CN114807373A

CN114807373A - Human BMP3 and NDRG4 gene methylation detection kit

Info

Publication number: CN114807373A
Application number: CN202210610762.8A
Authority: CN
Inventors: 许嘉森; 吴诗扬; 刘芳; 刘志明
Original assignee: Surexam Bio Tech Co Ltd
Current assignee: Surexam Bio Tech Co Ltd
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-07-29

Abstract

The invention relates to a human BMP3 and NDRG4 gene methylation detection kit, which comprises enzyme digestion reaction liquid, wherein the enzyme digestion reaction liquid comprises enzyme digestion buffer solution and methylation dependent restriction endonuclease, and also comprises primers respectively designed aiming at CpG islands of BMP3 genes and/or NDRG4 genes and fluorescence PCR reaction liquid of partial double-stranded linear DNA probes. The detection kit can realize enzyme digestion reaction and fluorescence PCR reaction in one tube in sequence to detect the methylation states of BMP3 and NDRG4 genes, and has the characteristics of simple and rapid operation, high sensitivity, good specificity and easy automation.

Description

Human BMP3 and NDRG4 gene methylation detection kit

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a human BMP3 and NDRG4 gene methylation detection kit.

Technical Field

Disorders of the epigenetic program are a significant cause of colorectal cancer development (Liu R et al, Mutat Res,2019,779: 45-57). DNA methylation changes are the most important epigenetic changes that control the transcription and expression of specific genes and play an important role in colorectal carcinogenesis. In the early and late stages of carcinogenesis, silencing of methylated genes often occurs in focal lesions of the adenoma-carcinoma cascade. Thus, methylation changes drive the development and progression of colorectal cancer. Detection of gene methylation may help identify individuals with early colorectal cancer, and may help improve disease outcome.

BMP3, bone morphogenic protein 3, whose gene encodes a secreted ligand for transforming growth factor beta (TGF- β) superfamily proteins, has an important function in bone formation. BMP3 gene methylation can be detected in a variety of tumors, including colorectal, pancreatic, gastric, lung, breast, and biliary tract cancers (Kisiel JB et al, J Mol Biomark Diagn,2013,4: 1000145). In colorectal Cancer, aberrant methylation of the BMP3 gene down-regulates BMP3 expression, and inactivation of BMP3 is an early and common event in colorectal Cancer development (Loh K et al, Genes Chromosomes Cancer,2008,47: 449-60). Studies have shown that BMP3 methylation in stool samples has an overall sensitivity and specificity for colorectal cancer diagnosis of 70% and 89%, respectively (Liu R et al, Mutat Res,2019,779: 45-57).

NDRG4, N-Myc downstream regulatory 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 functions as an oncogene, which is critical for astrocyte survival; in malignant meningiomas, NDRG4 plays a carcinogenic role by inhibiting apoptosis through inhibition of p53 expression. However, NDRG4 has a cancer-suppressing effect in other tumors such as colorectal cancer, gastric cancer, breast cancer, pancreatic cancer. NDRG4 promoter methylation can be detected in gastric, colorectal, breast and pancreatic cancers and can lead to down-regulation of NDRG4 expression. Studies have shown that NDRG4 methylation has an overall sensitivity and specificity for colorectal cancer diagnosis of 76% and 82%, respectively (Liu R et al, Mutat Res,2019,779: 45-57).

In view of the high diagnostic value of BMP3 and NDRG4 in colorectal cancer screening respectively, the application of joint detection of BMP3 and NDRG4 gene methylation in colorectal cancer diagnosis is also concerned in recent years. Studies have shown that the combination of the methylation status of the BMP3 and NDRG4 genes in stool samples is a very useful marker with sensitivity and specificity for colorectal cancer diagnosis of 89% and 87%, respectively (Liu R et al, Mutat Res,2019,779: 45-57). As can be seen, the combined detection of BMP3 and NDRG4 gene methylation has better diagnostic performance in colorectal cancer screening.

The current methods for detecting methylation of BMP3 and NDRG4 genes are mainly fluorescence PCR methods based on bisulfite conversion. The method comprises the steps of treating a DNA sample by using bisulfite, converting unmethylated cytosine into uracil, then using the DNA sample treated by the bisulfite as a template, performing fluorescence PCR by using a specific primer and a fluorescent probe, and judging the methylation state according to a detected fluorescence signal, such as Chinese patent applications CN201510486088.7 and CN 201710090789.8. Although the kits described in the above two chinese patent applications have good sensitivity and/or specificity, they fail to solve the drawbacks caused by bisulfite conversion, such as: the DNA sample is easily degraded due to severe transformation conditions; DNA purification and recovery are needed after transformation, and the operation steps are complicated; incomplete conversion may cause bias in detection results; and the like. Therefore, it is necessary to provide a methylation detection kit for BMP3 and NDRG4 genes, which does not need bisulfite conversion, is simple and rapid to operate and can accurately detect the methylation.

Disclosure of Invention

Based on the above, the invention provides a human BMP3 and NDRG4 gene methylation detection kit, which utilizes methylation dependent restriction enzyme to combine with a specially designed multiplex fluorescence PCR system, can realize enzyme digestion reaction and multiplex fluorescence PCR reaction in parallel at one time to detect BMP3 and NDRG4 gene methylation, and has the characteristics of simple and rapid operation, high sensitivity and good specificity.

