CN112094912A

CN112094912A - Plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules and application thereof

Info

Publication number: CN112094912A
Application number: CN202011112824.XA
Authority: CN
Inventors: 顾月清; 刘云龙; 胡子健
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2020-12-18

Abstract

The invention discloses a plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules and application thereof. A combination of plasma-free DNA methylation genes for identifying benign and malignant lung nodules selected from at least any two of SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO 1. The invention discloses application of a plasma free DNA methylation gene combination as a detection target in preparation of a reagent for identifying benign and malignant lung nodules. In the technical scheme of the invention, a methylation relative quantitative method is adopted, GAPDH is mainly used as an internal reference, a methylation relative quantitative reference product is constructed, and then a relative methylation standard curve is made. The methylation degree of the lung malignant tumor related gene can be obtained through a standard curve, and compared with quantitative methylation detection, the quantitative methylation detection method is more sensitive, improves the detection accuracy, reduces the false positive rate, and has higher clinical value for the diagnosis of benign and malignant lung nodules.

Description

Plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules and application thereof

Technical Field

The invention belongs to the field of biological detection, and relates to a plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules and application thereof.

Background

The 2011 random contrast research results of the National Lung Screening Trial (NLST) show that compared with the X-ray chest radiography, the mortality rate of Lung cancer can be reduced by 20% by Screening high risk population with low-dose chest CT. However, the low-dose helical CT can only identify lung nodules, but has low differential diagnosis sensitivity on benign and malignant lung nodules, namely a false positive rate of 96.4%, which brings some trouble to patients. According to the Chinese diagnosis and treatment expert consensus on pulmonary sarcoidosis, sarcoidosis is a systemic granulomatous disease of which the etiology and pathogenesis are not clear, mainly causes diseases of middle-aged and young people, and has a slightly higher female morbidity than male; the incidence and clinical manifestations are different between different regions and races, the annual incidence in Sweden reaches 160/10 ten thousand, the annual incidence in caucasian America is 49.8/10 ten thousand, the annual incidence in African America is 141/10 ten thousand, the annual incidence in Japan is 1.01/10 ten thousand, and China is lack of epidemiological data of sarcoidosis. Typical pulmonary sarcoidosis is manifested by enlargement of the mediastinal and symmetrical double-portal lymph nodes, with or without intrapulmonary shading; it is often accompanied by eye and skin diseases, and may also affect tissues and/or organs such as liver, spleen, lymph node, salivary gland, heart, nervous system, bone and muscle. The diagnosis of sarcoidosis mainly depends on clinical, image and pathological data for comprehensive judgment. Based on the fact that the tissue biopsy of the affected part is definitely non-caseous necrotic epithelioid granuloma, the patient can be diagnosed with sarcoidosis after clinical and imaging performance is combined, except other causes. The affected tissues and/or organs, clinical manifestations, treatment response and prognosis of patients with different sarcoidosis are quite heterogeneous, most patients have good prognosis, part of patients have self-limiting disease courses, about 25 percent of patients have chronic and progressive disease courses, and finally irreversible diseases such as pulmonary fibrosis, liver cirrhosis, lethal arrhythmia, blindness and the like are caused, so that the quality of life and the service life of the patients are seriously influenced.

The currently recommended differential diagnosis method for pulmonary nodules is (1) stage I and II sarcoidosis: the identification of tuberculosis infection, lymphoproliferative diseases, IgG4 related diseases, malignant tumors and the like is required; (2) stage III sarcoidosis needs to be identified with various occupational lung diseases, pulmonary tuberculosis and the like; (3) stage iv sarcoidosis requires the identification of pulmonary fibrosis due to various etiologies, such as various occupational pulmonary fibrosis, idiopathic pulmonary fibrosis, other secondary pulmonary fibrosis due to various causes, and the like.

Early lung cancer usually appears as a lung nodule, but the lung nodule comprises both benign tumor and malignant tumor, so the identification of benign and malignant lung nodule has very important significance for early lung cancer discovery. At present, the differential diagnosis of benign and malignant lung nodules mainly depends on CT images and histopathological biopsy, and is judged according to the subjective experience of doctors, the wound is large, patients cannot tolerate the disease, and the like. Other tumor markers include carcinoembryonic antigen, alpha-fetoprotein, carbohydrate antigen 125, carbohydrate antigen 199, neuron-specific enolase, cytokeratin 21-1, and the like. However, the clinical sensitivity of the existing tumor marker for detecting malignant nodules alone is too low to meet the clinical requirements, and the combined detection of multiple tumor markers can improve the clinical sensitivity and accuracy of early diagnosis of lung nodules. In view of the important auxiliary effect of the tumor marker on early diagnosis of benign and malignant lung nodules, one or more efficient tumor indexes can be searched to improve the judgment rate of the benign and malignant lung nodules.

