Human MGMT methylation detection reagent based on digital PCR

Abstract

The invention discloses a human MGMT methylation detection reagent based on digital PCR (Polymerase Chain Reaction). The reagent comprises an MGMT upstream primer F1 with a sequence as shown in SEQ ID NO.1, an MGMT downstream primer R1 with a sequence as shown in SEQ ID NO.2, an MGMT probe P1 with a sequence as shown in SEQ ID NO.3, an ACTB upstream primer F with a sequence as shown in SEQ ID NO.4, an ACTB downstream primer R with a sequence as shown in SEQ ID NO.5 and an ACTB probe P with a sequence as shown in SEQ ID NO.6, the MGMT probe P1 and the ACTB probe P are respectively marked with different detection groups. The detection reagent, the kit and the detection method are high in sensitivity, high in specificity and good in detection accuracy.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a human MGMT methylation detection reagent based on a digital PCR method. Background Art

[0002] The MGMT gene is located on chromosome 10, with a total length of 170 kb, containing 5 exons and 4 introns. Among them, the first exon is the promoter region, which is rich in GC sequences; the remaining 4 exons are all coding regions, which can encode 207 amino acids of O6-methylguanine-DNA methyltransferase. O6-methylguanine-DNA methyltransferase is mainly distributed in the cytoplasm. It mainly protects cells from damage by alkylating agents by irreversibly transferring the alkylation group from O6-mG to the cysteine ​​residue at position 145 of the MGMT protein, and repairs the DNA damaged by alkylating agents to maintain the stability of the genome in the cell. In this process, MGMT acts as both a methyltransferase and a methyl receptor protein to complete the transfer reaction alone.

[0003] In recent decades, DNA methylation has been the subject of intensive research. The methylation status of the MGMT gene promoter is closely related to the expression level of the MGMT protein. Promoter methylation is the most common abnormality of the MGMT gene, which often occurs in the CpG island of the MGMT gene promoter. Methylation of the MGMT promoter will cause changes in chromatin structure, prevent transcription factors from binding, lead to gene silencing, reduce MGMT protein expression, and thus lose the function of repairing DNA. MGMT gene promoter methylation has become an important indicator for judging the prognosis of patients with brain gliomas, and is also an indicator of good response to alkylating chemotherapy drugs.

[0004] Current methods for detecting MGMT gene promoter methylation include: 1. Methylation-specific PCR (MS-PCR): After the template is converted by bisulfite, the methylated cytosine in the template remains unchanged, while the unmethylated cytosine is converted to uracil. Two sets of primers are designed based on the sequence differences between the methylated template and the unmethylated template after conversion for PCR amplification. Finally, the amplified fragment is detected by gel electrophoresis to determine whether the gene is methylated. This method is prone to false positives when the bisulfite conversion is incomplete.

[0005] 2. Fluorescence quantitative method (Methylight): After the template is converted by bisulfite, specific primers and TaqMan fluorescent probes are designed for amplification, and finally nucleic acid quantitative analysis is performed based on the intensity of the fluorescent signal. However, this method requires the preparation of a fluorescent standard curve and may result in false positives.

[0006] 3. Restriction enzyme digestion method (RE-PCR): first treat the template to be tested with a methylation-sensitive restriction endonuclease, and then design primers using the DNA sequences on both sides of the methylation site for PCR amplification. Although this method is low-cost and simple to operate, it can only reflect the methylation status of the CpG island at the restriction endonuclease recognition site; and the number of available restriction endonucleases is small, so it can only reflect the methylation status of some sites; in addition, this method is prone to incomplete enzyme digestion and false positives, affecting the accuracy of the results.

[0007] 4. Bisulfite sequencing: This method is the "gold standard" for detection. It establishes an accurate methylation map of a single DNA chain of a genomic DNA molecule by subjecting the template converted by bisulfite to PCR amplification, purification, cloning, sequencing and other steps in sequence, and can clarify the methylation status of each CpG site. However, the entire detection process is time-consuming and cumbersome, and the detection cost is also high.

