Multichannel methylation gene fluorescent quantitative PCR reaction liquid kit

Abstract

The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) reaction liquid kit for multichannel methylation genes. The invention provides a multichannel methylation gene fluorescent quantitative PCR (Polymerase Chain Reaction) premix solution, which is prepared from the following effective components: KCl, Glycol, MgCl2, dNTPs (Deoxyribose Nucleic Acid) and DNA (Deoxyribose Nucleic Acid) polymerase, and the use ratio of the KCl to the Glycol to the MgCl2 to the dNTPs to the DNA polymerase is (60 to 80 mmol) to (0.5 to 1 L) to (6 to 8 mmol) to (600 to 800 [mu] mol) to (0.04 to 0.13) * 10 <-6 > U. The invention develops a universal premixed solution suitable for different gene methylation QPCR (Quantitative Polymerase Chain Reaction) and preparation of concentration.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a multi-channel methylation gene fluorescence quantitative PCR reaction solution kit. Background Art

[0002] Multiplex methylation gene fluorescence quantitative PCR reaction refers to the amplification reaction of DNA template with methylation primers and probes of multiple genes in the same PCR reaction solution after sulfite conversion to obtain the methylation products of each gene, such as using gene methylation as a marker for early screening and detection of lung cancer. However, to obtain ideal results, the composition and matching of QPCR reaction solution are very important when selecting primers and probes of multiple genes for amplification reaction.

[0003] PCR, also known as polymerase chain reaction, is a technology used to amplify specific DNA fragments. It allows the number of DNA fragments to grow exponentially under the guidance of the primer nucleotide sequence. The PCR process is roughly divided into three steps: denaturation-annealing-extension: 1. Denaturation: (90℃-96℃): The double-stranded DNA template breaks hydrogen bonds under the action of heat to form single-stranded DNA; 2. Annealing: (60℃-65℃): The system temperature is reduced, the primer binds to the DNA template, and a local double strand is formed; 3. Extension: (70℃-75℃): Under the action of Taq enzyme (at around 72℃, the activity is best), dNTP is used as a raw material, starting from the 3′ end of the primer and extending in the direction from 5′→3′ end to synthesize a DNA chain complementary to the template. After a denaturation-annealing-extension process, the original one DNA double strand becomes two; after two times, it becomes four; and so on. As long as a few dozen cycles are experienced, a large number of identical DNA fragments can be generated. After amplification is completed, the amplified DNA needs to be detected, for example, by using gel electrophoresis or real-time fluorescence quantitative PCR (qPCR) to record the amplification results.

[0004] qPCR introduces fluorescent groups during the amplification process. As the amount of DNA increases exponentially, the fluorescent signal in the reaction system will also increase. By detecting the strength of the fluorescent signal, the DNA amplification can be quantitatively analyzed.

[0005] At present, qPCR can be roughly divided into two types according to different fluorescent substances. One is to use fluorescent dyes, mainly SYBR GreenⅠ dyes, which can bind to double-stranded DNA and show fluorescent signals after binding. When there is no binding, the fluorescent signal is almost undetectable. As the number of copies increases, the fluorescent signal increases. Another method is to use fluorescent probes, also known as TaqMan probe method. The principle is to design a fluorescent probe that can specifically bind to the target gene according to the target gene. The probe contains two groups: a fluorescent group and a quenching group. Under normal circumstances, the fluorescent group cannot emit fluorescence due to the presence of the quenching group. During amplification, the probe binds to the DNA template, and the two groups are cut when the probe position is amplified, so that the fluorescent group can emit fluorescence. Similarly, as the number of copies increases, the fluorescent signal increases. With the help of this quantitative relationship, people can draw the amplification curve of PCR, which is a curve in which the fluorescent signal changes with the number of cycles. With the help of this curve (the ideal curve should show an "S" type of growth), the situation of PCR can be analyzed, so this technology is becoming more and more widely used.

