Human MLH1 methylation detection reagent based on digital PCR

Abstract

The invention discloses a human MLH1 methylation detection reagent based on digital PCR (Polymerase Chain Reaction). The reagent comprises a methylation primer probe combination of MLH1, and the combination comprises an MLH1 upstream primer with a sequence as shown in SEQ ID NO.1, an MLH1 downstream primer with a sequence as shown in SEQ ID NO.2 and an MLH1 probe with a sequence as shown in SEQ ID NO.3. The invention further discloses a kit for detecting the methylation of the human MLH1 based on the digital PCR. The invention further discloses a human MLH1 methylation detection kit and a human MLH1 methylation detection method based on digital PCR. The reagent, the kit and the detection method are high in specificity and good in accuracy.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a human MLH1 methylation detection reagent based on a digital PCR method. Background Art

[0002] The MLH1 gene is an important component of the DNA mismatch repair (MMR) gene and is expressed in a variety of tissues. The MLH1 gene contains 19 exons and is located on chromosome 3p22.2. The promoter region of the MLHI gene is a high-density CpG island distribution area that is easily methylated. After the CpG island in the promoter region is methylated, the MLH1 gene is silenced, resulting in the inactivation of the DNA mismatch repair gene, so that the mismatched gene cannot be repaired in time, causing the loss of MMR protein expression, thereby leading to the occurrence of colorectal tumors or endometrial cancer. Therefore, the detection of MLH1 methylation is helpful for the diagnosis of colorectal tumors or endometrial cancer, but no relevant digital PCR detection kits have been developed yet.

[0003] The detection methods for determining MLH1 gene promoter methylation in the prior art 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.

[0004] 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, which is cumbersome and may result in false positives.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] Therefore, it is urgent to develop a human MLH1 methylation detection kit based on a new method to overcome the problems existing in the existing technology. Summary of the invention

[0009] 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.

[0010] 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.

[0011] 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 errors in the interpretation of 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.

[0012] 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.

[0013] Based on this, one of the purposes of the present invention is to provide a human MLH1 methylation detection reagent based on digital PCR, the reagent comprising a MLH1 methylation primer probe combination, the MLH1 methylation primer probe combination comprising: MLH1 upstream primer, the sequence is shown in SEQ ID NO.1; MLH1 downstream primer, the sequence is shown in SEQ ID NO.2; MLH1 probe, the sequence is shown in SEQ ID NO.3.

[0014] In one embodiment, the reagent further comprises an ACTB primer-probe combination; the ACTB primer-probe combination comprises: ACTB upstream primer, the sequence is shown in SEQ ID NO.4; ACTB downstream primer, the sequence is shown in SEQ ID NO.5; ACTB probe, the sequence is shown in SEQ ID NO.6; The MLH1 probe and ACTB probe are respectively labeled with different detection groups.

[0015] 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; In a preferred embodiment, the MLH1 probe is labeled with ROX, and the ACTB probe is labeled with HEX.

[0016] In another embodiment, the MLH1 probe and ACTB probe are further labeled with a quencher group, respectively, and the quencher group is selected from one or more of DABCYL, BHQ1, BHQ2, ECLIPSE, and TAMRA.

[0017] Another object of the present invention is to provide a human MLH1 methylation detection kit based on digital PCR, wherein the kit comprises the reagents as shown above.

[0018] In one embodiment, the kit further comprises dNTPs, a buffer, salt ions and a polymerase.

[0019] 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.

[0020] 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.

[0021] 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).

[0022] 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.

[0023] In certain preferred embodiments, provided compositions include Tris-HCl, ammonium sulfate, and magnesium chloride.

[0024] 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.

[0025] 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.

[0026] Another object of the present invention is to provide a method for detecting human MLH1 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 MLH1 methylation detection reagent.

[0027] In one embodiment, the PCR amplification procedure is: 98°C for 2 min; 98°C for 15 sec, 54°C for 30 sec, and 30 to 50 cycles; the fluorescence photography temperature is 58°C.

