CN109694912B

CN109694912B - Application of methylation sites, nucleic acid composition for detecting methylation, kit and detection method thereof

Info

Publication number: CN109694912B
Application number: CN201910150962.8A
Authority: CN
Inventors: 阙艳鹏; 钱纯亘; 郑刚; 胡鹍辉
Original assignee: Shenzhen Yhlo Biotech Co Ltd
Current assignee: Shenzhen Yhlo Biotech Co Ltd
Priority date: 2019-02-28
Filing date: 2019-02-28
Publication date: 2022-06-10
Anticipated expiration: 2039-02-28
Also published as: CN109694912A

Abstract

The invention relates to application of methylation sites, a nucleic acid composition for detecting methylation, a kit and a detection method thereof. The methylation site is used as a biomarker in the preparation of a lung cancer detection reagent, a lung cancer detection kit or a lung cancer detection device, and the methylation site is chr7: 19730212. The false positive rate of the detection kit developed by taking the methylation sites as the biomarkers for lung cancer detection is low.

Description

Application of methylation sites, nucleic acid composition for detecting methylation, kit and detection method thereof

Technical Field

The invention relates to the field of molecular biology, in particular to application of methylation sites, a nucleic acid composition for detecting methylation, a kit and a detection method thereof.

Background

Lung cancer is one of the most rapidly growing malignancies that threaten human health and life. In many countries, the incidence and mortality of lung cancer have been reported to be significantly higher in recent 50 years, with lung cancer incidence and mortality in men accounting for the first of all malignancies, in women accounting for the second, and mortality accounting for the second. The etiology of lung cancer is not completely clear up to now, and a large amount of data show that a large amount of smoking for a long time has a very close relationship with the occurrence of lung cancer. In addition, carcinogens in atmospheric pollution and smoke can also contribute to lung cancer.

There are three main types of lung cancer diagnosis: x-ray examination and fiberbronchoscopy. X-ray examination is performed by fluoroscopy or positive chest X-ray examination to detect lung shadows, but is often difficult to visualize for early cancers less than 2cm in diameter. The carcinoembryonic antigen test has limited specificity and sensitivity, and thus has little diagnostic value for early cases. Although the accuracy of the fiberbronchoscopy is high, the fiberbronchoscopy has certain pain and is difficult to accept by patients.

DNA methylation refers to the covalent bonding of a methyl group to the cytosine 5' carbon of a genomic CpG dinucleotide under the action of DNA methyltransferase. DNA methylation is one of the DNA modifications and is an important component of epigenetics. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression. Changes in DNA methylation status are an important cause of lung cancer. The methylation of the lung cancer related gene can be detected, so that the method can be used for early screening and diagnosis of the lung cancer. However, the conventional kit for detecting methylation of lung cancer related genes has a high false positive rate, and is not favorable for accurate detection of lung cancer.

Disclosure of Invention

Based on the above, there is a need to provide an application of methylation sites as biomarkers, and a detection kit developed based on the methylation sites has a low false positive rate for detecting lung cancer.

In addition, it is necessary to provide a nucleic acid composition for detecting methylation, a kit and a detection method thereof.

The application of the methylation sites as biomarkers in the preparation of lung cancer detection reagents, lung cancer detection kits or lung cancer detection devices is characterized in that the methylation sites are chr7: 19730212.

After a great deal of research on biomarkers of lung cancer, the fact that the methylation mutation condition of the 19730212 th site of the 7 th chromosome has high correlation with the lung cancer is unexpectedly found, so that the methylation mutation condition can be used as the biomarkers to be applied to the preparation of lung cancer detection reagents, lung cancer detection kits or lung cancer detection devices. Through test verification, the false positive rate of the detection kit developed by taking the methylation sites as the biomarkers for lung cancer detection is lower than 5%, and the accurate detection of the lung cancer is facilitated.

A nucleic acid composition for detecting methylation comprising:

an amplification primer pair, wherein the amplification primer pair is designed aiming at the 19730022-19730301 locus region on the No. 7 chromosome of a target gene; and

A methylation detection probe designed for the gene of interest that is methylated at position 19730212 on chromosome 7.

In one embodiment, the forward primer of the amplification primer pair has the sequence shown in SEQ ID No. 1.

In one embodiment, the reverse primer of the amplification primer pair has the sequence shown in SEQ ID No. 2.

In one embodiment, the methylation detection probe has a sequence shown in SEQ ID No. 3.

In one embodiment, the kit further comprises a non-methylation detection probe designed for the target gene which is not methylated at the 19730212 th site on the 7 th chromosome.

In one embodiment, the methylated detection probe and the unmethylated detection probe each have a fluorophore attached thereto, and the fluorophore of the methylated detection probe is different from the fluorophore of the unmethylated detection probe.

In one embodiment, the sequence of the unmethylated detection probe is shown in SEQ ID No. 4.

A kit for detecting methylation comprises the nucleic acid composition for detecting methylation.