In order to achieve the above object, the technical solution of the present invention includes the following.

The human BMP3 and/or NDRG4 gene methylation detection kit comprises an enzyme digestion reaction solution, wherein the enzyme digestion reaction solution contains methylation-dependent restriction enzyme;

also comprises primers respectively designed aiming at CpG islands of BMP3 gene and/or NDRG4 gene and fluorescent PCR reaction liquid of a partial double-stranded linear DNA probe;

the partial double-stranded linear DNA probe of each gene comprises a fluorescent probe with a long base number and a quenching probe with a short base number; the 5 'end of the fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the fluorescent probe is marked with a fluorescent quenching group; the quenching probe is completely complementary with the 5 'end of the fluorescent probe, and the 3' end is marked with a fluorescent quenching group; the fluorescent reporter group is different for different genes.

In some of these embodiments, primers to the BMP3 gene are shown in SEQ ID No.1 to SEQ ID No.2, and/or primers to the NDRG4 gene are shown in SEQ ID No.5 to SEQ ID No. 6.

In some of these embodiments, the probe for BMP3 gene comprises a fluorescent probe as shown in SEQ ID No.3 and a quenching probe as shown in SEQ ID No. 4; and/or the probe aiming at the NDRG4 gene comprises a fluorescent probe shown as SEQ ID NO.7 and a quenching probe shown as SEQ ID NO. 8.

In one embodiment, the fluorescent reporter comprises FAM, VIC, ROX, or CY 5; the fluorescence quenching group is BHQ. When no template is amplified in the fluorescent PCR system, the fluorescent probe is combined with the quenching probe, so that the fluorescent reporter group and the quenching group are very close to each other, and a fluorescent signal is effectively quenched; when template amplification exists in the system, the fluorescent probe is preferentially and stably combined with an amplification product and separated from the quenching probe to release a fluorescent signal in a PCR reaction.

In some of these embodiments, the methylation dependent restriction enzyme is used to digest methylated BMP3 and NDRG4 gene DNA selected from any one of MspJI, FspEI and LpnPI in a DNA sample; preferably, the methylation-dependent restriction enzyme is MspJI, and the dosage of the MspJI is 1-2U/reaction; more preferably, the MspJI is used in an amount of 1.5U/reaction.

The enzyme digestion reaction liquid also comprises enzyme digestion buffer solution, enzyme activity liquid and water without nuclease; the enzyme activity solution is used for stimulating the digestion of methylation-dependent restriction enzyme, and is a solution prepared by dissolving short double-stranded oligonucleotide in 10mM Tris-HCl (pH value of 8.0), wherein the short double-stranded oligonucleotide is of a stem-loop structure and comprises two methylation sites; preferably, the sequence of the short double-stranded oligonucleotide is shown in SEQ ID NO.13, and the enzyme activity solution is a solution containing the short double-stranded oligonucleotide with the concentration of 150 +/-1 nM.

In some of these embodiments, the cleavage buffer is an aqueous solution (pH 7.9 at 25 ℃) containing 50. + -.2 mM potassium acetate, 20. + -.1 mM Tris-acetate, 10. + -.1 mM magnesium acetate and 100. + -.5. mu.g/ml recombinant albumin.

The kit also comprises fluorescent PCR reaction liquid, wherein the fluorescent PCR reaction liquid comprises PCR buffer solution, dNTP, primers, probes, DNA polymerase, betaine, tetramethylammonium chloride and water without nuclease.

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 DNA polymerase, and reduce the secondary structure of GC-rich template, thereby promoting PCR amplification of GC-rich template.

In some embodiments, the tetramethylammonium chloride is present in the fluorescent PCR reaction solution at a concentration of 10mM to 20 mM; preferably, the concentration of tetramethylammonium chloride is 15 ± 1 mM. The addition of tetramethylammonium chloride removes non-specific priming and reduces false binding, thereby improving the specificity of the PCR reaction and the specificity of hybridization.

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 aiming at the CpG island of the ACTB gene, and are preferably shown in SEQ ID NO.8 to SEQ ID NO. 12.

In some embodiments, the enzyme digestion reaction solution and the fluorescence PCR reaction solution are packaged in the same PCR amplification tube by layers through hot-melt materials, and by matching with other designs, a one-tube enzyme digestion/fluorescence PCR system can simplify detection operation.

The hot-melt material is fully refined paraffin with the melting point of 70-72 ℃.

In some of these embodiments, a negative quality control and a positive quality control are also included. The negative quality control product consists of BMP3 and NDRG4 gene unmethylated WBC DNA, BSA and TE buffer solution; the positive quality control product consists of BMP3 gene methylated cell strain DNA, NDRG4 gene methylated cell strain DNA, WBC DNA, BSA and TE buffer solution. The negative quality control material and the positive quality control material 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 detection results.

Another object of the present invention is to provide a method for using the above kit.