DNA methylation is a very important tumor biomarker, and methylation is a base modification on DNA and plays an important role in regulating and controlling gene expression. DNA methylation is the most widely and deeply studied important part of epigenetics and is closely related to the development and development of lung cancer. The DNA methylation refers to a chemical modification phenomenon that S-adenosylmethionine (SAM) is used as a methyl donor under the catalytic action of DNA methyltransferases (DNMTs) and a methyl group is added to the C5 position of CpG nucleotide cytosine of the DNA, wherein about 3% -6% of cytosine exists in a form of 5-methylcytosine, and methylation change of the DNA mainly occurs in a CpG nucleotide sequence and comprises two states of hypermethylation and hypomethylation, and the main mechanism is that DNA methylation enables a regulatory factor compound to be close to a nucleosome and histone deacetylation with positive charge and DNA negative charge are reacted, so that a chromosome structure is compact and can not be transcribed, cancer suppressor genes are subjected to deep methylation, chromosome instability and cell cycle disorder, and the DNA further develops into malignant tumors. Therefore, the occurrence and development of tumors are often accompanied by methylation of abnormal DNA, and a proper methylation detection site is found, so that the benign and malignant part of the lung nodule can be diagnosed.

ctDNA is a cfDNA present in blood, which originates from tumor cells. Generally, most tumor cells in vivo release some of their own DNA. ctDNA has multiple origins, most commonly being (1) apoptotic or necrotic tumor cells, (2) active release of tumor cells, (3) circulating tumor cells and in exosome vesicles. ctDNA has a higher sensitivity as a biomarker than CTC detection compared to circulating tumor cells. Since the changes of ctDNA are consistent with the tumor genome, ctDNA may be a potential substitute for the tumor genome, and has no tissue heterogeneity, playing a great role in the diagnosis of lung cancer. The kit tries to use a minimally invasive ctDNA methylation detection method of liquid biopsy, so that the identification of benign and malignant lung nodules is simpler, the tolerance of patients is high, and the kit is easy to popularize.

The current methods for detecting DNA methylation mainly comprise the following methods: compared with methylation specificity PCR, a bisulfite sequencing method, a high-resolution dissolution curve method, a fluorescence quantitative method, a chip method, high-throughput sequencing, a flight mass spectrometry method and the like, the real-time fluorescence quantitative PCR method has the advantages of low cost, high popularization rate, rapidness, high sensitivity and good specificity, so that the kit selects the method, and has higher clinical application value.

Disclosure of Invention

The invention aims to solve the problem that clinical diagnosis of benign and malignant pulmonary nodules is difficult and invasive, and provides a plasma free DNA methylation gene combination for identifying the benign and malignant pulmonary nodules.

Another object of the present invention is to provide a kit for identifying benign and malignant pulmonary nodules. The relative methylation degree of the lung cancer cell is detected, and a plurality of lung malignant tumor related genes are subjected to combined detection, so that the lung cancer cell has the great advantages of high clinical sensitivity and high clinical specificity in the aspects of differential diagnosis of benign and malignant lung nodules and early lung cancer diagnosis.

A combination of plasma-free DNA methylation genes for identifying benign and malignant lung nodules selected from at least any two of SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO 1.

As a preferred aspect of the invention, the combination comprises at least SOX17 and SCT.

As a further preferred aspect of the present invention, the combination is selected from any one of the following: combination 1: SOX17 and SCT; and (3) combination 2: SOX17, SCT, and SHOX 2; and (3) combination: SCT, SHOX2, HOXA7 and CDO 1; and (4) combination: SOX17, SCT, SHOX2, HOXA 7; and (3) combination 5: SOX17, SCT, SHOX2, HOXA7, and CDO 1; and (4) combination 6: SOX17, SCT, RASSF1A, SHOX2, HOXA7, and CDO 1.

The Genebank numbers in the ncbi (national Center for Biotechnology information) website of SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1 genes are respectively: SOX 17: NC _000008.11(54457935..54460892), SCT: NC _000011.10(626095..627692), RASSF 1A: NC _000003.12(50329786..50340836), SHOX 2: NC _000003.12 (158095905..158106420), HOXA 7: NC _000007.14(27153716..27156675) and CDO 1: NC _000005.10 (115804733.. 115816659).

The invention discloses application of a plasma free DNA methylation gene combination as a detection target in preparation of a reagent for identifying benign and malignant lung nodules.

A composition comprises a primer and a probe for detecting the combination of the free DNA methylation genes in the blood plasma of the invention by real-time fluorescence quantitative PCR.

As a preferable embodiment of the present invention, the sequences of the primers and probes for detecting SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1 are as follows:

TABLE 1

Wherein the length of the primer is between 15 and 30nt, and the target gene only hybridizes with the DNA region after bisulfite conversion under strict conditions.

The above probes were designed based on TaqMan (TM), are between 15-30nt in length, and hybridize under stringent conditions to only the bisulfite converted DNA region of the target gene. Furthermore, the specific probe is modified by fluorescent dye, the fluorescent group marked at the 5 'end of the specific TaqMan probe is any one of organic fluorescent dye or inorganic dye, and the quenching group marked at the 3' end of the specific TaqMan probe is any one of organic dye. Preferably, the organic fluorescent dye is FAM, VIC, ROX, TRT, Cy3, Cy5, HEX and JOE, and the quenching group is MGB, BHQ-1, BHQ-2 and BHQ-3.