A method for detecting methylation, comprising the steps of:

extracting a target gene in a sample to be detected;

carrying out sulfite treatment on the target gene; and

performing fluorescent quantitative PCR amplification reaction on the target gene after sulfite treatment by using a nucleic acid composition, and performing detection analysis according to a reaction result, wherein the nucleic acid composition comprises an amplification primer pair and a methylation detection probe, the amplification primer pair is designed for 19730022-19730301 locus regions on No. 7 chromosome of the target gene, and the methylation detection probe is designed for the target gene with the methylation of 19730212 locus on No. 7 chromosome.

Drawings

FIG. 1 is an amplification curve of a positive control;

FIG. 2 is an amplification curve of a negative control;

FIG. 3 is an amplification curve of a blank quality control product.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

In one embodiment, the methylation site is used as a biomarker in preparation of a lung cancer detection reagent, a lung cancer detection kit or a lung cancer detection device, and the methylation site is chr7: 19730212. I.e., position 19730212 on chromosome seven. The methylation condition of the target gene of the sample to be detected can be detected through the methylation site.

The research unexpectedly finds that the methylation mutation conditions of the chr7:19730212 of the lung cancer patients are obviously different from those of the healthy people, and the methylation mutation frequency of the chr7:19730212 of the lung cancer patients is obviously increased. Therefore, chr7:19730212 can be used as a biomarker for detecting early lung cancer, and is used for early diagnosis, treatment prediction, disease course detection or relapse monitoring of lung cancer and the like.

In one embodiment, biomarkers are biochemical markers that can mark changes or changes that may occur in the structure or function of systems, organs, tissues and cells.

In one particular example, the test sample contains CTC cells. The target gene is CtDNA, and the methylation site is located on the 19730212 th site on the seventh chromosome of the CTC cell. The methylation sites are not limited to the detection of methylation in CTC cells, and may be used to detect methylation in other cells, for example, in cancer tissue sections.

CTC (Circulating Tumor Cell) is a generic term for various Tumor cells present in peripheral blood. CTCs are found in peripheral blood in different morphologies, both in free individual CTCs and in cell aggregates (CTM Circulating Tumor Microemboli). Tumor cells undergo Epithelial-Mesenchymal Transition (EMT Epithelial mesenchyme Transition) during their entry into the peripheral blood circulation, so the cell types that make up CTCs include Epithelial cell phenotype, Mesenchymal cell phenotype, mixed Epithelial and Mesenchymal cell phenotype. CtDNA (circulating tumor DNA) is a unique DNA sequence in CTC cells. Researches show that the development condition of the tumor can be observed, the early tumor can be diagnosed and the tumor treatment effect can be tracked and monitored by detecting the change of the CtDNA.

After a great deal of research on biomarkers of lung cancer, the fact that the methylation mutation condition of the 19730212 th site of the 7 th chromosome has high correlation with the lung cancer is unexpectedly found, so that the methylation mutation condition can be used as the biomarkers to be applied to the preparation of lung cancer detection reagents, lung cancer detection kits or lung cancer detection devices. Tests prove that the false positive rate of the detection kit developed by taking the methylation sites as biomarkers for detecting the lung cancer is lower than 5%, and the accurate detection of the lung cancer is facilitated.

The lung cancer detection kit developed by taking the methylation sites as biomarkers can obtain a result with high specificity and low false positive rate for lung cancer detection only by detecting the methylation condition of chr7:19730212, and is simple and convenient to operate.

The nucleic acid composition for detecting methylation of one embodiment comprises an amplification primer pair and a methylation detection probe, wherein the amplification primer pair is designed for the 19730022-19730301 locus region on the 7 th chromosome of a target gene, and the methylation detection probe is designed for the target gene methylated at the 19730212 locus on the 7 th chromosome.

In one specific example, the gene of interest is CtDNA. The sequence of the 19730022-19730301 locus region on the 7 th chromosome of the target gene is shown as SEQ ID No. 5. Specifically, the sequence shown as SEQ ID No.5 is: 5'-GTCATGGTGGACGGATCACAAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTATGATCCCAGCTATTCCGGAGGCTGAGGCAGGAGAATGGCGTGAACCTGGGAGGCGAGGCTTGCAGTGAGCCAAGATCGCGCCACTGCATTCCAGCCTGGGTGACGGAGCAAGACTCCGTCTCAAAAAAGAAAAAAAAAAAAAAGAACACAAAGGGATAAAGCAATT-3' are provided.

In one specific example, the sequence of the forward primer of the amplification primer pair is shown as SEQ ID No. 1. Specifically, the sequence shown as SEQ ID No.1 is: 5'-GGAGAATGGCGTGAACCTG-3' are provided.

In one specific example, the sequence of the reverse primer of the amplification primer pair is shown as SEQ ID No. 2. Specifically, the sequence shown as SEQ ID No.2 is: 5'-TGCTTTATCCCTTTGTGTTC-3' are provided.

In one specific example, the methylation detection probe has the sequence shown in SEQ ID No. 3. Specifically, the sequence shown as SEQ ID No.3 is: 5'-TCGGTTGTAGCgCGGTGACGTAAGGT-3' are provided.

In one embodiment, the nucleic acid composition further comprises an unmethylated detection probe. The non-methylation detection probe is designed aiming at the target gene of which the 19730212 th site on the 7 th chromosome is not methylated. In one specific example, the sequence of the unmethylated detection probe is shown in SEQ ID No. 4. The sequence shown as SEQ ID No.4 is 5'-TCGGTTGTAGCaCGGTGACGTAAGGT-3'.

