Kit for diagnosing non-small cell lung cancer as well as use method and application thereof

Abstract

The invention relates to a kit for diagnosing non-small cell lung cancer as well as a use method and application thereof, and belongs to the field of biological medicines. The kit comprises reagents required by RNA extraction and reverse transcription, and a PCR amplification system containing primers and probes for respectively and specifically targeting SFTPA1 and SFTPA2 genes, wherein the PCR amplification system comprises primers and probes for respectively and specifically targeting SFTPA1 and SFTPA2 genes; the 5'end of the probe is provided with a fluorophore, and the 3 'end of the probe is provided with a quenching agent; the probe and the primer are designed based on specific regions of SFTPA1 and SFTPA2 genes, so that high specificity and high sensitivity of the probe and the primer in gene expression detection are ensured. The use method of the kit comprises the following steps: extracting RNA (Ribonucleic Acid) in a detection sample, reversely transcribing the RNA into cDNA (Complementary Deoxyribose Nucleic Acid), detecting the expression levels of the SFTPA1 and SFTPA2 genes by utilizing a qPCR (Quantitative Polymerase Chain Reaction) technology or a dPCR technology, and finally, preliminarily diagnosing whether a detection object suffers from the non-small cell lung cancer or not by comparing the expression differences of the SFTPA1 and SFTPA2 genes of the detection object and healthy people and non-small cell lung cancer patients. The invention provides a reliable tool for early diagnosis and individualized treatment of the non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a kit for diagnosing non-small cell lung cancer, its use method and applications, specifically including a kit for distinguishing healthy people from non-small cell lung cancer patients by simultaneously detecting the differential expression of SFTPA1 gene and SFTPA2 gene, and its use method and applications. Background Art

[0002] Non-small cell lung cancer (NSCLC) is one of the cancers with a high mortality rate among patients globally, and its early diagnosis is crucial for the prognosis of patients. However, the current early diagnosis methods still have problems of insufficient sensitivity and specificity, resulting in many patients being in the advanced stage of the disease when diagnosed. The detection of gene markers has shown great potential in the early diagnosis of cancer, especially as a non-invasive method for detecting gene expression through blood, which can provide a simple and reliable approach for the early diagnosis of non-small cell lung cancer.

[0003] In the prior art, the diagnostic methods for non-small cell lung cancer (NSCLC) mainly rely on imaging examinations and tissue biopsies. However, with the development of molecular biology, methods based on detecting the expression levels of specific genes have gradually become effective means for early diagnosis. Some existing gene detection methods mainly focus on the expression analysis of a single or a few genes, such as the detection of mutant genes like EGFR, KRAS, ALK, etc. These genes play a crucial role in the molecular typing and targeted therapy of NSCLC. However, these methods often rely on the expression level of a single gene or require the extraction of RNA from tissue samples, with complex operations and high costs. At the same time, due to their specific expression in lung diseases, the SFTPA1 and SFTPA2 genes have gradually become a research hotspot. The SFTPA1 and SFTPA2 genes encode surfactant protein A1 (SP-A1) and surfactant protein A2 (SP-A2) respectively, which are important components of pulmonary surfactant. They are mainly expressed in type II alveolar epithelial cells and act together to maintain the stability of the surface tension on the alveolar surface, prevent alveoli from collapsing during exhalation, and ensure normal lung function. In addition, they play an important role in innate immunity, can recognize and bind to pathogens such as bacteria and viruses, promote the phagocytosis of macrophages, regulate the pulmonary inflammatory response, and enhance the anti-infection ability of the lungs. Studies have shown that the abnormal expression of the SFTPA1 and SFTPA2 genes is closely related to lung diseases such as pneumonia and acute respiratory distress syndrome (ARDS). As important molecular markers for the early diagnosis of some lung diseases, the combined detection of SFTPA1 and SFTPA2 helps to distinguish normal populations from diseased populations and provides a scientific basis for the early diagnosis and individualized treatment of diseases. However, the current detection of the expression of the SFTPA1 and SFTPA2 genes mainly stays at the protein level, and there is still a lack of a mature method for detecting the expression of the SFTPA1 and SFTPA2 genes at the RNA level, especially a method for the early diagnosis of non-small cell lung cancer through blood samples. Summary of the Invention

