A primer-probe combination, kit and application for detecting the benign and malignant nature of pulmonary nodules

Abstract

The present invention relates to the field of gene detection, and particularly to a primer-probe combination, a kit and their applications for detecting the benign and malignant nature of pulmonary nodules. The primer-probe combination includes primer pairs and probes for detecting gene mutation sites and methylation sites, and can achieve simultaneous detection covering the EGFR L858R mutation site, the EGFR 19Del mutation site, and the methylation sites of the HOXA7, CCNA1, and ZNF808 genes at one time. Thus, the present invention is a simple, rapid and accurate combined detection scheme for DNA methylation and gene mutation to assist in differentiating the benign and malignant nature of pulmonary nodules; at the same time, the primer-probe combination can achieve rapid pre-cancer screening, and also has application values such as accurate medication guidance and prognosis and metastasis risk prediction.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and particularly to a primer-probe combination, a kit and an application thereof for detecting the benign and malignant nature of pulmonary nodules. Background Art

[0002] Lung cancer is a common malignant tumor in the respiratory system, characterized by high incidence and mortality rates. In recent years, with the improvement of medical standards, although the survival period of patients can be effectively extended, patients with advanced lung cancer still have a high risk of death after metastasis and invasion. Reports show that early-stage lung cancer has no specific clinical symptoms, and most patients are easily overlooked. By the time symptoms appear and they seek medical treatment, they are already in the middle and late stages, increasing the difficulty of treatment and affecting the prognosis. Therefore, early and accurate screening of lung cancer is of great significance. CT examination is currently the main method for clinical diagnosis of malignant tumors, which can clearly show tiny internal lesions in the human body. However, its radiation is harmful to the human body, and it can only be done once a year. The detection sensitivity and specificity of serological markers such as cyfra21-1, NSE, SCC, and CEA are not high, and they cannot be evaluated for patients who were negative before treatment, so they are not suitable for application. Therefore, in the face of the increasingly severe cancer diagnosis and treatment situation today, many experts and scholars are calling for the development of new detection indicators, especially in the field of early cancer screening.

[0003] Epigenetic modification is defined as heritable changes in gene activity that do not involve changes in the underlying DNA sequence. These modifications fine-tune the gene expression program through epigenetic factors and are the main molecular mechanisms controlling key biological processes such as cell differentiation and embryonic development. Moreover, strong evidence indicates that epigenetic reprogramming is the driving force behind the dynamic transcriptomic heterogeneity of cancer. Changes in DNA methylation can occur in the early stage of cancer, and DNA methylation is associated with a large number of abnormal changes in cancer cells. Hypermethylation in the gene promoter region is considered to have carcinogenic and prognostic effects. Therefore, DNA methylation analysis can effectively identify clinically significant tumor methylation markers. Previous studies have found that there are multiple genes in lung cancer, such as hypermethylation or hypomethylation of SHOX2, DAPK, HOXA7, RASSF1A, ITGA4, FHIT, GSTP1, which all have certain value in the diagnosis or prognosis evaluation of lung cancer.

[0004] The epidermal growth factor receptor (EGFR) plays a key role in cell growth, proliferation, and differentiation. When the EGFR gene mutates, it may lead to uncontrolled cell growth, thus triggering malignant tumors such as lung cancer. Detection of EGFR gene mutations not only helps to guide the treatment plan for lung cancer, but also can predict the response of lung cancer patients to certain treatment plans. In addition, detection of EGFR gene mutations can also predict the risk of recurrence and metastasis of lung cancer. Therefore, detection of EGFR gene mutations is of great significance for the diagnosis and treatment of lung cancer.

[0005] In recent years, the emergence of high-throughput technologies such as whole-genome bisulfite sequencing (WGBS) and methyl-sensitive restriction enzyme sequencing (MRE-Seq) has accelerated and expanded people's understanding of the epigenetic mechanisms of tumorigenesis, revealing a large number of potentially valuable cancer-specific epigenetic markers or features, which can be used for tumor diagnosis, prognosis prediction, or assessment of its response to treatment. However, high-throughput technologies are limited by problems such as high cost, long cycle, and large sample size required. Therefore, it is necessary to develop a new lung cancer kit to provide a richer, simpler, and cheaper multi-omics detection solution. Summary of the Invention

[0006] The object of the present invention is to provide a primer-probe combination, a kit, and its application for detecting the benign and malignant of pulmonary nodules to solve the problems existing in the above-mentioned prior art. The present invention provides a simple, fast, and accurate combined detection solution for DNA methylation and gene mutation to assist in differentiating the benign and malignant of pulmonary nodules. The primer-probe combination provided by the present invention can simultaneously detect the EGFR L858R mutation site, the EGFR 19Del mutation site, the methylation site of the HOXA7 gene, the methylation site of the CCNA1 gene, and the methylation site of the ZNF808 gene. Thus, the present invention provides a richer, simpler, and cheaper multi-omics detection solution.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a primer-probe combination for detecting the benign and malignant of pulmonary nodules, including primer pairs and probes for detecting gene mutation sites and methylation sites;

[0009] The gene mutation sites include the EGFR L858R site and the EGFR 19Del site; the methylation sites include the methylation site of the HOXA7 gene, the methylation site of the CCNA1 gene, and the methylation site of the ZNF808 gene;