Further, the methylated detection probe and the unmethylated detection probe are both connected with a fluorophore, and the fluorophore of the methylated detection probe is different from the fluorophore of the unmethylated detection probe. Through the double fluorescence signals, the specificity and the accuracy of detection can be improved, and the false positive rate is reduced.

In one embodiment, the fluorescent group is selected from one of FAM, VIC, FITC, and HEX.

In one embodiment, a quenching group is attached to both the methylated and unmethylated detection probes. Further, the quenching group is selected from TAMRA, BHQ-1 and Dabcyl.

In one embodiment, the 5 'end of the methylated detection probe and the 5' end of the unmethylated detection probe are both linked to a fluorophore, and the 3 'end of the methylated detection probe and the 3' end of the unmethylated detection probe are both linked to a quencher.

In one specific example, the fluorophore attached to the 5' end of the methylation detection probe is FAM. The fluorescent group connected to the 5' end of the non-methylation detection probe is VIC. Both the 3 'end of the methylated detection probe and the 3' end of the unmethylated detection probe are connected with quenching groups, which are TAMRA.

The nucleic acid composition for detecting methylation comprises an amplification primer pair and a methylation detection probe, wherein the amplification primer pair is designed aiming at the 19730022-19730301 locus region on the No. 7 chromosome of a target gene, and the methylation detection probe is designed aiming at the target gene methylated at the 19730212 locus on the No. 7 chromosome, so that the nucleic acid composition is low in false positive rate and high in accuracy when used for detecting lung cancer. Tests prove that the lung cancer detection kit containing the nucleic acid composition has a false positive rate of less than 5% and specificity of no less than 95% in the detection of lung cancer, and is beneficial to accurate and effective detection of lung cancer.

The kit for detecting methylation of one embodiment includes the nucleic acid composition for detecting methylation of the above embodiment.

In one embodiment, the kit further comprises at least one of a positive quality control material, a negative quality control material, a DNA polymerase and a PCR reaction solution.

In one embodiment, the positive quality control is CTC cells in peripheral blood of a tumor stage III patient or peripheral blood of a tumor stage III patient. Further, the positive quality control material is CTC cells in peripheral blood of a patient with stage III lung cancer or peripheral blood of a patient with stage III lung cancer.

In one embodiment, the negative quality control is CTC cells in normal human serum, peripheral blood of a tumor stage I patient, or peripheral blood of a tumor stage I patient. Further, the negative quality control material is CTC cells in peripheral blood of a patient with stage I lung cancer or peripheral blood of a patient with stage I lung cancer.

In one particular example, the DNA polymerase is Taq DNA polymerase.

In one embodiment, the PCR reaction solution comprises Tris buffer, dNTP and Mg²⁺. Further, PCThe R reaction solution comprises Tris buffer solution, 0.1-0.5 mmol/L dNTP and 3.1-3.9 mmol/L Mg²⁺. Wherein dNTP is deoxyribonucleoside triphosphate, including dATP, dGTP, dTTP, dCTP.

In one embodiment, the kit further comprises a CtDNA extraction reagent. Further, the CtDNA extraction reagent comprises cell lysate, adsorbent and eluent.

Wherein the cell lysate is used for the lysis of CTC cells to release DNA. Further, the cell lysate is a cell lysate purchased from Biotechnology engineering (Shanghai) Inc.

The adsorbent is used to adsorb DNA released from CTC cells. Further, the adsorbent is a magnetic bead. The adsorbent is not limited to magnetic beads, and may be other adsorbents, for example, a nanopore adsorbent microsphere.

The eluent is used to elute the DNA from the magnetic beads. Further, the eluent is an organic solvent. Further, the eluent includes at least one of ethanol and chloroform. The eluent is not limited to the above-mentioned reagent, and may be another organic solvent, for example, phenol.

In one embodiment, the CtDNA extraction reagent further comprises a decontamination reagent and a reconstitution reagent.

Wherein the impurity removal reagent is used for removing impurities such as protein, grease, RNA, saccharides, inorganic salt and the like in DNA released by CTC cells. Further, the impurity removing reagent is at least one selected from absolute ethyl alcohol and trichloromethane.

The re-solubilization reagent is used to re-fuse the eluted DNA. Further, the redissolving reagent is sterile TE solution or sterile double distilled water. The reconstitution reagent is not limited to the above-mentioned reagent, and may be other reconstitution reagents, for example, sterile enzyme-removed water.

In one embodiment, the kit further comprises a sulfite conversion reagent. The sulfite conversion reagent is used for converting unmethylated 5 'cytosine in the gene DNA to be detected into uracil, and the methylated 5' cytosine is not changed, so that Bis-DNA is obtained finally. Cytosine (C) in the gene DNA to be detected can be converted into uracil (U), and the possibility of deamination of 5-methylcytosine into uracil is extremely low, so that cytosine in the DNA sample treated by bisulfite can only be 5-methylcytosine. After DNA methylation, because the existence of 5-methylcytosine does not influence base pairing, compared with the traditional Polymerase Chain Reaction (PCR) amplification process, methylation information can be lost, and DNA methylation information can be effectively retained through sulfite conversion.