The use method of the kit comprises the following steps:

(1) obtaining a DNA sample to be detected;

(2) enzyme digestion/fluorescent PCR reaction: adding 1 mul of DNA sample to be detected into the enzyme digestion reaction solution, and carrying out enzyme digestion reaction and fluorescence PCR reaction in sequence on a fluorescence PCR instrument, wherein the reaction conditions are as follows: enzyme digestion is carried out for 1 hour at 37 ℃, inactivation is carried out for 20 minutes at 65 ℃, and 1 cycle is carried out; pre-denaturation at 95 ℃ for 5 min for 1 cycle; denaturation at 95 ℃ for 20 seconds, annealing/extension at 62 ℃ for 30 seconds (fluorescence signal acquisition at this step), 40 cycles.

The invention mainly has the following beneficial effects:

(1) the invention adopts a methylation dependent restriction enzyme combined fluorescent PCR method to realize methylation detection.

And the methylation dependent restriction enzyme of the invention is preferably MspJI selected from the group consisting of a methylated BMP3 sequence and a methylated NDRG4 sequence, and the enzyme can recognize ^m CNNR sites, effecting p-methylation in nucleic acid sequences ^m Cleavage at N9/N13 on the 3' side of the CNNR site. When the enzyme is used for methylation detection of BMP3 and NDRG4 genes by aiming at the specific primers and probes designed by the invention, the enzyme has a better and more stable detection effect on amplification efficiency, and can effectively and accurately detect unmethylated sequences and methylated sequences of BMP3 and NDRG4 genes.

(2) The probe in the kit adopts a partial double-stranded linear DNA probe system consisting of a longer fluorescent probe and a shorter quenching probe, the probe system can release a fluorescent signal only under the condition that an amplification product is generated when being applied to a fluorescent PCR reaction, and the fluorescent probe can be combined with the quenching probe to effectively quench the self-fluorescent signal of the probe under the condition that no amplification product is generated; the proper probe designed according to the invention effectively reduces background signals and improves the accuracy of detection. Has high sensitivity. .

(3) The primer and the probe designed by the invention are matched with a proper fluorescent PCR reaction solution, so that triple fluorescent PCR reaction can be realized, BMP3 gene methylation, NDRG4 gene methylation and reference genes can be detected in a single tube by one-time reaction, reagent consumables are greatly saved, the detection time is shortened, and the detection result is accurate.

(4) Betaine and tetramethylammonium chloride are added into the fluorescent PCR reaction solution. Wherein, the betaine can reduce the dependence of denaturation temperature on basic group, improve the stability of DNA polymerase, and can reduce the secondary structure of GC-rich template to promote the PCR amplification of GC-rich template, and the tetramethylammonium chloride can remove non-specific initiation and reduce error combination, 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.

Drawings

FIG. 1 is a graph showing the fluorescence PCR amplification curves of the partially double-stranded linear DNA probe system of the present invention and a conventional fluorescent probe for detecting methylation of BMP3 and NDRG4 in example 3. Wherein (a) the partially double-stranded linear DNA probe system of the present invention detects unmethylated WBC DNA; (b) the partial double-stranded linear DNA probe system detects the DNA of BMP3 and NDRG4 methylated cell strains; (c) detection of unmethylated WBC DNA with conventional fluorescent probes; (d) the DNA of BMP3 and NDRG4 methylated cell lines is detected by a traditional fluorescent probe.

FIG. 2 is a graph showing the fluorescence PCR amplification of a 1ng DNA sample with a 1% ratio of methylated DNA according to the invention in example 5.

FIG. 3 is a fluorescent PCR amplification curve chart of the negative quality control product of the present invention.

FIG. 4 is a fluorescent PCR amplification curve chart of the positive quality control of the present invention.

Detailed Description

The practice of the present invention 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 maniotis, molecular cloning, a laboratory manual, 3 rd edition (2002). The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various chemicals used in the examples are commercially available.

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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention relates to a human BMP3 and NDRG4 gene methylation detection kit, which comprises enzyme digestion reaction liquid and fluorescence PCR reaction liquid, wherein the enzyme digestion reaction liquid and the fluorescence PCR reaction liquid are packaged in the same PCR amplification tube in a layered mode through hot-melt materials.

The enzyme digestion reaction solution contains proper methylation-dependent restriction enzyme.

The methylation dependent restriction endonuclease can recognize a methylation site and can cut double-stranded DNA near the 3' side of methylated cytosine, and when a DNA sample to be detected is treated by the methylation dependent restriction endonuclease, the methylation dependent restriction endonuclease can recognize a methylated nucleic acid sequence and generate enzyme cutting activity, but cannot recognize and cut an unmethylated nucleic acid sequence, so that the methylated nucleic acid sequence is cut into small fragments by enzyme, and the unmethylated nucleic acid sequence is completely reserved; and then, carrying out fluorescence PCR reaction on the DNA sample to be detected after enzyme digestion by using a fluorescence PCR reaction solution prepared by specific primers which are designed aiming at the two sides of the enzyme digestion site or the enzyme digestion site of the nucleic acid sequence of the region to be detected and a methylation-dependent restriction endonuclease recognition region contained in the nucleic acid sequence and probes positioned at the enzyme digestion site. Compared with a fluorescence PCR method based on bisulfite conversion, the method avoids the DNA degradation caused by bisulfite conversion or the bias of detection results caused by incomplete conversion, thereby improving the detection sensitivity; the fluorescence PCR detection can be directly carried out without purification and recovery steps after the digestion of the DNA sample, so that the operation steps are reduced, the detection cost and the detection time are saved, and the automation is facilitated.