[0010] The nucleotide sequences of the primer pair for detecting the EGFR L858R site are shown in SEQ ID NO.1-2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3;

[0011] The nucleotide sequences of the primer pairs for detecting the EGFR 19Del locus are shown in SEQ ID NO.4-5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6;

[0012] The nucleotide sequences of the primer pairs for detecting the methylation locus of the HOXA7 gene are shown in SEQ ID NO.10-11, and the nucleotide sequence of the probe is shown in SEQ ID NO.12;

[0013] The nucleotide sequences of the primer pairs for detecting the methylation locus of the CCNA1 gene are shown in SEQ ID NO.13-14, and the nucleotide sequence of the probe is shown in SEQ ID NO.15;

[0014] The nucleotide sequences of the primer pairs for detecting the methylation locus of the ZNF808 gene are shown in SEQ ID NO.16-17, and the nucleotide sequence of the probe is shown in SEQ ID NO.18.

[0015] Preferably, for the probes for detecting the EGFR L858R locus, the EGFR 19Del locus, the methylation locus of the HOXA7 gene, the methylation locus of the CCNA1 gene, and the methylation locus of the ZNF808 gene, the 5' ends are all labeled with a fluorescent group and the 3' ends are all labeled with a quenching group.

[0016] Preferably, the fluorescent group is one of FAM, VIC, HEX, ROX, and CY5; the quenching group is one of MGB, Super Quencher 1, BHQ1, BHQ2, and BHQ3.

[0017] Preferably, the 12th to 15th bases of the probe for detecting the EGFR L858R locus are a random mismatch sequence; the 11th to 14th bases of the probe for detecting the EGFR 19Del locus are a random mismatch sequence.

[0018] The present invention provides the application of the above primer-probe combination in the preparation of a kit for differentiating the benign and malignant nature of pulmonary nodules.

[0019] The present invention provides a kit for differentiating the benign and malignant nature of pulmonary nodules, and the kit includes the above primer-probe combination.

[0020] The present invention provides the application of the above primer-probe combination in the preparation of a kit for early screening of lung cancer.

[0021] The present invention provides a kit for early screening of lung cancer, and the kit includes the above primer-probe combination.

[0022] The present invention provides the application of the above primer-probe combination in constructing a lung cancer risk assessment model.

[0023] The present invention provides a lung cancer risk assessment model. Using the above primer-probe combination to perform fluorescence amplification detection on a test sample, the melting curve Rm of the EGFR L858R gene mutation, the melting curve Rm of the EGFR 19Del gene mutation, the Ct value of the methylation site of the HOXA7 gene, the Ct value of the methylation site of the CCNA1 gene, and the Ct value of the methylation site of the ZNF808 gene are obtained;

[0024] Taking the melting curve Rm of the EGFR L858R gene mutation, the melting curve Rm of the EGFR 19Del gene mutation, the Ct value of the methylation site of the HOXA7 gene, the Ct value of the methylation site of the CCNA1 gene, and the Ct value of the methylation site of the ZNF808 gene as input variables, a lung cancer risk assessment model is constructed; the lung cancer risk assessment model calculates the risk value P according to the following equation:

[0025] P = -0.146 * the melting curve Rm of the EGFR L858R gene mutation + 0.145 * the melting curve Rm of the EGFR 19Del gene mutation - 0.359 * the Ct value of the methylation site of the CCNA1 gene - 0.466 * the Ct value of the methylation site of the HOXA7 gene - 0.306 * the Ct value of the methylation site of the ZNF808 gene + 48.037;

[0026] When P > -0.64, the sample test result is positive; when P ≤ -0.64, the sample test result is negative.

[0027] The present invention discloses the following technical effects:

[0028] The present invention provides a primer-probe combination for detecting the benign and malignant nature of lung nodules. Based on the self-developed J-STAR-2M multi-color amplification melting curve method technology, under the condition of the same amplification program, using Taq DNA polymerase and the primer-probe combination, compatible detection of gene mutations and gene methylation sites can be achieved.

[0029] This primer-probe combination includes primer pairs and probes for detecting gene mutation sites and methylation sites, and can achieve simultaneous detection of the EGFR L858R mutation site, the EGFR 19Del mutation site, and the methylation sites of the HOXA7, CCNA1, and ZNF808 genes in one detection. It can be seen that the present invention is a simple, fast, and accurate combined detection scheme for DNA methylation and gene mutations to assist in differentiating the benign and malignant nature of lung nodules; at the same time, this primer-probe combination can achieve rapid pre-cancer screening, and also has application values such as precise drug guidance and prognosis and metastasis risk prediction.

[0030] The present invention also provides a kit, which contains the above primer-probe combination and is divided into two reaction tubes according to the gene mutation site and gene methylation site, namely reaction tube 1 and reaction tube 2. Among them, reaction tube 1 is a detection tube for EGFRL858R mutation site and EGFR 19Del mutation site, and reaction tube 2 is a detection tube for methylation of HOXA7, CCNA1 and ZNF808 genes. The present invention adopts the freeze-drying technology to freeze-dry substances such as enzymes, primers, probes, enzyme protectants, monovalent and divalent cation compounds, and buffers in reaction tube 1 and reaction tube 2. After freeze-drying, they are sealed in a vacuum light-shielding bag, which can achieve room-temperature transportation and stable storage at room temperature.