The kit for detecting methylation comprises the nucleic acid composition of the embodiment, can detect the middle and late stages of lung cancer, can screen and diagnose the early stage of lung cancer, and has the advantages of low false positive rate, high accuracy and strong specificity; meanwhile, the kit can be used for detecting the course of the lung cancer patient, provides genotype reference for the later treatment of the lung cancer patient, and can be used for evaluating the treatment effect of the lung cancer.

With the completion of human genome sequencing and the rapid development of high-throughput sequencing technology, gene screening is becoming the direction of lung cancer diagnosis. The first generation of DNA sequencing technology used the comprehensive application of the chain termination method pioneered in 1975 by Sanger (Sanger) and Coelson (Coulson), and the chemical method (chain decomposition method) invented in 1976-1977 by Mark Simm (Maxam) and Gilbert (Gilbert). And in 1977 the first genomic sequence was determined. The core principle of the method is as follows: because the 2 'and 3' of the ddNTP do not contain hydroxyl, a certain proportion of ddNTP with radioisotope labels is respectively added into a DNA synthesis reaction system, and the DNA sequence of the molecule to be detected can be determined according to the position of an electrophoresis zone after gel electrophoresis and autoradiography. The first generation sequencing technology has the defects of high sequencing cost, long sequencing time of a single gene, low flux and the like, and seriously influences the real large-scale application of the first generation sequencing technology. In the second generation sequencing technology, the magnetic beads with DNA are treated with a polymerase and single-stranded collectin before sequencing, and then the magnetic beads are placed on a PTP plate. The plate was specially designed with a number of wells about 44 μm in diameter, each well being capable of holding only one magnetic bead, and each magnetic bead was fixed in position by this method for sequencing by pyrosequencing. A magnetic bead having a diameter smaller than that of the hole of the PTP plate was placed in the hole to initiate the sequencing reaction. If dNTPs can be paired with a sequence to be detected, a pyrophosphate group is released after synthesis, and the released pyrophosphate group reacts with ATP sulfurylase in a reaction system to generate ATP. The generated ATP and luciferase are oxidized together to enable the luciferin molecules in the sequencing reaction to turn over fluorescence, the emitted fluorescence is recorded by a CCD camera on the other side of the PTP plate, and finally, the computer is used for optical signal processing to obtain the final sequencing result. Second generation sequencing technologies are difficult to detect for gene methylation.

Therefore, one embodiment of the present invention also provides a methylation detection method, which can perform non-disease diagnosis and therapeutic detection of methylation of a target gene, and can better develop a drug for inhibiting methylation of a cellular gene by detecting methylation of a target gene. Specifically, the detection method includes the following steps S110 to S130:

and S110, extracting the target gene of the sample to be detected.

In one embodiment, the sample to be tested is a blood product for testing.

In one embodiment, the sample contains CTC cells and the target gene is CtDNA.

In a specific example, the method for extracting the target gene of the sample to be tested comprises the following steps: the CtDNA extraction reagent in the kit of the above embodiment is used for extracting the DNA of the circulating tumor cells. The CtDNA extraction reagent in the kit according to the above embodiment is not limited to the one, and a commercially available CtDNA extraction reagent may be used.

In one embodiment, before S110, obtaining the circulating tumor cells in the test sample is further included.

In one embodiment, the method for obtaining the circulating tumor cells in the sample to be tested is to separate the circulating tumor cells from the sample to be tested by using a microfluidic chip. The method for obtaining the circulating tumor cells in the sample to be tested is not limited to the above-mentioned method, but may be other methods, for example, flow cytometric separation to select the circulating tumor cells.

S120, carrying out sulfite treatment on the target gene.

In one embodiment, the step of subjecting the gene of interest to a sulfite treatment comprises: mixing 22-28 mul of target gene with 22-28 mul of NaOH aqueous solution of 2.9mol/L, and incubating for 28-32 min at 40-44 ℃; adding 13-17 mu L of hydroquinone for continuous incubation; and (3) incubating until the incubation liquid is light yellow, adding sodium bisulfite solution, mixing uniformly to make the total volume be 300 mu L, adding 150 mu L of paraffin oil or vaseline, and carrying out dark water bath at 48-52 ℃ for 15-17 h to obtain DNA treated by sulfite. Wherein the sodium bisulfite solution is an aqueous solution of sodium bisulfite with pH of 4.9-5.1 and 3.4-3.8 mol/L.

S130, carrying out fluorescent quantitative PCR amplification reaction on the target gene treated by the sulfite by adopting a nucleic acid composition, and carrying out detection analysis according to a reaction result, wherein the nucleic acid composition comprises an amplification primer pair and a methylation detection probe, the amplification primer pair is designed aiming at the 19730022-19730301 locus region on the No. 7 chromosome of the target gene, and the methylation detection probe is designed aiming at the target gene methylated at the 19730212 locus on the No. 7 chromosome.