[0008] 5. Methylation-sensitive high-resolution melting (MS-HRM) method: After the template is converted by bisulfite, a set of primers that do not target CpG sites are designed for amplification. Due to the different contents of cytosine and guanine in the PCR amplification product, the Tm value of the melting curve changes. The methylation status of the sample can be detected based on the change in the Tm value. However, this method can only detect the overall methylation level of the genome, and cannot detect the DNA methylation status of specific sites.

[0009] Therefore, there is an urgent need to develop a human MGMT methylation detection kit based on a new method to overcome the problems existing in the existing technology. Summary of the invention

[0010] Digital PCR technology is called the "third generation" PCR technology in the industry. Unlike qPCR, digital PCR technology disperses the diluted reaction system into multiple separated tiny independent reaction units (droplets), each of which contains or does not contain the target molecule. After PCR amplification is completed, the fluorescent signals of all droplets are identified and counted to calculate the number of negative and positive droplets, and the concentration of the target molecule is calculated by the Poisson distribution principle, thereby achieving absolute quantification of the target molecule. The result of digital PCR is not dependent on the standard curve and is not affected by the efficiency of PCR amplification. In addition, the digital PCR detection system uses droplet processing to separate rare detection fragments from a large amount of complex background, further improving the detection sensitivity and accuracy.

[0011] In the present invention, the D600 series digital PCR analysis system produced by MyBio is mainly used. The digital PCR analysis system uses vibration droplet generation technology to generate droplets and spread them into reaction wells. The reaction wells after amplification are photographed as a whole, each droplet is judged to have fluorescence or not, and the number of droplets with fluorescence and the number of droplets without fluorescence are counted, thereby achieving quantitative detection of the target sample.

[0012] Since the digital PCR analysis system of the present invention needs to take a picture of the reaction well as a whole, if the threshold of the intensity of the fluorescence signal in the droplet is not clearly divided, it will lead to the misinterpretation of the positive and negative droplets, affecting the accuracy of the detection results. Therefore, it is necessary to distinguish the fluorescence signal of the target (positive droplet) in the reaction unit from other background signals or non-specific signals (negative droplets), which the present invention has already achieved.

[0013] In addition, the primer amplification efficiency and the specificity of the primer probe will affect the performance of the detection reagent. For example, when the amplification efficiency between reaction units is uneven or the specificity of the primer probe is relatively poor, fluorescent signal crosstalk or insufficient detection signal aggregation will occur, thereby affecting the accuracy of the detection result.

[0014] Based on this, one of the purposes of the present invention is to provide a human MGMT methylation detection reagent based on digital PCR, the reagent comprising an MGMT primer probe combination and an ACTB primer probe combination; The MGMT primer-probe composition comprises: MGMT upstream primer F1, the sequence is shown in SEQ ID NO.1; MGMT downstream primer R1, the sequence is shown in SEQ ID NO.2; MGMT probe P1, the sequence is shown in SEQ ID NO. 3; The ACTB primer-probe composition comprises: ACTB upstream primer F, the sequence is shown in SEQ ID NO.4; ACTB downstream primer R, the sequence is shown in SEQ ID NO.5; ACTB probe P, the sequence is shown in SEQ ID NO.6; The MGMT probe P1 and ACTB probe P are respectively labeled with different detection groups.

[0015] In one embodiment, the MGMT primer-probe composition further comprises: MGMT upstream primer F2, the sequence is shown in SEQ ID NO.7; MGMT downstream primer R2, the sequence is shown in SEQ ID NO.8; In another embodiment, the detection group is selected from one or more of ALEX-350, FAM, VIC, TET, CALFluorGold540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CALFluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, Cy3, Cy5, Cy5.5, and Quasar 705.

[0016] In a preferred embodiment, the MGMT probe P1 is labeled with ROX, and the ACTB probe P is labeled with HEX.

[0017] In another embodiment, the MGMT probe P1 and ACTB probe P are also labeled with a quencher group, respectively, and the quencher group is selected from one or more of DABCYL, BHQ1, BHQ2, ECLIPSE, and TAMRA.

