CN116814790A - Application of PITX2 gene as a marker in detecting lung cancer - Google Patents

Abstract

The invention discloses the application of PITX2 gene as a marker in detecting lung cancer. The present invention found that the gene related to the diagnosis of early lung cancer is the PITX2 gene, which is DNA and RNA extracted from lung cancer tissues and adjacent normal tissues. Using whole-gene methylation sequencing and expression analysis methods, another group of lung cancer tissues and Original results after screening and verification of normal human tissue DNA. In lung tissue, the PITX2 gene detected lung cancer with a sensitivity of 74% and a specificity of 94%. In plasma, the PITX2 gene detected lung cancer with a sensitivity of 58% and a specificity of 85%. It can be seen that a single PITX2 gene can detect lung cancer in plasma or lung tissue. The invention can screen and assist diagnosis of early lung cancer and has important application value.

Description

Application of PITX2 gene as a marker in the detection of lung cancer

Technical Field

The invention belongs to the field of biomedicine, and specifically relates to the application of PITX2 gene as a marker in detecting lung cancer.

Background Art

Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Histologically, NSCLC is divided into adenocarcinoma (AC), squamous cell carcinoma (SCC), and large cell carcinoma. Compared with SCLC, non-small cell lung cancer grows and spreads more slowly. Most NSCLC patients are diagnosed at an advanced stage. Despite the widespread use of lung cancer screening methods such as computed tomography (CT) and MRI, these technologies can detect small nodules of 2-3 mm, but still cannot assess their specific characteristics, i.e., benign or malignant. Many problems associated with these methods (such as high rates of false positive results, patients' fear of radiation, excessive costs, and the need for long follow-up) further limit the effectiveness of these methods. An important reason for the late diagnosis of lung cancer is the lack of reliable biomarkers. The widely used blood protein diagnostic biomarkers for lung cancer, such as serum protein antigen (CEA) and neuron-specific enolase (NSE), have detection rates of only 26% and 21-39%, respectively. Circulating tumor DNA (ctDNA), circulating tumor cells, serum noncoding RNA, and exosomes from blood have been explored. Among them, the use of ctDNA for lung cancer detection has attracted much attention. Incorporating reliable biomarkers into lung cancer screening programs using ctDNA will help identify patients at an early stage.

DNA methylation is an important epigenetic modification that plays a vital role in regulating gene expression and modifying chromatin conformation. DNA methylation is an early event in the development of cancer, so diagnostic biomarkers that detect DNA methylation changes in the regulatory regions of tumor suppressor genes can be used as one of the most promising diagnostic and prognostic tools. The development of reliable early detection methods for NSCLC using DNA methylation markers will become a focus of translational cancer research.

Early diagnosis of tumors usually depends on two key factors: high sensitivity of the test indicators and non-invasive, simple detection methods. With the rapid development of science and technology, tumor markers have developed into a new field, new challenge and hope for tumor diagnosis and treatment. Tumor markers can be detected in body fluids or tissues and can reflect the presence, degree of differentiation, prognosis estimation and treatment effect of tumors.

Summary of the invention

The purpose of the present invention is to diagnose lung cancer at an early stage.

The present invention firstly protects the use of PITX2 gene as a marker in preparing a lung cancer detection kit; the Gene ID of PITX2 gene is 5308.

The present invention also protects a kit for screening lung cancer, which may include a primer probe set for detecting the methylation level of the PITX2 gene; the Gene ID of the PITX2 gene is 5308.

The kit may specifically consist of the primer probe set for detecting the methylation level of the PITX2 gene.

Any of the above-mentioned primer probe sets for detecting the methylation level of the PITX2 gene may be primer probe set PITX2-1, primer probe set PITX2-2 or primer probe set PITX2-3.

Any of the above primer-probe sets PITX2-1 consists of primer PITX2-F1 shown in SEQ ID NO: 1, primer PITX2-R1 shown in SEQ ID NO: 2, and probe PITX2-P1 shown in SEQ ID NO: 3.