[0028] In another embodiment, the method further comprises: after performing PCR amplification reaction on the droplets in the reaction well, reacting at 110° C. for 10 minutes.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The invention discloses a primer and a probe for detecting the methylation rate of the MLH1 gene in a sample. The primer and probe reagent, the kit and the detection method of the invention have high specificity and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The test results of the kit of the present invention and the kit of the reference example MLH1 are shown in the figure; in the figure, 9 and 5 are blank controls, 11 and 7 are positive controls 1, and 12 and 8 are positive controls 2; 9, 11, and 12 are PCR reactions performed using the reagents of the reference example, and 5, 7, and 8 are PCR reactions performed using the reagents of Example 2 of the present invention; Figure 2 The specific detection result of the kit of the present invention; Figure 3 is the test result of the sensitivity of the kit of the present invention; Figure 4 The test result of the precision of the kit of the present invention; Figure 5 The stability test result of the kit of the present invention; Figure 6It is the detection linear relationship of different methylation rates of the kit of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be further described and illustrated below in conjunction with the embodiments of the present invention.

[0032] 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.

[0033] In order to study the methylation of the MLH1 gene, existing samples usually select diseased tissues, peripheral blood samples or stool samples. Among the above samples, diseased tissues are difficult to obtain, and the content of tumor DNA in peripheral blood samples or stool samples is low. How to accurately detect these low-content tumor DNA is a technical difficulty in this field.

[0034] Based on this, the present invention chooses to use digital PCR technology with high detection sensitivity to develop a human MLH1 methylation detection reagent. However, during the development process, the primer-probe combination obtained according to the conventional design method is applied to the digital PCR instrument to detect peripheral blood samples or FFPE samples (tissue section samples fixed by formalin and embedded in paraffin). There is still a problem of insufficient sensitivity. 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 sample 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 MLH1 methylation, it is necessary to limit the length of the amplicon of the detection target. In the present invention, the CpG site of the MLH1 gene most related to cancer is located in the gene promoter region from -197 to -392 bp; therefore, the design of primers and probes is selected within this region.

[0035] The conventional TaqMan probe design length is 25~32bp, and the annealing temperature (Tm value) is 55~65℃; if the probe is too short, the probe Tm value will be too low and cannot hybridize to the target sequence, and if it is too long, the probe specificity will be reduced; after bisulfite treatment, the unmethylated C base on the detection target will become a U base, resulting in a decrease in the overall CG content. In order to maintain the Tm value of the probe within the range of 55~65℃, the design length of the probe needs to be increased. However, in the detection of SNP or tumor mutations, in order to improve specificity, the probe is often designed to be shorter. The existing technology usually modifies the probe by means of locked nucleic acid to increase the Tm value, but the modification will increase the synthesis cost of the probe. In addition, too much modification will make it difficult to hydrolyze the probe, thereby reducing the generation of fluorescent signals.

[0036] The present invention selects to design the primer probe in the most relevant region, and considering the presence of fragmented DNA, in order to improve the detection sensitivity, the design length of the probe is controlled as much as possible. Since the probe length is short and the Tm value is too low, it is difficult for the probe to hybridize to the target sequence. For this reason, the present invention designs tailing sequences on the primer and the probe respectively, and improves the Tm value of the probe by complementarity of the primer tailing sequence and the probe tailing sequence. In summary, the present invention can achieve the improvement of the probe specificity while ensuring a suitable Tm value.

[0037] In the following exemplary experiment, 30 mg of nucleic acid sample was extracted from paraffin sections and treated with bisulfite.

[0038] The optional nucleic acid extraction method is the method described in the Tiangen Paraffin-embedded Tissue DNA Extraction Kit product manual; the optional bisulfite conversion method is the method described in the ZYMO EZ DNA Methylation-Gold™ Kit product manual.

[0039] Example 1 A human MLH1 methylation detection reagent based on digital PCR, the reagent comprising a primer probe combination as shown in the following table.

[0040] Table 1

[0041] In this embodiment, the 5' end of the MLH1 upstream primer contains 13 bp that do not match the detection target sequence, but are partially complementary to the 3' end of the probe. The 3' end of the MLH1 probe contains 13 bases that do not match the detection target sequence, but are complementary to the sequence at the 5' end of the corresponding upstream primer. The upstream primer and the downstream primer of the present invention are both designed to cover the area with the CpG island. Considering that whether the CpG is methylated will affect the final amplification efficiency, the upstream primer and the downstream primer of the present invention both set the coverage area of ​​the CpG island at a position outside the 3' end 4bp, thereby reducing the impact on the amplification efficiency as much as possible. At the same time, the MLH1 probe is designed to cover 1 CpG island. If the sample is an unmethylated MLH1 gene, the MLH1 methylation probe cannot be complementary to it and will not emit light. Therefore, the design position of the primer probe of the present invention is very important. The design of the primer needs to avoid the poor amplification efficiency and poor detection sensitivity caused by inadequate design of the CpG coverage position, and avoid the problem of nonspecific amplification; the design of the probe needs to consider the lack of specificity and low sensitivity due to too long length, and avoid the problem of insufficient Tm value due to too short length.