[0006] Hot-start PCR is often used to enhance the specificity of PCR amplification. Hot-start PCR mainly uses an enzyme modifier (such as an antibody, affinity ligand, aptamer or chemical modifier) ​​to inhibit the activity of DNA polymerase at room temperature. This modification reduces the binding ability between primers and templates, and between primers during the preparation of the PCR reaction system, thereby avoiding nonspecific amplification. Since the activity of DNA polymerase is inhibited at room temperature, hot-start technology provides great convenience for preparing multiple PCR reaction systems at room temperature without sacrificing the specificity of the PCR reaction. When the reaction system is prepared, in the initial heating step of the reaction, the enzyme modifier is released at high temperature (usually above 90°C), so that the DNA polymerase is activated. The activation time and temperature vary depending on the DNA polymerase and the hot-start modifier. For some polymerases, activation and pre-denaturation are combined into one step.

[0007] Multiplex PCR can achieve the simultaneous amplification of multiple fragments of different target genes in the same reaction tube. Multiplex PCR not only means saving time, reagents and samples, but also makes it possible to compare multiple amplicons at the same time. Comparison between single PCR and multiplex PCR: In single PCR, each reaction contains only one primer pair to amplify one target fragment. In multiplex PCR, multiple primer pairs are used in the same reaction to amplify multiple target fragments. Therefore, there are considerable requirements for the design of primers and probes for each target gene in multiplex PCR: Under the same reaction conditions, each gene should maintain its original amplification efficiency during amplification, and also maintain compatibility with other genes, that is, no interference.

[0008] When there are multiple primer pairs in one reaction tube, such as in multiplex PCR, nonspecific amplification and reduced amplification efficiency are the most concerned issues, because it is impossible to optimize the reaction for all primer pairs and fragments. Therefore, primer design is crucial to reduce nonspecific amplification caused by mismatches. Primer sequences should be unique to the target fragments, and the Tm values ​​of all primers should be within 5°C. Before performing multiplex PCR, each primer pair should be verified for its specificity and amplification efficiency in a single reaction. Moreover, amplicons of different sizes should be distinguishable in gel electrophoresis for identification. In addition to primer design and amplicon size, hot-start DNA polymerase and specially optimized PCR buffer composition are also necessary for multiplex PCR, which can contribute to the success rate and specificity of amplification.

[0009] Although multiplex PCR is commonly used for endpoint PCR, it is becoming increasingly popular for real-time quantitative PCR due to its ability to perform multiple labeling and detection. Multiplex real-time quantitative PCR is also commonly used for the detection of genetic markers for identification. Summary of the invention

[0010] The purpose of the present invention is to provide a preparation method for a multi-channel methylation gene fluorescence quantitative PCR reaction solution kit.

[0011] In a first aspect, the present invention provides a multi-channel methylation gene fluorescence quantitative PCR premix, the effective components of which are composed of KCl, Glycerol, MgCl2, dNTPs and DNA polymerase.

[0012] In the premixed solution described above, the ratio of KCl, Glycerol, MgCl2, dNTPs, and DNA polymerase is: 60-80mmol: 0.5-1L: 6-8mmol: 600-800μmol: (0.04-0.13)*10 -6 U.

[0013] The premix solution consists of an aqueous solution of the active ingredient.

[0014] In a second aspect, the present invention provides a use of the premix described in the first aspect in preparing a PCR reaction system.

[0015] In the application described above, the PCR reaction system is a fluorescent quantitative PCR reaction system;

[0016] Furthermore, the fluorescence quantitative PCR reaction system is a multi-channel methylation gene fluorescence quantitative PCR reaction system.

[0017] In a third aspect, the present invention provides a PCR reaction system, which comprises the premixed solution described in the first aspect and n primer probe sets;

[0018] n is a natural number greater than 1;

[0019] n primer probe sets correspond to n detected genes.

[0020] Each of the primer probe sets is composed of a primer pair for detecting a certain gene and a fluorescent probe. The fluorescent groups of the fluorescent probes in each primer probe set are different.

[0021] In the above-mentioned PCR reaction system, the concentration of the active ingredient provided by the premix in the PCR reaction system is as follows:

[0022] The concentration of KCl is 60-80 mM;

[0023] The concentration of Glycerol is 0.5-1%;

[0024] The concentration of MgCl2 is 6-8mM;

[0025] The concentration of the dNTPs is 600-800 μM;

[0026] The concentration of the DNA polymerase is 0.04-0.13 U / μL.

[0027] The PCR reaction system is composed of the premixed solution described in the first aspect and a mixed solution containing n primer probe sets;

[0028] The mixed solution containing n primer probe sets consists of n primer probe sets, a template and a solvent (TE buffer).