The composition is applied to preparation of a kit for identifying benign and malignant lung nodules.

A kit for identifying benign and malignant pulmonary nodules, comprising the composition of the invention.

Preferably, the kit further comprises primers and probes for detecting the reference gene GAPDH by fluorescent quantitative PCR; preferably, primers for detecting the GAPDH gene by fluorescent quantitative PCR are shown as SEQ ID NO.37 and 38, and a TaqMan probe is shown as SEQ ID NO. 39. The kit of the invention takes the internal reference gene as reference to quantitatively detect the relative methylation of each target gene.

As a preferred aspect of the present invention, the kit further comprises: the kit comprises a PCR reaction system, a negative quality control product and a positive quality control product, wherein the negative quality control product is human genome DNA converted by bisulfite or sterile enzyme-free water, and the positive quality control product is artificially synthesized plasmid DNA which is constructed according to a DNA detection target and is diluted to a certain concentration (1 x 10^4 copies/mu L).

The reaction system is that each reaction volume is 20-50 mu L, and each detection system contains 10-25 mu L of premixed solution of DNA polymerase and PCR reaction buffer solution per reaction; 6 types of lung malignant tumor related genes and internal reference GAPDH through bisulfite conversion DNA upstream and downstream primer and probe set, wherein each gene and internal reference GAPDH upstream and downstream primer concentration is 0.2 mu m-0.6 mu m/reaction, each gene and internal reference GAPDH probe is 0.2 mu m-0.6 mu m/reaction; test sample DNA template: 5 mu L of the solution; positive control 5 μ L; negative control: 5 μ L.

Preferably, the pre-mixed solution of the DNA polymerase and the PCR reaction buffer solution in the kit comprises dNTP, Tris-HCl, potassium chloride, ammonium sulfate, magnesium chloride and the like in the PCR buffer solution of the hot start Taq DNA polymerase.

The sample detected by the kit is derived from plasma of a human body, is not limited to the plasma, and also comprises fresh tissues, paraffin-embedded tissue sections, alveolar lavage fluid or pleural effusion; the DNA of the tested sample is complete genome, cell-free DNA or circulating tumor DNA which is treated by a bisulfite conversion kit.

The method for identifying the benign and malignant pulmonary nodules by using the kit comprises the following steps:

extracting and purifying plasma cfDNA by using a DNA extraction kit;

converting and purifying the extracted cfDNA by using a bisulfite conversion kit;

and (3) carrying out qPCR amplification by using the treated bisDNA as a template and adopting the detection target primer probe set, and the internal reference primer and the internal reference probe of the internal reference gene.

Further, a plurality of nucleic acid fragment detections are carried out in one reaction tube, and a plurality of waveband fluorescent signal detections are carried out in one reaction tube simultaneously, so that different DNA fragments are distinguished through different fluorescent signals; or, different nucleic acid fragment detection can be carried out in different reaction tubes, and single-waveband fluorescent signal detection can be carried out in one reaction tube at the same time, so that different DNA fragments can be distinguished through fluorescent signals; alternatively, multiple nucleic acid fragment detection may be performed in different reaction tubes, and fluorescence signal detection may be performed in multiple wavelength bands simultaneously in one reaction tube, so that different DNA fragments may be distinguished by fluorescence signals. For example, triple detection, three target probes label FAM, VIC and ROX dyes, respectively.

Further, a kit target verification link constructs each detection target and a relative methylation quantitative reference plasmid of GAPDH, and performs gradient dilution to construct a relative methylation degree standard curve equation.

Further, the reaction procedure for performing real-time fluorescent PCR was as follows:

one cycle is as follows: 30s at 95 ℃;

then 50 cycles: 15s at 95 ℃ and 30s at 60 ℃;

setting a Ct threshold value in a linear amplification interval after the PCR reaction is finished;

and (3) data analysis: firstly, taking a CT value of a GAPDH gene as an internal reference, and taking three repeatedly detected delta CT mean; secondly, when the detection result of the internal reference site GAPDH is in a normal range, and the detection result delta CT mean value of any one or more target points in the target gene is brought into a standard curve equation, the relative methylation degree value of each target is obtained and is brought into regression equations of different combinations, and whether the sample is positive or not is judged according to cut-off values of ROC curves.

Further, performing a binomial regression fit on the target gene combination, wherein the performance indexes of the combined detection comprise: clinical sensitivity, clinical specificity and AUC values of the area under the receiver operating characteristic curve.

Has the advantages that:

a large number of clinical experiments show that the kit can improve the detection sensitivity of the biomarker to 10 copies/microliter, and the improvement of the detection sensitivity is realized by optimizing specific nucleotide sequence primers and DNA probes, screening suitable target gene detection sites and optimizing each target combination.