The detection kit adopts a one-tube enzyme digestion/fluorescence PCR system, namely, a hot-melt material with the melting point of 70-72 ℃ is used for separating enzyme digestion reaction liquid from fluorescence PCR reaction liquid, the enzyme digestion reaction liquid is positioned on the upper layer of the hot-melt material, and the enzyme digestion and endonuclease inactivation of DNA to be detected can be realized under the condition of not melting the hot-melt material; after the endonuclease is inactivated, directly entering PCR reaction, when the temperature is raised to 95 ℃, isolating enzyme digestion reaction liquid and a hot-melt material of fluorescence PCR reaction from melting and floating to the upper layer of a reaction system (the density of the hot-melt material is lower than that of the reaction liquid), and mixing the fluorescence PCR reaction liquid with an enzyme digestion product on the upper layer to realize fluorescence PCR detection of the enzyme digestion product. The one-tube enzyme digestion/fluorescence PCR system realizes enzyme digestion reaction and fluorescence PCR reaction in the same tube in sequence, and compared with the prior art, the operation is simple and rapid, and the automation is easy.

The present invention will be further illustrated with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

Example 1 preparation of the methylation detection kit for human BMP3 and NDRG4 genes

The human BMP3 and NDRG4 gene methylation detection kit comprises an enzyme digestion/fluorescence PCR reaction system, a negative quality control product and a positive quality control product. The preparation of the kit comprises the following steps:

(1) designing and preparing primers and probes: and respectively designing a plurality of specific primers and probes aiming at the CpG island of the BMP3 gene, the CpG island of the NDRG4 gene and the CpG island of the ACTB gene by combining the information of the cutting sites of methylation dependent restriction endonucleases. Wherein the probes are all designed into a partial double-stranded linear DNA probe system and comprise a longer fluorescent probe and a shorter quenching probe; the 5 'end of the fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the fluorescent probe is marked with a fluorescent quenching group; the quenching probe is completely complementary with the 5 'end of the fluorescent probe, and the 3' end is marked with a fluorescent quenching group; the fluorescent reporter group comprises FAM, VIC, ROX or CY 5; the fluorescence quenching group is BHQ. And performing a preliminary experiment on each primer and probe, comparing the performances such as sensitivity, specificity and the like, and finally preferably selecting the primers and probes of the kit disclosed by the invention, wherein the primers and probes are shown as SEQ ID NO.1 to SEQ ID NO.12 in Table 1. The primer and the probe are stored in a mother solution of 100 mu M, and a working solution of 10 mu M is prepared for standby according to the detection requirement.

TABLE 1 kit primer and Probe sequences

(2) Preparing a fluorescent PCR reaction solution: preparing the fluorescent PCR reaction solution according to the preparation scheme of the fluorescent PCR reaction solution, and storing for later use. The preparation scheme of the fluorescent PCR reaction solution is specifically shown in Table 2.

TABLE 2 fluorescent PCR reaction solution preparation protocol

Name of reagent	Per reaction (ul)
		PCR buffer solution	10
dNTP(10mM)	4
		Primer (10. mu.M)	Adding 1 μ l each
Probe (10 μ M)	Adding 1 μ l each
		Betaine (5M)	1
Tetramethylammonium chloride (200mM)	3
		DNA polymerase (5U/. mu.l)	0.4
Nuclease-free water	Adding water to 40 μ l
		Total volume	40

(3) Selection of methylation dependent restriction enzymes: methylation dependent restriction enzymes were selected based on the sequence of methylated BMP3 and the sequence of methylated NDRG 4. The methylation-dependent restriction endonuclease is any one of MspJI, FspEI and LpnPI; preferably, the methylation dependent restriction enzyme is MspJI. In the invention, the MspJI dosage is 1-2U/reaction; in this example, MspJI was used in an amount of 1.5U/reaction.

(4) Preparation of enzyme active solution: the enzyme activity solution is used for stimulating the digestion of methylation-dependent restriction enzyme, and is a solution prepared by dissolving short double-stranded oligonucleotide in 10mM Tris-HCl (pH value of 8.0), wherein the short double-stranded oligonucleotide is of a stem-loop structure and comprises two methylation sites; preferably, the short double-stranded oligonucleotide sequence is: CTGC ^m CAGGATCTTTTTTGATC ^m CTGGCAG (SEQ ID NO.13, superscript therein) ^m Representing methylation), the enzyme activity solution is a solution containing a double-stranded oligonucleotide with a concentration of 150 + -1 nM short.

(5) Preparation of enzyme digestion buffer solution: the preparation was carried out according to the composition of the digestion buffer. The digestion buffer consisted of: 50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 100. mu.g/ml recombinant albumin, nuclease-free water. The pH value of the enzyme digestion buffer solution is about 7.9 at 25 ℃.