Any of the above primer-probe sets PITX2-2 consists of primer PITX2-F2 shown in SEQ ID NO:4, primer PITX2-R2 shown in SEQ ID NO:5 and probe PITX2-P2 shown in SEQ ID NO:6.

Any of the above primer-probe sets PITX2-3 consists of primer PITX2-F3 shown in SEQ ID NO:7, primer PITX2-R3 shown in SEQ ID NO:8 and probe PITX2-P3 shown in SEQ ID NO:9.

Any of the above-mentioned kits may also include a primer probe set for detecting the methylation level of an internal reference gene.

Any of the above-mentioned kits may specifically consist of any of the above-mentioned primer probe sets for detecting the methylation level of the PITX2 gene and any of the above-mentioned primer probe sets for detecting the methylation level of the internal reference gene.

The internal reference gene may be the ACTB gene. The Gene ID of the ACTB gene is 60.

Any of the above-mentioned primer probe sets for detecting the methylation level of the internal reference gene may be primer probe set ACTB-1 or primer probe set ACTB-2.

The primer probe set ACTB-1 consists of primer ACTB-F1 shown in SEQ ID NO: 10, primer ACTB-R1 shown in SEQ ID NO: 11, and probe ACTB-P1 shown in SEQ ID NO: 12.

The primer probe set ACTB-2 consists of primer ACTB-F2 shown in SEQ ID NO: 13, primer ACTB-R2 shown in SEQ ID NO: 14, and probe ACTB-P2 shown in SEQ ID NO: 15.

Any of the above-mentioned probes (such as the probe for detecting the internal reference gene and the probe for detecting the methylation level of the PITX2 gene) has a fluorescent label at one end and a fluorescent quenching label at the other end.

The detection object of any of the above-mentioned kits can specifically be genomic DNA of lung nodule tissue, plasma cfDNA or fecal cfDNA.

Any of the above-mentioned kits may further include a data processing system; the data processing system converts the methylation level of the PITX2 gene into dCT _PITX2 of the subject to be tested, so as to determine whether the subject to be tested is a lung cancer patient;

The calculation method of the dCT _PITX2 of the test subject is as follows: chemically modifying the genomic DNA, plasma cfDNA or fecal cfDNA of the test subject's lung nodule tissue, and then using it as a template, using the primers and probes in claim 3 and/or 6 to perform fluorescent PCR amplification, collecting fluorescent signals, and obtaining the CT values of PITX2 and ACTB, respectively, which are recorded as CT _PITX2 and CT _ACTB ; if the amplification curve is not an "S" type or the CT value is blank, the CT value is recorded as 45; further calculating the dCT value of PITX2, dCT _PITX2 = CT _PITX2 - CT _ACTB ;

The determination method is as follows: if the PITX2 gene of the subject is methylated, the subject is a lung cancer patient; otherwise, the subject is not a lung cancer patient; whether the PITX2 gene is methylated is determined by comparing the dCT and dCT critical values of the PITX2 gene of the sample to be tested;

If the dCT of the PITX2 gene of the subject is less than the dCT critical value, then the PITX2 gene of the subject is methylated; if the dCT of the PITX2 gene of the subject is greater than or equal to the dCT critical value, then the PITX2 gene of the subject is not methylated.

When the detection object of any of the above-mentioned kits is the genomic DNA of lung nodule tissue, the dCT critical value is an average statistical dCT value after comparing the PITX2 gene dCT values of lung cancer tissue and adjacent normal tissue confirmed by a large number of cases or recorded in the database, that is, the critical value (threshold), which is a critical dCT value that can maximally distinguish between lung cancer and non-lung cancer.

When the detection object of any of the above-mentioned kits is plasma cfDNA, the dCT critical value is an average statistical dCT value after comparing the dCT values of the PITX2 gene in the blood of confirmed lung cancer patients and healthy volunteers confirmed by a large number of cases or recorded in the database, that is, the critical value (threshold), which is a critical dCT value that can maximally distinguish between lung cancer and non-lung cancer.

The present invention also protects the use of PITX2 gene as a marker in early screening of lung cancer.

Any of the above-mentioned lung cancers may be non-small cell lung cancer or small cell lung cancer.

