Human bmp3 and ndrg4 gene methylation detection kit

Abstract

The present application relates to a human BMP3 and NDRG4 gene methylation detection kit, the kit comprises a restriction enzyme reaction solution, the restriction enzyme reaction solution comprises a restriction enzyme buffer, a methylation-dependent restriction enzyme, and further comprises a fluorescent PCR reaction solution of primers and a partial double-stranded linear DNA probe designed for a CpG island of a BMP3 gene and / or a NDRG4 gene. The detection kit can realize the sequential restriction enzyme reaction and fluorescent PCR reaction in one tube to detect the methylation state of the BMP3 and NDRG4 genes, and has the characteristics of simple and rapid operation, high sensitivity, good specificity and easy automation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a human BMP3 and NDRG4 gene methylation detection kit. TECHNICAL BACKGROUND

[0002] Disturbance of epigenetic program is an important cause of colorectal cancer (Liu R et al., Mutat Res, 2019, 779:45-57). DNA methylation alteration is the most important epigenetic change, which controls the transcription and expression of specific genes and plays an important role in the occurrence of colorectal cancer. The silencing of methylated genes often occurs in the early and late stages of carcinogenesis in the focal lesions of adenoma-carcinoma cascade. Therefore, methylation alteration drives the occurrence and progression of colorectal cancer. Detection of gene methylation may help to identify individuals with early colorectal cancer and may help to improve the disease outcome.

[0003] BMP3, i.e. bone morphogenetic protein 3, is a gene encoding a secreted ligand of transforming growth factor beta (TGF-beta) superfamily proteins, which has an important function in bone formation. BMP3 gene methylation can be detected in a variety of tumors, including colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, breast cancer and cholangiocarcinoma (Kisiel JB et al., J Mol Biomark Diagn, 2013, 4:1000145). In colorectal cancer, abnormal methylation of the BMP3 gene can down-regulate the expression of BMP3, and BMP3 inactivation is an early and common event in the occurrence of colorectal cancer (Loh K et al., Genes Chromosomes Cancer, 2008, 47:449-60). Studies have shown that the overall sensitivity and specificity of fecal sample BMP3 methylation in the diagnosis of colorectal cancer are 70% and 89%, respectively (Liu R et al., Mutat Res, 2019, 779:45-57).

[0004] NDRG4, N-Myc downstream regulated gene 4, is a member of the NDRG gene family, which is involved in cell proliferation, differentiation, development and stress (Shi HH et al., BMB Rep, 2020, 53:658-663). NDRG4 plays multiple roles in human malignancies (Shi HH et al., BMB Rep, 2020, 53:658-663). In glioblastoma, NDRG4 acts as an oncogene, which is essential for the survival of astrocytes; in malignant meningioma, NDRG4 plays an oncogenic role by inhibiting p53 expression to inhibit apoptosis. However, NDRG4 has a tumor suppressor role in other tumors such as colorectal cancer, gastric cancer, breast cancer, and pancreatic cancer. NDRG4 promoter methylation can be detected in gastric cancer, colorectal cancer, breast cancer, and pancreatic cancer, and can lead to down-regulation of NDRG4 expression. Studies have shown that the overall sensitivity and specificity of NDRG4 methylation in the diagnosis of colorectal cancer are 76% and 82%, respectively (Liu R et al., Mutat Res, 2019, 779:45-57).

[0005] Given the high diagnostic value of BMP3 and NDRG4 for colorectal cancer screening, respectively, in recent years, the combined detection of BMP3 and NDRG4 gene methylation in the diagnosis of colorectal cancer has also attracted much attention. Studies have shown that the combination of BMP3 and NDRG4 gene methylation status in fecal samples is a very useful marker, with a sensitivity and specificity of 89% and 87%, respectively, in the diagnosis of colorectal cancer (Liu R et al., Mutat Res, 2019, 779:45-57). It can be seen that the combined detection of BMP3 and NDRG4 gene methylation has better diagnostic performance in colorectal cancer screening.

[0006] Currently, the main method for detecting BMP3 and NDRG4 gene methylation is fluorescence PCR based on bisulfite conversion. This method uses bisulfite to treat DNA samples, converting unmethylated cytosine to uracil, and then using specific primers and fluorescent probes for fluorescence PCR with the bisulfite-treated DNA sample as the template. The methylation status is determined according to the detection of fluorescent signals, such as Chinese patent applications CN201510486088.7 and CN201710090789.8. Although the kits described in the above two Chinese patent applications have good sensitivity and / or specificity, they fail to address the shortcomings caused by bisulfite conversion, such as: the conversion conditions are harsh and can easily cause DNA sample degradation; DNA purification and recovery are required after conversion, which is a tedious operation; incomplete conversion can cause biased test results; etc. Therefore, it is necessary to provide a BMP3 and NDRG4 gene methylation detection kit that does not require bisulfite conversion, is simple and rapid to operate, and is accurate in detection. SUMMARY

[0007] Based on this, the application provides a human BMP3 and NDRG4 gene methylation detection kit, which utilizes methylation-dependent restriction enzymes and a specially designed multiplex fluorescent PCR system to simultaneously detect BMP3 and NDRG4 gene methylation by simultaneously performing enzyme digestion and multiplex fluorescent PCR reactions, and has the characteristics of simple and rapid operation, high sensitivity and good specificity.

[0008] To achieve the above-mentioned purposes, the technical solutions of the application include the following.

[0009] The human BMP3 and / or NDRG4 gene methylation detection kit comprises an enzyme digestion reaction solution, and the enzyme digestion reaction solution comprises methylation-dependent restriction enzymes.