[0029] In an embodiment of the present invention, the PCR reaction system is a multi-channel methylation gene fluorescence quantitative PCR reaction system, for example a 6-channel methylation gene fluorescence quantitative PCR reaction system, wherein the n primer probe sets are 6 primer probe sets, which are respectively used to detect 6 genes: GRIK2, HOXA1, HOXA9, HIST1H3A, SHOX2 and ACTB.

[0030] Each 25 μL multi-channel methylation gene fluorescence quantitative PCR reaction system is composed of 8.6 μL of the first part of the premix solution (Table 1) and 16.4 μL of the second part of the mixed solution containing 6 primer probe sets (Table 2).

[0031] The premixed solution used in the first part is composed of dNTPs, MgCl2, potassium chloride, Glycerol and Taqman DNA polymerase. The mixed solution containing 6 primer probe sets used in the second part is composed of 6 primer probe sets, template and TE buffer. The specific primers and probes are dissolved in TE buffer (10mM Tris-1mM EDTA, PH8.0) to prepare a solution containing 2-5 target genes and 1 internal reference gene primer probe. The working concentration and ratio of primers and probes are relatively normal.

[0032] In a fourth aspect, the present invention provides a PCR reaction kit, comprising the premixed solution described in the first aspect and n primer probe sets;

[0033] n is a natural number greater than 1; each of the primer probe sets consists of a primer pair and a fluorescent probe for detecting a certain gene.

[0034] In the above PCR reaction kit, the premix and the primer probe set are packaged independently.

[0035] In a fifth aspect, the present invention provides a use of the premix described in the first aspect, the PCR reaction system described in the third aspect, or the kit described in the fourth aspect in any of the following or in the preparation of a product having any of the following functions:

[0036] 1) PCR amplification;

[0037] 2) Detect the methylation level of the gene to be tested.

[0038] The above PCR amplification can further be fluorescent quantitative PCR amplification;

[0039] Furthermore, the fluorescent quantitative PCR amplification is multiplex methylation gene fluorescent quantitative PCR amplification.

[0040] The six-channel fluorescence quantitative PCR instruments used in the above PCR amplification were Quant Studio 5 (Applied Biosystem, Thermo Fisher Scientific, USA) and Quant Gene 9600 (Bori Technology Co., Ltd., Hangzhou, China).

[0041] In a sixth aspect, the present invention provides a method for detecting the methylation level of a gene to be tested, comprising the following steps:

[0042] 1) converting the DNA molecule to be tested by sulfite to obtain a converted DNA molecule;

[0043] 2) Using the converted DNA molecule as a template, performing fluorescent quantitative PCR amplification using the PCR reaction system described in the third aspect, and determining the methylation level of the gene to be tested in the DNA molecule to be tested according to the amplification result.

[0044] In an embodiment of the present invention, the gene to be tested is at least one of GRIK2, HOXA1, HOXA9, HIST1H3A, SHOX2 and ACTB.

[0045] The present invention mainly explores the components and proportions of the premix kit of the first part to work with various target gene composition screening and diagnostic kits, including dNTP, MgCl2, KCL, Glycerol concentration and Taq DNA polymerase dosage. The present invention develops a universal premix component and concentration preparation suitable for different gene methylation QPCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is used to detect the optimal dNTP concentration in the fluorescence quantitative PCR reaction of multiple methylation genes by gel electrophoresis. DETAILED DESCRIPTION

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] Example 1. Preparation of a multi-channel methylation gene fluorescence quantitative PCR reaction kit and establishment of a detection method

[0050] 1. Preparation of multiplex methylation gene fluorescence quantitative PCR reaction kit

[0051] For each QPCR reaction, it is composed of the first part of QPCR master mix (Table 1) and the second part of the mixture containing primers and probes (Table 2).

[0052] 1. Design and synthesis of marker genes and related primers used for lung cancer detection

[0053] As this embodiment, the methylation of the marker gene for detecting lung cancer is used as a working system template. The inventor extracted DNA and RNA from lung cancer tissue (i.e., surgical tissue of lung cancer patients (confirmed by pathology, as tumor-positive samples)) and normal adjacent tissue (normal adjacent tissue during surgery of the same case, as tumor-negative samples), using whole-gene methylation sequencing and expression analysis, combined with a variety of databases and comprehensive clinical information, through large-scale screening, probes were designed to enrich the methylation sites of related genes. By comparing the differences in methylation levels, markers SHOX2, HOXA1, GRIK2, HOXA9 and HIST1H3A for lung cancer detection were obtained. ACTB was used as an internal reference gene.

