CN117187388A - Application of GRIK2 gene as marker in preparation of lung cancer detection kit - Google Patents
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Abstract
The application discloses application of GRIK2 gene as a marker in preparation of a lung cancer detection kit. The application discovers that the gene related to early lung cancer diagnosis is GRIK2 gene, which is DNA and RNA extracted from lung cancer tissues and paracancerous normal tissues, and the gene is subjected to screening verification by using DNA of another group of lung cancer tissues and normal human tissues by using a whole-gene methylation sequencing and expression analysis method. In lung tissue, the sensitivity of GRIK2 gene to detect lung cancer reaches 71% and the specificity is 87%. In plasma, the sensitivity of GRIK2 gene to detect lung cancer reaches 55% and the specificity is 80%. It follows that a single GRIK2 gene is able to detect lung cancer in plasma or lung tissue. The application can screen and assist diagnosis for early lung cancer, and has important application value.
Description
Technical Field
The application belongs to the field of biomedicine, and particularly relates to application of GRIK2 gene serving as a marker in preparation of a lung cancer detection kit.
Background
Lung cancer is classified into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC). Histologically, NSCLC is classified as Adenocarcinoma (AC), squamous cell carcinoma (squamous cell carcinoma, SCC), and large cell carcinoma. Non-small cell lung cancer grows and spreads slower than SCLC. Most NSCLC patients are diagnosed with advanced stages. Although lung cancer screening methods such as computed tomography CT and MRI are widely used, these techniques can find small nodules of 2-3 mm, but their specific features, i.e., benign or malignant, cannot be assessed. Many of the problems associated with these methods (e.g., high rate of false positive results, fear of radiation by the patient, excessive expense, long follow-up time) further limit the effectiveness of these methods. An important reason for the late diagnosis of lung cancer is the lack of reliable biomarkers. Serum protein antigen (CEA), neuron Specific Enolase (NSE) and the like are widely applied to blood protein diagnosis biomarkers of lung cancer, and the detection rates are only 26% and 21-39% respectively. Wherein tumor DNA from blood circulation (ctDNA), circulating tumor cells, serum non-coding RNAs and exosomes from blood have been explored. Among them, the use of ctDNA for lung cancer detection is of great interest, and the incorporation of reliable biomarkers into lung cancer screening programs using ctDNA would help to identify patients early.
DNA methylation is an important epigenetic modification that plays a critical role in regulating gene expression and modifying chromatin conformation. DNA methylation is an early event in the progression of cancer, and thus diagnostic biomarkers to detect DNA methylation changes in the regulatory region of tumor suppressor genes can be used as one of the most promising diagnostic and prognostic tools. Use of vimentin (ColoSureTM) and SEPT9 in colorectalAbnormal DNA methylation on genes is two good examples. Hypermethylation of CEBPA is a biomarker for prognosis of acute myeloid leukemia. Therefore, the development of reliable early detection methods for NSCLC using DNA methylation markers would be an important point in the research of transformed cancers.
Early diagnosis of tumors generally depends on two key factors: high sensitivity of the inspection index and a noninvasive and simple detection method. With the rapid development of technology, the search for tumor markers has evolved into a new field of tumor diagnosis and treatment, a new challenge and hope. The tumor markers can be detected in body fluid or tissue, and can reflect the existence, differentiation degree, prognosis estimation and treatment effect of tumors.
Disclosure of Invention
The application aims to find markers for early lung cancer, which are more effective in screening and diagnosis.
The application firstly protects the application of GRIK2 gene as a marker in preparing a lung cancer detection kit; gene ID of GRIK2 Gene was 2898.
The application also protects a kit for screening lung cancer, which can comprise a primer probe group for detecting the methylation level of the GRIK2 gene; gene ID of GRIK2 Gene was 2898.
The kit can specifically consist of the primer probe set for detecting the methylation level of the GRIK2 gene.
Any of the primer probe sets for detecting the methylation level of the GRIK2 gene can be a primer probe set GRIK2-1, a primer probe set GRIK2-2 or a primer probe set GRIK2-3.
Any of the primer probe sets GRIK2-1 consists of the nucleotide sequence shown in SEQ ID NO:1, primer GRIK2-F1 shown in SEQ ID NO:2 and the primer GRIK2-R1 shown in SEQ ID NO:3, and the probe GRIK2-P1 shown in the formula I.