The non-small cell lung cancer may be lung adenocarcinoma or lung squamous cell carcinoma.

The present invention finds that the gene related to the diagnosis of early lung cancer is the PITX2 gene, which is DNA and RNA extracted from lung cancer tissue and adjacent normal tissue, and the original result is obtained after screening and verification with another group of lung cancer tissue and normal human tissue DNA using whole gene methylation sequencing and expression analysis. In lung tissue, the sensitivity of PITX2 gene in detecting lung cancer reaches 74%, and the specificity is 94%. In plasma, the sensitivity of PITX2 gene in detecting lung cancer reaches 58%, and the specificity is 85%. It can be seen that a single PITX2 gene can detect lung cancer in plasma or lung tissue, and the operation is simple, time-consuming, and has high sensitivity and specificity. The PITX2 gene can also be combined with a target gene known for detecting lung cancer to diagnose early lung cancer, which can effectively improve the detection rate. The present invention can screen and assist in the diagnosis of early lung cancer, and has important application value.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

In the following embodiments, "sample to be tested" refers to the nucleic acid sample to be tested; specifically, the sample to be tested can be separated blood cells, one or more cells separated from blood, cell lines, lung lavage fluid, tissue sections, surgical tissues, biopsy tissues, paraffin-embedded tissues, body fluids, feces, urine, plasma, serum, whole blood, etc. gDNA, cfDNA and ctDNA.

In the following embodiments, "lung cancer" includes adenocarcinoma and squamous cell carcinoma, which are common malignant tumors in the respiratory tract, especially non-small cell lung cancer. The early symptoms are not obvious, and it takes about 5 to 7 years from the beginning of cancer development to the clinical discovery of a mass; the 5-year survival rate of early lung cancer is more than 90%, while it is only 10% in the late stage.

In the following examples, "target nucleic acid" or "target gene" refers to a nucleic acid fragment of the human PITX2 gene, i.e., a methylated DNA-specific fragment of the human PITX2 gene. Highly specific primers are designed to amplify different target fragments, and highly specific probes are used for identification, and the designed primers and probes are complementary to the sites to be detected.

In the following examples, "probe" refers to a single-stranded nucleic acid with a known nucleotide sequence, whose nucleotide sequence structure is substantially complementary to the target nucleic acid and can form a double strand with the "target nucleic acid". The 5' end of the probe can carry a fluorescent group and/or the 3' end can carry a quenching group marker. The combination of primers and probes with methylation-specific sequences in sample DNA enables molecular markers to detect the disease - lung cancer.

In the method of the present invention, the extracted DNA needs to be chemically modified to obtain the converted DNA fragments as the test sample. Bisulfite, bisulfite or hydrazine salt modification can be applied to this chemical modification, so as to convert cytosine in the DNA sample into uracil, while 5' methylcytosine remains unchanged, distinguish methylated or unmethylated gene fragments, and can be identified and PCR amplified using specifically designed primers and probes.

One skilled in the art can use quantitative measurements to determine the methylation level of a specific CpG position, referring to the methylation level exceeding a certain cutoff value, where the cutoff value can be a value representing the average or median methylation level of a given population, or it is preferably an optimal cutoff value.

The method of the present invention is to carry out two-channel fluorescence quantitative probe PCR amplification reactions in a reaction tube, obtain the Ct value of the PITX2 gene through the fluorescence signal, subtract the Ct value of the internal reference gene ACTB from the Ct value of the PITX2 gene of the lung cancer positive clinical sample to obtain the dCt (△Ct) value of the PITX2 gene of the sample, and compare it with the dCT threshold (critical value) of the PITX2 gene methylation obtained from a large number of known lung cancer tissues and control tissue DNA samples. If it is less than the threshold, it means that the PITX2 gene is methylated, that is, the methylation state of the PITX2 gene is judged.