[0054] According to the nucleotide sequences of the above genes, primers and probes were designed respectively (the specific sequences are shown in Table 3 below), and synthesized by Nanjing GenScript Biotech Co., Ltd.

[0055] 2. Multiplex methylation gene fluorescence quantitative PCR reaction kit

[0056] The kit includes a QPCR reagent (PCR reaction system), and the QPCR reagent is composed of a QPCR premix as shown in Table 1 and a mixed solution containing a QPCR primer probe set as shown in Table 2;

[0057] For each 25 μL sample QPCR reagent, it is composed of a first part of 8.6 μL of QPCR premix solution (Table 1) and a second part of 16.4 μL of a mixed solution containing a primer probe set (Table 2).

[0058] The QPCR premix shown in Table 1 is composed of substances with the following working concentrations: dNTPs concentration is 600-800 μM, MgCl2 concentration is 6-8 mM, potassium chloride concentration is 60-80 mM, Glycerol concentration is 0.5-1% (volume percentage), Taq DNA polymerase concentration is 0.04-0.13 U / μL. The Tag polymerase is Accurate Tag HSDNA polymerase (CM0008, 5 U / μL, AGL Biotech, China).

[0059] The QPCR primers and probes shown in Table 2 consist of primer pairs for 6 groups of genes, corresponding to 5 target genes and 1 internal reference gene;

[0060] Each primer pair consists of an upstream primer, a downstream primer and a fluorescent probe; the upstream primer and the downstream primer are used to amplify the methylated gene to be tested, and the fluorescent probe is used to bind to the methylated gene to be tested for quantification;

[0061] The 5' end of the fluorescent probe in each primer pair is labeled with a different fluorescent group such as CY5.0, CY5.5, FAM, ROX, TAMRA or VIC, and the 3' end is labeled with an MGB quenching group; and the fluorescent probes in each primer pair are labeled with different fluorescent groups.

[0062] Table 1 shows the composition of the QPCR premix

[0063] Component name Storage solution concentration Working concentration Example Final Concentration Volume (μL) KCl 1M 60-80mM 70mM 1.75 Glycerol 20% (volume percentage) 0.5-1％ 0.75％ 0.95 MgCl2 50mM 6-8mM 7mM 3.5 dNTPs 10mM 600-800μM 700μM 1.75 DNA enzyme 5U / μL 0.04-0.13 U / μL 0.13 U / μL 0.65 Total volume 8.6

[0064] In the above table, the first column is the components of the QPCR premix, the second column is the initial storage solution concentration (the storage solution of each component is obtained by dissolving each component in water to obtain a storage solution of the corresponding concentration), the third column is the working concentration range adapted to each scenario, the fourth column is the concentration used in the embodiments of the present invention, and the fifth column is the volume of the storage solution added when used in the embodiments.

[0065] Table 2 shows the mixed solution containing the QPCR primer probe set.

[0066]

[0067] In the above table, the first column is the components of QPCR primers and probes, the second column is the starting concentration, that is, the concentration of the primer or probe storage solution, specifically the primer storage solution obtained by dissolving the primer or probe in TE (PH 8.0), and the third column is the final concentration of each component in the first column in the QPCR reagent, that is, the working concentration. The fourth column is the starting concentration added volume, of which the five target genes are SHOX2, HOXA1, GRIK2, HOXA9, HIST1H3A, and the one internal reference gene is ACTB.

[0068] Table 3 shows the sequences of the primers and probes for the genes.

[0069]

[0070]

[0071] The third column in the above table is sequence 1 to sequence 18 from top to bottom.

[0072] 2. Establishment of a multiplex fluorescence quantitative PCR reaction detection method for methylation genes

[0073] 1. Test sample DNA

[0074] Extract genomic DNA from the sample to be tested.

[0075] The above-mentioned example samples to be tested can be lung cancer tissue (i.e., surgical tissue of lung cancer patients confirmed by pathology), or human lung cancer cell lines (A549 cells and H1299 cells, respectively) as tumor-positive samples, or normal adjacent tissues (normal adjacent tissues of the same case during surgery) as tumor-negative samples.