[0031] The probe in reaction tube 1 of the kit provided by the present invention is a blocking modified fluorescent probe. By setting random bases and hairpin structures at the 5' end of the probe, in the absence of mismatch, its binding stability with the DNA template is stronger than that of the nucleotide probe with the DNA template; in the case of mismatch, the binding stability of the blocking modified fluorescent probe with the DNA template is weaker than that of the nucleotide probe with the DNA template. Therefore, for a single-base mismatch, the blocking modified fluorescent probe has a larger melting temperature difference compared to the nucleotide probe, thereby achieving the inhibition of wild-type amplification signals and improving the detection sensitivity and specificity of mutants under the condition of a high proportion of wild-type background. The probe in reaction tube 2 of the kit of the present invention is a short-sequence peptide nucleic acid modified probe. By designing peptide nucleic acid modification at the methylation site, when methylation exists at the peptide nucleic acid modification site, the binding affinity of the peptide nucleic acid modified probe with the DNA template is stronger than that of the ordinary Taqman probe, thereby improving the detection sensitivity of the methylation site. In addition, the peptide nucleic acid modification can increase the melting temperature of the probe, enable the shortening of the probe sequence, and further enhance the specificity of the probe.

[0032] Moreover, the experimental results of the present invention prove that the kit provided by the present invention can effectively distinguish patients with malignant lung nodules from subjects with benign lung nodules, and achieve the detection of the benign and malignant nature of lung nodules with low invasiveness, low cost, high sensitivity and high specificity. The total sensitivity for detecting blood samples of malignant lung nodules can reach 89.61%, and the specificity for detecting blood samples of benign lung nodules can reach 95.30%. It can reduce the detection of false negative results and has high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0034] Figure 1The freeze-dried effect diagrams of reaction tube 1 and reaction tube 2 in the kit provided by the present invention. Among them, A is the in-situ freeze-drying example diagram of reaction tube 1; B is the in-situ freeze-drying example diagram of reaction tube 2;

[0035] Figure 2 The results diagrams of positive and negative controls; among them, A is the detection result of the amplification positive control of reaction tube 1, 1 is the positive peak diagram of EGFR19Del mutation, 2 is the positive peak diagram of EGFR L858R mutation, 3 is the melting curve peak of the internal reference GAPDH gene; B is the detection result of the amplification negative control of reaction tube 1, 4 is the melting curve peak of the internal reference GAPDH gene, the red line is the Cy5 channel, and the curve of this channel is straight without a melting curve peak, indicating that the EGFR 19Del mutation is negative. The blue line is the FAM channel, and the curve of this channel is straight without a melting curve peak, indicating that the EGFR L858R mutation is negative; C is the detection result of the amplification positive control of reaction tube 2, a is the amplification curve of the methylation site of the ZNF808 gene, b is the amplification curve of the methylation site of the HOXA7 gene, c is the amplification curve of the internal reference ACTB gene site, d is the amplification curve of the methylation site of the CCNA1 gene, and the red horizontal line is the threshold line automatically analyzed by the instrument; D is the detection result of the amplification negative control of reaction tube 2, e is the amplification curve of the internal reference ACTB gene site, the red horizontal line is the threshold line automatically analyzed by the instrument, the blue horizontal line is the FAM channel, and the curve of this channel is straight without an amplification curve, indicating that the methylation of the HOXA7 gene site is negative. The yellow line is the ROX channel, and the curve of this channel is straight without an amplification curve, indicating that the methylation of the ZNF808 gene site is negative;

[0036] Figure 3 The detection result of lung cancer patient sample 1 (p = 2.312); among them, A is the amplification result of reaction tube 1, 1 is the positive peak diagram of EGFRL858R mutation, 2 is the melting curve peak of the internal reference GAPDH gene, and the red horizontal line is the Cy5 channel, and the curve of this channel is straight without a melting curve peak, indicating that the EGFR 19Del mutation is negative; B is the amplification result of reaction tube 2, a is the amplification curve of the methylation site of the HOXA7 gene; b is the amplification curve of the internal reference ACTB; c is the amplification curve of the methylation site of the CCNA1 gene, d is the ROX channel, and the curve of this channel is straight without an amplification curve, indicating that the methylation of the ZNF808 gene site is negative. The orange horizontal line is the ROX channel, and the curve of this channel is straight without an amplification curve, indicating that the methylation of the ZNF808 gene site is negative;

[0037] Figure 4Detection result of sample 2 for lung cancer patients (p = 4.3056); among them, A is the amplification result of reaction tube 1, 1 is the positive peak of EGFR19Del mutation, 2 is the melting curve peak of the internal reference GAPDH gene, the blue line is the ROX channel, and the curve of this channel is straight without an amplification curve, indicating negative methylation at the ZNF808 gene locus; B is the amplification result of reaction tube 2, a is the amplification curve of the internal reference ACTB, b is the amplification curve of the methylated site of the ZNF808 gene, c is the amplification curve of the methylated site of the CCNA1 gene, d is the FAM channel, and the curve of this channel is straight without an amplification curve, indicating negative methylation at the HOXA1 gene locus. The orange horizontal line is the threshold line automatically analyzed by the instrument.

[0038] Figure 5 It is the clinical ROC curve of the kit. Among them, A is the comparison of the ROC curves of 226 clinical samples with mutations at the EGFR 19Del and EGFR L858R loci and methylated sites of the CCNA1, HOXA7, and ZNF808 genes, and B is the ROC curve of the risk p value of malignant lung nodules (AUC = 0.953).