In one embodiment, the nucleic acid composition further comprises an unmethylated detection probe. The non-methylation detection probe is designed aiming at the target gene of which the 19730212 th site on the 7 th chromosome is not methylated.

Furthermore, the methylated detection probe and the unmethylated detection probe are both connected with a fluorophore, and the fluorophore of the methylated detection probe is different from the fluorophore of the unmethylated detection probe. Through the double fluorescence signals, the specificity and the accuracy of detection can be improved, and the false positive rate is reduced.

In one specific example, the sequence of the unmethylated detection probe is shown in SEQ ID No. 4. The sequence shown as SEQ ID No.4 is 5'-TCGGTTGTAGCaCGGTGACGTAAGGT-3'. Further, the fluorophore attached to the 5' end of the methylation detection probe is FAM. The fluorescent group connected to the 5' end of the non-methylation detection probe is VIC. Both the 3 'end of the methylated detection probe and the 3' end of the unmethylated detection probe are connected with quenching groups, which are TAMRA.

In one embodiment, the reaction system for performing the fluorescence quantitative PCR amplification reaction comprises 0.1 ng/L-1 ng/L of a sample to be detected, 0.1 mu mol/L-2 mu mol/L of an amplification primer pair, 1.3U-1.7U of TaqDNA polymerase, 0.1-0.5 mmol/L dNTP and 3.1-3.9 mmol/L Mg²⁺And 0.1-2 mu mol/L methylation detection probe. Furthermore, the reaction system also comprises a non-methylation detection probe of 0.1 to 2 mu mol/L.

In one embodiment, the reaction procedure for performing the fluorescent quantitative PCR amplification reaction is: pre-denaturation at 94-95 ℃ for 2-5 min for 1 cycle; denaturation at 94-95 ℃ for 15-20 s, annealing at 53-58 ℃ for 15-20 s, and extension at 68-72 ℃ for 15-20 s for 50-60 cycles, and collecting a fluorescence signal after each cycle.

In one embodiment, the step of performing a detection analysis based on the reaction result comprises: and determining the methylation degree of the target gene in the sample to be detected by determining the Ct value of the fluorescence channel where the methylation detection probe is located. Specifically, the smaller the Ct value of the fluorescence channel where the methylation detection probe is located, the more methylated circulating tumor cells are in the sample to be detected. In one specific example, the smaller the Ct value of the FAM fluorescence channel, the more methylated circulating tumor cells are in the test sample.

Further, the step of performing detection analysis according to the reaction result further comprises: and (3) judging the non-methylation degree of the target gene in the sample to be detected by judging the Ct value of the fluorescence channel where the non-methylation detection probe is positioned. Specifically, the larger the Ct value of the fluorescence channel where the unmethylated detection probe is located, the more unmethylated circulating tumor cells are in the sample to be detected. In one specific example, the larger the Ct value of the VIC fluorescence channel, the more non-methylated circulating tumor cells in the test sample.

In one embodiment, before the step of S140, the method further comprises the step of preparing a positive quality control: taking tumor tissue slices of patients with stage III lung cancer, performing primary culture, after proliferation, digesting and diluting until the cell density is 10⁵Per mL to 10⁸Cell suspension per mL.

In one embodiment, before the step of S140, the method further comprises the step of preparing a negative quality control product: taking tumor tissue section of lung cancer stage I patient, performing primary culture, and after proliferation, digesting and dilutingReleased until the cell density is 10⁵Per mL to 10⁸Cell suspension per mL.

In one embodiment, the platform for the quantitative fluorescence PCR detection comprises a conventional quantitative fluorescence detection platform, such as the quantitative fluorescence detection platform of Roche, the quantitative fluorescence detection platform of Bio-Rad, the quantitative fluorescence detection platform of AgiIent, and the quantitative fluorescence detection platform of ABI.

The methylation detection method can accurately detect the methylation degree of a target gene, has low false positive detection rate, high accuracy and strong specificity, can be used for carrying out non-disease treatment and diagnostic detection on the methylation condition of the gene, and can be applied to the development of medicaments for inhibiting the methylation of the target gene.

Further, the sample to be tested according to the above embodiment is a blood sample, and generally, about 10 CTC cells are contained in 10mL of whole blood of a patient with a primary cancer. In the case where the amount of CTC cells is too small, it is difficult to control the number of CTC cells in the sample to be tested. The above embodiment detects the copy number of DNA by a dual fluorescence channel instead of detecting the number of cells. The more the DNA is detected, the smaller the Ct value is, the more the CTC cells in the sample to be detected are represented, the simple operation is realized, and the accurate result is obtained.

The following are specific examples:

example 1

Extracting DNA of CTC cells in sample to be detected

(1) And (3) screening CTC cells from a sample to be detected by a Hylo-Dva280 type micro-fluidic chip, wherein the sample to be detected is whole blood.