[0076] After homogenization, the above samples were extracted with a blood / cell / tissue genomic DNA extraction kit (Beijing Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. # DP304-03) to obtain the genomic DNA of the sample to be tested.

[0077] Human whole-genome methylated DNA (EpiTect PCR Control DNA Set, Qiagen Cat No. / ID:59695, where methylated DNA is represented by +vectr) is a positive control DNA standard. Human whole-genome unmethylated DNA (EpiTect PCR Control DNA Set, Qiagen Cat No. / ID:59695, where unmethylated DNA is represented by -vectr) is a negative control DNA standard. The samples used in the embodiments of the present invention are mainly standards of human whole-genome methylated DNA and human whole-genome unmethylated DNA.

[0078] 2. Bisulfite conversion of methylated DNA

[0079] The genomic DNA of the above-mentioned sample to be tested was subjected to bisulfite modification treatment using the EZ DNA Methylation-DirectTM Kit (ZYMORESEARCH, D5001 / D5002), and the converted DNA was obtained after purification for later use.

[0080] 3. Multiplex methylation gene fluorescence quantitative PCR reaction

[0081] The 6-channel fluorescence quantitative PCR instruments used were Quant Studio 5 (Applied Biosystem, ThermoFisher Scientific, USA) and Quant Gene 9600 (Bori Technology Co., Ltd., Hangzhou, China).

[0082] Using the converted DNA obtained in 2 above as a template, QPCR reagents were prepared according to the concentrations in Table 1 and Table 2 in 1 above, and multiple methylation gene fluorescence quantitative PCR reactions (QPCR) were performed.

[0083] The amplification sequence set for the above QPCR is: the first stage: 95°C for 5 min, 1 cycle; the second stage: 95°C for 15 sec; 63°C for 30 sec; 45 cycles; the third stage: collecting fluorescence signals at 63°C, with a temperature change rate of 1.6C / sec.

[0084] The fluorescence signal of the above PCR reaction was detected to obtain the fluorescence CT values ​​of SHOX2, HOXA1, GRIK2, HOXA9, HIST1H3A and ACTB, respectively. The CT value after deducting the CT value of the internal reference ACTB was the dCT value of the gene. If the dCT value of the gene was ≤ the dCT critical value of the gene, the gene was methylated in the sample; if the dCT value of the gene was > the dCT critical value of the gene, the gene was not methylated in the sample.

[0085] The dCT critical value of each gene is an average statistical dCT value after comparing the dCT values ​​of lung cancer tissue (tumor-positive sample) and adjacent normal tissue (tumor-negative sample) confirmed by a large number of cases, that is, the critical value (threshold), which is a critical dCT value that can best distinguish tumors from non-tumors. In the embodiment of the present invention, the dCT critical values ​​of each target gene are: GRIK2: 5, HOXA: 7, HOXA: 6, HIST1HA: 10, SHOX: 6.

[0086] The lung cancer tissue confirmed by pathology and the positive control DNA constitute the dCT value of the positive sample group. If the amplification curve is not an "S" type or the CT value is blank, the CT value is recorded as 45.

[0087] Example 2: Optimization of the optimal dNTP concentration in the multi-channel methylation gene fluorescence quantitative PCR (MMSP) reaction using gel electrophoresis

[0088] This experiment is to prove the working condition of multiple PCR reactions with different target gene combinations under various dNTP concentrations. The gel electrophoresis identification method can directly observe whether the size of the PCR reaction product meets the design and whether there are non-specific bands.

[0089] 1. The sample DNA tested was from lung cancer tissue (i.e., surgical tissue of lung cancer patients (as tumor-positive samples) and adjacent normal tissue (as tumor-negative samples of adjacent normal tissue during surgery in the same case). Human lung cancer cell line A549 cells were used as tumor-positive samples. After the samples were homogenized, genomic DNA was extracted using a blood / cell / tissue genomic DNA extraction kit (Beijing Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat.# DP304-03).

[0090] 2. Bisulfite conversion of methylated DNA

[0091] The DNA molecules obtained in 1 above were subjected to sulfite conversion using a ZYMO conversion kit to obtain converted DNA.