[0010] The fluorescent PCR reaction solution also comprises primers and partial double-stranded linear DNA probes designed for the CpG islands of the BMP3 gene and / or the NDRG4 gene;

[0011] The partial double-stranded linear DNA probe for each gene comprises a fluorescent probe with a long number of bases and a quencher probe with a short number of bases; the fluorescent probe is labeled with a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; the quencher probe is completely complementary to the 5' end of the fluorescent probe and is labeled with a fluorescent quencher group at the 3' end; and the fluorescent reporter groups for different genes are different.

[0012] In some embodiments, the primers for the BMP3 gene are as shown in SEQ ID NO. 1 to SEQ ID NO. 2, and / or the primers for the NDRG4 gene are as shown in SEQ ID NO. 5 to SEQ ID NO. 6.

[0013] In some embodiments, the probe for the BMP3 gene comprises a fluorescent probe as shown in SEQ ID NO. 3 and a quencher probe as shown in SEQ ID NO. 4; and / or the probe for the NDRG4 gene comprises a fluorescent probe as shown in SEQ ID NO. 7 and a quencher probe as shown in SEQ ID NO. 8.

[0014] In one embodiment, the fluorescent reporter group comprises FAM, VIC, ROX or CY5; and the fluorescent quencher group is BHQ. When there is no template amplification in the fluorescent PCR system, the fluorescent probe is combined with the quencher probe, so that the fluorescent reporter group and the quencher group are extremely close, effectively quenching the fluorescent signal; when there is template amplification in the system, the fluorescent probe is preferentially combined with the amplification product, separated from the quencher probe, and releases the fluorescent signal in the PCR reaction.

[0015] In some embodiments, the methylation-dependent restriction enzyme used to digest the methylated BMP3 and NDRG4 genes in the DNA sample is selected from any one of MspJI, FspEI and LpnPI; preferably, the methylation-dependent restriction enzyme is MspJI, and the amount of MspJI used is 1-2 U per reaction; more preferably, the amount of MspJI used is 1.5 U per reaction.

[0016] The enzyme digestion reaction solution further comprises an enzyme digestion buffer, an enzyme activity solution, and nuclease-free water; the enzyme activity solution is used to stimulate the digestion of the methylation-dependent restriction enzyme, and is a solution of short double-stranded oligonucleotides dissolved in 10 mM Tris-HCl (pH 8.0), wherein the short double-stranded oligonucleotides are in a stem-loop structure and comprise two methylation sites; preferably, the sequence of the short double-stranded oligonucleotides is as shown in SEQ ID NO. 13, and the enzyme activity solution is a solution containing short double-stranded oligonucleotides at a concentration of 150 ± 1 nM.

[0017] In some embodiments, the enzyme digestion buffer is an aqueous solution containing 50 ± 2 mM potassium acetate, 20 ± 1 mM Tris-acetate, 10 ± 1 mM magnesium acetate, and 100 ± 5 μg / ml recombinant albumin (pH 7.9 at 25°C).

[0018] The fluorescent PCR reaction solution further comprises a PCR buffer, dNTPs, primers, a probe, a DNA polymerase, betaine, tetramethylammonium chloride, and nuclease-free water.

[0019] In some embodiments, the concentration of betaine in the fluorescent PCR reaction solution is 100 mM-150 mM; preferably, the concentration of betaine is 125 ± 5 mM. The addition of betaine can reduce the dependence of denaturation temperature on bases, improve the stability of the DNA polymerase, and reduce the secondary structure of GC-rich templates to facilitate the PCR amplification of GC-rich templates.

[0020] In some embodiments, the concentration of tetramethylammonium chloride in the fluorescent PCR reaction solution is 10 mM-20 mM; preferably, the concentration of tetramethylammonium chloride is 15 ± 1 mM. The addition of tetramethylammonium chloride can remove non-specific initiation and reduce mispairing, thereby improving the specificity of the PCR reaction and the specificity of hybridization.

[0021] In some embodiments, the fluorescent PCR reaction solution further comprises an internal reference gene primer and a probe. The internal reference gene primer and the probe are designed for the CpG island of the ACTB gene, and preferably, are as shown in SEQ ID NO. 8-SEQ ID NO. 12.

[0022] In some embodiments, the enzyme cutting reaction solution and the fluorescent PCR reaction solution are layered and packaged in the same PCR amplification tube by a hot melt material, and in combination with other designs, a one-tube enzyme cutting / fluorescent PCR system can be realized to simplify the detection operation.

[0023] The hot melt material is a fully refined paraffin with a melting point of 70-72 DEG C.

[0024] In some embodiments, negative quality control and positive quality control are further included. The negative quality control is composed of WBC DNA, BSA and TE buffer which are unmethylated with BMP3 and NDRG4 genes; the positive quality control is composed of cell strain DNA which is methylated with BMP3 gene, cell strain DNA which is methylated with NDRG4 gene, WBC DNA, BSA and TE buffer. The negative quality control and the positive quality control can ensure the effectiveness of the kit, and can better prevent the generation of false positive results and false negative results, thereby ensuring the accuracy and reliability of the detection results.

[0025] Another object of the present application is to provide a use method of the above-mentioned kit.