[0039] Figures 6 - 12 It is the evaluation of the detection limit of the kit. Among them, Figure 6 It is the positive peak map of the EGFR L858R mutation gradient detection. 1 is 1% MT, 2 is 0.5% MT, 3 is 0.1% MT, and 4 is 0.05% MT. Figure 7 It is the positive peak map of the EGFR 19Del mutation gradient detection. 1 is 1% MT, 2 is 0.5% MT, 3 is 0.1% MT, and 4 is 0.05% MT. Figure 8 It is the melting curve peak of the internal reference GAPDH gene. Figure 9 It is the gradient amplification curve of the methylated site of the HOXA7 gene. 1 is 5% methylation ratio, 2 is 1% methylation ratio, 3 is 0.5% methylation ratio, and 4 is 0.1% methylation ratio. Figure 10 It is the gradient amplification curve of the methylated site of the CCNA1 gene. 1 is 5% methylation ratio, 2 is 1% methylation ratio, 3 is 0.5% methylation ratio, and 4 is 0.1% methylation ratio. Figure 11 It is the gradient amplification curve of the methylated site of the ZNF808 gene. 1 is 5% methylation ratio, 2 is 1% methylation ratio, 3 is 0.5% methylation ratio, and 4 is 0.1% methylation ratio. Figure 12 It is the amplification curve of the internal reference ACTB. Detailed implementation manners

[0040] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0041] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0044] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0045] Example 1 Screening of Detection Targets

[0046] According to the databases of the National Center for Biotechnology Information (NCBI), TCGA, and MEXPRESS in the United States, 20 methylation regions of 10 genes with significant differences between lung cancer tissues and benign lung nodules were screened (see Table 1), and primers and probes were designed respectively for clinical sample detection. Among them, there were 21 malignant tissue samples of lung nodules and 32 benign tissue samples of lung nodules. According to the clinical test results, after statistical analysis, three methylation regions with higher AUC were preferably selected (see Table 2), namely cg03232620 (HOXA7), cg13711394 (CCNA1), and cg11224334 (ZNF808) as the methylation detection targets of the present invention.

[0047] Table 1 Candidate Methylation Sites

[0048]

[0049] Table 2 Preferred Detection Sites of Gene Methylation in Malignant Lung Nodules

[0050]

[0051]

[0052] Note: “+” represents the sense strand of DNA; “-” represents the antisense strand of DNA.

[0053] The detection sites of mutant genes selected in the present invention are EGFR L858R and EGFR 19Del. The specific information of mutant gene sites is shown in Table 3.

[0054] Table 3 Detection gene mutation sites of malignant lung nodules

[0055]

[0056] Example 2 Design of primers

[0057] Primers and probes were designed according to the detection targets screened in Example 1. For the blocking modified fluorescent probe involved in this example, that is, the original oligonucleotide fluorescent probe was introduced with random mismatch sequences, and the probe was modified by any one or more of thiolation modification, deoxyuracil modification, deoxyinosine modification, 2-methoxy modification, and phosphorylation modification. Its 5′ end was labeled with a fluorescent group, and its 3′ end was labeled with a quenching group. The fluorescent group was one of FAM, VIC, HEX, ROX, CY5 or other fluorescent groups with luminescence characteristics, and the quenching group was one of MGB, Super Quencher 1, BHQ1, BHQ2, BHQ3 or other chemical groups with fluorescence quenching function. For the short sequence peptide nucleic acid modified probe involved in this example, peptide nucleic acid modification was designed at the corresponding positions of the bases covering the methylation sites to improve the probe affinity. Its 5′ end was labeled with a fluorescent group, and its 3′ end was labeled with a quenching group. The fluorescent group was one of FAM, VIC, HEX, ROX, CY5 or other fluorescent groups with luminescence characteristics, and the quenching group was MGB. The primer and probe sequences are shown in Table 4. The primer probes were synthesized by Shanghai Langjing Biotechnology Co., Ltd., as shown in Table 4 specifically.

[0058] Table 4 Primer and probe sequences

[0059]

[0060]

[0061] Note: In the primer name, "F" indicates the upstream primer, "R" indicates the downstream primer, and "P" indicates the probe; VIC indicates that the probe is fluorescently labeled with VIC; ROX indicates that the probe is fluorescently labeled with ROX; FAM indicates that the probe is fluorescently labeled with FAM; CY5 indicates that the probe is fluorescently labeled with CY5; ACTB and GAPDH are internal reference genes; the double-underlined bases are peptide nucleic acid-modified bases, and N is a randomly mismatched base.

[0062] Example 3 Development of the Kit

[0063] This example is based on the self-developed J-STAR-2M multi-color amplification melting curve method technology. Under the same amplification program conditions, it can achieve the compatibility detection of gene mutations and gene methylation sites.