(2) A commercial magnetic bead method DNA extraction kit (purchased from Biotechnology engineering (Shanghai) Co., Ltd.) is adopted, and the DNA in the CTC cells is extracted according to the use instruction of the kit, and the specific operation is as follows:

(a) And (3) loading the collected CTC cells into a sample tube, diluting to 50 mu L by using a buffer solution with a salt balance function, adding 430 mu L of cell lysate, shaking for resuspension, and standing for 5min in a 65 ℃ water bath at constant temperature. After cooling to room temperature, 20. mu.L of RNaseA was added, mixed by a vortex shaker and left for 2 min. Adding 400 mu L of magnetic bead diluent and 10 mu L of magnetic bead mother liquor into the sample tube, shaking and uniformly mixing the mixture by a vortex oscillator for 20s, and standing the mixture for 1 min.

(b) Placing the sample tube on a magnetic separator for 30s, absorbing and discarding supernatant after the magnetic bead components are completely adsorbed to the tube wall, taking the sample tube out of the magnetic separator, adding 400 mu L of buffer solution for cleaning the magnetic beads into the sample tube, uniformly mixing for 10s by a vortex oscillator, placing the sample tube on the magnetic separator again for 30s, discarding the supernatant components after the magnetic bead components are completely adsorbed to the tube wall, and taking the sample tube out of the magnetic separator.

(c) Adding 700 mu L of ethanol water solution with the mass percentage of 70% into the sample tube, and gently blowing and stirring the mixture by using a pipette and uniformly mixing the mixture. And (3) placing the sample tube on a magnetic frame for 30s, taking out the sample tube from the magnetic frame after the magnetic bead components are completely adsorbed to the tube wall, and absorbing and discarding the supernatant. And repeating the step of washing the ethanol aqueous solution with the mass percentage of 70 percent once. And opening the cover of the sample tube, and drying in an air box at about 25 ℃ for 10min until no liquid remains in the sample tube.

(d) mu.L of TE solution was added to the sample tube, and heated in a water bath at 65 ℃ for 7 min. Mixing uniformly for 4 times during heating. And taking out the sample tube, placing the sample tube on a magnetic frame, observing that all magnetic bead components are adsorbed on the tube wall, carefully absorbing the supernatant into a new centrifugal tube, and obtaining the DNA of the CTC cells in the sample to be detected, namely the DNA to be processed.

Example 2

DNA methylation treatment of CTC cells in a sample to be tested

(1) An aqueous sodium bisulfite solution having a concentration of 3.6mol/L was prepared, and titrated with an aqueous NaOH solution of 3mol/L to a pH of 5.0.

(2) 25 mu L of DNA to be treated is taken, 2.5 mu L of 3mol/L NaOH aqueous solution is added, after water bath at 42 ℃ for 30min, 15 mu L of hydroquinone is added, and the water bath is kept until the solution becomes light yellow. The above aqueous sodium bisulfite solution was added to make the total volume of the solution 300. mu.L. The EP tube was protected from light, slowly inverted and mixed, and 150. mu.L of vaseline was added. Wrapping with tinfoil to avoid light, and performing water bath at 50 ℃ for 16h to obtain methylated DNA, namely the DNA to be detected.

Example 3

Kit for detecting methylation

The kit comprises a nucleic acid composition, a positive quality control product, a negative quality control product, a blank quality control product, DNA polymerase and PCR reaction liquid;

the nucleic acid composition is shown in table 1, the nucleic acid composition comprises an amplification primer pair, a methylation detection probe and a non-methylation detection probe, the amplification primer pair is designed for the 19730022-19730301 th site region on chromosome 7 of a target gene, the methylation detection probe is designed for the target gene with methylation at the 19730212 th site on chromosome 7, the non-methylation detection probe is designed for the target gene with methylation at the 19730212 th site on chromosome 7, the target gene is CtDNA, the sequences of the 19730022-19730301 th site regions on chromosome 7 in the target gene are shown in SEQ ID No.5, specifically, the sequences shown in SEQ ID No.5 are: 5'-GTCATGGTGGACGGATCACAAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTATGATCCCAGCTATTCCGGAGGCTGAGGCAGGAGAATGGCGTGAACCTGGGAGGCGAGGCTTGCAGTGAGCCAAGATCGCGCCACTGCATTCCAGCCTGGGTGACGGAGCAAGACTCCGTCTCAAAAAAGAAAAAAAAAAAAAAGAACACAAAGGGATAAAGCAATT-3', respectively;

The preparation process of the positive quality control product comprises the following steps: biopsy of tumor tissue from stage III Lung cancer patients was performed in DMEM medium (Hyclone) at 37 ℃ with 5% CO₂After culturing for 48 hours in a concentration cell culture chamber (purchased from Thermo Fisher Scientific) to proliferate cells, digesting the cells diluted with 2.5% by mass of trypsin solution diluted with ultrapure water to a cell suspension having a cell density of 10^ 5/mL, and treating 1mL of the cell suspension according to the procedure of example 1 to obtain the DNA to be treated. Finally, diluting the DNA to be treated by using 1mL of calibrator diluent (purchased from life science company) containing heat inactivated bovine serum to obtain a positive quality control;

the preparation process of the negative quality control product comprises the following steps: biopsy of lung cancer stage I patients was prepared in DMEM medium (purchased from Hyclone) at 37 deg.C with 5% CO₂Cell culture chamber (purchased from Thermo F)isher Scientific), culturing for 48h to proliferate cells, digesting and diluting the cells with 2.5% trypsin solution by mass percent diluted with ultrapure water to obtain cell suspension with the cell density of 10^ 5/mL, taking 1mL of the cell suspension, treating the cell suspension according to the operation of example 1 to obtain DNA to be treated, and finally diluting the DNA to be treated with 1mL of calibrator diluent (purchased from life science company) containing heat-inactivated bovine serum to obtain a negative quality control product;

Blank quality control products are as follows: dilutions of calibrator (purchased from life science) containing newborn bovine serum;

the DNA polymerase is Taq DNA polymerase.