Considering the needs of clinical detection, the use of liquid biopsy methods can effectively reduce the harm to patients. When real-time fluorescence PCR is used for detection, the probe is connected with a fluorescent group suitable for judging the methylated DNA fragments of different genes. One end of the probe is labeled with a fluorescent group, and the other end is labeled with a quenching group; wherein the quenching group can quench the fluorescence emitted by the fluorescent group. When the PCR amplification reaction is carried out, the base with the fluorescent group is cut off by the forward exo-cutting activity of the polymerase, and the free fluorescent group is no longer affected by the quenching group, and a fluorescent signal of a certain wavelength can be emitted under the action of the excitation light. With the continuous accumulation of PCR products, the fluorescent signal is continuously enhanced, so that the presence of specific methylated DNA can be detected. As a preferred embodiment of the present invention, the fluorescent group labeled with the detection probe can be VIC, ROX, FAM, Cy5, Cy5.5, HEX, TET, JOE, NED or TAMRA, etc.; and the quenching group can be BHQ, MGB or Dabcy1. The present invention is suitable for the multi-channel PCR detection technology commonly used in clinical detection, and realizes multi-channel fluorescence detection in one reaction tube.

In the following examples, all patients gave informed consent and the experiments were ethically approved.

In the following examples, human whole-genome methylated DNA standard (EpiTect PCR Control DNA Set, Qiagen Cat No./ID: 59695, where methylated DNA is represented by +ve ctr) is used as positive control DNA. Human whole-genome unmethylated DNA standard (EpiTect PCR Control DNA Set, Qiagen Cat No./ID: 59695, where unmethylated DNA is represented by -ve ctr) or water are both negative control DNA.

Example 1: Obtaining markers for lung cancer detection

The inventors of the present invention extracted DNA and RNA from 16 cases of clinical stage I-IV lung cancer tissues (i.e., surgical tissues of lung cancer patients (confirmed by pathology, C), as tumor-positive samples) and adjacent normal tissues (the adjacent normal tissues of the same case during surgery (confirmed by pathology, A), as tumor-negative samples), and used whole-genome methylation sequencing and expression analysis methods, combined with multiple databases and comprehensive clinical information for large-scale screening. Afterwards, by comparing the differences in methylation levels, CpG islands related to lung cancer were obtained (specifically including: 1. Designing probes to enrich the methylation sites of related genes, designing multiple pairs of primers for each CpG island, and amplifying single-band primer pairs using methylation-specific PCR gel electrophoresis; 2. Using a series of dilutions of human whole-genome methylation DNA standards, it was confirmed that the amount of PCR amplification products was proportional to the amount of the standard; 3. After screening and verification by another group of lung cancer tissues and normal human tissue DNA, 3 pairs of CpG primer pairs were found to be related to lung cancer), thereby obtaining markers for lung cancer detection. The markers used for lung cancer detection consist of two genes: PITX2 (Gene ID: 5308) and ACTB (Gene ID: 60).

Based on the nucleotide sequences of the PITX2 gene and the ACTB gene, the primers and probes used in Table 1 were designed and synthesized by Nanjing GenScript Biotech Co., Ltd.

Table 1

Note: "F" in the primer name indicates upstream primer, "R" indicates downstream primer, and "P" indicates probe; ROX indicates that the probe is labeled with ROX fluorescent group; CY5 indicates that the probe is labeled with CY5 fluorescent group; MGB indicates that the other end of the probe is quenched by the fluorescent MGB group; ACTB (GeneID: 60) is the internal reference gene.

Example 2: Detection of PITX2 gene as a marker in lung cancer tissue (C) and adjacent normal tissue (A)

1. Dilute the upstream primer, downstream primer and probe of PITX2 and ACTB in Table 1 with water respectively.

2. The samples to be tested (27 clinical lung adenocarcinoma (LUAD) paired samples (lung cancer tissue (represented by C) and adjacent normal tissue (represented by A), 4 clinical lung squamous cell carcinoma (LUSC) paired samples (lung cancer tissue (represented by C) and adjacent normal tissue (represented by A), 2 human lung cancer cell lines (A549 cells and H1299 cells, respectively)) were homogenized, and then genomic DNA was extracted using a blood/cell/tissue genomic DNA extraction kit (Beijing Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. # DP304-03) to obtain the genomic DNA of the samples to be tested.

