improved the specificity of the system and eliminated false positives. At the same time, when detecting AdV positive templates, there was no significant difference in the Ct value, that is, the sensitivity of the system was not reduced. In summary, by adding fluorine-containing compounds to the multiple detection system of adenovirus, influenza B virus, respiratory syncytial virus, influenza A virus, Mycoplasma pneumoniae, rhinovirus and new coronavirus, the specificity of the system can be improved; at the same time, the optimal sensitivity of the system was further maintained when adding 5mM and 10mM NaF.

[0104] Example 3 The additive of the present invention is used to detect multiple mutations G719X in exon 18 of the EGFR gene

[0105] By adding different concentrations of NaBF4 to the EGFR gene exon 18 multiple mutation G719X system, the appropriate concentration of NaBF4 was investigated. The fixed components of the system are shown in Table 7:

[0106] Table 7

[0107] Fixed ingredients 1× concentration Tris buffer (pH 8.3) 30mM <![CDATA[MgCl2]]> 3mM dNTPs 0.5mM <![CDATA[(NH4)2SO4]]> 35mM Mutant Primer R1 225nM Mutant Primer R2 225nM Mutant Primer R3 225nM Mutation probe P(ROX) 225nM Upstream primer F1 45nM Upstream primer F2 450nM Takara Taq DNA Polymerase 1U Thermolabile UDG 0.1U TMAC 30mM

[0108] Based on the above fixed components, different concentrations of NaBF4 were added as shown in Table 8 to prepare 4 reaction systems.

[0109] Table 8

[0110]

[0111]

[0112] Prepare 4 PCR reaction systems according to the above table, each containing a fixed component and a variable component. Divide the prepared PCR reaction systems into 8 PCR tubes, 8 tubes for each system; finally, add equal amounts of templates: 2 tubes are NTC (no template control), 3 tubes are Neg (negative control, no mutant), and the remaining 3 tubes are 3 test templates containing different mutation types of G719X (named X1, X2, and X3, all of which are mixed templates containing mutants and wild types).

[0113] The prepared PCR reaction system was placed on an ABI 7500 fluorescence quantitative PCR instrument for PCR amplification. The program used was: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 15 seconds, 61°C annealing and extension for 30 seconds, no fluorescence signal collection, 15 cycles; 94°C denaturation for 15 seconds, 56°C annealing and extension for 30 seconds, fluorescence signal collection, 30 cycles; 10°C to terminate the reaction.

[0114] Result analysis:

[0115] Figure 7 The amplification results of the no-template control and negative control of the four reaction systems are shown (ROX channel).

[0116] Figure 8 The amplification results of the X3 template of the four reaction systems are shown (ROX channel). Table 9 below compares the qPCR amplification results of the four reaction systems.

[0117] Table 9

[0118]

[0119] from Figure 7 It can be seen that even if the conventional additive TMAC is used, in system ① (NaBF4=0mM), false positive fluorescence signals appear in the ROX channel in the NTC and Neg reactions, and in the three reaction systems of system ② (NaBF4=5mM), system ③ (NaBF4=10mM), and system ④ (NaBF4=15mM), the false positive fluorescence signals in Neg are eliminated by adding NaBF4. Combined with the data in Table 9, it can be seen that by adding fluorine-containing compounds, false positive results caused by primer-template mismatch can be eliminated, and false positive results caused by primer dimers can be eliminated at the same time.

[0120] Compared with system ① (NaBF4 = 0mM), system ② (NaBF4 = 5mM) has no false positives, and there is no significant difference in Ct values ​​when detecting the test templates X1 / X2 / X3, indicating that NaBF4 at a concentration of 5mM not only helps to improve the specificity of the system, but also ensures the sensitivity of the system. When the concentration of NaBF4 is greater than 5mM, as its concentration increases, the positive fluorescence signal of the ROX channel is somewhat suppressed when detecting the test templates (such as X2 / X3), which is reflected in the delayed trend of the Ct value.