(6) Preparation of enzyme digestion reaction liquid: preparing the enzyme digestion reaction solution according to the preparation scheme of the enzyme digestion reaction solution, and storing for later use. The preparation scheme of the enzyme digestion reaction solution is specifically shown in Table 3.

TABLE 3 preparation scheme of enzyme digestion reaction solution

Name of reagent	Per reaction (ul)
		Enzyme digestion buffer solution	1
Enzyme active liquid	0.4
		MspJI(5U/μl)	0.3
Nuclease-free water	Adding water to 9 μ l
		Total volume	9

(7) Preparation of enzyme digestion/fluorescence PCR reaction system: and (3) uniformly mixing and centrifuging the prepared fluorescent PCR reaction solution in the step (2), adding 40 mu l of the prepared fluorescent PCR reaction solution to the bottom of a PCR amplification tube, sealing the tube by using fully refined paraffin (manufacturer: Kunlun, model number 70#) with the melting point of 70-72 ℃, cooling and solidifying the fully refined paraffin, and adding 9 mu l of the prepared enzyme digestion reaction solution in the step (6) to the PCR amplification tube in which the fully refined paraffin is condensed. The enzyme digestion/fluorescence PCR reaction system can also be prepared in a PCR eight-tube.

(8) Preparing a negative quality control product and a positive quality control product: adding appropriate amount of BSA, BMP3 and NDRG4 gene unmethylated WBC DNA into TE buffer solution to prepare a negative quality control product, wherein the BSA concentration is 0.1. mu.g/. mu.l, and the BMP3 and NDRG4 gene unmethylated WBC DNA concentration is 10 ng/. mu.l; and adding appropriate amounts of BSA, BMP3 gene methylated cell line DNA, NDRG4 gene methylated cell line DNA and WBC DNA into TE buffer solution to prepare a positive quality control product, wherein the BSA concentration is 0.1. mu.g/. mu.l, the BMP3 gene methylated cell line DNA concentration is 0.1 ng/. mu.l, the NDRG4 gene methylated cell line DNA concentration is 0.1 ng/. mu.l, and the WBC DNA concentration is 9.8 ng/. mu.l.

(9) Subpackaging and assembling the kit: the specification of the kit is 24 persons/box, and the split charging and assembling scheme is shown in table 4.

TABLE 4 kit Split charging and Assembly protocol

The whole detection process of the detection kit comprises the following steps:

(1) enzyme digestion/fluorescent PCR reaction: adding 1 mu l of DNA sample to be detected into enzyme digestion reaction liquid (positioned on the upper layer of condensed fully refined paraffin) of an enzyme digestion/fluorescence PCR reaction system, and placing the enzyme digestion reaction liquid and the fluorescence PCR reaction liquid on a fluorescence PCR instrument in sequence, wherein the reaction conditions are as follows: enzyme digestion is carried out for 1 hour at 37 ℃, inactivation is carried out for 20 minutes at 65 ℃, and 1 cycle is carried out; pre-denaturation at 95 ℃ for 5 min for 1 cycle; denaturation at 95 ℃ for 20 seconds, annealing/extension at 62 ℃ for 30 seconds (fluorescence signal acquisition at this step), 40 cycles. The fluorescence channel of the fluorescence PCR instrument selects FAM, VIC and ROX channels.

(2) Interpretation of the detection results: and judging and reading the detection result according to the Ct value of the fluorescence signal detected by the fluorescence PCR instrument. If the Ct value of the reference gene ROX signal is greater than 30 or no Ct value, the detection result is invalid; if the Ct value of the internal reference gene ROX signal is less than or equal to 30, the detection result is valid, the Δ Ct values are respectively calculated by the formulas of "the Δ Ct value of the BMP3 gene is equal to the Ct value of the BMP3 gene FAM signal-the Ct value of the internal reference gene ROX signal" and "the Δ Ct value of the NDRG4 gene is equal to the Ct value of the NDRG4 gene VIC signal-the Ct value of the internal reference gene ROX signal", the Δ Ct values are taken as a negative and positive judgment standard, and the Δ Ct values are less than or equal to 9.0: negative, Δ Ct value greater than 9.0 or no Δ Ct value: and (4) positive. The specific test result judgment table is as follows:

TABLE 5 judgment table for test results of the kit

EXAMPLE 2 comparison of different methylation-dependent restriction enzymes

(1) Purpose of experiment

In the embodiment, different methylation-dependent restriction enzymes are used for detecting the methylation of the BMP3 and NDRG4 genes so as to compare the enzyme digestion effect of the different methylation-dependent restriction enzymes and the influence on the detection effect of the kit.

(2) Experimental methods

In this example, according to the preparation steps of the kit in example 1, MspJI, FspEI, and LpnPI were used as methylation-dependent restriction endonucleases to prepare three enzyme digestion/fluorescence PCR reaction systems, respectively; the three enzyme digestion/fluorescence PCR reaction systems are used for respectively carrying out enzyme digestion/fluorescence PCR reaction on 3 parts of unmethylated WBC DNA and cell strain DNA methylated by BMP3 and NDRG4 with the same concentration. Other components of the kit and the enzyme digestion/fluorescence PCR reaction were carried out according to the detection procedure in example 1, and the loading amount of DNA was 1. mu.l.