The PCR reaction solution comprises Tris buffer solution, dNTP and Mg²⁺。

TABLE 1 nucleic acid compositions

Example 4

The kit of example 3 was used to perform fluorescent quantitative PCR detection on the positive, negative, and blank quality controls, respectively, to obtain Ct values for each sample to be tested. The measurement results are shown in FIGS. 1 to 3, where "Cycle Number" indicates the Number of cycles, "Δ Rn" indicates the fluorescence value, "FAM" indicates the FAM channel, and "VIC" indicates the VIC channel in FIGS. 1 to 3. FIG. 1 shows an amplification curve of a positive control, with Ct values; FIG. 2 is an amplification curve of a negative quality control product, and the Ct value is 42; FIG. 3 is an amplification curve of a blank quality control product.

Wherein, the reaction system is as follows: 0.5ng/L sample to be tested, 1 mu mol/L amplification primer pair, 1.5U Taq DNA polymerase, 0.3mmol/L dNTP and 3.5mmol/L Mg²⁺1 mu mol/L of methylated detection probe and 1 mu mol/L of unmethylated detection probe; the sample to be tested is a positive quality control product, a negative quality control product or a blank quality control product;

the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5min for 1 cycle; denaturation at 94 ℃ for 17s, annealing at 55 ℃ for 15s, and extension at 70 ℃ for 20s for 60 cycles, and collecting a fluorescence signal after each cycle is finished;

The detection channels are FAM channels and VIC channels.

As can be seen from FIGS. 1 to 3, the amplification curves of the blank quality control substance in the FAM channel and the VIC channel are both straight lines, and the Ct values of the blank quality control substance in the two channels are UNDET. The negative quality control product has an amplification curve in the VIC channel, and the Ct value of the negative quality control product is 40-60. The positive quality control product has an amplification curve in an FAM channel, and the Ct value of the positive quality control product is 20-30.

Example 5

(1) And taking 15mL of sample to be detected, and carrying out CTC cell screening through a Hylo-Dva280 type micro-fluidic chip. The samples to be tested are 20 blood samples of lung cancer stage I patients, 20 blood samples of lung cancer stage II patients, 20 blood samples of lung cancer stage III patients, 20 blood samples of lung cancer stage IV patients (all with bronchoscope and pathological results), 20 blood samples of healthy people and 20 pure water samples.

(2) With reference to the procedure of example 1, a commercial magnetic bead method DNA extraction kit (purchased from bio-engineering (shanghai) corporation) was used to extract DNA from CTC cells according to the instructions of the kit, and the specific procedure was as follows:

(a) adding the collected CTC cells into a sample tube, adding 430 mu L of cell lysate into the sample tube, shaking for resuspension, closing a sample tube cover, and standing at a constant temperature of 65 ℃ for 5 min. After cooling to room temperature, 20. mu.L of RNaseA was added, mixed by a vortex shaker and left for 2 min. Adding 400 mu L of magnetic bead diluent and 10 mu L of magnetic bead mother liquor into the sample tube, shaking and uniformly mixing the mixture by a vortex oscillator for 20s, and standing the mixture for 1 min.

(b) And (2) placing the sample tube on a magnetic separator for 30s, sucking and discarding supernatant after the magnetic bead components are completely sucked to the tube wall, taking the sample tube out of the magnetic separator, adding 400 mu L of buffer solution for cleaning the magnetic beads into the sample tube, uniformly mixing for 10s by using a vortex oscillator, placing the sample tube on the magnetic separator again for 30s, after the magnetic bead components are completely sucked to the tube wall, discarding the supernatant components, and taking the sample tube out of the magnetic separator.

(c) Adding 700 mu L of ethanol-water mixed solution with the mass percentage of 70% into the sample tube, and gently blowing and mixing the mixture by using a pipette. And (3) placing the sample tube on a magnetic frame for 30s, taking out the sample tube from the magnetic frame after the magnetic bead components are completely adsorbed to the tube wall, and absorbing and discarding the supernatant. And the organic solution washing step was repeated once. And (3) opening the cover of the sample tube, placing the sample tube in an air box at about 25 ℃, and drying for 10min until no liquid remains in the sample tube.

(d) To the sample tube, 75. mu.L of TE solution was added and heated in a water bath at 65 ℃ for 7 min. In the heating process, mix well 4 times. And taking out the sample tube, placing the sample tube on a magnetic frame, observing that all magnetic bead components are adsorbed to the tube wall, and then sucking the supernatant to a new centrifugal tube to obtain the DNA of the CTC cells in the sample to be detected, namely the DNA to be processed.

(3) The DNA to be treated was methylated according to the procedure of example 2 to obtain a DNA to be tested.