In the technical scheme of the invention, a plurality of genes of 6 lung malignant tumor related genes SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1 can be combined to verify a lung malignant tumor patient and a benign nodule patient, and ROC curve fitting and two-term regression can be carried out. Experiments show that when cfDNA in plasma of a patient with lung malignant tumor is used for carrying out methylation detection on the multiple genes for verification, the methylation level of the methylation related genes of the lung malignant tumor is generally higher; in a negative verification experiment, cfDNA in plasma of a benign nodule patient is used for carrying out methylation detection on the multiple genes, and the result shows that no methylation or low methylation expression exists, so that compared with methylation qualitative detection, the kit uses a methylation relative quantitative method, and the false positive rate can be reduced. The relative methylation quantitative detection adopted by the kit is more accurate and effective than the qualitative methylation detection of the target gene of the conventional commercial kit. In summary, methylation of the 6 malignancy-associated genes SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1 are meaningful clinical indicators for good and malignant diagnosis, prognosis evaluation and efficacy monitoring of lung nodules. Therefore, the reagent kit provided by the invention provides a new rapid, reliable and accurate way for the differential diagnosis of benign and malignant lung nodules, and the reagent kit provided by the invention plays an important role in the field of medical detection.

The optimized TaqMan probe is adopted in the kit. The TaqMan probe is the most accurate and sensitive method for real-time fluorescent quantitative PCR, and obtains an internationally recognized nucleic acid molecule quantitative detection method, and the optimized and modified TaqMan probe such as MGB-TaqMan is a new technology developed on the basis of a common TaqMan probe, and has the advantages of low fluorescent background, higher resolution and stronger hybridization specificity. Good repeatability, shortened probe length and the like, and is particularly suitable for designing sequence probes containing high AT after bisulfite treatment. Therefore, the optimized modified TaqMan probe is used by the kit, so that the experimental method is more reliable, efficient, stable and repeatable.

In addition, in the technical scheme of the invention, a methylation relative quantitative method is adopted, GAPDH is mainly used as an internal reference, a methylation relative quantitative reference product is constructed, and then a relative methylation standard curve is prepared. The methylation degree of the genes related to the lung malignant tumor can be obtained through a standard curve, and compared with quantitative methylation detection, the methylation degree detection method is more sensitive, improves the detection accuracy, reduces the false positive rate, and has higher clinical value for diagnosing benign and malignant lung nodules.

In addition, in the technical scheme of the invention, single-gene double-target detection is adopted, each gene has two sets of primer probe sets for screening and optimizing methylation detection, the primer probe sets do not interfere with each other, and the clinical sensitivity and the clinical specificity of detection can be obviously improved.

Drawings

FIG. 1: is a SOX17 relative methylation quantitative standard curve equation (A) and a SCT relative methylation quantitative standard curve equation (B)

FIG. 2: benign nodule patient (A) and lung malignant tumor (B) SOX17 and SCT gene methylation detection amplification curve chart

FIG. 3: ROC curves for 6 combinations of genes related to lung malignant tumor methylation: ROC curve (a) for combination 1; ROC curve (B) for combination 2; ROC curve (C) for combination 3 and ROC curve (D) for combination 4; ROC curve (E) for combination 5; ROC curve (F) for combination 6;

Detailed Description

The present invention provides the following specific implementation examples to describe the embodiments of the present invention in detail, but the present embodiment is only an example, and the content related to the present invention may also be implemented or applied in other ways, and therefore, the protection scope of the present invention is not limited thereby. 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 experimental procedures in the following examples are conventional unless otherwise specified. Reagents and equipment used in the following examples are commercially available or commonly used in the industry unless otherwise specified.

Definition of

As used herein, "benign and malignant lung nodules" refers to solitary lung nodules that are single-shot, round or quasi-round opaque lesions less than or equal to 3cm in diameter surrounded by air-containing lung tissue. According to the CT performance of the chest, the lung nodules are divided into solid nodules and uniform high-density soft tissue shadows in ground glass nodules, the ground glass nodules are defined as density increasing areas which are circular or similar to circles on the CT, the boundaries are clear, and the display of the lung interstitial structure is not influenced. Lung nodules are mostly benign lesions, with the highest probability of malignancy for some solid nodules, including both cases. Early lung cancer exists in the form of lung nodules without obvious clinical symptoms, people often ignore the development of tumors, most patients are in the middle and late stage of the tumors when diagnosed, and effective treatment cannot be timely adopted.

As used herein, "lung malignancy" refers to a malignancy of the lung characterized by uncontrolled growth of cells in the lung tissue. Lung cancer includes small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

As used herein, "early stage lung cancer" means a cell that is in an early stage of, or predisposed to, transformation into a cancer cell. Or stage I or II lung cancer. Such cells may exhibit one or more phenotypic traits characteristic of cancer cells.

As used herein, "nucleic acid", "nucleic acid sequence", and the like, refer to a polynucleotide, which may be gDNA, plasmid DNA, cfDNA. "gDNA" refers to genomic DNA, plasmid DNA is in vitro recombinant DNA, cfDNA refers to cell-free DNA circulating in human blood. Combinations of these (i.e., recombinant nucleic acids that are partially gDNA and partially plasmid DNA) are also possible.