(3) Results and analysis of the experiments

The results of the measurements are shown in the following table (N denotes unmethylated WBC DNA, P denotes BMP3 and NDRG4 methylated cell line DNA).

TABLE 6 detection results of different methylation dependent endonucleases

From the detection results in the table, the enzyme digestion/fluorescence PCR reaction system prepared by MspJI, FspEI and LpnPI can realize accurate detection, all the unmethylated WBC DNA samples are detected to be negative, and all the BMP3 and NDRG4 methylated cell line DNA samples are detected to be double positive for methylation of BMP3 and NDRG 4. However, the Ct value of each sample detected by using the restriction enzyme/fluorescence PCR reaction system prepared by MspJI is lower than that of the restriction enzyme/fluorescence PCR reaction system prepared by FspEI and LpnPI, and the Ct value of each sample detected by using the restriction enzyme/fluorescence PCR reaction system prepared by MspJI is more stable, which indicates that the amplification efficiency of the restriction enzyme/fluorescence PCR reaction system prepared by MspJI is better and more stable, and the detection effect of the kit is better.

Example 3 partial double-stranded Linear DNA Probe System test Effect verification experiment

(1) Purpose of experiment

In the embodiment, the detection effect of the partial double-stranded linear DNA probe system is verified by comparing the detection result with the detection result of the traditional probe.

(2) Experimental methods

In this example, 3 DNA samples of unmethylated WBC DNA and methylated cell lines of BMP3 and NDRG4 were tested at the same concentration using the kit of example 1 prepared using a partially double-stranded linear DNA probe system and an enzymatic/fluorescent PCR reaction system set up with conventional fluorescent probes (base composition as set forth in example 1 for fluorescent probes SEQ ID Nos. 3, 7, and 11, but not including the quenching probe SEQ ID Nos. 4, 8, and 12), respectively, and the DNA loading was 1. mu.l. At the same time, 9 repeated detections were performed using the kit described in example 1 prepared from a partially double-stranded linear DNA probe system and an enzyme digestion/fluorescent PCR reaction system established from a conventional fluorescent probe, using nuclease-free water as a template. The enzyme digestion/fluorescence PCR reaction system established by the traditional fluorescence probe is the same as the kit disclosed by the invention in the embodiment 1 except that the composition of the probe is different from that of the kit disclosed by the embodiment 1.

(3) Results and analysis of the experiments

The results are shown in FIG. 1 and the following table (N for unmethylated WBC DNA, P for BMP3 and NDRG4 methylated cell line DNA, W1-W9 for nuclease-free water 1-9 replicates).

TABLE 7 detection results of different probes

From the above detection results, when the partial double-stranded linear DNA probe system and the traditional fluorescent probe are used for detecting DNA samples, all unmethylated WBC DNA samples are detected to be negative, and all BMP3 and NDRG4 methylated cell strain DNA samples are detected to be double positive in methylation of BMP3 and NDRG 4; however, the Ct value of each sample detected by the partial double-stranded linear DNA probe system is lower than that of the traditional fluorescent probe (shown in figure 1), and the Ct value of each sample detected by the partial double-stranded linear DNA probe system is more stable; the partial double-stranded linear DNA probe system has higher sensitivity and better and more stable detection effect. As can be seen from the results of 9 times of repeated detection of nuclease-free water in the above table, although the Ct value of the partial double-stranded linear DNA probe system can be detected from the 9 th repeated detection, the Ct value is still maintained above 39, and no endogenous false positive amplification is generated; the Ct value of the traditional fluorescent probe is detected from the 3 rd repeated detection, and is reduced along with the increase of the repeated detection times, and false positive amplification is generated when the 9 th repeated detection is carried out; thus, the partial double-stranded linear DNA probe system of the invention has better specificity. In addition, the background signal of the partial double-stranded linear DNA probe system of the invention is also low. In a word, the results prove that the partial double-stranded linear DNA probe system adopted by the invention has high sensitivity, good specificity and low background signal, is beneficial to improving the detection performance of the kit and ensures the accuracy and reliability of the detection result.

EXAMPLE 4 selection experiment of betaine concentration in fluorescent PCR reaction solution of the present invention

(1) Purpose of experiment

In this example, the optimal concentration of betaine in the fluorescent PCR reaction solution is screened by comparing the detection results of the fluorescent PCR reaction solutions containing different concentrations of betaine.

(2) Experimental methods

In this example, fluorescent PCR reaction solutions with betaine concentrations of 0mM, 100mM, 125mM, 150mM and 200mM were prepared according to the kit preparation method described in example 1, and five different enzyme digestion/fluorescent PCR reaction systems were formed with the enzyme digestion reaction solutions. The five enzyme digestion/fluorescence PCR reaction systems are used for respectively carrying out enzyme digestion/fluorescence PCR reaction on 3 parts of unmethylated WBC DNA and cell strain DNA methylated by BMP3 and NDRG4 with the same concentration. Other components of the kit and the enzyme digestion/fluorescence PCR reaction were carried out according to the detection procedure in example 1, and the loading amount of DNA was 1. mu.l.