[0064] This example provides a DNA mutation and methylation detection kit for the benign and malignant of pulmonary nodules, especially a freeze-dried DNA mutation and methylation detection kit for the benign and malignant of pulmonary nodules. According to different targets, it is divided into two reaction tubes. Reaction tube 1 is for the detection of EGFR L858R and EGFR 19Del mutation sites, and reaction tube 2 is for the methylation detection of HOXA7, CCNA1, and ZNF808 genes. The component reagents of reaction tube 1 and reaction tube 2 are shown in Table 5 and Table 6 respectively. Among them, the lyoprotectant can be one or a combination of several of ficoll, sucrose, trehalose, mannitol, betaine, potassium sorbate, polyvinylpyrrolidone, polyethylene glycol, agar, and gelatin, etc. The lyoprotectant can be 1%-15% trehalose, 0.1%-10% mannitol, 0.02%-5% polyvinylpyrrolidone. The preferred concentrations of the component reagents of reaction tube 1 and reaction tube 2 are shown in Table 7 and Table 8 respectively. At the same time, internal reference genes are used to indicate the quality of DNA extraction and bisulfite conversion. The mutation internal reference is the human ACTB gene, and the methylation internal reference is the human GAPDH gene. The nucleotide sequences of the primers and probes in this kit are shown in Table 4.

[0065] The present invention uses freeze-drying technology to lyophilize substances such as enzymes, primers, probes, enzyme protectants, monovalent and divalent cation compounds, and buffers in reaction tube 1 and reaction tube 2. After lyophilization, the freeze-dried effect diagrams of reaction tube 1 and reaction tube 2 are as Figure 1 shown, and then they are sealed in a vacuum light-proof bag, which can achieve room-temperature transportation and room-temperature stable storage. The freeze-drying procedure is shown in Table 9.

[0066] Table 5 Component Reagents of Reaction Tube 1 of the DNA Mutation and Methylation Kit for the Benign and Malignant of Pulmonary Nodules

[0067]

[0068]

[0069] Table 6 DNA mutations and methylation kit reaction tube 2-component reagents for benign and malignant pulmonary nodules

[0070] Component Preferred concentration range / volume percentage Tris-HCl 10 - 80 mM dNTPs 0.1 - 1 mM Glycerol-free Taq DNA polymerase 0.05 U / μL - 0.25 U / μL UNG / UDG enzyme 0.025 U / μL - 0.125 U / μL Lyoprotectant 1% - 30% v / v <![CDATA[MgCl2]]> 1.5 - 1 mM KCl 10 - 100 mM HOF 0.05 - 1 μM HOR 0.05 - 1 μM HOP 0.05 - 1 μM CCF 0.05 - 1 μM CCR 0.05 - 1 μM CCP 0.05 - 1 μM ZNF 0.05 - 1 μM ZNR 0.05 - 1 μM ZNP 0.05 - 1 μM ACF 0.05 - 1 μM ACR 0.05 - 1 μM ACP 0.05 - 1 μM

[0071] Table 7 DNA mutations and methylation kit reaction tube 1-component reagents for benign and malignant pulmonary nodules

[0072] Component Preferred concentration / volume percentage Tris-HCl 45 mM dNTPs 0.2 mM Glycerol-free Taq DNA polymerase 0.25 U / μL Mannitol 0.6% m / v Trehalose 10% m / v Polyvinylpyrrolidone 0.05% m / v UNG / UDG enzyme 0.1 U / μL <![CDATA[MgCl2]]> 2.5 mM KCl 50 mM 858F 0.05 μM 858R 0.4 μM 858P 0.2 μM 19F 0.08 μM 19R 0.48 μM 19P 0.24 μM GAF 0.1 μM GAR 0.3 μM GAP 0.15 μM Ultra-pure water Made up to 20 μL

[0073] Table 8 DNA mutations and methylation kit reaction tube 2-component reagents for benign and malignant pulmonary nodules

[0074] Component Preferred concentration / volume percentage Tris-HCl 45 mM dNTPs 0.2 mM Glycerol-free Taq DNA polymerase 0.25 U / μL Mannitol 0.6% m / v Trehalose 10% m / v Polyvinylpyrrolidone 0.05% m / v UNG / UDG enzyme 0.1 U / μL <![CDATA[MgCl2]]> 2.5 mM KCl 50 mM HOF 0.3 μM HOR 0.3 μM HOP 0.2 μM CCF 0.2 μM CCR 0.2 μM CCP 0.15 μM ZNF 0.25 μM ZNR 0.25 μM ZNP 0.15 μM ACF 0.3 μM ACR 0.3 μM ACP 0.15 μM Ultra-pure water Made up to 20 μL

[0075] Table 9 Freeze-drying procedure

[0076] Temperature Time Pressure -65℃ 2.5h 0.1 MPa -60℃ 5h 0.25 mbar -5℃ 2h 0.25 mbar 5℃ 1h 0.25 mbar 10℃ 1h 0.25 mbar 15℃ 1h 0.25 mbar 25℃ 3h 0.25 mbar

[0077] Example 4 Application of the kit developed in Example 3

[0078] The operating steps of this example are as follows: Extract the DNA of the subject's plasma sample, transform part of the DNA with a methylation conversion reagent, then purify the transformed DNA, and use the kit provided by the present invention to amplify the original DNA sample and the transformed DNA sample. By the J-STAR-2M multi-color amplification melting curve method technology, detect the methylation level and mutation situation of the target region in the sample, and then judge whether the plasma sample to be tested is a malignant pulmonary nodule.

[0079] 1. Collection of blood samples

[0080] A total of 75 blood samples of pulmonary nodules were collected, including blood samples of 43 subjects with pathologically confirmed malignant pulmonary nodules and blood samples of 32 subjects with benign pulmonary nodules. After centrifuging the collected blood samples at room temperature, the supernatant was taken to obtain plasma samples. The above 75 samples were used as test set samples.