[0018] Another object of the present invention is to provide a human MGMT methylation detection kit based on digital PCR, wherein the kit comprises the reagents shown above.

[0019] In one embodiment, the kit further comprises dNTPs, a buffer, salt ions and a polymerase.

[0020] Deoxynucleoside triphosphates, i.e., dNTPs, for example, dATP, dCTP, dGTP, dTTP, dITP, dUTP, α-thio-dNTP, biotin-dUTP, fluorescein-dUTP, digoxigenin-dUTP, 7-deaza-dGTP can be used. dNTPs are well known in the art and are commercially available.

[0021] The buffers and salts used in the present invention provide suitable stable pH and ionic conditions for nucleic acid synthesis, such as reverse transcriptase and DNA polymerase activity. A variety of buffers and salt solutions and modified buffers that can be used in the present invention are known in the art, including reagents not specifically disclosed herein.

[0022] Preferred buffers include, but are not limited to, Tris, TRICINE, BISTRICINE, HEPES, MOPS, TES, TAPS, PIPES, CAPS. In preferred embodiments, provided compositions include Tris. In some embodiments, compositions for use in the methods of the present invention include a buffer comprising Tris at a pH of about 8 to 9, and in certain preferred embodiments Tris is Tris-HCl (pH 8.0-9.0).

[0023] Preferred salt solutions include, but are not limited to, ammonium sulfate, magnesium chloride, potassium acetate, potassium sulfate, potassium chloride, ammonium chloride, ammonium acetate, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride solution, sodium acetate, lithium chloride, lithium acetate. In a preferred embodiment, a composition is provided that includes ammonium sulfate and magnesium chloride.

[0024] In certain preferred embodiments, provided compositions include Tris-HCl, ammonium sulfate, and magnesium chloride.

[0025] The present invention is not particularly limited, and common polymerases in the field of diagnostic reagents can be used. Exemplary polymerases can be polymerases derived from thermotolerant bacteria that are known in the art.

[0026] In another embodiment, the kit further comprises betaine, which is used to reduce the non-specificity of the reaction.

[0027] In another embodiment, the kit further comprises a blank control, a negative control and a positive control; the blank control is nuclease-free water; the negative control is non-methylated DNA; and the positive control is methylated DNA.

[0028] Another object of the present invention is to provide a method for detecting human MGMT methylation based on digital PCR, the method comprising the following steps: (1) After nucleic acid extraction from the sample to be tested, bisulfite conversion treatment is performed; (2) Mixing the converted sample nucleic acid with a PCR reaction premix to obtain a reaction system; (3) randomly distributing the reaction system into more than 5,000 droplets, some of which contain sample nucleic acid and others do not, and the droplets are spread evenly in the reaction wells; (4) performing PCR amplification on the droplets in the reaction wells; (5) Taking fluorescent photos of the reaction wells and obtaining the number of methylated nucleic acids in the sample to be tested based on the number of droplets with signals in the reaction wells; The PCR reaction premix includes the above-mentioned human MGMT methylation detection reagent.

[0029] In one embodiment, the PCR amplification procedure is: 95°C for 2 min; 95°C for 20 sec, 56°C for 60 sec, and 30 to 50 cycles; and the fluorescence photography temperature is 35°C.

[0030] In another embodiment, the sample to be tested is a FFPE sample (paraffin-embedded pathological tissue) or an EDTA anticoagulated peripheral whole blood sample.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The length of the amplicon formed after the primers in the detection kit of the present invention bind to the target is less than 160 bp, so the fragmentation degree of the detection sample is low, and the detection sensitivity of peripheral blood samples or FFPE samples can be effectively improved. The detection reagent and kit of the present invention have high specificity and good detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The LOB performance test results of the kit of the present invention; Figure 2 is the LOD performance test result of the kit of the present invention; Figure 3 It is the precision performance test result of the kit of the present invention; Figure 4 The test results of Example 3 and the reference example of the present invention are shown below: Figure 5 This is the test result of the kit of the present invention on clinical samples. DETAILED DESCRIPTION

[0033] The present invention will be further described and illustrated below in conjunction with the embodiments of the present invention.