(3) Results and analysis of the experiments

TABLE 8 detection results of fluorescent PCR reaction solutions containing betaine at different concentrations

From the detection results in the table, accurate detection can be realized by using the fluorescent PCR reaction solution with the betaine concentration of 100 mM-150 mM, all the unmethylated WBC DNA samples are detected to be negative, and all the BMP3 and NDRG4 methylated cell line DNA samples are detected to be double positive in methylation of BMP3 and NDRG 4; however, the Ct value of each sample detected by the fluorescent PCR reaction solution with betaine concentration of 125mM was lower than that detected by the fluorescent PCR reaction solution with betaine concentration of 100mM or 150mM, indicating that the detection effect of the fluorescent PCR reaction solution with betaine concentration of 125mM was better. When the fluorescent PCR reaction solution with the betaine concentration of 0mM is used, the Ct value detected by the fluorescent PCR reaction solution is high, even a single false positive result (such as a sample N-2) and an invalid result (such as a sample P-3) are generated, and accurate detection cannot be realized. When the fluorescent PCR reaction solution with the betaine concentration of 200mM is used, the Ct value detected by the reaction solution is also high, and an invalid result (such as a sample N-2) is generated, so that accurate detection cannot be realized. The results show that the addition of a proper amount of betaine helps to improve the detection effect of the fluorescent PCR reaction solution and ensure the accuracy and reliability of the detection result. In view of the above results, the betaine concentration of the fluorescent PCR reaction solution of the kit of the present invention is 100 mM-150 mM; preferably, the concentration of betaine is 125 mM.

The selection experiment design aiming at the concentration of the tetramethylammonium chloride in the fluorescent PCR reaction solution is similar to the experiment design; the result shows that the concentration of tetramethylammonium chloride in the fluorescent PCR reaction solution of the kit is 10 mM-20 mM; preferably, the concentration of tetramethylammonium chloride is 15mM, and specific data are omitted.

Example 5 sensitivity analysis experiment

(1) Purpose of experiment

In this example, the sensitivity of the present invention was analyzed by detecting different ratios of methylated DNA and different initial amounts of DNA samples.

(2) Experimental methods

In this example, unmethylated WBC DNA, BMP3, and NDRG4 methylated cell line DNA were selected to prepare DNA samples with initial amounts of methylated DNA ratios of 0%, 1%, 5%, and 10% of 10ng, 5ng, 1ng, and 0.1ng, respectively, as samples to be tested, and the human BMP3 and NDRG4 gene methylation detection kits described in this example 1 were used to perform the detection according to the detection procedure in example 1.

(3) Results and analysis of the experiments

The results of the measurements are shown in the following table.

TABLE 9 sensitivity analysis experiment detection results

As can be seen from the detection results in the table above, the detection method can detect the methylation of BMP3 and NDRG4 genes (shown in figure 2) when the DNA content is as low as 1ng and the proportion of methylated DNA is as low as 1%, and proves that the detection sensitivity of the invention is high. The fluorescence PCR amplification curve of the negative quality control substance and the positive quality control substance is shown in FIG. 3 and FIG. 4.

Example 6 comparative experiment with similar products

(1) Purpose of experiment

In the embodiment, the accuracy of the kit is verified by comparing the detection result of the methylation detection 'gold standard' bisulfite sequencing method with the detection result of the Cologuard kit.

(2) Experimental methods

In this example 100 stool DNA samples were collected as test samples, 9 of which were from healthy subjects, 25 of which were from colorectal benign lesions, and the remaining 66 of which were from colorectal cancer patients. Appropriate amounts of the 100 DNA samples collected above were tested using the kit of the present invention, bisulfite sequencing, and Cologuard kit (FDA approved fecal DNA intestinal cancer screening kit, available from Exact Sciences Corporation). The detection process of the kit of the invention is carried out according to the detection steps in example 1; and (3) completing the American Gilg organism by using a bisulfite sequencing method, and detecting the detection process of the Cologuard kit according to the product instruction.

(3) Results and analysis of the experiments

The results of the measurements are shown in tables 10 and 11.

TABLE 10 comparison of the results of the detection of the kit of the invention and the bisulfite sequencing method

TABLE 11 comparison table of the test results of the kit of the present invention and the Cologuard kit

From the detection results in the table, the coincidence rate of the detection result of the kit and the detection result of the methylation detection 'gold standard' bisulfite sequencing method is 100%, and the detection accuracy of the kit is proved. Meanwhile, the coincidence rate of the detection results of the kit and the Cologuard kit is 96%, the Cologuard kit detects 30 negative cases, 2 positive cases of BMP3 methylation and 2 positive cases of NDRG4 methylation in healthy people and colorectal benign lesion DNA samples, and detects 7 negative cases and 59 positive cases of BMP3 and/or NDRG4 methylation in colorectal cancer DNA samples, the kit only detects 1 positive case of BMP3 methylation and 1 positive case of NDRG4 methylation in healthy people and colorectal benign lesion DNA samples, and the rest 32 negative cases, the kit only detects 5 negative cases in colorectal cancer DNA samples, and the rest 61 positive cases of BMP3 and/or NDRG4 methylation, which shows that compared with the Cologuard kit, the kit provided by the invention has higher sensitivity and specificity for colorectal cancer screening, and reduces the false negative rate and the false positive rate. In addition, the detection process comparing the kit with the Cologuard kit shows that the Cologuard kit adopts a bisulfite-based fluorescence PCR method to detect the methylation of BMP3 and NDRG4, the detection process relates to the bisulfite conversion of a DNA sample to be detected, the required reagents are various, and the operation steps are complicated.