[0092] 3. Multiple methylation gene PCR (MMSP) reaction

[0093] According to the method of Example 1, 8.6 μL of the reagents shown in Table 1 and 16.4 μL of the reagents in Table 2 were mixed to obtain 25 μL of different gene detection QPCR reagents, wherein only the amount of dNTP was different in Table 1 of this experiment, and the final concentrations of the other components and examples were the same as those in Table 1 of Example 1, and the final concentrations of dNTP in the QPCR reagent were 800 μM, 600 μM, 400 μM and 200 μM, respectively; the primer probes used in Table 2 of this experiment were not completely the same as those in Table 2 of Example 1, and were as follows: primer probes corresponding to the three genes ACTB, HIST1H3A and HOXA1, corresponding to the configuration of the three-gene detection QPCR reagent; or primer probes corresponding to the two genes GRIK2 and HOXA9, corresponding to the configuration of the two-gene detection QPCR reagent; or primer probes corresponding to the gene SHOX2, corresponding to the configuration of the single-gene detection QPCR reagent; except for the primer probes of the corresponding genes in Table 2, the rest were supplemented to 16.4 μL with TE buffer.

[0094] Using the converted DNA obtained in 2 above as a template and the above-mentioned different gene detection QPCR reagents, a multichannel methylation gene fluorescence quantitative PCR reaction (QPCR) was performed.

[0095] The quality and concentration of dNTPs are closely related to the efficiency of PCR amplification. In particular, the concentrations of the four dNTPs should be equal (equimolar preparation). If the concentration of any one of them is different from the others (too high or too low), it will cause mismatching. Too low a concentration will reduce the yield of PCR products. dNTPs can bind to Mg2+, reducing the concentration of free Mg2+.

[0096] This experiment is to prove the methylation status of target genes under various dNTP concentrations in multiple PCR reactions composed of different target genes. After multiple methylation gene fluorescence quantitative PCR reactions with different dNTP dosages of 800μM, 600μM, 400μM and 200μM for three different target gene combinations, the amplified products were separated by electrophoresis gel, showing the following: Figure 1As shown, 1.50bpLadder, a marker of DNA fragment size; 2. dNTP 800μM; corresponding to 3 gene tests: ACTB (154bp); HIST1H3A (132bp); HOXA1 (111bp); 3. dNTP 600μM; corresponding to 3 gene tests: ACTB (154bp); HIST1H3A (132bp); HOXA1 (111bp); 4. dNTP 400μM; corresponding to 3 gene tests: ACTB (154bp); HIST1H3A (132bp); HOXA1 (111bp); 5. dNTP 200μM; corresponding to 3 gene tests: ACTB (154bp); HIST1H3A (132bp); HOXA1 (111bp); 6. dNTP 800μM; corresponding to 2 gene detection: GRIK2 (124bp); HOXA9 (94bp); 7.dNTP 600μM; corresponding to 2 gene detection: GRIK2 (124bp); HOXA9 (94bp); 8.dNTP 400μM; corresponding to 2 gene detection: GRIK2 (124bp); HOXA9 (94bp); 9.dNTP 200μM; corresponding to 2 gene detection: GRIK2 (124bp); HOXA9 (94bp); 10.dNTP 800μM; corresponding to single gene detection: SHOX2 (120bp); 11.dNTP 600μM; corresponding to single gene detection: SHOX2 (120bp); 12.dNTP 400μM; corresponding to single gene detection: SHOX2 (120bp); 13.dNTP 200μM; corresponding to single gene detection: SHOX2 (120bp); according to the results of the intensity, uniformity and separation of the electrophoretic bands, it can be seen that 600-800μM dNTP can make all 3 genes amplified simultaneously (in lanes 2, 3, 4 and 5 dNTP concentrations), 2 genes amplified simultaneously (at 6, 7, 8 and 9 dNTP concentrations) and 1 gene amplified alone (at 10, 11, 12 and 13 dNTP concentrations) have similar amplification effects. Therefore, the median dNTP concentration can be positioned at 700μM.