[0026] The use method of the kit comprises the following steps:

[0027] (1) obtaining a DNA sample to be detected;

[0028] (2) enzyme cutting / fluorescent PCR reaction: adding 1 μl of the DNA sample to be detected into the enzyme cutting reaction solution, and sequentially performing enzyme cutting reaction and fluorescent PCR reaction on a fluorescent PCR instrument, and the reaction conditions are set as follows: 37 DEG C enzyme cutting for 1 hour, 65 DEG C inactivation for 20 minutes, 1 cycle; 95 DEG C pre-denaturation for 5 minutes, 1 cycle; 95 DEG C denaturation for 20 seconds, 62 DEG C annealing / elongation for 30 seconds (the fluorescence signal is collected at this step), 40 cycles.

[0029] The present application mainly has the following beneficial effects:

[0030] (1) The present application realizes methylation detection by using methylation-dependent restriction enzyme combined with fluorescent PCR.

[0031] The methylation-dependent restriction enzyme of the present application is MspJI which is preferred according to the methylated BMP3 sequence and the methylated NDRG4 sequence, and the enzyme can recognize m CNNR site, and realizes the detection of methylated nucleic acid sequence in mThe enzyme cutting at N9 / N13 on the 3' side of the CNNR site. The enzyme has better and more stable detection effect on the amplification efficiency when used for BMP3 and NDRG4 gene methylation detection by the specific primers and probes designed in the application, and can effectively and accurately detect the non-methylation sequence and methylation sequence of BMP3 and NDRG4 genes.

[0032] (2) The probe in the kit of the application adopts a partially double-stranded linear DNA probe system composed of a longer fluorescent probe and a shorter quenched probe. When applied to a fluorescent PCR reaction, the probe system can only release a fluorescent signal in the case of producing an amplification product, and the fluorescent probe can effectively quench the fluorescent signal of the probe itself in the case of no amplification product. The suitable probe designed according to the application effectively reduces the background signal and improves the accuracy of detection. It has high sensitivity.

[0033] (3) The primers and probes designed in the application, in combination with a suitable fluorescent PCR reaction solution, can realize triple fluorescent PCR reaction, and detect BMP3 gene methylation, NDRG4 gene methylation and an internal reference gene in a single tube at one time. Not only is the reagent consumable greatly saved, but also the detection time is shortened, and the detection result is accurate. In addition, the kit of the application is suitable for various sample types, and can be used for fecal DNA samples, plasma DNA samples, cell DNA samples, tissue DNA samples and the like.

[0034] (4) Betaine and tetramethylammonium chloride are added in the fluorescent PCR reaction solution of the application. The betaine can reduce the dependence of denaturation temperature on bases, improve the stability of DNA polymerase, and reduce the secondary structure of GC-rich templates to promote the PCR amplification of GC-rich templates. The tetramethylammonium chloride can remove non-specific initiation and reduce error binding, thereby improving the specificity of PCR reaction and the specificity of hybridization. Therefore, the addition of betaine and tetramethylammonium chloride helps to improve the sensitivity and specificity of fluorescent PCR detection. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The figure is a fluorescent PCR amplification curve for BMP3 and NDRG4 methylation detection by the partially double-stranded linear DNA probe system of the application and the traditional fluorescent probe in Example 3. Among them, (a) the partially double-stranded linear DNA probe system of the application detects unmethylated WBC DNA; (b) the partially double-stranded linear DNA probe system of the application detects BMP3 and NDRG4 methylated cell line DNA; (c) the traditional fluorescent probe detects unmethylated WBC DNA; (d) the traditional fluorescent probe detects BMP3 and NDRG4 methylated cell line DNA.

[0036] Figure 2The fluorescence PCR amplification curve of 1 ng DNA sample with 1% methylation DNA detected in Example 5 of the present application.

[0037] Figure 3 The fluorescence PCR amplification curve of the negative quality control of the present application.

[0038] Figure 4 The fluorescence PCR amplification curve of the positive quality control of the present application. DETAILED DESCRIPTION

[0039] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology, cell biology, immunology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Third Edition (2002). Unless otherwise indicated, the methods of experimentation set forth in the following examples were conducted in accordance with conventional techniques or as recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0041] The present application relates to a human BMP3 and NDRG4 gene methylation detection kit, comprising a restriction enzyme reaction solution and a fluorescence PCR reaction solution, which are layered and packaged in the same PCR amplification tube by hot melt material.

[0042] The restriction enzyme reaction solution contains a suitable methylation-dependent restriction enzyme.

[0043] The methylation-dependent restriction enzyme can recognize the methylation site and cut the double-stranded DNA near the 3' side of the methylated cytosine. When the enzyme is used to treat the DNA sample to be detected, the enzyme can recognize the methylated nucleic acid sequence and have the enzyme cutting activity, but cannot recognize and cut the non-methylated nucleic acid sequence, so that the methylated nucleic acid sequence is cut into small fragments, and the non-methylated nucleic acid sequence is kept intact; then, the specific primers located at both sides of the cutting site or at the cutting site and the probe located at the cutting site are used to prepare a fluorescent PCR reaction solution for the fluorescent PCR reaction of the DNA sample to be detected after the enzyme cutting, in which process, if the methylation modification does not occur in the detection region, the DNA sample to be detected is not cut and can be combined with the specific primers and the probe to generate a fluorescent signal, and if the methylation modification occurs in the detection region, the DNA sample to be detected is cut into small fragments and cannot be combined with the specific primers and the probe to generate no fluorescent signal. Compared with the fluorescent PCR method based on bisulfite conversion, the DNA degradation caused by bisulfite conversion or the detection result bias caused by incomplete conversion is avoided, so that the detection sensitivity is improved; after the DNA sample is cut, the fluorescent PCR detection can be directly performed without the purification and recovery steps, the operation steps are reduced, the detection cost and time are saved, and the automation is facilitated.