Example 7 comparison of results of different DNA sample types

(1) Purpose of experiment

In this example, the types of samples suitable for the kit of the present invention were analyzed by detecting different types of DNA samples.

(2) Experimental methods

In this example, 1 each of tissue DNA samples, plasma DNA samples, stool DNA samples, and intestinal lavage fluid (BLF) DNA samples of 8 colorectal cancer patients (nos. 1 to 8) was collected as a test sample, and the test was performed according to the test procedure in example 1 using the human BMP3 and NDRG4 gene methylation test kits described in example 1.

(3) Results and analysis of the experiments

The results of the measurements are shown in the following table.

TABLE 12 detection results of different DNA sample types

From the above table of test results, for 8 colorectal cancer patients who provided tissue DNA, plasma DNA, stool DNA and BLF DNA at the same time, the test results of 4 DNA samples were completely consistent, and the coincidence rate was 100%, indicating that the kit of the present invention is applicable to various DNA sample types, including tissue DNA, plasma DNA, stool DNA and BLF DNA.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Claims

1. The human BMP3 and/or NDRG4 gene methylation detection kit is characterized by comprising an enzyme digestion reaction solution, wherein the enzyme digestion reaction solution contains methylation-dependent restriction enzyme;

wherein the partially double-stranded linear DNA probe of each gene comprises a fluorescent probe with a long base number and a quenching probe with a short base number; the 5 'end of the fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the fluorescent probe is marked with a fluorescent quenching group; the quenching probe is completely complementary with the 5 'end of the fluorescent probe, and the 3' end is marked with a fluorescent quenching group; the fluorescent reporter group is different for different genes.

2. The kit for detecting methylation of human BMP3 and/or NDRG4 gene of claim 1, wherein primers for BMP3 gene are shown in SEQ ID No.1 to SEQ ID No.2, and/or primers for NDRG4 gene are shown in SEQ ID No.5 to SEQ ID No. 6.

3. The kit for detecting methylation of human BMP3 and/or NDRG4 gene of claim 1, wherein the probe for BMP3 gene comprises a fluorescent probe shown in SEQ ID No.3 and a quenching probe shown in SEQ ID No. 4; and/or the probe aiming at the NDRG4 gene comprises a fluorescent probe shown as SEQ ID NO.7 and a quenching probe shown as SEQ ID NO. 8.

4. The kit for detecting methylation of human BMP3 and/or NDRG4 gene according to claim 1, wherein the methylation dependent restriction enzyme is selected from any one of MspJI, FspEI and LpnPI; preferably, the methylation dependent restriction enzyme is MspJI.

5. The kit for detecting methylation of human BMP3 and/or NDRG4 gene according to claim 4, wherein the MspJI is used in an amount of 1-2U/reaction; more preferably, the MspJI is used in an amount of 1.5U/reaction.

6. The kit for detecting methylation of human BMP3 and/or NDRG4 gene according to claim 1, wherein the digestion reaction solution further comprises digestion buffer solution, enzyme activity solution and nuclease-free water; the enzyme activity solution is Tris-HCl buffer solution containing double-stranded oligonucleotide, and the double-stranded oligonucleotide is of a stem-loop structure and comprises two methylation sites; preferably, the double-stranded oligonucleotide sequence is shown in SEQ ID NO. 13.

7. The kit for detecting the methylation of human BMP3 and/or NDRG4 gene of claim 6, wherein the concentration of the double-stranded oligonucleotide in the enzymatic solution is 150. + -.1 nM, and/or the concentration of the double-stranded oligonucleotide in the reaction is 150. + -.1 nM

The enzyme digestion buffer solution is an aqueous solution containing 50 plus or minus 2mM potassium acetate, 20 plus or minus 1mM Tris-acetic acid, 10 plus or minus 1mM magnesium acetate and 100 plus or minus 5 mu g/ml recombinant albumin.

8. The kit for detecting methylation of human BMP3 and/or NDRG4 gene according to claim 1, wherein the fluorescent PCR reaction solution further comprises PCR buffer, dNTP, DNA polymerase, betaine, tetramethylammonium chloride and nuclease-free water;

the concentration of the betaine in the fluorescent PCR reaction solution is 100 mM-150 mM; preferably, the concentration of betaine is 125. + -.5 mM.

9. The kit for detecting methylation of human BMP3 and/or NDRG4 of claim 8, wherein 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.

10. The kit for detecting human BMP3 and/or NDRG4 gene methylation according to claim 1, wherein the enzyme digestion reaction solution and the fluorescence PCR reaction solution are packaged in the same PCR amplification tube in layers by a hot-melt material, and preferably, the hot-melt material is fully refined paraffin with a melting point of 70-72 ℃.