[0034] The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that for those skilled in the art, several modifications and improvements made on the basis of this invention and creation concept all belong to the protection scope of the present invention. The reagents used without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0035] Gliomas are the most common primary intracranial tumors with high malignancy and a median overall survival of less than 15 months. The annual incidence of gliomas in my country is 5-8 people per 100,000, and the 5-year mortality rate is second only to pancreatic cancer and lung cancer. It was found that about 30% of patients with gliomas lack MGMT (O6-methylguanine-DNA-methyltransferase) protein. MGMT protein is a DNA repair protein. The main function of MGMT protein is to protect cells from the mutagenic, carcinogenic and cytotoxic effects of alkylating agents by irreversibly transferring the alkylation group from O6-mG to the cysteine ​​residue at position 145 of the MGMT protein. The MGMT gene usually does not mutate or delete. This phenomenon of MGMT protein deficiency may be caused by gene modification changes, and gene methylation is the main type of epigenetic modification of this gene. In order to detect the methylation of the MGMT gene, existing samples usually select diseased tissues, cerebrospinal fluid samples or peripheral blood samples. Compared with the commonly used diseased tissues and cerebrospinal fluid samples, peripheral blood samples are easier to obtain, but the content of circulating tumor DNA (ctDNA) in peripheral blood samples is low. How to accurately detect these low-content ctDNA is a technical difficulty in this field.

[0036] Based on this, the present invention chooses to use digital PCR technology with high detection sensitivity to develop a human MGMT methylation detection reagent. However, during the development process, the primer-probe combination obtained according to the conventional design method is still not sensitive enough when it is used on a digital PCR instrument to detect peripheral blood samples or FFPE samples. After many explorations, it was found that the Taqman probe used in the conventional design method requires the upstream primer, probe and downstream primer to match the target, which results in a higher requirement for the length of the target-targets with a certain length or more can be detected, and targets below a certain length cannot be detected. However, the ctDNA fragments free in the peripheral blood samples have a high degree of fragmentation; FFPE samples are particularly prone to fragmentation due to a certain degree of damage to the genomic DNA caused by improper preparation or storage. Therefore, in order to improve the detection sensitivity of human MGMT methylation, it is necessary to limit the length of the amplicon of the detection target. According to established theory, tumor cell DNA mainly comes from apoptotic cells. During apoptosis, DNA enzymes cut genomic DNA in the internucleosomal region to produce fragments of 147 to 200 bp, with a peak value usually at about 166 base pairs. The amplicon lengths of the MGMT primer pair and the ACTB primer pair designed by the present invention are both less than 160 bp. In addition, the present invention selects the CpG site in the overlapping region of the MGMT promoter and the MGMT exon 1 to design the primer probe. The amplicon lengths of the designed MGMT primer pair and the ACTB primer pair are 100 bp and 85 bp, respectively.

[0037] The MGMT gene probe of the present invention preferably covers as many CpG islands as possible, for example 3, 4, 5, 6 CpG islands, etc. More preferably, the MGMT gene probe of the present invention covers 5 CpG islands, and this design scheme can ensure that as many CpG islands as possible are covered while controlling the length of the amplicon. Based on the characteristics of the end-point photography method of digital PCR, although multiple CpG islands are covered, it does not mean that the multiple CpG islands all need methylation to be detected, but the multiple CpG islands have the possibility of being detected at random. However, due to the insufficient length of the probe, when the degree of methylation is not high, it is not easy to detect, resulting in the digital PCR instrument detection results used in the early stage of the present invention showing a stratified non-aggregation phenomenon, which ultimately affects the accuracy of the detection results. The subsequent present invention designs a primer tailing sequence to cooperate with the probe tailing sequence, and utilizes the complementarity of the primer tailing sequence and the probe tailing sequence to improve the probe binding ability while also ensuring the specificity of the probe, solving the problem of stratification of the detection results and improving the detection accuracy.