[0097] Example 3: Optimal magnesium ion and Taq DNA polymerase dosage for multi-channel qPCR at 700uM dNTP concentration

[0098] Mg2+ has a significant effect on the specificity and yield of PCR amplification. In general PCR reactions, when the concentration of various dNTPs is 200umol / L, the Mg2+ concentration should be 1.5-2.0mmol / L. If the Mg2+ concentration is too high, the reaction specificity will be reduced, and non-specific amplification will occur. If the concentration is too low, the activity of Taq DNA polymerase will be reduced, and the reaction product will be reduced. Mg2+ has a significant effect on the specificity and yield of PCR amplification. In general PCR reactions, when the concentration of various dNTPs is 200umol / L, the Mg2+ concentration should be 1.5-2.0mmol / L. If the Mg2+ concentration is too high, the reaction specificity will be reduced, and non-specific amplification will occur. If the concentration is too low, the activity of Taq DNA polymerase will be reduced, and the reaction product will be reduced. However, in the multiple methylation gene fluorescence quantitative PCR reaction, the Mg2+ concentration will vary depending on the gene used.

[0099] 1. Obtain genomic DNA

[0100] The positive control used was a commercial standard DNA (MBA): human whole genome methylated DNA and human whole genome unmethylated DNA mixed in different ratios (100%, 67%, 33% and 0%).

[0101] Here, 100% refers to the methylated DNA of the entire human genome, and 0% refers to the 100% unmethylated DNA of the entire human genome.

[0102] 2. Bisulfite conversion of methylated DNA

[0103] Same as Example 1, Part 2.

[0104] 3. Multiplex methylation gene fluorescence quantitative PCR reaction

[0105] According to the method of Example 1, 8.6 μL of the corresponding reagent in Table 1 and 16.4 μL of the corresponding reagent in Table 2 were mixed to obtain 25 μL of different detection QPCR reagents, wherein only the concentrations of Mg2+ and DNA enzyme in Table 1 of this experiment were different, and the remaining components and the final concentrations of the examples were the same as those in Table 1 of Example 1. The final concentrations of MgCl2 in the QPCR reagent were 6 mM, 8 mM and 10 mM, respectively; the amounts of DNA enzyme in the QPCR reagent were 2.5 U (corresponding to a final concentration of 0.1 U / μL) and 1.0 U (corresponding to a final concentration of 0.04 U / μL), respectively. The primer probes used in Table 2 of this experiment were the same as those in Table 2 of Example 1.

[0106] The QPCR machine and reaction conditions were basically the same as those in Example 1, and the CT values ​​of the target genes GRIK2, HOXA1, HOXA9, HIST1H3A, SHOX2 and ACTB were obtained respectively. The CT value after deducting the CT value of the internal control ACTB was the dCT value of the gene. If it was ≤ the dCT critical value (the dCT critical value in the embodiment of the present invention is shown in Table 3), the subject was methylated based on the gene.

[0107] The results are shown in Table 4, expressed as dCT values, with dark grey indicating that the gene was methylated under this condition.

[0108] Table 4 shows the amount of magnesium ions and Taq DNA polymerase

[0109]

[0110]

[0111] The QPCR reaction results are as follows: when the MgCl2 concentration is 6-8 mM and the Taq DNA polymerase dosage is 2.5 U, the DNA at 33% MBD also shows methylation positive (dark gray) for all target genes, and when the DNA is at 0% MBD, that is, 100% of the human whole gene sulfite-converted non-methylated DNA shows methylation negative (white or light gray), proving that the optimal MgCl2 median concentration is 7 mM and the Taq DNA polymerase dosage is 2.5-3.0 U.

[0112] Example 4. Optimal concentrations of glycerol and potassium chloride in qPCR

[0113] Using the median dNTP concentration of 700 μM in Example 2 above, and the median MgCl2 concentration of 7 mM and the amount of Taq DNA polymerase used in Example 3, the working concentrations of glycerol and potassium chloride were optimized.

[0114] 1. Obtain genomic DNA

[0115] The test sample was commercial standard DNA: human whole genome methylated DNA and human whole genome unmethylated DNA were mixed at a ratio of 33%.

[0116] 2. Bisulfite conversion of methylated DNA

[0117] Same as Example 1, Part 2.