Any of the primer probe sets GRIK2-2 consists of the nucleotide sequence shown in SEQ ID NO:4, primer GRIK2-F2 shown in SEQ ID NO:5 and primers GRIK2-R2 and SEQ ID NO:6, and the probe GRIK2-P2 shown in the specification.
Any of the primer probe sets GRIK2-3 consists of the nucleotide sequence shown in SEQ ID NO:7, primer GRIK2-F3 shown in SEQ ID NO:8 and primers GRIK2-R3 and SEQ ID NO:9, and a probe GRIK 2-P3.
Any of the above kits may further comprise a primer probe set for detecting the methylation level of the reference gene.
Any one of the above-mentioned kits may specifically be composed of any one of the above-mentioned primer probe sets for detecting methylation level of the GRIK2 gene and any one of the above-mentioned primer probe sets for detecting methylation level of the reference gene.
The reference gene may be an ACTB gene. The Gene ID of the ACTB Gene was 60.
Any of the primer probe sets for detecting the methylation level of the reference gene can be a primer probe set ACTB-1 or a primer probe set ACTB-2.
The primer probe group ACTB-1 consists of SEQ ID NO:10, the primer ACTB-F1 shown in SEQ ID NO:11 and the primer ACTB-R1 shown in SEQ ID NO:12, and the probe ACTB-P1.
The primer probe group ACTB-2 consists of SEQ ID NO:13, the primer ACTB-F2 shown in SEQ ID NO:14 and the primer ACTB-R2 shown in SEQ ID NO:15, and the probe ACTB-P2.
One end of any of the probes (such as a probe for detecting a reference gene and a probe for detecting the methylation level of the GRIK2 gene) is provided with a fluorescent label, and the other end is provided with a fluorescence quenching label.
The detection object of any of the above kits may specifically be genomic DNA, plasma cfDNA or fecal cfDNA of lung nodule tissue.
Any of the above kits may further comprise a data processing system; the data processing system converts the methylation level of GRIK2 gene into dCT of the tested person GRIK2 The method is used for judging whether the tested person is a lung cancer patient or a lung cancer high risk group;
dCT of the subject GRIK2 The calculation method of (1) is as follows: chemically modifying genomic DNA, plasma cfDNA or fecal cfDNA of lung nodule tissue of a person to be tested, performing fluorescent PCR amplification by using the primer and probe according to claim 3 and/or 6 as a template, collecting fluorescent signals, respectively obtaining CT values of GRIK2 and ACTB, and sequentially marking as CT GRIK2 And CT ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not of the "S" type or the CT value is blank, the CT value is marked as 45; further calculate the dCT value of GRIK2, dCT GRIK2 ＝CT GRIK2 -CT ACTB ；
The judging method comprises the following steps: if the GRIK2 gene of the person to be tested is methylated, the person to be tested is a lung cancer patient or a lung cancer high risk group; otherwise, the person to be tested is not a lung cancer patient or a lung cancer high risk group; whether methylation of the GRIK2 gene occurs or not is achieved by comparing the dCT and dCT critical values of the GRIK2 gene of the sample to be tested;
if dCT of the GRIK2 gene of the tester is less than dCT threshold, methylation of the tester occurs based on the GRIK2 gene; if dCT of the GRIK2 gene of the testee is more than or equal to dCT critical value, the testee does not generate methylation based on the GRIK2 gene.
When the detection target of any of the above-described kits is genomic DNA of a lung nodule tissue, the dCT threshold is a value of dCT which is an average statistic after dCT of the GRIK2 gene of a lung cancer tissue and a paracancerous normal tissue confirmed by a relatively large number of cases or recorded in a database, that is, a threshold (threshold value) which is a threshold dCT value capable of maximally distinguishing lung cancer from non-lung cancer.
When the subject of any of the above kits is plasma cfDNA, the dCT threshold is a mean statistical dCT value, i.e., a threshold (threshold value), after dCT values of the GRIK2 gene in the blood of established lung cancer patients and healthy volunteers, confirmed by a relatively large number of cases or recorded in a database, which is a threshold dCT value capable of maximally distinguishing lung cancer from non-lung cancer.