[0081] 2. DNA extraction

[0082] The extraction, transformation and purification processes of the above blood samples are as follows:

[0083] 2.1 Extraction of DNA samples

[0084] Centrifuge 10 mL of whole blood samples at room temperature to obtain plasma samples. Use QIAamp Circulating Nucleic Acid Kit (50) (Cat. No. 55114) to extract plasma cfDNA. The plasma volume used is 4 - 5 mL. For specific operations, refer to the kit instructions. After extraction, elute with 40 μL of purified water. Use QubitTM Quantify the samples using the dsDNA Quantification Kit (Cat. No. Q33230).

[0085] 2.2 Bisulfite conversion and purification

[0086] Equally divide the extracted free DNA into two parts. Take one part for bisulfite conversion. The nucleic acid conversion kit used is the EZ DNA Methylation-Gold TM Kit (Cat. No. D5005). Refer to the kit instructions for the specific experimental operations. After the conversion is completed, elute with 10 μL of purified water. Use the Qubit TM ssDNA Quantification Kit (Cat. No. Q10212) to quantify the samples and store them at -20 °C for later use.

[0087] 2.3 PCR amplification

[0088] Prepare the reconstitution reaction solution according to Table 10. Mix 20 ng of untransformed plasma free DNA with ultrapure water to construct Reaction Tube 1 with the reconstitution reaction solution and add it to Reaction Tube 1; mix 20 ng of bisulfite-converted cfDNA with ultrapure water to construct Reaction Tube 2 with the reconstitution reaction solution and add it to Reaction Tube 2. Perform amplification detection according to the amplification program in Table 11.

[0089] Table 10 Preparation of the reconstitution reaction solution

[0090] Reconstitution reaction solution in Reaction Tube 1 Dosage Reconstitution reaction solution in Reaction Tube 2 Dosage CfDNA 20 ng CfDNA after bisulfite conversion 20 ng Ultra-pure water Made up to 20 μL with water Ultra-pure water Made up to 20 μL with water

[0091] Table 11 Reaction program *

[0092]

[0093] * Note: 1. Compatible instrument: Temperature control is carried out by the temperature control mode selection module; 2. Collect FAM / VIC / ROX / CY5 fluorescence signals during the third step of the PCR cycle at 50 °C and during the gradient heating in the melting curve analysis step.

[0094] After the reaction is completed, manually adjust the baseline and set an appropriate threshold. The baseline is usually the fluorescence signals of 3 - 15 cycles. Eliminate the samples with unsuccessful detections according to the requirements of quality control, and read the Ct values of the qualified samples. The quality control standards are shown in Table 12. If all samples meet the requirements, the results of the samples to be tested can be analyzed.

[0095] Table 12 Quality control of amplification data *

[0096]

[0097]

[0098] *Note: The above conditions must all be met in the same experiment; otherwise, the results of this experiment are invalid.

[0099] 3. Results

[0100] A total of 75 clinical samples were detected in the above experiment. The positive rates of EGFR L858R and EGFR 19Del gene mutations, and the positive rates of HOXA7, CCNA1, and ZNF808 methylation in clinically diagnosed benign and malignant pulmonary nodules are shown in Table 13.

[0101] Table 13 Positive rates of each locus of the test set samples

[0102]

[0103] For the test results of the above 75 clinical samples, the melting curve Rm of EGFR L858R and EGFR 19Del gene mutations, and the Ct values of HOXA7, CCNA1, and ZNF808 methylation sites were subjected to logistic regression with the clinical pathological diagnosis results to construct the positive prediction risk values for liver cancer of gene mutations and gene methylation, that is, the P value, P = -0.146 * EGFR L858R Rm value + 0.145 * EGFR19Del Rm value - 0.359 * CCNA1 Ct value - 0.466 * HOXA7 Ct value - 0.306 * ZNF808 Ct value + 48.037.

[0104] The risk values of the above sample test results were converted. Using MedCalc software, the melting curve Rm of EGFR L858R and EGFR19Del gene mutations, and the Ct values of HOXA7, CCNA1, and ZNF808 methylation sites, combined with the clinical pathological results, were used to construct an ROC curve and determine the optimal cut-off risk value. Specifically: The melting curve Rm of EGFR L858R and EGFR 19Del gene mutations, and the Ct values of HOXA7, CCNA1, and ZNF808 methylation sites were converted into one-dimensional P values according to the formula P = -0.146 * EGFR L858R Rm value + 0.145 * EGFR 19Del Rm value - 0.359 * CCNA1 Ct value - 0.466 * HOXA7 Ct value - 0.306 * ZNF808 Ct value + 48.037, and then the ROC curve analysis was performed with the P value and the clinical diagnosis results to determine the optimal Cutoff value, which is the threshold for determining whether the sample is positive. When the p value > -0.64, the sample test result is positive; when the p value ≤ -0.64, the sample test result is negative.

[0105] Example 5 Application of the kit developed in Example 3

[0106] 1. Sample collection

[0107] A total of 226 blood samples of pulmonary nodules were collected from regional hospitals in Tai'an, Nantong, Bengbu and other places. Among them, 77 cases were pathologically diagnosed as malignant nodules, and 149 cases were pathologically diagnosed as benign nodules. After the above samples were processed by blinding, they were used as verification set samples. This study was reviewed and met the development standards by the Ethics Committee of our hospital, and all patients signed informed consent documents.