[0038] Example 1 A human MGMT methylation detection reagent based on digital PCR, the reagent comprising a primer probe combination as shown in the following table.

[0039] Table 1

[0040] In this embodiment, MGMT upstream primer F1 and MGMT downstream primer R1 are MGMT methylation primer pairs, which can amplify the corresponding region of the methylated MGMT promoter. Both MGMT upstream primer F1 and MGMT probe P1 are designed with artificial tailing sequences that are not complementary or identical to the target, and the artificial tailing sequences of the two are complementary. Based on the tailing sequence, MGMT probe P1 can better bind to the target with the help of MGMT upstream primer F1, thereby improving the specificity of the detection.

[0041] The present invention selects the housekeeping gene (Housekeeping Gene) on the human genome, the β-actin gene (ACTB gene), as the internal reference gene. This type of gene is stably expressed in all cells. Therefore, the methylation rate of MGMT can be accurately calculated by the MGMT quantitative value / ACTB quantitative value.

[0042] Example 2 A human MGMT methylation detection reagent based on digital PCR, the reagent comprising a primer probe combination as shown in the following table.

[0043] Table 2

[0044] When the amplicon length is shorter, the design positions of the primers and probes are also correspondingly restricted. When the amplicon length is less than 160bp, the primers and probes of the MGMT gene are inevitably designed to cover several CpG islands, but primer coverage of CpG islands will affect the amplification efficiency. The present invention further designs a non-methylated primer pair MGMT upstream primer F2 and MGMT downstream primer R2, which can effectively avoid the influence of the primer methylation site on the amplification efficiency and improve the amplification efficiency.

[0045] Example 3 A human MGMT methylation detection kit based on digital PCR, the kit comprising the reagent of Example 1, and further comprising a PCR reaction buffer, wherein the PCR reaction buffer comprises dNTPs, a buffer, salt ions and a polymerase. The total volume of the PCR reaction system for detecting human MGMT methylation of the present invention is 25 μL. The specific configuration is shown in the following table.

[0046] Table 3

[0047] The 2×PCR reaction buffer at least includes: 3mmol / L MgCl2, 30mmol / L Tris-HCl with a pH value of 8.3, 0.5mmol / L dNTPs, 3U DNA polymerase and 70mmol / L (NH4)2SO4.

[0048] Example 4 A human MGMT methylation detection kit based on digital PCR, the kit comprising the reagent of Example 2, and further comprising a PCR reaction buffer, wherein the PCR reaction buffer comprises dNTPs, a buffer, salt ions and a polymerase. The total volume of the PCR reaction system for detecting human MGMT methylation of the present invention is 25 μL. The specific configuration is shown in the following table.

[0049] Table 4

[0050] The 2×PCR reaction buffer at least includes: 3mmol / L MgCl2, 30mmol / L Tris-HCl with a pH value of 8.3, 0.5mmol / L dNTPs, 3U DNA polymerase and 70mmol / L (NH4)2SO4.

[0051] Example 5 In order to further reduce the nonspecific amplification of the entire reaction, the present invention preferably adds 0.8M betaine to the reaction system of Example 3 or Example 4.

[0052] The reagents and kits of the present invention are used on a D 600 series digital PCR instrument of the Microbiota to complete the detection of MGMT gene methylation by preparing droplets and then performing PCR amplification. The specific amplification program is shown in Table 5. After the amplification, the fluorescence signal is collected under the conditions of a photographing temperature of 35° C. and an exposure time of 2 s for both the HEX channel and the ROX channel.

[0053] Table 5

[0054] In the present invention, the photographing temperature is set at 35° C. At this temperature, both positive and negative droplets in the reaction wells can be identified, and the signal-to-noise ratio is good.

[0055] Test Example 1 Performance Verification-LOB Using the reaction system of Example 3 and the reaction procedure of Table 5, the negative samples were tested 23 times. As a result, one reaction had a weak positive droplet, and the LOB was 1 copy / reaction, that is, the samples with 1 or less positive droplets in the test results were judged as negative samples. The specific results are shown in Figure 1 In the figure, the blue horizontal line is the threshold line, the droplets below the threshold line are negative droplets, and the droplets above the threshold line are positive droplets (same as the following embodiment figures).