The application also protects the application of the GRIK2 gene as a marker in early screening of lung cancer.
Any of the lung cancer score described above 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 carcinoma.
The application discovers that the gene related to early lung cancer diagnosis is GRIK2 gene, which is DNA and RNA extracted from lung cancer tissues and paracancerous normal tissues, and is obtained by screening through whole-gene methylation sequencing and expression analysis, and the original result is obtained after screening and verification through another group of lung cancer tissues and normal human tissues DNA. In lung tissue, the sensitivity of GRIK2 gene to detect lung cancer reaches 71% and the specificity is 87%. In plasma, the sensitivity of GRIK2 gene to detect lung cancer reaches 55% and the specificity is 80%. Therefore, the single GRIK2 gene can detect lung cancer in blood plasma or lung tissues, and has the advantages of simple operation, short time consumption and higher sensitivity and specificity. The GRIK2 gene can also be combined with a known target gene for detecting lung cancer to diagnose early lung cancer, so that the detection rate can be effectively improved. The application can screen and assist diagnosis for early lung cancer, and has important application value.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following examples, "sample to be tested" refers to a nucleic acid sample to be tested; specifically, the sample to be tested may be isolated blood cells, one or more of cells isolated from blood, cell lines, lung lavage fluid, tissue sections, surgical tissue, biopsy tissue, paraffin embedded tissue, body fluids, feces, urine, plasma, serum, whole blood, and the like gDNA, cfDNA, and ctDNA.
In the examples that follow, "lung cancer" includes adenocarcinomas and squamous carcinomas, which are common malignant tumors in the respiratory tract, particularly non-small cell lung cancer. Early symptoms are not obvious, and the period from the beginning of canceration to the clinical discovery of tumor is about 5-7 years; the survival rate of early lung cancer reaches more than 90% in 5 years, and only 10% in the later stage.
In the examples described below, the "target nucleic acid" or "target gene" refers to a nucleic acid fragment of the human GRIK2 gene, i.e., a methylated DNA-specific fragment of the human GRIK2 gene. Highly specific primers are designed to amplify different target fragments, highly specific probes are used for recognition, and the designed primers and probes can be complementary with the site to be detected.
In the following examples, a "probe" refers to a single-stranded nucleic acid having a known nucleotide sequence whose nucleotide sequence structure is substantially complementary to a target nucleic acid, and which can form a double strand with the "target nucleic acid". The 5 'end of the probe may carry a fluorophore and/or the 3' end may carry a quencher label. The combination of the primer and the probe with the methylation specific sequence in the sample DNA enables the molecular marker to detect the lung cancer.
In the method of the present application, the extracted DNA needs to be chemically modified to obtain a converted DNA fragment as a sample to be measured. Bisulphite, bisulphite or hydrazine salt modifications can be applied to such chemical modifications to convert cytosines in DNA samples to uracils, while 5' methylcytosines are unchanged, distinguishing methylated or unmethylated gene fragments, and PCR amplification recognition can be performed using specifically designed primers and probes.
The person skilled in the art can use quantitative measurements to determine the methylation level of a specific CpG position, the nail methylation level exceeding a certain threshold, wherein the threshold can be a value representing the average or median methylation level of a given population, or it is preferably the optimal threshold.
The method of the application is that two fluorescence quantitative probe PCR amplification reactions are carried out in a reaction tube, the Ct value of the GRIK2 gene is obtained through fluorescence signals, the Ct value of an internal reference gene ACTB is subtracted from the Ct value of the GRIK2 gene of a lung cancer positive clinical sample to become the dCT (delta Ct) value of the GRIK2 gene of the sample, compared with a threshold value (critical value) for obtaining methylation dCT of the GRIK2 gene from a large number of known lung cancer tissues and control tissue DNA samples, the methylation state of the GRIK2 gene is judged when the Ct value is smaller than the threshold value.