[0042] Example 2 The present invention preferably uses the housekeeping gene (Housekeeping Gene) on the human genome, the β-actin gene (ACTB gene), as an internal reference gene. This type of gene is stably expressed in all cells. Therefore, the methylation rate of MLH1 can be accurately calculated by the MLH1 methylation value / ACTB quantitative value. A human MLH1 methylation detection reagent based on digital PCR of the present invention includes a primer probe combination as shown in the following table.

[0043] Table 2

[0044] In this embodiment, the ACTB primer probe is designed to be complementary to the bisulfite-converted ACTB gene. The methylation rate of the present invention is calculated as the quantitative value of methylation of the MLH1 gene / the quantitative value of the ACTB internal reference gene.

[0045] Example 3 A human MLH1 methylation detection kit based on digital PCR, the kit comprising the reagent of Example 2 and 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 MLH1 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 8.5, 0.5mmol / L dNTPs, 3U LionTaq hot start enzyme and 70mmol / L (NH4)2SO4.

[0048] The MLH1 probe of the present invention does not or rarely emits fluorescence by polymerase hydrolysis, but emits fluorescence by changing the conformation of the probe after hybridization with the amplification product. Therefore, the amount of the downstream primer of the MLH1 gene used is 1 / 2 of the upstream primer.

[0049] Example 4 The kit of the present invention is applied to the D 600 series digital PCR instrument of Microbiota. The detection of MLH1 is completed by preparing microdroplets and then performing PCR amplification. The specific amplification procedure involved in the detection is shown in Table 5.

[0050] Table 5

[0051] The present invention can change the fluorescence intensity of hybridization by changing the photographing temperature of the product, and no fluorescence is emitted when the probe and the target sequence are completely unlinked. In the present invention, the photographing temperature is set at 58°C, at which temperature, both positive and negative droplets in the reaction well can be identified, and the signal-to-noise ratio is good.

[0052] Comparative Example 1 The probe of this control example was designed as the sequence (10 bp) without tail in Example 2, and the detection result was that no positive signal could be detected. It is speculated that this may be because the probe is too short and cannot be well complementary to the amplicon, so there is no detectable signal.

[0053] Comparative Example 2 The conventional primer and probe design method is used, and no tailing sequence is designed on the upstream primer and the probe. The specific primer and probe are shown in the following table.

[0054] Table 6

[0055] The primer probes of Example 2 and Control Example 2 were used to perform PCR reaction in the reaction system of Example 3 and the reaction program of Table 5. The concentration of the MLH1 downstream primer in the reaction system used in Control Example 2 was 500 nM. Figure 1 shown.

[0056] After the primer probe was tailed, the blank control did not produce false positives, and from the detection of positive samples, compared with the primer probe of Control Example 2, the primer probe designed by the present invention significantly opened up the threshold division of detection. It can be considered that the tailing part of the primer effectively helps the probe to target, thereby improving the accuracy of detection. In addition, because the complementary region between the probe and the target is shorter, the specificity of detection can be effectively improved compared with Control Example 2.

[0057] Test Example 1 Performance Verification - Specificity Using the reagents of Example 2, PCR reaction was performed under the reaction system of Example 3 and the reaction program of Table 5, and 20 methylated samples were tested. The test results are as follows: Figure 2 As shown. The detection results of the 20 unmethylated samples in this embodiment are all 0 copies / μL. Therefore, the detection specificity is good. In the figure, NTC is a blank control, the positive control is a MHL1 methylated sample with 10 copies / μL, and the negative control is an unmethylated sample.

[0058] Test Example 2 Performance Verification-Sensitivity Using the reagents of Example 2, PCR reaction was performed under the reaction system of Example 3 and the reaction program of Table 5, and the sensitivity sample with a methylation rate of 0.6% was tested 20 times. The test results are shown in the following table and Figure 3 In the figure, NTC is the blank control, the positive control is the MHL1 methylation sample with 10 copies / μL, the negative control is the unmethylated sample, and the remaining 20 samples are all sensitive samples with a methylation rate of 0.6%. The blue horizontal line in the figure is the threshold line, and the droplets below the threshold line are negative droplets, and the droplets above the threshold line are positive droplets, and their positive detection rates are all 100%.