3. Take the genomic DNA of the sample to be tested, and use EZ DNA Methylation-Direct ^TM KIT (ZYMORESEARCH, D5001/D5002) for bisulfite modification to obtain the transformed DNA of the subject to be tested.

4. Prepare the reaction system shown in Table 2 (25 μL in total), wherein the template is the transformed DNA of the test subject, the positive control DNA, the negative control DNA or the blank control, and the DNA enzyme is Accurate Tag HS DNA polymerase (CM0008, 5 μ/μl, AGL Biotechnology, China). Then perform fluorescence PCR amplification according to the reaction procedure to obtain the CT value. The fluorescence quantitative PCR instruments used are QuantStudio 5 (Appliedbiosystem, Thermo Fisher Scientific, USA)) and Quant Gene9600 (Bori Technology, Hangzhou, China).

The reaction procedure was as follows: first stage: 95°C for 5 min, 1 cycle; second stage: 95°C for 15 sec; 60°C for 30 sec; 45 cycles; third stage: collecting fluorescence signals at 58°C to obtain the CT values of PITX2 and ACTB, respectively, recorded as CT _PITX2 and CT _ACTB ; if the amplification curve was not "S"-shaped or the CT value was blank, the CT value was recorded as 45.

Table 2. Reaction system

The DNA sequences of the amplified regions of primer probe set PITX2-1 and primer probe set ACTB-1 after sulfite conversion are shown in Table 3.

Table 3

5. Further calculate the dCT of PITX2, recorded as dCT _PITX2 (dCT _PITX2 = CT _PITX2 - CT _ACTB ). The detection results of dCT _PITX2 are shown in Table 4.

Table 4. Statistical results of dCT _PITX2 in lung cancer tissue (C) and adjacent normal tissue (A)

Note: TE is 10mM Tris HCl-EDTA (10mM/1mM) buffer, used as blank control; "+" indicates PITX2 gene methylation, and "-" indicates PITX2 gene unmethylation.

6. Results of the determination of PITX2 gene as a marker in lung cancer tissue (C) DNA

(1) In Table 4, most lung cancer tissues (C), human lung cancer cell lines A549 cells and H1299 cells are gene methylation-positive DNA, and most adjacent normal tissues (A) are gene methylation-negative DNA. The DNA treated with BS was amplified by fluorescent PCR, and the CT value obtained by detection was the dCT of PITX2 after deducting the CT value of the internal control ACTB. The dCT value of the positive sample group was composed of the lung cancer tissue confirmed by pathology and the positive control DNA, and the adjacent normal tissue and the negative control DNA were composed of the dCT of the negative sample group.

(2) Whether the gene is methylated is determined by comparing the dCT value of PITX2 of the sample to be tested (denoted as dCT _PITX2 ): dCT _PITX2 = CT _PITX2 - CT _ACTB . That is, the CT value obtained by deducting the CT value of ACTB from the CT value of PITX2 detected. If the dCT of the PITX2 gene of the subject to be tested is less than the dCT critical value, then the subject to be tested is methylated based on the PITX2 gene. The dCT critical value is an average statistical dCT value after comparing the dCT values of the PITX2 gene of lung cancer tissues and normal tissues adjacent to the cancer confirmed by a large number of cases, that is, the critical value (threshold), which is a critical dCT value that can best distinguish between lung cancer and non-lung cancer.

The results showed that under the formulated PCR reaction conditions, the dCT critical value of PITX2 in tissues was 4.

According to the above steps, the "primers and probes of the primer probe set PITX2-1" were replaced with "primers and probes of the primer probe set PITX2-2" or "primers and probes of the primer probe set PITX2-3", and the "primers and probes of the primer probe set ACTB-1" were replaced with "primers and probes of the primer probe set ACTB-2", and the other steps remained unchanged. The results showed that under the PCR reaction conditions, the dCT critical value of PITX2 was also 4.

In this paper, dCT Th is the dCT critical value, and 4 is the dCT critical value of the tissue.

The DNA sequences of the amplified regions of primer probe set PITX2-2, primer probe set PITX2-3, and primer probe set ACTB-2 after sulfite conversion are shown in Table 3.