In view of the clinical testing needs, the liquid biopsy method can effectively reduce the damage to patients. When the real-time fluorescence PCR is used for detection, the probe is connected with a fluorescent group suitable for judging methylated DNA fragments of different genes. One end of the probe is marked with a fluorescent group, and the other end is marked with a quenching group; wherein the quenching group quenches fluorescence emitted by the fluorescent group. When the PCR amplification reaction is carried out, the forward exo-activity of the polymerase is utilized to cut off the base with the fluorescent group, the free fluorescent group is not influenced by the quenching group any more, and a fluorescent signal with a certain wavelength can be emitted under the action of the excitation light. As PCR products accumulate, the fluorescent signal increases continuously, so that the presence of specifically methylated DNA can be detected. As a preferred mode of the present application, the fluorescent group labeled with the detection probe may be VIC, ROX, FAM, cy, cy5.5, HEX, TET, JOE, NED, TAMRA or the like; and the quenching group may be BHQ, MGB or Dabcy1. The application is suitable for the commonly used multichannel PCR detection technology of the clinical detection at present, and realizes multichannel fluorescence detection in one reaction tube.
In the examples below, all patients had informed consent and the experiment was ethically approved.
In the examples below, a human whole-gene methylated DNA standard (EpiTect PCR Control DNA Set, qiagen CatNo./ID:59695, where methylated DNA is expressed as +ve ctr) was used as positive control DNA. Human whole-gene unmethylated DNA standard (EpiTect PCR Control DNA Set, qiagen CatNo./ID:59695, where unmethylated DNA is denoted by-ve ctr) or water was negative control DNA.
Example 1 obtaining markers for lung cancer detection
The inventor of the application uses DNA and RNA extracted from 16 clinical lung cancer tissues of stage I-IV (namely, the operation tissue (C) of a lung cancer patient is confirmed by pathology, and is used as a tumor positive sample) and a cancer side normal tissue (the cancer side normal tissue (A) is confirmed by pathology and is used as a tumor negative sample) in the same case operation, and uses whole gene methylation sequencing and expression analysis method to carry out large-scale screening by combining various databases and comprehensive clinical information. And then, comparing methylation level differences to obtain CpG islands related to lung cancer (specifically comprising 1 designing primers of the enriched methylation sites of GRIK2 genes, designing a plurality of pairs of primers for each CpG island, amplifying by a methylation specific PCR gel electrophoresis method to obtain a single primer pair, 2 verifying that the serial dilutions of a human whole-gene methylated DNA standard substance are used, using a Tagman enzyme probe PCR method to verify that the amplification product quantity is in direct proportion to the use level of the standard substance, and 3 performing screening verification on the DNA of another group of lung cancer tissues and normal human tissues, wherein 3 pairs of CpG primer pairs are related to lung cancer, thereby obtaining the marker for lung cancer detection. The markers for lung cancer detection consisted of 2 genes of GRIK2 (Gene ID: 2898) and ACTB (Gene ID: 60).
The primers and probes used in Table 1 were designed and synthesized by Nanjing Style Biotech company based on the nucleotide sequences of the GRIK2 gene and the ACTB gene, respectively.
TABLE 1
Note that: the primer name contains "F" as the upstream primer, "R" as the downstream primer, and "P" as the probe; VIC means that the probe is labeled with a VIC fluorophore; CY5 represents that the probe is labeled with a CY5 fluorophore; MGB means that the other end of the probe is quenched and marked by a fluorescent MGB group; ACTB (Gene ID: 60) is a reference Gene.
Example 2 detection of GRIK2 Gene as marker in lung cancer tissue (C) and paracancerous Normal tissue (A)
1. The upstream primer, downstream primer and probe of GRIK2 and ACTB in Table 1 were diluted with water, respectively.
2. Samples to be tested (27 cases of clinical lung adenocarcinoma (LUAD) paired samples (lung cancer tissue (represented by C) and paracancerous normal tissue (represented by a), 4 cases of clinical lung squamous carcinoma (luc) paired samples (lung cancer tissue (represented by C) and paracancerous normal tissue (represented by a), 2 cases of human lung cancer cell lines (a 549 cells, H1299 cells, respectively)) were taken for homogenization, and then genomic DNA was extracted using a blood/cell/tissue genomic DNA extraction kit (beijing tiangen biochemical technology (beijing) limited, cat.#dp304-03) to obtain genomic DNA of the samples to be tested.