[0118] 3. Multiplex methylation gene fluorescence quantitative PCR reaction

[0119] According to the method of Example 1, 8.6 μL of the corresponding reagent in Table 1 and 16.4 μL of the corresponding reagent in Table 2 were mixed to obtain 25 μL of different detection QPCR reagents, wherein only the concentrations of glycerol and potassium chloride were different in Table 1 of this experiment, and the remaining components and final concentrations were the same as those in Table 1 of Example 1. The final concentrations of glycerol in the QPCR reagent were 3%, 2%, 1%, 0.5% and 0, respectively; and the amounts of potassium chloride in the QPCR reagent were 100 mM, 80 mM, 60 mM, 40 mM, 20 mM and 0, respectively. The primer probes used in Table 2 of this experiment were the same as those in Table 2 of Example 1.

[0120] The QPCR machine and reaction conditions used were basically the same as those in Example 1, except that different concentrations of glycerol (3%, 2%, 1%, 0.5% and 0) and different concentrations of potassium chloride (100 mM, 80 mM, 60 mM, 40 mM, 20 mM and 0) were used for PCR amplification reaction.

[0121] The CT values ​​of target genes GRIK2, HOXA1, HOXA9, HIST1H3A, SHOX2 and ACTB were obtained respectively. The CT value after deducting the CT value of the internal control ACTB was the dCT value of the gene. If it was ≤ the dCT critical value (the critical value is shown in Table 3), the gene in the sample to be tested was methylated.

[0122] Table 5 shows the optimal concentrations of glycerol and potassium chloride in qPCR

[0123]

[0124]

[0125] The results are shown in Table 5. The CT / dCT values ​​of QPCR showed that when the Glycerol concentration was 0.5-1.0% (median 0.75%) and the KCl concentration was 60-80 mM (median 70 mM), each target gene exhibited the lowest CT / dCT value, indicating that this condition was the optimal amplification condition.

[0126] Therefore, in the six-channel methylation gene fluorescence quantitative PCR reaction solution, except for the primer probe, the optimal concentration of each component is: dNTPs concentration is 700μM, MgCl2 concentration is 7mM, potassium chloride concentration is 70mM, Glycerol concentration is 0.75%, and Taq DNA polymerase concentration is 0.13U / μL.

Claims

1. A multi-channel methylation gene fluorescent quantitative PCR premix, whose effective components are composed of KCl, Glycerol, MgCl2, dNTPs and DNA polymerase.

2. The premix according to claim 1, characterized in that: The ratio of KCl, Glycerol, MgCl2, dNTPs and DNA polymerase is: 60-80mmol: 0.5-1L: 6-8mmol: 600-800μmol: (0.04-0.13)*10 -6 U.

3. Use of the premix according to claim 1 or 2 in preparing a PCR reaction system.

4. The use according to claim 3, characterized in that: The PCR reaction system is a fluorescent quantitative PCR reaction system; Furthermore, the fluorescence quantitative PCR reaction system is a multi-channel methylation gene fluorescence quantitative PCR reaction system.

5. A PCR reaction system, comprising the premix according to claim 1 or 2 and n primer probe sets; n is a natural number greater than 1; each primer probe set consists of a primer pair and a fluorescent probe for detecting a certain gene.

6. The PCR reaction system according to claim 5, characterized in that: The concentration of the active ingredients provided by the premix in the PCR reaction system is as follows: The concentration of KCl is 60-80 mM; The concentration of Glycerol is 0.5-1%; The concentration of MgCl2 is 6-8mM; The concentration of the dNTPs is 600-800 μM; The concentration of the DNA polymerase is 0.04-0.13 U / μL.

7. A PCR reaction kit, comprising the premix according to claim 1 or 2 and n primer probe sets; n is a natural number greater than 1; each of the primer probe sets consists of a primer pair and a fluorescent probe for detecting a certain gene.

8. The PCR reaction kit according to claim 7, characterized in that: The premix solution and the primer probe set are packaged independently.

9. Use of the premix according to claim 1 or 2, or the PCR reaction system according to claim 5 or 6, or the kit according to claim 7 or 8 in any of the following or in the preparation of a product having any of the following functions: 1) PCR amplification; 2) Detect the methylation level of the gene to be tested.

10. A method for detecting the methylation level of a gene to be tested, comprising the following steps: 1) converting the DNA molecule to be tested by sulfite to obtain a converted DNA molecule; 2) Using the converted DNA molecule as a template, performing fluorescent quantitative PCR amplification using the PCR reaction system of claim 5 or 6, and determining the methylation level of the gene to be tested in the DNA molecule to be tested according to the amplification result.

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