[0004] To solve the deficiencies in the prior art, the present invention provides a method for distinguishing healthy populations from non-small cell lung cancer patients by detecting the expression levels of the SFTPA1 gene and the SFTPA2 gene in blood samples, and discloses the corresponding kit and its preparation method. The kit of the present invention has the advantages of simple operation, high sensitivity, strong specificity, etc., and is suitable for the early diagnosis of non-small cell lung cancer.

[0005] Specifically, the present invention relates to a kit for diagnosing non-small cell lung cancer, its usage method and uses, belonging to the field of biological medicine. The kit includes reagents required for RNA extraction and reverse transcription, and a PCR amplification system containing primers and probes specifically targeting the SFTPA1 and SFTPA2 genes respectively; the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher; the probe and primers are designed based on the specific regions of the SFTPA1 and SFTPA2 genes to ensure high specificity and high sensitivity in gene expression detection. The usage steps of the kit are as follows: extract RNA from the test sample and reverse transcribe it into cDNA, then use qPCR technology or dPCR technology to detect the expression levels of the SFTPA1 and SFTPA2 genes, and finally, by comparing the expression differences of the SFTPA1 and SFTPA2 genes between the test subject and healthy people and non-small cell lung cancer patients, preliminarily diagnose whether the test subject has non-small cell lung cancer. The present invention provides a reliable tool for the early diagnosis and individualized treatment of non-small cell lung cancer

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] On the one hand, the present invention provides the use of a biomolecular marker composition for preparing a reagent for judging whether a subject has non-small cell lung cancer. The biomolecular marker composition includes the SFTPA1 and SFTPA2 genes, and the SFTPA1 gene and SFTPA2 gene respectively contain the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing

[0008] On the other hand, the present invention provides the use of a biomolecular marker composition for preparing a reagent for evaluating the treatment effect of non-small cell lung cancer. The primer or / and probe specifically targets the SFTPA1 or SFTPA2 gene, and the SFTPA1 gene and SFTPA2 gene respectively contain the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing

[0009] On the other hand, the present invention provides the use of a primer or / and probe for preparing a reagent for judging whether a subject has non-small cell lung cancer. The primer or / and probe specifically targets the SFTPA1 or SFTPA2 gene, and the SFTPA1 gene and SFTPA2 gene respectively contain the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing

[0010] On the other hand, the present invention provides a kit for diagnosing non-small cell lung cancer, which kit comprises primers and / or probes specifically targeting the SFTPA1 or SFTPA2 gene, and the SFTPA1 gene and the SFTPA2 gene respectively have the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing.

[0011] Further, the probe targeting the SFTPA1 gene comprises the sequences shown in SEQ ID NO:3-4 in the sequence listing; the primer targeting the SFTPA1 gene comprises the sequences shown in SEQ ID NO:7-8 in the sequence listing;

[0012] Further, the probe targeting the SFTPA2 gene comprises the sequences shown in SEQ ID NO:5-6 in the sequence listing; the primer targeting the SFTPA2 gene comprises the sequences shown in SEQ ID NO:9-10 in the sequence listing.

[0013] In some specific embodiments, the kit comprises probes (SEQ ID NO:3-6) and primers (SEQ ID NO:7-10) specifically targeting the SFTPA1 and SFTPA2 genes respectively.

[0014] Further, the kit further comprises DNA polymerase, dNTPs, MgCl2, reaction buffer; the kit further comprises reagents required for RNA extraction and reverse transcription, and the reagents required for RNA extraction include lysis solution, washing solution, elution solution, etc.; the reagents required for reverse transcription include reverse transcriptase, reverse transcription buffer and dNTPs.