3. Taking genomic DNA of a sample to be tested, and adopting EZ DNAMethylation-Direct TM KIT (ZYMO RESEARCH, D5001/D5002) is bisulphite modified to obtain the DNA transformed by the tester.
4. A reaction system (25. Mu.L total) shown in Table 2 was prepared, wherein the template was DNA transformed by the subject, DNA of a human lung cancer cell line (as a positive control), negative control DNA or a blank control, and the DNase was Accurate Tag HSDNA polymerase (CM 0008, 5. Mu/. Mu.L, AGL Bio Inc., china). And then performing fluorescent PCR amplification according to a reaction program to obtain CT values. The fluorescent quantitative PCR instruments used were Quantum studio 5 (applied biosystem, thermo Fisher Scientific, USA) and Quantum Gene 9600 (Boy technologies, hangzhou, china).
The reaction procedure is: the first stage: 5min at 95 ℃ for 1 cycle; and a second stage: 15sec at 95 ℃; 30sec at 60 ℃;45 cycles; and a third stage: collecting fluorescence signals at 58 deg.C to obtain CT values of GRIK2 and ACTB, respectively, and sequentially recording as CT GRIK2 And CT ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not "S" shaped or the CT value is blank, the CT value is noted as 45.
TABLE 2 reaction System
DNA sequences of amplified regions of the primer probe group GRIK2-1 and the primer probe group ACTB-1 after sulfite conversion are shown in Table 3.
TABLE 3 Table 3
5. Further calculation of dCT for GRIK2, noted dCT GRIK2 (dCT GRIK2 ＝CT GRIK2 -CT ACTB )。dCT GRIK2 The results of the measurement are shown in Table 4.
TABLE 4 dCT in lung cancer tissue (C) and paracancerous Normal tissue (A) GRIK2 Statistics of (2)
Note that: TE was 10mM Tris HCl-EDTA (10 mM/1 mM) buffer as a blank control; "+" indicates methylation of the GRIK2 gene, and "-" indicates unmethylation of the GRIK2 gene.
6. Determination result of GRIK2 Gene as marker in lung cancer tissue (C) DNA
(1) In Table 4, most of the lung cancer tissues (C), human lung cancer cell lines A549 and H1299 were gene-methylation positive DNA, and most of the paracancestral tissues (A) were gene-methylation negative DNA. And (3) performing fluorescence PCR amplification on the DNA treated by the BS, detecting a CT value obtained by detection, and subtracting the CT value of the internal control ACTB to obtain the CT value which is dCT of GRIK 2. The positive sample group dCT values are formed by the lung cancer tissues and positive control DNA confirmed by pathology, and the negative sample group dCT is formed by the paracancerous normal tissues and negative control DNA.
(2) Whether the gene is methylated or not is determined by comparing the dCT value of GRIK2 (dCT GRIK2 ) To realize: dCT GRIK2 ＝CT GRIK2 -CT ACTB . Namely, the CT value obtained by detecting GRIK2 is subtracted by the CT value of ACTB, if dCT of the GRIK2 gene of the subject is detected<dCT, the tester is methylated based on the GRIK2 gene. Whereas the dCT threshold is a statistically average dCT value after dCT of the GRIK2 gene of the lung cancer tissue and the paracancerous normal tissue confirmed by a relatively large number of cases, i.e., a threshold value (threshold value), which is a critical dCT value capable of maximally distinguishing lung cancer from non-lung cancer.
The results showed that the critical value of dCT for GRIK2 in tissue was 4 under the composed PCR reaction conditions.
According to the above steps, the "primer and probe of the primer probe set GRIK 2-1" is replaced with the "primer and probe of the primer probe set GRIK 2-2" or the "primer and probe of the primer probe set GRIK 2-3", the "primer and probe of the primer probe set ACTB-1" is replaced with the "primer and probe of the primer probe set ACTB-2", and the other steps are unchanged. The results showed that the dCT threshold for GRIK2 was also 4 under the conditions of the PCR reaction.
Herein, dCT Th is dCT threshold, dCT Th is dCT threshold of 4 as organization.
DNA sequences of amplified regions of the primer probe group GRIK2-2, the primer probe group GRIK2-3 and the primer probe group ACTB-2 after sulfite conversion are shown in Table 3.