(4) The kit in the embodiment 1 is adopted to carry out fluorescence quantitative PCR detection on DNA to be detected, the reaction system and the reaction program refer to the embodiment 4, Ct values of samples to be detected in FAM channels and VIC channels are counted, and the determination results are detailed in tables 2-3.

TABLE 2 Ct values of samples to be tested in FAM channel

Ct value	0～15	16～30	30～45	45～60	UNDET	Total of
							Stage IV samples (example)	16	3	1	0	0	20
Stage III specimens (examples)	8	7	3	2	0	20
							Phase II sample (example)	5	6	8	1	0	20
Phase I sample (example)	1	0	6	11	2	20
							Normal human sample (example)	0	0	0	1	19	20
Pure water sample (example)	0	0	0	0	20	20

TABLE 3 Ct value of each sample to be tested in VIC channel

Ct value	0～15	16～30	30～45	45～60	UNDET	In total
							Stage IV samples (example)	2	1	4	8	5	20
Stage III specimens (examples)	2	3	9	4	2	20
							Phase II sample (example)	1	1	13	2	3	20
Phase I sample (example)	8	7	3	2	1	20
							Normal human sample (example)	0	0	0	3	17	20
Pure water sample (example)	0	0	0	0	20	20

As can be seen from tables 2-3, the number of cases with Ct values of 0-15 in FAM channels of samples in stages I-IV increases in sequence, which indicates that the methylation degree of CTC cells in samples in the middle and late stages of lung cancer is higher. The number of cases with the Ct value of 45-60 in the VIC channel of the stage IV sample is larger than that of the stage I sample, possibly because the number of CTC cells in the stage IV sample is more than that of the stage I sample.

Example 6

(1) CTC cells were screened from 50 test samples, including 30 blood samples of patients diagnosed with stage III and IV lung cancer and 20 blood samples of healthy persons, according to the procedure of step (1) of example 5.

(2) Referring to the procedure of example 1, DNA in equivalent amount of CTC cells was extracted using a magnetic bead method blood DNA extraction kit from Shanghai Biotech, to obtain DNA to be treated.

(4) The kit of example 1 is used to perform fluorescent quantitative PCR detection on DNA to be detected, and the reaction system and the reaction program are referred to example 4. The results are shown in Table 4. Sensitivity, specificity and false positives were calculated:

the sensitivity is the number of lung cancer samples with positive detection results/total lung cancer samples;

the specificity is the number of lung cancer samples with negative detection result/total normal samples;

the false positive rate is the number of lung cancer samples with positive test results/total number of normal samples.

TABLE 450 test results of samples tested

	Patient sample	Healthy human sample
			Positive (one)	29	1
Negatives (one)	1	19
			Sensitivity (%)	96.7％	/
Specificity (%)	/	95％
			False positive rate (%)	/	5％

As can be seen from Table 4, when the kit of the above embodiment detects methylation of CTC cells in a sample to be detected, the sensitivity can reach 96.7%, the specificity can reach 95%, and the false positive rate is as low as 5%.

In summary, the lung cancer detection kit containing the nucleic acid composition of the above embodiment can detect methylation of CTC cells of a sample to be detected, and has good performance indexes such as detection sensitivity and specificity. Meanwhile, the kit can detect only a whole blood sample, does not need body fluid biopsy or tissue slice detection, can be used for early screening and diagnosis of lung cancer, can reveal the treatment effect of a lung cancer patient, and provides genotype reference for treatment of the lung cancer patient.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not 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.

Sequence listing

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Claims

1. The application of the methylation locus as a biomarker in the preparation of a lung cancer detection reagent, a lung cancer detection kit or a lung cancer detection device is characterized in that the methylation locus is the 191 th locus in a sequence shown as SEQ ID No.5 on the 7 th chromosome.

2. A nucleic acid composition for detecting methylation, comprising:

the amplification primer pair is designed aiming at a target gene with a sequence shown as SEQ ID No.5 on the No. 7 chromosome; and

a methylation detection probe designed for methylation at position 191 of the gene of interest.

3. The nucleic acid composition for detecting methylation of claim 2, wherein the forward primer of the amplification primer pair has the sequence shown in SEQ ID No. 1.

4. The nucleic acid composition for detecting methylation according to claim 2, wherein the reverse primer of the amplification primer pair has the sequence shown as SEQ ID No. 2.

5. The methylation detection nucleic acid composition of claim 2, wherein the methylation detection probe has a sequence shown in SEQ ID No. 3.

6. The methylation detection nucleic acid composition of any one of claims 2-5, further comprising a non-methylation detection probe designed for the no methylation at position 191 of the gene of interest.

7. The methylation detection nucleic acid composition of claim 6, wherein the methylation detection probe and the non-methylation detection probe are both linked to a fluorophore, and the fluorophore of the methylation detection probe is different from the fluorophore of the non-methylation detection probe.

8. The methylation detection nucleic acid composition of claim 6, wherein the unmethylated detection probe has the sequence shown in SEQ ID No. 4.

9. A kit for detecting methylation, comprising the nucleic acid composition for detecting methylation of any one of claims 2 to 8.