In the present invention, detection of methylation of circulating tumor DNA involves extracting DNA from a sample, treating with a bisulfite conversion kit and purifying such that unmethylated cytosine (C) in the DNA duplex is deaminated to uracil (U) while methylated cytosine (mC) remains unchanged. Thus, in the subsequent PCR amplification, uracil is gradually replaced by thymine (T). The specific primer probe set provided by the invention utilizes the methylation difference to detect the methylation level of the target region in the biomarker gene. When the target region of the target gene is not methylated, the specific primer probe set used cannot be effectively paired and combined with the target region (treated by bisulfite) as a template in a PCR amplification reaction to amplify a product; when the target gene is methylated, the primer probe set is effectively paired with the target region (treated with bisulfite) as a template in a PCR amplification reaction, and the reaction results in the production of an amplification product. Through multiple tests, two groups of primer probe sets aiming at biomarker genes are screened out, and the primer probe sets can be used independently or jointly to help identify the benign and malignant state of lung nodules in a subject.

The 6 lung malignant tumor-associated abnormal methylation genes involved in the present invention mainly include SOX17(SRY-related HMG-box 17), SCT (secretin), RASSF1A (Ras association domain family 1A), SHOX2(Short status family ox 2), HOXA7((Homeobox A7) and CDO1(Cysteine dioxygenase type 1).

The invention designs primer probe sets aiming at two regions of each gene of the 6 genes, detects the methylation degree of each gene, but labels the same fluorescent dye aiming at the probes of the two regions, but does not interfere with each other. The single-gene double-target detection can improve the clinical sensitivity of detection. The probes in the target regions of different genes can be used for labeling different fluorescent dyes so as to achieve the aim of multiple detection. The PCR reaction was monitored in real time using a fluorescent probe. The 5' end of the used probe reports fluorescent groups, and the fluorescent groups are one or more of Cy5, FAM, TET, Rox Texas Red, HEX, Cy3 and JOE.

As used herein, "sensitivity" refers to the lowest analyte concentration that can be detected, otherwise known as the limit of detection, and "specificity" of a detection method of the kit refers to the ability to detect negatives on non-targets and unmethylated targets. The term "clinical sensitivity" refers to the ratio of the number of patients with disease detected in the test (true positive rate) to the total number of patients, and the term "clinical specificity" refers to the ratio of the number of patients with benign lung nodules detected in the test (true negative rate)

The invention provides a plasma free DNA methylation detection kit for identifying benign and malignant pulmonary nodules, which is mainly used for relatively quantifying the methylation degree of lung malignant tumor related genes.

The present invention will be described in detail below with reference to specific embodiments and further details of the technical solutions.

Example 1: design of primer probe group of 6 lung malignant tumor related genes

The lung malignancy-associated genes SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1 and internal reference GAPDH were queried by the National Center for Biotechnology Information (NCBI), and CpG islands were predicted by Methyl Primer Express v1.0 software or "MethPrimer" from ABI, Inc., where the default CpG islands span at least 200bp, GC content > 50%, and CpG frequency > 0.6. And finding 50bp positions on the upstream and downstream of the position of the CpG island to carry out sequence interception, and downloading and storing a standard sulfurized sequence, namely converting cytosine except CpG dinucleotide into uracil.

Designing up and down stream primers and probes aiming at sequences of genes SOX17, SCT, RASSF1A, SHOX2, HOXA7, CDO1 and internal reference GAPDH related to lung malignant tumor, wherein the up and down stream primers and probes are designed on the sequences of 6 lung malignant tumor related genes and internal reference GAPDH by using Methyl Primer Express v1.0 or Meth Primer or using Primer Premier 5. And the primer probe specificity was verified using NCBI. The upstream and downstream primer and probe sets for 6 methylation-related genes of malignant lung tumor and the internal reference gene GAPDH are shown in Table 1, and the primer probe sequences shown in Table 1 were synthesized by Biotechnology engineering (Shanghai) GmbH.

Example 2: constructing a target positive reference product, an internal reference gene GAPDH reference product and a relative methylation standard linear equation of a constructed target of the kit is obtained by converting a target sequence plasmid into escherichia coli, inserting a plasmid pUC57 into a positive sequence shown in table 2 after SCT/SOX17, 4 other methylation related genes of the lung malignant tumor and GAPDH detection target are converted to construct a recombinant plasmid, wherein the resistance is ampicillin, and the recombinant plasmid is converted into escherichia coli top 10. The above parts are completed by bio-engineering (Shanghai) corporation.

TABLE 2 detection of specific Gene positive sequences after transformation of target for detection by the kit of the invention

And E, culturing escherichia coli:

escherichia coli was cultured in a solid LB medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, ampicillin 50. mu.g/mL, agar 15g/L, ampicillin concentration 50. mu.g/mL) at 37 ℃ for 24 hours, a single colony was picked up to 3mL of a liquid LB medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, ampicillin 50. mu.g/mL) at 37 ℃ at 210r/min, and after 12 hours of culture, 100. mu.L to 10mL of the liquid LB medium was aspirated for expansion culture (1%) under the following culture conditions: culturing at 37 deg.C for 2h at a speed of 210 r/min. And centrifuging at 12000r/min for 1min to collect the Escherichia coli thallus.