(3) Determining whether a sample is derived from a lung cancer patient is determined by comparing the dCT and dCT thresholds of the GRIK2 gene in the sample (e.g., lung nodules): if the GRIK2 gene dCT value of the sample to be tested is less than dCT threshold value, which indicates that the sample is methylated, the sample to be tested is positive, namely, the sample is from or possibly from a lung cancer patient; if the dCT value of the GRIK2 gene of the sample to be tested is more than or equal to the dCT critical value, the sample is not methylated, and the sample to be tested is negative, namely, the sample is not from or possibly not from a lung cancer patient.
By detecting the methylation degree of the GRIK2 gene, 22 methylation positive detection cases are found in lung cancer tissues of 31 paired samples, and the sensitivity or detection rate is 71%; in the lung cancer side normal tissue sample, 4 cases of methylation positive detection are detected, and the specificity is 87%. Therefore, the GRIK2 gene methylation detection method provided by the application can distinguish lung cancer tissues (C) from normal tissues (A). Methylation of the single gene found for GRIK2 has been shown to be associated with lung cancer tumorigenesis. If the GRIK2 gene and other genes are detected cooperatively, the detection rate and the identification rate of lung cancer tumors can be improved.
The above is a method for determining methylation of a sample or determining whether a sample is derived from a lung cancer patient by measuring the GRIK2 gene alone. In practice, the GRIK2 gene can also be used for combined early screening and diagnosis of lung cancer in combination with other known methylation status of target genes for detecting lung cancer.
Example 3 sensitivity experiment
The samples to be tested were 100% MetBisDNA (100% + ve ctr DNA), 10% MetBisDNA (10% + ve ctr DNA plus 90% -ve ctr DNA), 1% MetBisDNA (1% + ve ctr DNA plus 99% -ve ctr DNA), 0% MetBisDNA (100% -ve ctr DNA) and TE (negative control without DNA, only TE buffer), respectively. The 5 samples to be tested are serial dilutions of the sample.
Each sample to be tested was subjected to the following experiment:
1. the "primer and probe of primer probe set GRIK 2-1" and the "primer and probe of primer probe set ACTB-1" in Table 1 were diluted with water, respectively.
2. And taking a sample to be detected, and carrying out bisulphite modification by adopting EZ DNAMethylation-DirectTM KIT to obtain DNA converted by the sample to be detected.
3. Preparing a reaction system (total 25 mu L) shown in Table 2, wherein the template is DNA converted by a sample to be tested, positive control DNA or negative control DNA; PCR amplification was then performed according to the reaction procedure. The reaction procedure is: the first stage: 5min at 95 ℃ for 1 cycle; and a second stage: 15sec at 95 ℃; 30sec at 60 ℃;45 cycles; and a third stage: collecting fluorescence signals at 58 deg.C to obtain CT values of GRIK2 and ACTB, respectively, and sequentially recording as CT GRIK2 And CT ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not "S" shaped or the CT value is blank, the CT value is noted as 45. Further, the dCT value of the GRIK2 gene (dCT GRIK2 )，dCT GRIK2 ＝CT GRIK2 -CT ACTB 。
4. According to the method of step 6 in example 1, it was judged whether 5 samples to be tested, i.e., 5 serially diluted samples, were positive or negative.
The results of 5 samples to be tested are shown in Table 5. The result shows that the method provided by the application can detect whether the GRIK2 gene is methylated or not, and the minimum detection limit is 10% MetBisDNA, which is equivalent to 0.5ng cfDNA.
TABLE 5
Note that: "+" indicates methylation of the GRIK2 gene, and "-" indicates unmethylation of the GRIK2 gene.
According to the above steps, the "primer and probe of the primer probe set GRIK 2-1" is replaced with the "primer and probe of the primer probe set GRIK 2-2" or the "primer and probe of the primer probe set GRIK 2-3", the "primer and probe of the primer probe set ACTB-1" is replaced with the "primer and probe of the primer probe set ACTB-2", and the other steps are unchanged.
The results show that the primer probe combination consisting of any one of the 3 primer probe groups of GRIK2 and any one of the 2 primer probe groups of ACTB can detect whether the GRIK2 gene is methylated or not, and the minimum detection limit is 10 percent
MetBisDNA corresponds 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, pathologically confirmed lung cancer patients and 140 whole blood samples from healthy volunteers, respectively.