[0015] Further, a method for preparing a kit for early diagnosis of non-small cell lung cancer, the method comprising the following steps:

[0016] (1) According to the specific sequences of the SFTPA1 and SFTPA2 genes, use bioinformatics tools to analyze and design probe sequences (SEQ ID NO:3-SEQ ID NO:6) to ensure specific binding of the probes to the target genes; the length of each probe is controlled between 18 and 25 base pairs to balance binding specificity and amplification efficiency; the melting temperature (Tm) of the probes is optimized between 50°C and 65°C through calculation and experimental verification; synthesize the designed oligonucleotide probes and label the 5' end with a fluorescent group (FAM) and the 3' end with a quencher (TAMRA) to achieve detection of fluorescence signals.

[0017] (2) Design specific primers (SEQ ID NO:7 - SEQ ID NO:10) for amplifying the target sequence based on the sequences of SFTPA1 and SFTPA2 genes. It should be noted that when designing the primers, the binding regions of the probes need to be considered to ensure that the amplified products can be effectively recognized by the probes.

[0018] (3) Mix the synthesized probes with the corresponding primers, Taq DNA polymerase, dNTPs, magnesium ion buffer and other components of the reaction system according to the optimized concentration ratio to form an amplification reaction system.

[0019] (4) Aliquot the optimized amplification reaction system into independent vials or test tubes according to the amount required for a single reaction, and attach standard operating instructions. The kit also contains reagents for RNA extraction from blood samples and reverse transcription, ensuring that users can easily obtain high-quality cDNA from the samples for subsequent detection.

[0020] On the other hand, the present invention provides a system for diagnosing early non-small cell lung cancer. The system includes a data analysis module. The data analysis module is used to analyze the detection values of the biomolecular marker composition in the test sample. The biomolecular marker composition includes SFTPA1 and SFTPA2 genes, and the SFTPA1 gene and SFTPA2 gene respectively contain the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 in the sequence listing.

[0021] Furthermore, the data analysis module compares the detection values of the biomolecular marker composition in the test sample with normal values and / or abnormal values.

[0022] In some specific embodiments, when the detection value of the biomolecular marker composition ≤ 0.40903, it is diagnosed that the test subject does not have non-small cell lung cancer; or when the detection value of the biomolecular marker composition ≥ 0.40903, it is diagnosed that the test subject has non-small cell lung cancer.

[0023] Furthermore, the system uses fluorescence real-time PCR or digital PCR to detect the values of the biomolecular marker composition.

[0024] In some specific embodiments, the kit can detect the expression levels of SFTPA1 and SFTPA2 genes in blood samples by fluorescence quantitative PCR (qPCR) or digital PCR (dPCR) methods, and compare them with those of normal people and non-small cell lung cancer patients respectively. When the detection value of the biomolecular marker composition ≤ 0.40903, it is diagnosed that the test subject does not have non-small cell lung cancer; or when the detection value of the biomolecular marker composition ≥ 0.40903, it is diagnosed that the test subject has non-small cell lung cancer. To preliminarily diagnose whether the test subject has non-small cell lung cancer.

[0025] It should be noted that the detected values are the relative expression levels of SFTPA1 and SFTPA2 genes at the secretion level.

[0026] In some preferred embodiments, the detection effect of fluorescence quantitative PCR is better.

[0027] On the other hand, the present invention provides a use of SFTPA1 and SFTPA2 genes for preparing a reagent for evaluating the prognosis of non-small cell lung cancer, that is, the mortality rate of non-small cell carcinoma patients with higher expression levels of SFTPA1 and SFTPA2 genes is lower.