[0044] The detection kit provided by the application adopts a one-tube enzyme cutting / fluorescent PCR system, that is, a hot melt material with a melting point of 70-72℃ is used to separate the enzyme cutting reaction solution and the fluorescent PCR reaction solution, the enzyme cutting reaction solution is located in the upper layer of the hot melt material, and the enzyme cutting and the enzyme inactivation of the DNA to be detected can be realized without melting the hot melt material; after the enzyme inactivation, the PCR reaction is directly performed, when the temperature rises to 95℃, the hot melt material separating the enzyme cutting reaction solution and the fluorescent PCR reaction melts and floats to the upper layer of the reaction system (the density of the hot melt material is lower than that of the reaction solution), the fluorescent PCR reaction solution is mixed with the upper layer enzyme cutting product, and the fluorescent PCR detection of the enzyme cutting product is realized. The one-tube enzyme cutting / fluorescent PCR system realizes the enzyme cutting reaction and the fluorescent PCR reaction in the same tube in sequence, and compared with the prior art, the operation is simple and fast, and the automation is easy.

[0045] The application will be further described below with reference to the examples. It should be understood that the following examples are given only for the purpose of illustration, and are not used to limit the scope of the application. Those skilled in the art can make various modifications and replacements to the application without departing from the purpose and spirit of the application.

[0046] Example 1 Preparation of the human BMP3 and NDRG4 gene methylation detection kit

[0047] The human BMP3 and NDRG4 gene methylation detection kit comprises an enzyme cutting / fluorescent PCR reaction system, negative quality control and positive quality control. The preparation of the kit comprises the following steps:

[0048] (1) Design and preparation of primers and probes: according to the information of methylation-dependent restriction enzyme cutting sites, a plurality of specific primers and probes are designed for the CpG islands of the BMP3 gene, the CpG islands of the NDRG4 gene and the CpG islands of the ACTB gene. Among them, the probes are designed as a partial double-stranded linear DNA probe system, which comprises a longer fluorescent probe and a shorter quenched probe; the fluorescent probe is labeled with a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end; the quenched probe is completely complementary to the 5' end of the fluorescent probe and is labeled with a fluorescent quencher group at the 3' end; the fluorescent reporter group comprises FAM, VIC, ROX or CY5; the fluorescent quencher group is BHQ. Each primer and probe is pre-tested to compare the sensitivity, specificity and other properties, and finally the primers and probes of the kit are selected, which are specifically shown in SEQ ID NO. 1 to SEQ ID NO. 12, and are shown in Table 1. The primers and probes are stored in a 100 μM stock solution, and a 10 μM working solution is prepared for standby according to the detection requirements.

[0049] Table 1 Sequences of primers and probes of the kit

[0050]

[0051]

[0052] (2) Preparation of fluorescent PCR reaction solution: the fluorescent PCR reaction solution is prepared according to the fluorescent PCR reaction solution preparation scheme of the present application, and is stored for standby. The fluorescent PCR reaction solution preparation scheme is specifically shown in Table 2.

[0053] Table 2 Fluorescent PCR reaction solution preparation scheme

[0054] Reagent name Per reaction (μl) PCR buffer 10 dNTPs (10 mM) 4 Primers (10 μM) Add 1 μl of each Probes (10 μM) Add 1 μl of each Betaine (5 M) 1 Tetramethylammonium chloride (200 mM) 3 DNA polymerase (5 U / μl) 0.4 Nuclease free water Add water to 40 μl Total volume 40

[0055] (3) Selection of methylation-dependent restriction enzyme: according to the methylation BMP3 sequence and the methylation NDRG4 sequence, a methylation-dependent restriction enzyme is selected. The methylation-dependent restriction enzyme is any one of MspJI, FspEI and LpnPI; preferably, the methylation-dependent restriction enzyme is MspJI. In the present application, the amount of MspJI used is 1-2 U per reaction; in this embodiment, the amount of MspJI used is 1.5 U per reaction.

[0056] (4) Preparation of enzyme active solution: The enzyme active solution is used to stimulate the digestion of methylation-dependent restriction enzyme, and is prepared by dissolving short double-stranded oligonucleotides in 10 mM Tris-HCl (pH 8.0), wherein the short double-stranded oligonucleotides are in a stem-loop structure and contain two methylation sites; preferably, the short double-stranded oligonucleotide sequence is: CTGC m CAGGATCTTTTTTGATC m CTGGCAG (SEQ ID NO. 13, wherein the superscript m represents methylation), and the enzyme active solution is a solution containing short double-stranded oligonucleotides at a concentration of 150 ± 1 nM.

[0057] (5) Preparation of enzyme digestion buffer: The enzyme digestion buffer is prepared according to the composition of the enzyme digestion buffer. The composition of the enzyme digestion buffer is as follows: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / ml recombinant albumin, nuclease-free water. The pH value of the enzyme digestion buffer is about 7.9 at 25°C.

[0058] (6) Preparation of enzyme digestion reaction solution: The enzyme digestion reaction solution is prepared according to the enzyme digestion reaction solution preparation scheme of the present application, and is stored for standby use. The enzyme digestion reaction solution preparation scheme is specifically as shown in Table 3.