1. The "primer and probe of primer probe set GRIK 2-1" and the "primer and probe of primer probe set ACTB-1" in Table 1 were diluted with water, respectively.
2. 8ml of the sample to be tested is taken out of an EDTA anticoagulation vacuum blood collection tube, and is subjected to centrifugation twice within 2h (first 1600g centrifugation for 15min and second 15000g centrifugation for 15 min) to obtain cell-free plasma.
3. And (3) respectively taking the cell-free plasma, and extracting free DNA by using a plasma free DNA centrifugation kit (D3182-03S, meyer' S patches) to obtain cfDNA of the plasma of the person to be tested.
4. Respectively taking cfDNA of blood plasma of a person to be tested, and applying EZ DNAMethylation-Direct TM KIT (Cat.no.D5002, zymo Research, USA) was bisulphite modified to obtain cfDNA transformed by the tester.
5. The reaction system shown in Table 2 (25. Mu.L total) was prepared, wherein the template was cfDNA transformed by the tester, 100% MetBisDNA (100% + ve ctr DNA) (as positive control), 10% MetBisDNA (10% + ve ctr DNA plus 90% -ve ctr DNA) (as positive control), or TE (as negative control); PCR amplification was then performed according to the reaction procedure. The reaction procedure is: the first stage: 5min at 95 ℃ for 1 cycle; and a second stage: 15sec at 95 ℃; 30sec at 60 ℃;45 cycles; and a third stage: collecting fluorescence signals at 58 deg.C to obtain CT values of GRIK2 and ACTB, respectively, and sequentially recording as CT GRIK2 And CT ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not "S" shaped or the CT value is blank, the CT value is noted as 45. Further, the dCT value of the GRIK2 gene (dCT GRIK2 )，dCT GRIK2 ＝CT GRIK2 -CT ACTB Namely, the CT value of the GRIK2 gene obtained by detection is subtracted from the CT value of the internal control ACTB.
6. Whether the gene is methylated or not is determined by comparing the dCT value of GRIK2 (dCT GRIK2 ) To realize: dCT GRIK2 ＝CT GRIK2 -CT ACTB . dCT if the subject GRIK2 gene<dCT, the tester is methylated based on the GRIK2 gene. Whereas the dCT threshold is a statistically average dCT value, i.e., a threshold value (threshold value), after dCT values of the GRIK2 gene in the blood of relatively large numbers of established lung cancer patients and healthy volunteers, which is a critical dCT value capable of maximally distinguishing between lung cancer and non-lung cancer.
The results showed that the critical value of dCT for GRIK2 in blood was 6 under the composed PCR reaction conditions.
Herein, dCT Th is dCT critical value, dCT Th is dCT critical value of 6 as blood.
Determining whether a sample is derived from a lung cancer patient is determined by comparing dCT and dCt thresholds of the GRIK2 gene in the sample (e.g., blood): if the GRIK2 gene dCT value of the sample to be tested is less than dCT threshold value, which indicates that the sample is methylated, the sample to be tested is positive, namely, the sample is from or possibly from a lung cancer patient; if the dCT value of the GRIK2 gene of the sample to be tested is more than or equal to the dCT critical value, the sample is not methylated, and the sample to be tested is negative, namely, the sample is not from or possibly not from a lung cancer patient.
The results of the partial tests are shown in Table 6. The result shows that 17 methylation positive samples are detected in whole blood samples of 31 lung cancer patients, and the sensitivity or detection rate is 55%; in whole blood samples of 160 healthy volunteers, 32 methylation positives were detected, i.e., false positive rate was 20% and specificity was 80%. It can be seen that a single GRIK2 gene is able to distinguish lung cancer tissue (C) from normal tissue (A) in a plasma cfDNA sample, except that the detection effect of the plasma is slightly lower than that of lung tissue. However, the acquisition of plasma is essentially noninvasive and is not an alternative to obtaining lung tissue. When the sample to be detected is blood plasma, extensive early screening of lung cancer can be performed by detecting the GRIK2 gene. Of course, the GRIK2 gene may also be combined with target genes known for detecting lung cancer, together with the combined early screening and diagnosis of lung cancer.