(3) Whether the sample is from a lung cancer patient is determined by comparing the dCT and dCT critical values of the PITX2 gene in the sample (such as a lung nodule): if the dCT value of the PITX2 gene of the sample to be tested is less than the dCT critical value, it means that the sample is methylated, and the sample to be tested is positive, that is, it is from or may be from a lung cancer patient; if the dCt value of the PITX2 gene of the sample to be tested is ≥ the dCT critical value, the sample is not methylated, and the sample to be tested is negative, that is, it is not from or may not be from a lung cancer patient.

By detecting the methylation level of the PITX2 gene, 23 cases of methylation-positive detection were found in 31 paired samples of lung cancer tissues, with a sensitivity or detection rate of 74%; in normal tissue samples adjacent to lung cancer, 2 cases of methylation-positive detection were found, with a specificity of 94%. It can be seen that the PITX2 gene methylation detection method provided in this application can distinguish between lung cancer tissue (C) and normal tissue (A). If the PITX2 gene and other genes are detected in coordination, it will help to improve the detection rate and identification rate of lung cancer tumors.

The above is a method for determining the methylation of a sample or determining whether the sample is from a lung cancer patient by measuring the PITX2 gene alone. In practical applications, the PITX2 gene can also be combined with the methylation status of other target genes known to be used for detecting lung cancer for joint early screening and diagnosis of lung cancer.

Example 3: Sensitivity experiment

The samples to be tested are 100% MetBisDNA (100% +ve ctr DNA), 10% MetBisDNA (10% +ve ctr DNA added to 90% -ve ctr DNA), 1% MetBisDNA (1% +ve ctr DNA added to 99% -ve ctr DNA), 0% MetBisDNA (100% -ve ctr DNA) and TE (negative control without DNA, only TE buffer). These 5 samples to be tested are serially diluted samples.

Each sample to be tested is subjected to the following experiments:

1. Dilute the "primers and probes of primer probe set PITX2-1" and "primers and probes of primer probe set ACTB-1" in Table 1 with water respectively.

2. Take the sample to be tested and use EZ DNAMethylation-DirectTM KIT to perform bisulfite modification to obtain the DNA converted from the sample to be tested.

3. Prepare the reaction system shown in Table 2 (25 μL in total), in which the template is the DNA transformed from the sample to be tested, the positive control DNA or the negative control DNA; then perform PCR amplification according to the reaction procedure. The reaction procedure is: the first stage: 95°C for 5 min, 1 cycle; the second stage: 95°C for 15 sec; 60°C for 30 sec; 45 cycles; the third stage: collect the fluorescence signal at 58°C, and obtain the CT values of PITX2 and ACTB, respectively, which are recorded as CT _PITX2 and CT _ACTB ; if the amplification curve is not "S" type or the CT value is blank, the CT value is recorded as 45. Further calculate the dCT value of the PITX2 gene (recorded as dCT _PITX2 ), dCT _PITX2 = CT _PITX2 - CT _ACTB .

4. According to the method of step 6 in Example 1, determine whether the five samples to be tested (ie, the five serially diluted samples) are positive or negative.

The results of the five samples to be tested are shown in Table 5. The results show that the method provided by the present invention can detect whether the PITX2 gene is methylated, and the minimum detection limit is 10% MetBisDNA, equivalent to 0.5ng cfDNA.

Table 5

Note: “+” indicates PITX2 gene methylation, and “-” indicates PITX2 gene unmethylation.

According to the above steps, replace the "primers and probes of primer probe set PITX2-1" with "primers and probes of primer probe set PITX2-2" or "primers and probes of primer probe set PITX2-3", and replace the "primers and probes of primer probe set ACTB-1" with "primers and probes of primer probe set ACTB-2", and keep the other steps unchanged.

The results showed that the primer-probe combination consisting of any one of the three primer-probe sets of PITX2 and any one of the two primer-probe sets of ACTB can detect whether the PITX2 gene is methylated, and the minimum detection limit is 10%.

MetBisDNA, equivalent to 0.5ng cfDNA.