[0059] Table 3 Enzyme digestion reaction solution preparation scheme

[0060] Reagent name Per reaction (μl) Cleavage buffer 1 Enzyme active solution 0.4 MspJI (5 U / μl) 0.3 Nuclease free water Add water to 9 μl Total volume 9

[0061] (7) Preparation of enzyme digestion / fluorescent PCR reaction system: The fluorescent PCR reaction solution prepared in step (2) is mixed and centrifuged, 40 μl of which is added to the bottom of a PCR amplification tube and is sealed with full-refined paraffin wax (manufacturer: Kunlun, model 70#) having a melting point of 70-72°C. After the full-refined paraffin wax is cooled and solidified, 9 μl of the enzyme digestion reaction solution prepared in step (6) is added to the PCR amplification tube in which the full-refined paraffin wax has been cooled and solidified. The enzyme digestion / fluorescent PCR reaction system can also be prepared in a PCR octuple tube.

[0062] (8) Preparation of negative and positive quality control: Add appropriate amount of BSA, BMP3 and NDRG4 gene unmethylated WBC DNA into TE buffer to prepare negative quality control, wherein the concentration of BSA is 0.1 μg / μl, and the concentration of BMP3 and NDRG4 gene unmethylated WBC DNA is 10 ng / μl; add appropriate amount of BSA, BMP3 gene methylated cell strain DNA, NDRG4 gene methylated cell strain DNA and WBC DNA into TE buffer to prepare positive quality control, wherein the concentration of BSA is 0.1 μg / μl, the concentration of BMP3 gene methylated cell strain DNA is 0.1 ng / μl, the concentration of NDRG4 gene methylated cell strain DNA is 0.1 ng / μl, and the concentration of WBC DNA is 9.8 ng / μl.

[0063] (9) Sub-packing and assembly of kit: The kit specification is 24 persons per box, and the sub-packing and assembly scheme is shown in Table 4.

[0064] Table 4 Sub-packing and assembly scheme of kit

[0065]

[0066] The whole detection process of the detection kit comprises the following steps:

[0067] (1) Enzymatic digestion / fluorescent PCR reaction: 1 μl of the DNA sample to be detected is added to the enzyme digestion reaction solution (located in the upper layer of the condensed full refined paraffin) of the enzyme digestion / fluorescent PCR reaction system, and is placed on a fluorescent PCR instrument to sequentially perform enzyme digestion reaction and fluorescent PCR reaction, and the reaction conditions are set as follows: 37℃ enzyme digestion for 1 hour, 65℃ inactivation for 20 minutes, 1 cycle; 95℃ pre-denaturation for 5 minutes, 1 cycle; 95℃ denaturation for 20 seconds, 62℃ annealing / elongation for 30 seconds (fluorescent signal is collected at this step), 40 cycles. The fluorescent channel of the fluorescent PCR instrument is selected as FAM, VIC and ROX channels.

[0068] (2) Interpretation of detection results: The detection results are interpreted according to the fluorescent signal Ct value detected by the fluorescent PCR instrument. If the Ct value of the ROX signal of the internal reference gene is greater than 30 or there is no Ct value, the detection result is invalid; if the Ct value of the ROX signal of the internal reference gene is less than or equal to 30, the detection result is valid, then the ΔCt value is calculated by the formula “BMP3 gene ΔCt value = Ct value of BMP3 gene FAM signal - Ct value of internal reference gene ROX signal” and “NDRG4 gene ΔCt value = Ct value of NDRG4 gene VIC signal - Ct value of internal reference gene ROX signal”, and the ΔCt value is used as the negative and positive judgment standard. ΔCt value less than or equal to 9.0: negative, ΔCt value greater than 9.0 or no ΔCt value: positive. The specific detection result judgment table is as follows:

[0069] Table 5 Kit detection result judgment table

[0070]

[0071]

[0072] Example 2 Comparative experiment of different methylation-dependent restriction enzymes

[0073] (1) Purpose of experiment

[0074] In this example, BMP3 and NDRG4 gene methylation was detected using different methylation-dependent restriction enzymes to compare the effects of different methylation-dependent restriction enzymes and the effects on kit detection.

[0075] (2) Experimental method

[0076] In this example, three enzyme digestion / fluorescent PCR reaction systems were prepared using MspJI, FspEI, and LpnPI as methylation-dependent restriction enzymes according to the preparation steps of the kit in Example 1; three enzyme digestion / fluorescent PCR reactions were performed on three portions of the same concentration of unmethylated WBC DNA and BMP3 and NDRG4 methylated cell strain DNA using the three enzyme digestion / fluorescent PCR reaction systems described above. The other components of the kit and the enzyme digestion / fluorescent PCR reaction were performed according to the detection steps in Example 1, and the DNA loading amount was 1 μl.

[0077] (3) Experimental results and analysis

[0078] The detection results are shown in the table below (N represents unmethylated WBC DNA, and P represents BMP3 and NDRG4 methylated cell strain DNA).

[0079] Table 6 Detection results of different methylation-dependent restriction enzymes

[0080]

[0081]

[0082] From the above table detection results can be known, using MspJI, FspEI and LpnPI preparation of enzyme digestion / fluorescent PCR reaction system can realize accurate detection, all unmethylated WBC DNA samples were detected as negative, all BMP3 and NDRG4 methylation of cell strain DNA samples were detected as BMP3 and NDRG4 methylation double positive. However, using MspJI preparation of enzyme digestion / fluorescent PCR reaction system detected Ct value of each sample is lower than FspEI and LpnPI preparation of enzyme digestion / fluorescent PCR reaction system, and its detected Ct value of each sample is more stable, indicating that the amplification efficiency of MspJI preparation of enzyme digestion / fluorescent PCR reaction system is better and more stable, and the detection effect of the kit is better.

[0083] Example 3 Partial double-stranded linear DNA probe system detection effect verification experiment

[0084] (1) Purpose of the experiment

[0085] In this embodiment, the detection effect of the partial double-stranded linear DNA probe system is verified by comparing with the detection results of the traditional probe.