[0056] Test Example 2 Performance Verification-LOD Using the reaction system of Example 3 and the reaction procedure of Table 5, the lowest detection limit samples (<0.5% methylation) were tested repeatedly for multiple times, where 0.2% and 0.5% methylation samples were tested 10 times respectively. The specific results are shown in Figure 2 In the figure, 0.2% and 0.5% methylation samples were detected normally in both the internal reference and target channels 10 times, and the minimum detection limit of the kit of the present invention was 0.5%.

[0057] Test Example 3 Performance Verification-Precision Using the reaction system of Example 3 and the reaction procedure of Table 5, the sample with a methylation rate of 5% was tested 22 times. The specific results are shown in Figure 3 and Table 6. Among them, serial number 1 is a blank sample (NTC), and serial numbers 2 to 23 are samples with a methylation rate of 5%.

[0058] Table 6

[0059] Among them, uACTB is the unmethylated ACTB sequence, and mMGMT is the methylated MGMT sequence. It can be calculated from Table 6 that the average methylation ratio of 22 repeated tests is 4.89%, the standard deviation (SD value) is 0.26%, and the intra-assay precision (CV value) is 5.35%, which is a good precision.

[0060] Test Example 4 Performance Verification-Thermal Stability The thermal stability of the kit of the present invention was evaluated using the reaction system of Example 3 and the reaction program of Table 5. The blank control (nuclease-free water), negative control (unmethylated DNA), and positive control (containing methylated DNA) were incubated at 37°C for 3, 5, and 7 days. The test results are shown in the following table. There was no non-specific amplification in the blank control (NTC) and the negative control (NC), and the detection rate of the positive sample (PC) was 100%. The quantitative deviation was small (actual / theoretical <20%), the amplification efficiency was not significantly reduced, and the thermal stability was good.

[0061] Table 7

[0062] Test Example 5 Performance Verification - Freeze-Thaw Stability The freeze-thaw stability of the kit of the present invention was evaluated using the reaction system of Example 3 and the reaction program of Table 5. The blank control (nuclease-free water), negative control (unmethylated DNA), and positive control (containing methylated DNA) were repeatedly frozen and thawed 3, 5, and 7 times. The results are shown in the following table. There was no non-specific amplification in the blank control (NTC) and the negative control (NC), and the detection rate of the positive sample (PC) was 100%. The quantitative deviation was small (actual / theoretical <20%), the amplification efficiency was not significantly reduced, and the freeze-thaw stability was good.

[0063] Table 8

[0064] Comparison example The primers and probes were designed according to the conventional design method, wherein the upstream primer of MGMT and the probe of MGMT are shown in the following table, no artificial tailing sequence was used, and the remaining sequences were the same as those in Example 2.

[0065] Table 9

[0066] The conventional primer probe set (control example) and the optimized primer probe set (Example 3) were used to perform PCR amplification on methylated and unmethylated samples using the reaction system in Table 4 and the reaction program in Table 5. The concentration of the MGMT downstream primer in the reaction system used in the control example was 500 nM. The test results are as follows Figure 4 shown.

[0067] The results show that both the control example and Example 2 can effectively detect MGMT methylation, but the methylation samples of the traditional primer probe group have stratification, which is presumably due to the detection of sequences that are not fully methylated at the 5 detection sites. Although these sequences can be detected, their positive fluorescence signals are lower than those of sequences that are fully methylated at the 5 sites. The methylation samples of the optimized primer probe group do not have stratification, so the probe of the present invention has strong binding force and high specificity. Since the positive signal values ​​are more concentrated, it is more conducive to digital statistics, resulting in more accurate detection results.

[0068] Test Example 6 Testing of clinical samples The clinical samples were tested using the reaction system of Example 3 and the reaction procedure of Table 5. The test results are shown in the following table and Figure 5 As shown, sample No. 1 is a blank control, sample No. 2 is a positive control, and samples No. 3 to No. 8 are clinical samples. Samples with more than one positive droplet detected in the target channel are MGMT methylated samples.