Example 4: Lung cancer detection using blood as a test sample

The samples to be tested were 31 whole blood samples from ethically approved and pathologically confirmed lung cancer patients and 60 whole blood samples from healthy volunteers.

1. Dilute the "primers and probes of primer probe set PITX2-1" and "primers and probes of primer probe set ACTB-1" in Table 1 with water respectively.

2. Take 8 ml of the sample to be tested and put it into EDTA anticoagulation vacuum blood collection tube, centrifuge it twice within 2 hours (the first time at 1600g for 15 min, the second time at 15000g for 15 min) to obtain cell-free plasma.

3. Take the cell-free plasma respectively, and use the plasma free DNA centrifugation kit (D3182-03S, Meiji Company, Guangzhou) to extract free DNA to obtain cfDNA of the plasma of the test subject.

4. Take plasma cfDNA from the subjects respectively, and perform bisulfite modification using EZ DNA Methylation-Direct ^TM KIT (Cat. no. D5002, Zymo Research, USA) to obtain the converted cfDNA of the subjects.

5. Prepare the reaction system shown in Table 2 (25 μL in total), wherein the template is the transformed cfDNA of the test subject, 100% MetBisDNA (100% +ve ctr DNA) (as a positive control), 10% MetBisDNA (10% +ve ctr DNA added with 90% -ve ctr DNA) (as a positive control) or TE (as a negative control); then perform PCR amplification according to the reaction procedure. The reaction procedure is: the first stage: 95°C for 5 min, 1 cycle; the second stage: 95°C for 15 sec; 60°C for 30 sec; 45 cycles; the third stage: collect the fluorescence signal at 58°C, and obtain the CT values of PITX2 and ACTB, respectively, which are recorded as CT _PITX2 and CT _ACTB ; if the amplification curve is not "S" type or the CT value is blank, the CT value is recorded as 45. The dCT value of the PITX2 gene (denoted as dCT _PITX2 ) was further calculated: dCT _PITX2 = CT _PITX2 - CT _ACTB , ie, the CT value obtained by deducting the CT value of the internal control ACTB from the CT value of the detected PITX2 gene.

6. Whether the gene is methylated is determined by comparing the dCT value of PITX2 of the sample to be tested (denoted as dCT _PITX2 ): dCT _PITX2 = CT _PITX2 - CT _ACTB . If the dCT of the PITX2 gene of the subject to be tested is less than the dCT critical value, then the subject to be tested is methylated based on the PITX2 gene. The dCT critical value is an average statistical dCT value after comparing the dCT values of the PITX2 gene in the blood of a large number of confirmed lung cancer patients and healthy volunteers, that is, the critical value (threshold), which is a critical dCT value that can best distinguish lung cancer from non-lung cancer.

The results showed that under the formulated PCR reaction conditions, the dCT critical value of PITX2 in blood was 6.

Herein, dCT Th is the dCT critical value, and 6 is the dCT critical value of blood.

Whether the sample comes from a lung cancer patient is determined by comparing the dCT and dCt critical values of the PITX2 gene in the sample (such as blood): if the dCT value of the PITX2 gene of the sample to be tested is < the dCT critical value, it means that the sample is methylated, and the sample to be tested is positive, that is, it comes from or may come from a lung cancer patient; if the dCt value of the PITX2 gene of the sample to be tested is ≥ the dCT critical value, the sample is not methylated, and the sample to be tested is negative, that is, it does not come from or may not come from a lung cancer patient.

Some of the test results are shown in Table 6. The results showed that among the whole blood samples of 31 lung cancer patients, 18 were detected positive for methylation, with a sensitivity or detection rate of 58%; among the whole blood samples of 60 healthy volunteers, 9 were detected positive for methylation, that is, the false positive rate was 15% and the specificity was 85%. It can be seen that a single PITX2 gene can distinguish lung cancer tissue (C) from normal tissue (A) in plasma cfDNA samples, but the detection effect of plasma is slightly lower than that of lung tissue. However, the acquisition of plasma is basically non-invasive and is irreplaceable compared to obtaining lung tissue. When the sample to be tested is plasma, extensive early screening for lung cancer can be performed by detecting the PITX2 gene. Of course, the PITX2 gene can also be combined with target genes known for detecting lung cancer to jointly perform early screening and diagnosis of lung cancer.