[0086] (2) Experimental method

[0087] In this embodiment, 3 identical concentrations of unmethylated WBC DNA and BMP3 and NDRG4 methylated cell strain DNA were detected by using the kit prepared by the partial double-stranded linear DNA probe system described in Example 1 and the enzyme digestion / fluorescent PCR reaction system established by the traditional fluorescent probe (the base composition is the same as the fluorescent probe SEQ ID NO. 3, 7, 11 described in Example 1, but does not contain the quencher probe SEQ ID NO. 4, 8, 12), and the DNA sample amount was 1 μl. At the same time, 9 repeated detections were carried out using nuclease-free water as the template by using the kit prepared by the partial double-stranded linear DNA probe system described in Example 1 and the enzyme digestion / fluorescent PCR reaction system established by the traditional fluorescent probe. The enzyme digestion / fluorescent PCR reaction system established by the traditional fluorescent probe is the same as the kit of the present application described in Example 1 in terms of the composition of the probe, the preparation method and process, except that the composition of the probe is different from the kit of the present application described in Example 1.

[0088] (3) Experimental results and analysis

[0089] The detection results are shown in the following table (N represents unmethylated WBC DNA, P represents BMP3 and NDRG4 methylated cell strain DNA, W1-W9 represents the 1st-9th repeated detection of nuclease-free water). Figure 1

[0090] Table 7 Detection results of different probes

[0091] ​

[0092]

[0093] From the above table, it can be seen that when the partial double-stranded linear DNA probe system of the present application and the traditional fluorescent probe are used to detect DNA samples, all unmethylated WBC DNA samples are detected as negative, and all BMP3 and NDR4 methylated cell strain DNA samples are detected as BMP3 and NDR4 methylation double positive. However, the Ct value of each sample detected by the partial double-stranded linear DNA probe system of the present application is lower than that of the traditional fluorescent probe ( Figure 1 ), and the Ct value of each sample detected is more stable; it shows that the sensitivity of the partial double-stranded linear DNA probe system of the present application is higher, and the detection effect is better and more stable. From the above table, it can be seen that although the partial double-stranded linear DNA probe system of the present application can detect the Ct value from the 9th repeated detection, the Ct value is still maintained above 39, and no endogenous false positive amplification is generated; while the traditional fluorescent probe detects the Ct value from the 3rd repeated detection, and the Ct value decreases with the increase of the number of repeated detection, and false positive amplification is generated at the 9th repeated detection; it shows that the specificity of the partial double-stranded linear DNA probe system of the present application is better. In addition, the background signal of the partial double-stranded linear DNA probe system of the present application is also lower. In summary, the above results prove that the partial double-stranded linear DNA probe system used in the present application has high sensitivity, good specificity and low background signal, which helps to improve the detection performance of the kit and ensure the accuracy and reliability of the detection results.

[0094] Example 4 Selection experiment of betaine concentration in the fluorescent PCR reaction solution of the present application

[0095] (1) Purpose of the experiment

[0096] In this embodiment, the detection results of the fluorescent PCR reaction solution containing different concentrations of betaine are compared to screen the optimal concentration of betaine in the fluorescent PCR reaction solution.

[0097] (2) Experimental method

[0098] In this embodiment, the fluorescent PCR reaction solution with betaine concentration of 0 mM, 100 mM, 125 mM, 150 mM and 200 mM is prepared according to the kit preparation method described in Example 1, and five different enzyme digestion / fluorescent PCR reaction systems are formed with the enzyme digestion reaction solution. The above five enzyme digestion / fluorescent PCR reaction systems are used to perform enzyme digestion / fluorescent PCR reaction on 3 portions of the same concentration of unmethylated WBC DNA and BMP3 and NDR4 methylated cell strain DNA. The other components of the kit and the enzyme digestion / fluorescent PCR reaction are carried out according to the detection steps in Example 1, and the DNA loading amount is 1 μl.​

[0099] (3) Experimental results and analysis

[0100] The detection results are shown in the following table (N represents unmethylated WBC DNA, and P represents BMP3 and NDRG4 methylated cell line DNA).

[0101] Table 8 Detection results of fluorescent PCR reaction solution containing different concentrations of betaine

[0102]

[0103]

[0104] From the detection results in the above table, it can be seen that the fluorescent PCR reaction solution with a betaine concentration of 100 mM to 150 mM can achieve accurate detection, all unmethylated WBC DNA samples are detected as negative, and all BMP3 and NDRG4 methylated cell line DNA samples are detected as BMP3 and NDRG4 methylation double positive; however, compared with the fluorescent PCR reaction solution with a betaine concentration of 100 mM or 150 mM, the Ct value of each sample detected by the fluorescent PCR reaction solution with a betaine concentration of 125 mM is lower, indicating that the detection effect of the fluorescent PCR reaction solution with a betaine concentration of 125 mM is better. When the fluorescent PCR reaction solution with a betaine concentration of 0 mM is used, the detected Ct value is higher, which even leads to the generation of individual false positive results (such as sample N-2) and invalid results (such as sample P-3), and accurate detection cannot be achieved. When the fluorescent PCR reaction solution with a betaine concentration of 200 mM is used, the detected Ct value is also higher, which also leads to the generation of invalid results (such as sample N-2), and accurate detection cannot be achieved. The above results show that the addition of an appropriate amount of betaine helps to improve the detection effect of the fluorescent PCR reaction solution and ensure the accuracy and reliability of the detection results. In view of the above results, the betaine concentration of the fluorescent PCR reaction solution in the kit of the present application is 100 mM to 150 mM; preferably, the concentration of betaine is 125 mM.