[0108] 2. DNA sample extraction

[0109] Centrifuge 10 mL of whole blood samples at room temperature to obtain plasma samples. Use QIAamp Circulating Nucleic Acid Kit (50) (Cat. No. 55114) to extract plasma cfDNA. The plasma volume used is 4 - 5 mL. For specific operations, refer to the kit instructions. After extraction, elute with 40 μL of purified water. Use Qubit TM dsDNA Quantification Kit (Cat. No. Q33230) for sample quantification.

[0110] 3. Bisulfite conversion and purification treatment

[0111] Equally divide the extracted free DNA into two parts. Take one part for bisulfite conversion. The nucleic acid conversion kit used is EZ DNA Methylation - Gold TM Kit (Cat. No. D5005). For specific experimental operations, refer to the kit instructions. After conversion, elute with 10 μL of purified water. Use Qubit TM ssDNA Quantification Kit (Cat. No. Q10212) for sample quantification, and store it at -20 °C for later use.

[0112] 4. Amplification analysis

[0113] Mix about 20 ng of unconverted plasma free DNA with ultrapure water to 20 μL, and then add it to the in - situ lyophilized reaction tube 1 containing EGFRL858R and EGFR 19Del primer probes; mix 20 ng of bisulfite - converted free DNA with ultrapure water to 20 μL, and add it to the in - situ lyophilized reaction tube 2 containing HOXA7, CCNA1 and ZNF808 methylation site detection primer probes. Tighten the tube caps and move them to the detection area. Refer to the amplification reaction procedure in Table 11 for fluorescence amplification analysis.

[0114] A total of 226 clinical samples were detected in the above experiment. The EGFR L858R and EGFR 19Del gene mutations were positive, and the methylation of HOXA7, CCNA1, and ZNF808 was positive. The proportions in clinically diagnosed benign and malignant lung nodules are shown in Table 14. The optimal Cut off values for lung-related gene mutations and methylation sites are shown in Table 15. The receiver operating characteristic curve is shown in Figure 5 .

[0115] Table 14 Detection sensitivity and specificity of samples in the validation set

[0116]

[0117] Table 15 Optimal Cut off values for lung-related gene mutations and methylation sites

[0118] Detection site Optimal Cutoff value EGFRL858R ≥4.73 EGFR19Del ≥4.05 HOXA7 ≤38.14 CCNA1 ≤39.45 ZNF808 ≤37.77

[0119] As can be seen from Table 14, Table 15, and Figure 5 it can be seen that the AUC of the receiver operating curve for the risk p value of malignant lung nodules is 0.953. The sensitivity of this kit is 89.61% (95% CI: 80.82% - 94.64%), the specificity is 95.30% (95% CI: 90.62% - 97.71%), and the total coincidence rate is 93.36% (95% CI: 89.34% - 95.94%).

[0120] Example 6 Case presentation of Example 5

[0121] Two lung cancer blood samples (Sample 1 and Sample 2) in the samples received in Example 5 were presented, and the results are as Figures 3 - 4 shown. In Sample 1, the EGFR L858R mutation was positive, and the methylation sites of the HOXA7 gene and the CCNA1 gene were positive; both the EGFR L858R site mutation and the ZNF808 gene site methylation were negative. In Sample 2, the EGFR 19Del site mutation was positive, and the methylation of the ZNF808 gene and the CCNA1 gene site were positive; both the EGFR L858R site mutation and the HOX1A gene site methylation were negative.

[0122] At the same time, the melting curve Rm values of EGFR L858R and EGFR 19Del gene mutations, and the Ct values of the methylation sites of HOXA7, CCNA1, and ZNF808 were substituted into the formula: P = -0.146 * EGFR L858R Rm value + 0.145 * EGFR 19Del Rm value - 0.359 * CCNA1 Ct value - 0.466 * HOXA7 Ct value - 0.306 * ZNF808 Ct value + 48.037. The p value obtained for sample 1 was 2.312, and the p value for sample 2 was 4.3056, indicating that the corresponding patients of sample 1 and sample 2 were both lung cancer patients, which was consistent with the clinical diagnosis results.

[0123] Example 7 Determination of Detection Limit

[0124] 1. Extraction of cell line gDNA:

[0125] Using a Blood / Cell / Tissue Genomic DNA Extraction Kit (Tiangen Biochemical, DP304), the gDNA of the lung cancer NCI-1975 cell line and the gDNA of the HFF-1 cell line, which is negative for lung cancer and negative for EGFR L858R and EGFR 19Del mutations, were extracted respectively. Qubit TM dsDNA Quantification Kit (Cat.No.Q33230) was used for sample quantification.

[0126] 2. Bisulfite conversion:

[0127] 500 ng of the extracted gDNA of the HFF-1 cell line and the NCI-H1975 cell line were taken respectively, and a Bisulfite Conversion Kit (EZ DNA Methylation Kit, ZYMO) was used for bisulfite conversion. After purification and recovery, Qubit TM ssDNA Quantification Kit (Cat.No.Q10212) was used for sample quantification.