Table 6

Note: TE is 10mM Tris HCl-EDTA (10mM/1mM) buffer, used as blank control; "+" indicates PITX2 gene methylation, and "-" indicates PITX2 gene unmethylation.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims (10)

1. The application of the PITX2 gene as a marker in the preparation of lung cancer detection kits; the GeneID of the PITX2 gene is 5308. 2. A kit for screening lung cancer, including a primer probe set for detecting the methylation level of the PITX2 gene; The GeneID of the PITX2 gene is 5308. 3. The kit according to claim 2, characterized in that: the primer probe set for detecting PITX2 gene methylation level is primer probe set PITX2-1, primer probe set PITX2-2 or primer Probe set PITX2-3; The primer probe set PITX2-1 consists of the primer PITX2-F1 shown in SEQ ID NO: 1, the primer PITX2-R1 shown in SEQ ID NO: 2, and the probe PITX2-P1 shown in SEQ ID NO: 3; The primer probe set PITX2-2 consists of the primer PITX2-F2 shown in SEQ ID NO: 4, the primer PITX2-R2 shown in SEQ ID NO: 5, and the probe PITX2-P2 shown in SEQ ID NO: 6; The primer probe set PITX2-3 is composed of the primer PITX2-F3 shown in SEQ ID NO:7, the primer PITX2-R3 shown in SEQ ID NO:8, and the probe PITX2-P3 shown in SEQ ID NO:9. 4. The kit according to claim 2 or 3, characterized in that: the kit further includes a primer probe set for detecting the methylation level of the internal reference gene. 5. The kit according to claim 4, characterized in that: the internal reference gene is the ACTB gene; the GeneID of the ACTB gene is 60. 6. The kit according to claim 4, characterized in that: the primer probe set for detecting the methylation level of the internal reference gene is the primer probe set ACTB-1 or the primer probe set ACTB-2; The primer probe set ACTB-1 consists of the primer ACTB-F1 shown in SEQ ID NO: 10, the primer ACTB-R1 shown in SEQ ID NO: 11, and the probe ACTB-P1 shown in SEQ ID NO: 12; The primer probe set ACTB-2 is composed of the primer ACTB-F2 shown in SEQ ID NO: 13, the primer ACTB-R2 shown in SEQ ID NO: 14, and the probe ACTB-P2 shown in SEQ ID NO: 15. 7. The kit according to claim 3 or 6, characterized in that: one end of the probes has a fluorescent label, and the other end has a fluorescence quenching label. 8. The kit according to claim 2, characterized in that: the detection object of the kit is genomic DNA, plasma cfDNA or fecal cfDNA of pulmonary nodule tissue. 9. The kit according to any one of claims 2 to 8, characterized in that: the kit also includes a data processing system; the data processing system converts the methylation level of the PITX2 gene into the dCT of the subject _PITX2 , used to determine whether the person being tested is a lung cancer patient; The calculation method of the subject's dCT _PITX2 is as follows: chemically modifying the genomic DNA, plasma cfDNA or fecal cfDNA of the subject's pulmonary nodule tissue, and then using it as a template, using claims 3 and/or 6 Perform fluorescent PCR amplification with the primers and probes, collect the fluorescence signals, and obtain the CT values of PITX2 and ACTB respectively, which are recorded as CT _PITX2 and CT _ACTB in sequence; if the amplification curve is not "S" shaped or the CT value is blank, then The CT value is recorded as 45; further calculate the dCT value of PITX2, dCT _PITX2 =CT _PITX2 -CT _ACTB ; The judgment method is: if the PITX2 gene of the subject is methylated, the subject is a lung cancer patient; otherwise, the subject is not a lung cancer patient; Whether the PITX2 gene is methylated is determined by comparing the dCT and dCT critical value of the PITX2 gene of the sample to be tested; If the dCT of the PITX2 gene of the subject is less than the dCT critical value, the subject will be methylated based on the PITX2 gene. 10. Application of PITX2 gene as a marker in early screening of lung cancer.