[0105] The experimental design for selecting the concentration of tetramethylammonium chloride in the fluorescent PCR reaction solution of the present application is similar to the above experimental design; the results show that the concentration of tetramethylammonium chloride in the fluorescent PCR reaction solution in the kit of the present application is 10 mM to 20 mM; preferably, the concentration of tetramethylammonium chloride is 15 mM, and the specific data is omitted.

[0106] Example 5 Sensitivity analysis experiment

[0107] (1) Purpose of the experiment

[0108] In this embodiment, different methylation DNA proportions and different initial amounts of DNA samples are detected to analyze the sensitivity of the present application.

[0109] (2) Experimental method

[0110] In this embodiment, unmethylated WBC DNA and BMP3 and NDRG4 methylated cell strain DNA were selected to prepare DNA samples with a methylation DNA ratio of 0%, 1%, 5%, and 10%, respectively, and the initial amount was 10 ng, 5 ng, 1 ng, and 0.1 ng, respectively, as the to-be-tested samples, and the human BMP3 and NDRG4 gene methylation detection kit described in Example 1 was used to detect according to the detection steps in Example 1.

[0111] (3) Experimental results and analysis

[0112] The detection results are shown in the following table.

[0113] Table 9 Sensitivity analysis experiment detection results

[0114]

[0115] From the above table, it can be seen that the present application can detect BMP3 and NDRG4 gene methylation when the DNA content is as low as 1 ng and the methylation DNA ratio is as low as 1% (as shown in the table), which proves that the detection sensitivity of the present application is high. The fluorescence PCR amplification curve of the negative quality control and the positive quality control is shown in FIGS. 1 and 2, respectively. Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 1 Figure

[0116] Example 6 Comparison experiment of similar products

[0117] (1) Experimental purpose

[0118] In this embodiment, the detection results of the methylation detection "gold standard" bisulfite sequencing method and the Cologuard kit were compared to verify the accuracy of the kit.

[0119] (2) Experimental method

[0120] In this embodiment, 100 fecal DNA samples were collected as to-be-tested samples, of which 9 were from healthy persons, 25 were from colorectal benign lesions, and the remaining 66 were from colorectal cancer patients. The above-mentioned 100 DNA samples were taken in an appropriate amount and detected using the kit of the present application, the bisulfite sequencing method, and the Cologuard kit (FDA-approved fecal DNA intestinal cancer screening kit, purchased from Exact Sciences Corporation). The detection process of the kit of the present application was carried out according to the detection steps in Example 1; the bisulfite sequencing method was entrusted to Meiji Bio to complete, and the detection process of the Cologuard kit was carried out according to the product instructions. ​​​

[0121] (3) Experimental results and analysis

[0122] The detection results are shown in Tables 10 and 11.

[0123] Table 10 Comparison table of detection results of the kit of the present application and bisulfite sequencing method

[0124]

[0125] Table 11 Comparison table of detection results of the kit of the present application and Cologuard kit

[0126]

[0127] From the above table, the coincidence rate of the detection results of the kit of the present application and the methylation detection "gold standard" bisulfite sequencing method is 100%, which proves the detection accuracy of the kit of the present application. At the same time, the coincidence rate of the detection results of the kit of the present application and the Cologuard kit is 96%. The Cologuard kit detected 30 negative, 2 BMP3 methylation positive and 2 NDRG4 methylation positive in healthy and colorectal benign lesion DNA samples, and 7 negative and 59 BMP3 and / or NDRG4 methylation positive in colorectal cancer DNA samples. However, the kit of the present application only detected 1 case of BMP3 methylation positive and 1 case of NDRG4 methylation positive in healthy and colorectal benign lesion DNA samples, and the remaining 32 cases were negative. In colorectal cancer DNA samples, it only detected 5 negative, and the remaining 61 cases were BMP3 and / or NDRG4 methylation positive. This shows that compared with the Cologuard kit, the kit of the present application is more sensitive and specific for colorectal cancer screening, reducing the false negative rate and false positive rate. In addition, by comparing the detection processes of the kit of the present application and the Cologuard kit, it is found that the Cologuard kit uses a bisulfite-based fluorescent PCR method for BMP3 and NDRG4 methylation detection, and its detection process involves bisulfite conversion of the DNA sample to be tested, and requires a large number of reagents and complicated operation steps. However, the kit of the present application only needs to add the DNA sample to be tested to the enzyme digestion / fluorescent PCR reaction system for detection. Compared with the Cologuard kit, the kit of the present application is simpler and faster to operate, and is easy to automate.

[0128] Example 7 Comparison experiment of detection results of different DNA sample types

[0129] (1) Purpose of the experiment

[0130] In this embodiment, the detection of different types of DNA samples is used to analyze the sample types suitable for the kit of the present application.

[0131] (2) Experimental method

[0132] In this embodiment, 8 colorectal cancer patients (No. 1-8) were collected for 1 tissue DNA sample, 1 plasma DNA sample, 1 fecal DNA sample, and 1 intestinal lavage fluid (BLF) DNA sample as test samples, and the human BMP3 and NDRG4 gene methylation detection kit of Example 1 was used to detect according to the detection steps in Example 1.

[0133] (3) Experimental results and analysis

[0134] The detection results are shown in the following table.