[0128] 3. Preparation of detection limit reference products:

[0129] EGFR L858R gDNA standard (CBP10408, Cobioer), EGFR 19Del DNA standard (CBP10334, Cobioer), and gDNA of the HFF-1 cell line were taken respectively. The above gDNA was diluted to 10 ng / μL using TE. Then, using 10 ng / μL of the gDNA of the HFF-1 cell line, the above EGFR L858R gDNA standard and EGFR 19Del DNA standard were diluted to 10 ng / μL 1% MT, 10 ng / μL 0.5% MT, 10 ng / μL 0.1% MT, and 10 ng / μL 0.05% MT respectively.

[0130] Take the bisulfite-converted NCI-1975 gDNA and HFF-1 gDNA, and dilute them to 10 ng / μL respectively using TE. Then, using the 10 ng / μL HFF-1 gDNA after bisulfite conversion, dilute the 100% methylated bisulfite-converted NCI-1975 gDNA to 10 ng / μL 5%, 10 ng / μL 1%, 10 ng / μL 0.5%, and 10 ng / μL 0.1%.

[0131] 4. Amplification analysis:

[0132] Refer to the amplification system setting scheme in Table 10 below to configure the amplification reaction solution, and refer to the amplification reaction procedure in Table 11 to perform fluorescence amplification analysis. The amplification results are shown as Figure 6 shown.

[0133] Under the condition of 20 ng / reaction addition amount, the kit of the present invention is repeated 10 times to detect the EGFR L858R site with 0.1% MT, and 100% detection can be achieved (Rm≥4.73). When detecting the EGFR L858R site with 0.05% MT, 80% detection can be achieved. Therefore, the detection sensitivity of the EGFR L858R site of the kit of the present invention is 0.1%. Under the condition of 20 ng / reaction addition amount, the kit of the present invention is repeated 10 times to detect the EGFR 19Del site with 0.1% MT, and 100% detection can be achieved (Rm≥4.05). When detecting the EGFR 19Del site with 0.05% MT, 70% detection can be achieved. Therefore, the detection sensitivity of the EGFR 19Del site of the kit of the present invention is 0.1%.

[0134] Under the condition of 20 ng / reaction addition amount, the kit of the present invention is repeated 10 times to detect the bisulfite-converted NCI-1975 gDNA with a ratio of 0.5%. The gene loci of HOXA7, CCNA1, and ZNF808 can all be detected normally. When detecting the bisulfite-converted NCI-1975 gDNA with a ratio of 0.1%, the methylation site of the HOXA7 gene can achieve 60% detection (Ct≤38.14), the methylation site of the CCNA1 gene can achieve 80% detection (Ct≤39.45), and the methylation site of the ZNF808 gene can achieve 40% detection (Ct≤37.77). Therefore, the sensitivity of the above methylation sites is 0.5% methylation ratio under the condition of 20 ng.

[0135] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer-probe combination for detecting the benignity and malignancy of pulmonary nodules, characterized in that, Primer pairs and probes for detecting gene mutation sites and methylation sites The gene mutation sites include the EGFR L858R site and the EGFR 19Del site; the methylation sites include the HOXA7 gene methylation site, the CCNA1 gene methylation site, and the ZNF808 gene methylation site The nucleotide sequences of the primer pair for detecting the EGFR L858R site are shown in SEQ ID NO.1-2, and the nucleotide sequence of the probe is shown in SEQ ID NO.3 The nucleotide sequences of the primer pair for detecting the EGFR 19Del site are shown in SEQ ID NO.4-5, and the nucleotide sequence of the probe is shown in SEQ ID NO.6 The nucleotide sequences of the primer pair for detecting the HOXA7 gene methylation site are shown in SEQ ID NO.10-11, and the nucleotide sequence of the probe is shown in SEQ ID NO.12 The nucleotide sequences of the primer pair for detecting the CCNA1 gene methylation site are shown in SEQ ID NO.13-14, and the nucleotide sequence of the probe is shown in SEQ ID NO.15 The nucleotide sequences of the primer pair for detecting the ZNF808 gene methylation site are shown in SEQ ID NO.16-17, and the nucleotide sequence of the probe is shown in SEQ ID NO.18 2. The primer-probe combination according to claim 1, wherein For the probes for detecting the EGFR L858R site, the EGFR 19Del site, the HOXA7 gene methylation site, the CCNA1 gene methylation site, and the ZNF808 gene methylation site, the 5' ends are all labeled with a fluorescent group, and the 3' ends are all labeled with a quenching group 3. The primer-probe combination according to claim 2, wherein The fluorescent group is one of FAM, VIC, HEX, ROX, and CY5; the quenching group is one of MGB, Super Quencher 1, BHQ1, BHQ2, and BHQ3 4. The primer-probe combination according to claim 1, wherein The 12th to 15th bases of the probe for detecting the EGFR L858R site are random mismatch sequences; the 11th to 14th bases of the probe for detecting the EGFR 19Del site are random mismatch sequences 5. Use of the primer-probe combination according to any one of claims 1-4 in the preparation of a kit for differentiating the benign and malignant of lung nodules 6. A kit for identifying the benign and malignant nature of pulmonary nodules, characterized in that, The kit includes the primer-probe combination according to any one of claims 1-4 7. Use of the primer-probe combination according to any one of claims 1-4 in the preparation of a kit for early screening of lung cancer 8. Use of the primer-probe combination according to any one of claims 1-4 in the construction of a lung cancer risk assessment model

Images