TABLE 6
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Note that: TE was 10mM Tris HCl-EDTA (10 mM/1 mM) buffer as a blank control; "+" indicates methylation of the GRIK2 gene, "-" indicates unmethylation of the GRIK2 gene, and UD is under detection, i.e., no CT value.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
- Application of GRIK2 gene as marker in preparing lung cancer detection kit; gene ID of GRIK2 Gene was 2898.
- 2. A kit for screening lung cancer, comprising a primer probe group for detecting the methylation level of a GRIK2 gene;gene ID of GRIK2 Gene was 2898.
- 3. The kit of claim 2, wherein: the primer probe group for detecting the methylation level of the GRIK2 gene is a primer probe group GRIK2-1, a primer probe group GRIK2-2 or a primer probe group GRIK2-3;primer probe group GRIK2-1 consists of SEQ ID NO:1, primer GRIK2-F1 shown in SEQ ID NO:2 and the primer GRIK2-R1 shown in SEQ ID NO:3, a probe GRIK2-P1 shown in the specification;primer probe group GRIK2-2 consists of SEQ ID NO:4, primer GRIK2-F2 shown in SEQ ID NO:5 and primers GRIK2-R2 and SEQ ID NO:6, a probe GRIK 2-P2;primer probe group GRIK2-3 consists of SEQ ID NO:7, primer GRIK2-F3 shown in SEQ ID NO:8 and primers GRIK2-R3 and SEQ ID NO:9, and a probe GRIK 2-P3.
- 4. A kit according to claim 2 or 3, wherein: the kit further comprises a primer probe set for detecting the methylation level of the reference gene.
- 5. The kit of claim 4, wherein: the reference gene is ACTB gene; the Gene ID of the ACTB Gene was 60.
- 6. The kit of claim 4, wherein: the primer probe group for detecting the methylation level of the reference gene is a primer probe group ACTB-1 or a primer probe group ACTB-2;the primer probe group ACTB-1 consists of SEQ ID NO:10, the primer ACTB-F1 shown in SEQ ID NO:11 and the primer ACTB-R1 shown in SEQ ID NO:12, a probe ACTB-P1;the primer probe group ACTB-2 consists of SEQ ID NO:13, the primer ACTB-F2 shown in SEQ ID NO:14 and the primer ACTB-R2 shown in SEQ ID NO:15, and the probe ACTB-P2.
- 7. The kit of claim 3 or 6, wherein: one end of the probe is provided with a fluorescent label, and the other end is provided with a fluorescence quenching label.
- 8. The kit of claim 2, wherein: the detection object of the kit is genomic DNA, plasma cfDNA or fecal cfDNA of lung nodule tissue.
- 9. The kit of any one of claims 2 to 8, wherein: the kit further comprises a data processing system; the data processing system converts the methylation level of GRIK2 gene into dCT of the tested person GRIK2 The method is used for judging whether the tested person is a lung cancer patient or a lung cancer high risk group;dCT of the subject GRIK2 The calculation method of (1) is as follows: chemically modifying genomic DNA, plasma cfDNA or fecal cfDNA of lung nodule tissue of a person to be tested, performing fluorescent PCR amplification by using the primer and probe according to claim 3 and/or 6 as a template, collecting fluorescent signals, respectively obtaining CT values of GRIK2 and ACTB, and sequentially marking as CT GRIK2 And CT ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not of the "S" type or the CT value is blank, the CT value is marked as 45; further calculate the dCT value of GRIK2, dCT GRIK2 ＝CT GRIK2 -CT ACTB ；The judging method comprises the following steps: if the GRIK2 gene of the person to be tested is methylated, the person to be tested is a lung cancer patient or a lung cancer high risk group; otherwise, the person to be tested is not a lung cancer patient or a lung cancer high risk group;whether methylation of the GRIK2 gene occurs or not is achieved by comparing the dCT and dCT critical values of the GRIK2 gene of the sample to be tested;if dCT of the subject GRIK2 gene is less than dCT threshold, the subject undergoes methylation based on the GRIK2 gene.
- Application of GRIK2 gene as marker in early lung cancer screening.
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