[0135] Table 12 Detection results of different DNA sample types

[0136]

[0137]

[0138] From the above table, for the 8 colorectal cancer patients who provided tissue DNA, plasma DNA, fecal DNA and BLF DNA, the detection results of the four DNA samples were completely consistent, with a coincidence rate of 100%, indicating that the kit is suitable for various DNA sample types, including tissue DNA, plasma DNA, fecal DNA and BLF DNA.

[0139] The above-described embodiments only express several embodiments of the present application, which are described in more detail and in detail, but cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims. SEQUENCE LISTING <110> YISHENG BIOTECHNOLOGY CO., LTD. <120> Human BMP3 and NDRG4 gene methylation detection kit <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 agctggtttg gagttcaa 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 agaaagagca gcctgctc 18 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 cgcgcggcgc gtcggctccc ggcg 24 <210> 4 <211> 12 <212> DNA <213> Artificial Sequence <400> 4 acgcgccgcg cg 12 <210> 5 <211> 16 <212> DNA <213> Artificial Sequence <400> 5 acacccgcca aaccca 16 <210> 6 <211> 16 <212> DNA <213> Artificial Sequence <400> 6 gcggagatgc ggacga 16 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <400> 7 cttcggcgcc gccgacgcgc gggt 24 <210> 8 <211> 12 <212> DNA <213> Artificial Sequence <400> 8 gcggcgccga ag 12 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <400> 9 acgatggagg ggaagacg 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <400> 10 ccggctattc tcgcagct 18 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <400> 11 cgccgcgctc gtcgtcgaca acgg 24 <210> 12 <211> 12 <212> DNA <213> Artificial Sequence <400> 12 acgagcgcgg cg 12 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <400> 13 ctgcmcagga tcttttttga tcmctggcag 30

Claims

1. A human BMP3 or NDRG4 gene methylation detection kit, characterized in that, The kit includes an enzyme digestion reaction solution containing a methylation-dependent restriction endonuclease selected from any one of MspJI, FspEI, and LpnPI. It also includes fluorescent PCR reaction solutions containing primers targeting the BMP3 or NDRG4 gene and some double-stranded linear DNA probes; Each gene's partial double-stranded linear DNA probe comprises a long fluorescent probe and a short quenching probe; the fluorescent probe is labeled with a fluorescent reporter group at its 5' end and a fluorescent quenching group at its 3' end; the quenching probe is completely complementary to the fluorescent probe at its 5' end and is labeled with a fluorescent quenching group at its 3' end; the fluorescent reporter group is different for different genes. Primers targeting the BMP3 gene are shown in SEQ ID NO. 1 to SEQ ID NO. 2, or primers targeting the NDRG4 gene are shown in SEQ ID NO. 5 to SEQ ID NO. 6; Probes targeting the BMP3 gene include a fluorescent probe as shown in SEQ ID NO.3 and a quenching probe as shown in SEQ ID NO.4; or probes targeting the NDRG4 gene include a fluorescent probe as shown in SEQ ID NO.7 and a quenching probe as shown in SEQ ID NO.

8.

2. The human BMP3 or NDRG4 gene methylation detection kit according to claim 1, characterized in that, The methylation-dependent restriction endonuclease is MspJI.

3. The human BMP3 or NDRG4 gene methylation detection kit according to claim 1, characterized in that, The amount of MspJI used is 1-2 U / reaction.

4. The human BMP3 or NDRG4 gene methylation detection kit according to claim 3, characterized in that, The amount of MspJI used was 1.5 U / reaction.

5. The human BMP3 or NDRG4 gene methylation detection kit according to claim 1, characterized in that, The enzyme digestion reaction solution also includes an enzyme digestion buffer, an enzyme activity solution, and nuclease-free water; the enzyme activity solution is a Tris-HCl buffer containing double-stranded oligonucleotides, which have a stem-loop structure and contain two methylation sites.

6. The human BMP3 or NDRG4 gene methylation detection kit according to claim 5, characterized in that, The double-stranded oligonucleotide sequence is shown in SEQ ID NO.

13.

7. The human BMP3 or NDRG4 gene methylation detection kit according to claim 5 or 6, characterized in that, The concentration of the double-stranded oligonucleotides in the enzyme activity solution during the reaction is 150 ± 1 nM, and / or The enzyme digestion buffer is an aqueous solution containing 50±2 mM potassium acetate, 20±1 mM Tris-acetic acid, 10±1 mM magnesium acetate and 100±5 μg / ml recombinant albumin.

8. The human BMP3 or NDRG4 gene methylation detection kit according to claim 1, characterized in that, The fluorescent PCR reaction solution also contains PCR buffer, dNTPs, DNA polymerase, betaine, tetramethylammonium chloride, and nuclease-free water; The concentration of betaine in the fluorescent PCR reaction solution is 100 mM to 150 mM.

9. The human BMP3 or NDRG4 gene methylation detection kit according to claim 8, characterized in that, The concentration of betaine is 125±5 mM.

10. The human BMP3 or NDRG4 gene methylation detection kit according to claim 8, characterized in that, The concentration of the tetramethylammonium chloride in the fluorescent PCR reaction solution is 10 mM to 20 mM.

11. The human BMP3 or NDRG4 gene methylation detection kit according to claim 9, characterized in that, The concentration of the tetramethylammonium chloride is 15 ± 1 mM.

12. The human BMP3 or NDRG4 gene methylation detection kit according to claim 1, characterized in that, The enzyme digestion reaction solution and the fluorescent PCR reaction solution are packaged in layers using a heat-melting material within the same PCR amplification tube.

13. The human BMP3 or NDRG4 gene methylation detection kit according to claim 12, characterized in that, The hot-melt material is fully refined paraffin wax with a melting point of 70℃~72℃.

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