[0121] In summary, adding fluorine-containing compounds to the system for detecting multiple key mutations G719X in exon 18 of the EGFR gene can improve the specificity of the system; at the same time, the optimal sensitivity of the system is further maintained when 5mM NaBF4 is added.

Claims

1. A PCR reaction system, comprising: Reagents necessary for PCR reaction; and Fluorine-containing compounds, wherein the fluorine-containing compounds are selected from alkali metal fluorides, hydrofluoric acid, alkali metal salts of fluoroboric acid, The fluorine-containing compound is prepared to have a concentration of 4 mM to 24 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

2. The reaction system according to claim 1, wherein The fluorine-containing compound is one or more selected from the group consisting of sodium fluoride, potassium fluoride, lithium fluoride, hydrofluoric acid, sodium fluoroborate, potassium fluoroborate or lithium fluoroborate.

3. The reaction system according to claim 1 or 2, wherein The fluorine-containing compound is prepared to have a concentration of 8 mM to 24 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

4. The reaction system according to claim 1 or 2, wherein The fluorine-containing compound is prepared to have a concentration of 8 mM to 17 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

5. The reaction system according to claim 1 or 2, wherein The fluorine-containing compound is prepared to have a concentration of 8 mM to 12 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

6. A kit for PCR, comprising: DNA polymerase, dNTPs, buffers, divalent cations, and fluorinated compounds; and Optionally, primers for amplifying the template, Wherein, the fluorine-containing compound is selected from alkali metal fluorides, hydrofluoric acid, alkali metal salts of fluoroboric acid, The fluorine-containing compound is prepared to have a concentration of 4 mM to 24 mM in the reaction system in terms of free fluorine ions and any combination of fluorine ions.

7. The kit according to claim 6, wherein The fluorine-containing compound is one or more selected from the group consisting of sodium fluoride, potassium fluoride, lithium fluoride, hydrofluoric acid, sodium fluoroborate, potassium fluoroborate or lithium fluoroborate.

8. The kit according to claim 6 or 7, wherein The fluorine-containing compound is prepared to have a concentration of 8 mM to 24 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

9. The kit according to claim 6 or 7, wherein The fluorine-containing compound is prepared to have a concentration of 8 mM to 17 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

10. The kit according to claim 6 or 7, wherein The fluorine-containing compound is prepared to have a concentration of 8 mM to 12 mM in terms of free fluorine ions and any combination of fluorine ions in the reaction system.

11. The kit according to claim 6 or 7, wherein The PCR is multiplex PCR or qPCR.

12. Use of an additive for improving the specificity of PCR, wherein: The additive is a fluorine-containing compound, and the additive is selected from alkali metal fluorides, hydrofluoric acid, alkali metal salts of fluoroboric acid, And wherein the fluorine-containing compound is prepared to have a concentration of 4 mM to 24 mM in the reaction system in terms of free fluorine ions and any combination of fluorine ions.

13. The use according to claim 12, wherein the additive is selected from one or more of the group consisting of sodium fluoride, potassium fluoride, lithium fluoride, hydrofluoric acid, sodium fluoroborate, potassium fluoroborate or lithium fluoroborate.

14. The use according to claim 12 or 13, wherein the additive is formulated to have a concentration of 8 mM to 24 mM in terms of free fluoride ions and any combination of fluoride ions in the reaction system.

15. The use according to claim 12 or 13, wherein the additive is formulated to have a concentration of 8 mM to 17 mM in the reaction system in terms of free fluoride ions and any combination of fluoride ions.

16. The use according to claim 12 or 13, wherein the additive is formulated to have a concentration of 8 mM to 12 mM in terms of free fluoride ions and any combination of fluoride ions in the reaction system.

17. The use according to claim 12 or 13, wherein The PCR is multiplex PCR or qPCR.

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