[0059] Table 7

[0060] Test Example 3 Performance Verification-Precision Using the reagents of Example 2, PCR reaction was performed under the reaction system of Example 3 and the reaction program of Table 5, and 20 precision samples were tested. The methylation rate of the precision samples was 5.4%. The test results are shown in the following table and Figure 4 In the figure, NTC is the blank control, the positive control is the MHL1 methylation sample with 10 copies / μL, the negative control is the unmethylated sample, and the remaining 20 samples are precision samples with a methylation rate of 5.4%. The blue horizontal line in the figure is the threshold line, and the droplets below the threshold line are negative droplets, and the droplets above the threshold line are positive droplets. The detection precision (CV value) is 9.18%.

[0061] Table 8

[0062] Test Example 4 Performance Verification-Stability The reaction system of Example 3 of the present invention was placed in an environment of 37°C for thermal acceleration, and then the stability of the thermally accelerated reagent was measured using samples with methylation rates of 20%, 10%, and 5%. The measurement results are shown in the following table and Figure 5 As shown, 8 samples were tested in each group, including NTC (blank control), negative control and 2 samples with methylation rates of 20%, 10% and 5% respectively. The blue horizontal line in the figure is the threshold line, and the droplets below the threshold line are negative droplets, and the droplets above the threshold line are positive droplets. After testing, the thermal stability is good.

[0063] Table 9

[0064] The reaction system of Example 3 of the present invention was placed in an environment of -20°C and subjected to freeze-thaw treatment. Then, the stability of the reagents after freeze-thaw was measured using samples with methylation rates of 20%, 10%, and 5%. The results are shown in the following tables and Figure 5 As shown, 8 samples were tested in each group, namely NTC (blank control), negative control and 2 samples with methylation rates of 20%, 10% and 5% respectively. The blue horizontal line in the figure is the threshold line, the droplets below the threshold line are negative, and the droplets above the threshold line are positive. After testing, the freeze-thaw stability is good.

[0065] Table 10

[0066] The results show that the reaction system of the present invention can be stably detected after 3 days and 5 days of thermal acceleration at 37°C, or after 5 and 7 freeze-thaw cycles at -20°C.

[0067] Test Example 5 Performance Verification - Linear Relationship Using the reagents of Example 2, PCR reaction was performed under the reaction system of Example 3 and the reaction program of Table 5, and samples with methylation rates of 70% to 1.1% were tested. The test results are shown in the following table and Figure 6 As shown in the table, NTC is the blank control, the positive control is the MHL1 methylation sample with 10 copies / μL, the negative control is the unmethylated sample, and sample numbers 5 to 11 are samples with methylation rates diluted 2 times. After repeating the test 2 to 3 times for each gradient dilution sample, the average value was calculated and the linear relationship was fitted to obtain R 2 ≥0.98, the detection linear relationship of the kit of the present invention is good.

[0068] Table 11

[0069] 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 MLH1 methylation detection reagent based on digital PCR, characterized in that The kit includes a primer-probe combination for MLH1 methylation. The MLH1 methylation primer probe combination includes: MLH1 upstream primer, the sequence is shown in SEQ ID NO.1; MLH1 downstream primer, the sequence is shown in SEQ ID NO.2; MLH1 probe, the sequence is shown in SEQ ID NO.

3.

2. The human MLH1 methylation detection reagent according to claim 1, characterized in that Also included is an ACTB primer-probe combination; the ACTB primer-probe combination includes: ACTB upstream primer, the sequence is shown in SEQ ID NO.4; ACTB downstream primer, the sequence is shown in SEQ ID NO.5; ACTB probe, the sequence is shown in SEQ ID NO.6; The MLH1 probe and ACTB probe are respectively labeled with different detection groups.

3. The human MLH1 methylation detection reagent according to claim 2, 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, CALFluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, Cy3, Cy5, Cy5.5, and Quasar 705; preferably, the MLH1 probe is labeled with ROX, and the ACTB probe is labeled with HEX.

4. The human MLH1 methylation detection reagent according to claim 2, characterized in that The MLH1 probe and ACTB probe 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 MLH1 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 MLH1 methylation detection kit according to claim 5, characterized in that The kit also includes dNTPs, buffer, salt ions and polymerase.

7. The human MLH1 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 MLH1 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 MLH1 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: 98°C for 2 min; 98°C for 15 sec, 54°C for 30 sec, 30-50 cycles; the fluorescence photography temperature is 58°C.

10. The method according to claim 8, characterized in that After the PCR amplification reaction is performed on the microdroplets in the reaction wells, the reaction is carried out at 110° C. for 10 minutes.

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