[0069]

[0070] According to the clinical results, samples 736, 225, 1550, and 693 were MGMT+ (methylated), and samples 8244 and 8447 were MGMT- (unmethylated), and the test results were consistent with the clinical results. Among them, sample 8244 had a false positive droplet, which was within the LOB range, so the sample was MGMT- (unmethylated).

[0071] Although the present invention is described herein with reference to the illustrative embodiments of the present invention, the above embodiments are only preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above embodiments. It should be understood that those skilled in the art can design many other modifications and embodiments, which will fall within the scope and spirit of the principles disclosed in this application.

Claims

1. A human MGMT methylation detection reagent based on digital PCR, characterized in that The reagent comprises an MGMT primer-probe combination and an ACTB primer-probe combination; The MGMT primer-probe composition comprises: MGMT upstream primer F1, the sequence is shown in SEQ ID NO.1; MGMT downstream primer R1, the sequence is shown in SEQ ID NO.2; MGMT probe P1, the sequence is shown in SEQ ID NO. 3; The ACTB primer-probe composition comprises: ACTB upstream primer F, the sequence is shown in SEQ ID NO.4; ACTB downstream primer R, the sequence is shown in SEQ ID NO.5; ACTB probe P, the sequence is shown in SEQ ID NO.6; The MGMT probe P1 and ACTB probe P are respectively labeled with different detection groups.

2. The human MGMT methylation detection reagent according to claim 1, characterized in that The MGMT primer-probe composition further comprises: MGMT upstream primer F2, the sequence is shown in SEQ ID NO.7; MGMT downstream primer R2, the sequence is shown in SEQ ID NO.

8.

3. The human MGMT methylation detection reagent according to claim 1, characterized in that The detection group is selected from one or more of ALEX-350, FAM, VIC, TET, CAL FluorGold540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL FluorRed 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, Cy3, Cy5, Cy5.5, and Quasar 705; preferably, the MGMT probe P1 is labeled with ROX, and the ACTB probe P is labeled with HEX.

4. The human MGMT methylation detection reagent according to claim 1, characterized in that The MGMT probe P1 and ACTB probe P are also respectively labeled with a quenching group, and the quenching group is selected from one or more of DABCYL, BHQ1, BHQ2, ECLIPSE, and TAMRA.

5. A human MGMT methylation detection kit based on digital PCR, characterized in that The kit comprises the reagent shown in any one of claims 1 to 4.

6. The human MGMT methylation detection kit according to claim 5, characterized in that The kit also includes dNTPs, buffer, salt ions and polymerase.

7. The human MGMT methylation detection kit according to claim 5, characterized in that The kit also includes a blank control, a negative control and a positive control; the blank control is nuclease-free water; the negative control is non-methylated DNA; and the positive control is methylated DNA.

8. A method for detecting human MGMT methylation based on digital PCR, characterized in that The method comprises the following steps: (1) After nucleic acid extraction from the sample to be tested, bisulfite conversion treatment is performed; (2) Mixing the converted sample nucleic acid with a PCR reaction premix to obtain a reaction system; (3) randomly distributing the reaction system into more than 5,000 droplets, some of which contain sample nucleic acid and others do not, and the droplets are spread evenly in the reaction wells; (4) performing PCR amplification on the droplets in the reaction wells; (5) Taking fluorescent photos of the reaction wells and obtaining the number of methylated nucleic acids in the sample to be tested based on the number of droplets with signals in the reaction wells; The PCR reaction premix comprises the human MGMT methylation detection reagent according to any one of claims 1 to 4.

9. The method according to claim 8, characterized in that The PCR amplification procedure is: 95°C for 2 min; 95°C for 20 sec, 56°C for 60 sec, 30-50 cycles; the fluorescence photography temperature is 35°C.

10. The method according to claim 8, characterized in that The sample to be tested is a FFPE sample or an EDTA anticoagulated peripheral whole blood sample.

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