Extracting plasmids in escherichia coli:

plasmid extraction is carried out by using a small extraction kit of Tiangen DP103-02 plasmid, and the extraction steps refer to the instruction part. And (4) carrying out quantitative detection on the extracted plasmid by using Nano Drop to obtain a positive reference substance with high concentration of SHOX 2. The copy number of the target sequence is calculated as follows:

copy number of target sequence (g) × 6.23 × 10²³/660X plasmid DNA Length

The detection sensitivity and specificity of the kit are as follows (SCT/SOX17 is an example)

Meanwhile, the kit also provides a positive quality control product and a negative quality control product, and the specific steps are as follows: the positive quality control product is plasmid recombinant DNA confirmed by sequencing, and the concentration is 1 multiplied by 10⁴copies/. mu.L; the negative quality control material is human genome DNA converted by bisulfite or water without DNase.

Preparing a reference SCT/SOX17 relative methylation degree by taking GAPDH as a reference, wherein the reference mainly comprises 100 percent of methylation degree (10 percent)⁵copies/ul(SCT): 10⁵copies/ul(SOX17)：10⁵copies/ul(GAPDH))、10％(10⁴copies/ul(SCT):10⁴copies/ul(SOX17):10⁵ copies/ul(GAPDH))、1％(10³copies/ul(SCT):10³copies/ul(SOX17):10⁵copies/ul(GAPDH))、0.1％ (10²copies/ul(SCT):10²copies/ul(SOX17):10⁵copies/ul(GAPDH))、0.01％(10¹copies/ul(SCT)：10¹copies/ul(SOX17):10⁵copies/ul (GAPDH)). The relative methylation reference products of other 4 lung malignant tumor genes and the preparation method of SCT/SOX17 are not repeated

The qPCR experiment was performed using the above-described DNA template diluted in gradient, and the reaction system was as follows:

TABLE 3SCT/SOX17 relative methylation detection reaction System

5G qPCR Premix from dNTP, Mg²⁺Taq DNA polymerase, Tris-HCI and KCl, purchased from Tianjing (Shanghai) science and technology Ltd. Note: the primer probes were synthesized by Biotechnology, Shanghai, Inc., after providing sequences (see Table 1).

The above SOX17 upstream and downstream primers refer to two sets of primers of SOX17 primer combination 1 and combination 2. The SCT upstream and downstream primers refer to two groups of primers of SCT primer combination 1 and combination 2. Two groups of probes of SOX17 are labeled with FAM at the 5 'end and MGB at the 3' end; labeling 5 'ends of two groups of probes of SCT with VIC, and labeling 3' ends with MGB; GAPDH probe label 5 'end label ROX, 3' end label MGB; triple detection is achieved.

The primer probe sequences used are shown in Table 1.

Each detection group is provided with three multiple wells, and the qPCR reaction conditions are as follows:

TABLE 4SCT/SOX17 relative methylation and GAPDH detection reaction conditions

The obtained results are shown in the attached figures 1 and 2.

As can be seen from the results, the detection sensitivity was as low as 0.01% methylation, and was as low as 10 copies per microliter. The positive quality control substance is detected to be positive by a parallel experiment, and the negative quality control substance is detected to be negative by a template obtained by converting human genome DNA through bisulfite. The kit has good primer and probe specificity.

Example 3: clinical sensitivity and clinical specificity verification of 6 different lung malignant tumor methylation gene joint detection kits for detecting plasma cfDNA of benign tubercle patients and lung malignant tumor patients

This example judges the clinical sensitivity and clinical specificity of the selected gene combination in the kit by verifying the DNA methylation status of different combinations of 6 lung malignant tumor-associated methylation genes in lung malignant tumor plasma and the methylation status of plasma free DNA in normal human plasma.

Plasma free DNA extraction:

use of

Serum/Plasma Circulating DNA Kit (available from Nanjing Novowed Biotechnology Co., Ltd., product No. N902-01) was used according to the Kit instructions.

Plasma free DNA bisulfite conversion:

sulfite transformation was performed using the EZ DNA Methylation-DirectTM KIT (D5031) KIT from ZYMO RESEARCH Bio Inc. Amplification and detection:

relative methylation levels of different combinations of the above 6 marker genes were detected in plasma from 40 patients pathologically determined to be lung malignancy and 60 benign nodule individuals using a real-time fluorescent PCR assay.

The purified nucleic acid was qPCR amplified using two primer probe sets per gene (see table 1) to increase the clinical sensitivity of the assay. Setting three multiple wells, detecting internal reference GAPDH in each reaction, carrying out verification test in the same reaction system and conditions as those in example 2, analyzing data after the reaction is finished, and taking three repeatedly detected delta CT mean by taking a CT value of a GAPDH gene as an internal reference when a Ct threshold value is set in a linear amplification interval; then, when the detection result of the internal reference site GAPDH is within the normal range, and the detection result Δ CT mean value of any one or more target points in the target gene is brought into the standard curve equation, the relative methylation degree value of each target is obtained.

The present invention uses a commercially available software package (IBM SPSS Statistics 24 from IBM, GraphPad Prism 8.0.2 from GraphPad) for descriptive Statistics of plasma biomarker levels, Receiver Operating Characteristics (ROC) curves. Statistical differences were determined using the nonparametric Kruskal-Wallis test (ANOVA), followed by Dunn's multiple post-comparison tests. For all statistical comparisons, a P value <0.05 was considered statistically significant. The 6 marker genes were combined using logistic regression, resulting in an AUC of 0.9178 (95% CI: 0.8518-0.9839; P value: <0.0001) (FIG. 3). In order to make the monitoring and analysis method simpler, different markers are combined in multiple groups and a logistic regression model is established. The AUC and P values obtained are shown in Table 6, and the ROC graph is shown in FIG. 3.

Combination 1: SOX17 and SCT; and (3) combination 2: SOX17, SCT, and SHOX 2; and (3) combination: SCT, SHOX2, HOXA7 and CDO 1; and (4) combination: SOX17, SCT, SHOX2, and HOXA 7; and (3) combination 5: SOX17, SCT, SHOX2, HOXA7, and CDO 1; and (4) combination 6: SOX17, SCT, RASSF1A, SHOX2, HOXA7, and CDO 1.

TABLE 6 AUC and P values of ROC curves for different marker combinations

	Marker combination	AUC	P
				Single gene	SOX17	0.7662	0.0005
Single gene	SCT	0.7240	0.0035
				Combination 1	SOX17 and SCT	0.8171	＜0.0001
Combination 2	SOX17, SCT and SHOX2	0.8900	＜0.0001
				Combination 3	SCT, SHOX2, HOXA7 and CDO1	0.8958	＜0.0001
Combination 4	SOX17, SCT, SHOX2 and HOXA7	0.8854	＜0.0001
				Combination 5	SOX17, SCT, SHOX2, HOXA7 and CDO1	0.9178	＜0.0001
Combination 6	SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1	0.9178	＜0.0001

Generally, logistic regression models for 6 markers have slightly better clinical sensitivity and clinical specificity, but several other marker combinations are better candidates based on the procedure of the operational analysis and cost considerations.

The CT values of the genes are substituted into the equation according to the regression equation, cut-off values are obtained according to the ROC curve, and the combination of the detected biomarkers and the results of clinical sensitivity and clinical specificity are obtained according to the cut-off values and are shown in Table 7.

TABLE 7 combination of biomarkers tested and results of clinical sensitivity and clinical specificity

In order to achieve different expected uses and purposes, the detection kit provided by the invention can achieve different clinical sensitivity and clinical specificity indexes by adjusting cut-off values. In example 2, to improve clinical sensitivity, the cut-off value is not set to mean that the invention can only use the cut-off value, and different cut-off values can be selected as the determination criteria according to the ROC curve provided by the invention.

Example 4 clinical experiments verify that single-gene double-target detection and single-gene single-target clinical sensitivity and clinical specificity

Taking the combined test kit of SHOX2, CDO1, HOXA7 and SCT as an example, plasma samples of 30 lung malignant tumor patients and 30 benign lung nodule patients were divided into two equal parts (sample groups A/B), and the difference between single-gene double-target detection and single-gene single-target detection was verified, i.e., one sample was tested with two sets of primer probe sets for each gene in the combination of SHOX2, CDO1, HOXA7 and SCT, and the other sample was tested with one set of primer probe sets for each gene in the combination of SHOX2, CDO1, HOXA7 and SCT. The method comprises the following specific steps:

the two aliquots were processed according to the cfDNA extraction and sulfite conversion procedure of example 3, and real-time fluorescent quantitative PCR validation was performed with bisulfite-treated DNA template.

Two sets of primer probes (sample set a) using SHOX2, CDO1, HOXA7, and SCT genes:

reaction system a-1: the final volume composition of each well of the reaction system of the fluorescent quantitative PCR amplification is as follows: 10 μ L of 5G qPCR Premix, 0.8 μ L of SHOX2 specific primer probe combination 1, combination 2 mixture (10 μ M), wherein both sets of probes are labeled with FAM; CDO1 specific primer probe combination 1, combination 2 mix (10 μm) 0.8 μ L, where both sets of probes are labeled VIC; GAPDH specific primer probe mixture (10 μm) 0.8 μ L, where the GAPDH probe labeled ROX, added no enzyme water to 15 μ L, finally template 5 μ L, capped, and centrifuged instantaneously. The samples were tested in 3 duplicate wells (replicates) and tested according to the reaction conditions of example 1.

Reaction system a-2: the final volume composition of each well of the reaction system of the fluorescent quantitative PCR amplification is as follows: 10 μ L of 5G qPCR Premix, 0.8 μ L of HOXA7 specific primer probe combination 1, combination 2 mix (10 μ M), wherein both sets of probes are labeled with FAM; SCT specific primer probe combination 1, combination 2 mixed liquor (10 μm) 0.8 μ L, wherein, two groups of probes are labeled VIC; GAPDH specific primer probe mixture (10 μm) 0.8 μ L, where the GAPDH probe labeled ROX, added no enzyme water to 15 μ L, finally template 5 μ L, capped, and centrifuged instantaneously. The samples were tested in 3 duplicate wells (replicates) and tested according to the reaction conditions of example 1.

A single set of primer probes for SHOX2, CDO1, HOXA7, and SCT genes was used (sample set B):

reaction system B-1: the final volume composition of each well of the reaction system of the fluorescent quantitative PCR amplification is as follows: 10 μ L of 5G qPCR Premix, 0.8 μ L of SHOX2 specific primer probe combination 2 mix (10 μ M), wherein the probe labels FAM; CDO1 specific primer Probe combination 1 mix (10 μ M) 0.8 μ L, where the probes label VIC; GAPDH specific primer probe mixture (10 μm) 0.8 μ L, where the GAPDH probe labeled ROX, added no enzyme water to 15 μ L, finally template 5 μ L, capped, and centrifuged instantaneously. The samples were tested in 3 duplicate wells (replicates) and tested according to the reaction conditions in example 1.

Reaction system B-2: the final volume composition of each well of the reaction system of the fluorescent quantitative PCR amplification is as follows: 5G qPCR Premix 10 μ L, HOXA7 specific primer probe combination 1 mix (10 μ Μ)0.8 μ L, wherein the probe is labeled FAM; SCT specific primer Probe combination 1 mixture (10 μm) 0.8 μ L, wherein the probe is labeled VIC; GAPDH specific primer probe mixture (10 μm) 0.8 μ L, where the GAPDH probe labeled ROX, added no enzyme water to 15 μ L, finally template 5 μ L, capped, and centrifuged instantaneously. The samples were tested in 3 duplicate wells (replicates) and tested according to the reaction conditions in example 1.

The data were processed as in example 2, and the results are shown in Table 8:

TABLE 8 comparison of AUC values, clinical sensitivities and clinical specificities for the single-gene dual-target assay and the single-gene single-target assay

	AUC value	P value	Clinical sensitivity	Clinical specificity
					Sample set A (Single gene double target)	0.8956	＜0.0001	80.00％	80.00％
Sample set B (Single gene and target)	0.8878	＜0.0001	77.67％	80.00％

The results show that the AUC value of the benign and malignant lung nodule detected by the 4 methylation related genes of the single-gene double-target and single-gene single-target detection is above 0.8800, obviously, the clinical sensitivity of the single-gene double-target detection is higher, the clinical specificity is better, but the overall difference is not large, so that the cost and the clinical performance can be comprehensively considered in the application of the kit, and two detection modes can be selected.

While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

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Claims

1. A combination of plasma-free DNA methylation genes for identifying benign and malignant lung nodules, characterized by being selected from at least any two of SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO 1.

2. The plasma-free DNA methylation gene combination according to claim 1, wherein the combination comprises at least SOX17 and SCT.

3. The plasma-free DNA methylation gene combination according to claim 1, wherein the combination is selected from any one of the following: combination 1: SOX17 and SCT; and (3) combination 2: SOX17, SCT, and SHOX 2; and (3) combination: SCT, SHOX2, HOXA7 and CDO 1; and (4) combination: SOX17, SCT, SHOX2, HOXA 7; and (3) combination 5: SOX17, SCT, SHOX2, HOXA7, and CDO 1; and (4) combination 6: SOX17, SCT, RASSF1A, SHOX2, HOXA7, and CDO 1.

4. Use of the plasma free DNA methylation gene combination of any one of claims 1-3 as a detection target in preparation of a reagent for identifying benign and malignant lung nodules.

5. A composition comprising primers and probes for real-time fluorescent quantitative PCR detection of the combination of plasma free DNA methylated genes according to any one of claims 1 to 3.

6. The composition of claim 5, wherein the sequences of the primers and probes for detecting SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1 are as follows:

wherein, the fluorescent group marked at the 5 'end of the TaqMan probe is any one of organic fluorescent dye or inorganic dye, and the quenching group marked at the 3' end is any one of organic dye.

7. Use of the composition of any one of claims 4 to 6 for the preparation of a kit for identifying benign and malignant lung nodules.

8. A kit for identifying benign or malignant pulmonary nodules, comprising the composition according to any one of claims 4 to 6.

9. The lung cancer auxiliary diagnostic kit according to claim 8, characterized in that the kit further comprises primers and probes for detecting the reference gene GAPDH by fluorescent quantitative PCR; preferably, primers for detecting the GAPDH gene by fluorescent quantitative PCR are shown as SEQ ID NO.37 and 38, and a TaqMan probe is shown as SEQ ID NO. 39.

10. The kit for auxiliary diagnosis of lung cancer according to claim 8, wherein said kit further comprises: the kit comprises a PCR reaction system, a negative quality control product and a positive quality control product, wherein the negative quality control product is water without DNase or human genome DNA converted by bisulfite, and the positive quality control product is artificially synthesized plasmid DNA which is constructed according to a DNA detection target and is diluted to a certain concentration.