[0028] On the other hand, the present invention provides a use of SFTPA1 gene for preparing a reagent for evaluating the effect of drugs in treating non-small cell lung cancer. The drugs include one or more of Alectinib (tyrosine kinase inhibitor), Rifampin (a broad-spectrum antibacterial drug), and LDK-378 (anaplastic lymphoma tyrosine kinase inhibitor). These drugs are commonly used to treat non-small cell lung cancer. The results show that when any one of the above drugs is used alone, the expression level of SFTPA1 gene in non-small cell lung cancer patients can be increased. The increase in the expression level of SFTPA1 gene is helpful for the treatment of non-small cell lung cancer. Therefore, the SFTPA1 gene can be used to evaluate the therapeutic effect of the above drugs. By detecting its expression level, personalized treatment can be provided for patients.

[0029] The present invention also provides a use of SFTPA2 gene for preparing a reagent for evaluating the effect of drugs in treating non-small cell lung cancer. Through experimental data analysis, the expression level of SFTPA2 gene has a significant correlation with 30 drugs. Among them, CFI-400945 (a poly ADP-ribose polymerase 1 inhibitor) is the drug most related to the SFTPA2 gene. In addition, Barasertib (an Aurora kinase inhibitor) and BAY-1161909 (a PI3K / mTOR signaling pathway inhibitor) also show relatively high correlations. The use of these drugs can significantly up-regulate the expression of SFTPA2 gene, indicating that the up-regulation of SFTPA2 gene also has potential benefits for the treatment of non-small cell lung cancer. Therefore, the expression level of SFTPA2 gene can be used as a basis for evaluating drug efficacy and providing personalized treatment for patients.

[0030] Generally speaking, through multiple data analyses and experimental verifications, this patent systematically studied the expression and clinical application value of SFTPA1 and SFTPA2 genes in non-small cell lung cancer (NSCLC). First, the present invention screened single molecular markers and their combinations for potential diagnosis of non-small cell carcinoma. AUC curve analysis showed that in the tumor group (n = 45) and the control group (n = 30), the optimal threshold for the combined detection of SFTPA1 and SFTPA2 was 0.40903, with a corresponding sensitivity of 62.5% and a specificity of 78.57%, confirming the high accuracy of SFTPA1 and SFTPA2 in the early diagnosis of non-small cell lung cancer. Second, pan-cancer analysis of SFTPA1 and SFTPA2 was performed through the GEPIA2.0 and TIMER2.0 databases. The results showed that the two genes were significantly lowly expressed in non-small cell lung cancer tissues, with a p value < 0.05, showing statistical significance. Then, the prognostic survival analysis results showed that the high expression of SFTPA1 and SFTPA2 genes was significantly correlated with the overall survival (OS), and the HR values were both approximately 0.7, indicating that the high expression of SFTPA1 and SFTPA2 genes could effectively reduce the risk of patient death. In addition, drug sensitivity analysis based on the CellMiner database showed that the expression level of the SFTPA2 gene was positively correlated with Alectinib, Rifampin, and LDK-378 (R > 0.6), meaning that SFTPA2 plays an important role in drug response. In summary, the data and results provided by the present invention indicate that SFTPA1 and SFTPA2 genes, as potential biomarkers, have important clinical application values in the early screening, prognostic evaluation, and drug sensitivity prediction of non-small cell lung cancer. In addition, the present invention also provides a kit that can significantly improve the early diagnosis rate of non-small cell lung cancer, providing a simple and efficient diagnostic tool for clinical practice.

[0031] The beneficial effects of the present invention include:

[0032] 1. By detecting the expression of SFTPA1 gene and SFTPA2 gene in blood samples, the present invention diagnoses early non-small cell lung cancer, providing a highly sensitive and specific diagnostic method and its related kit;

[0033] 2. Compared with the prior art, the detection method provided by the present invention has the advantages of non-invasiveness, multi-gene combination, and simple operation;

[0034] 3. A kit and a detection method for the early diagnosis of non-small cell lung cancer provided by the present invention can significantly improve the early diagnosis rate of non-small cell lung cancer, providing a simple and efficient diagnostic tool for clinical practice. Description of the Drawings

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying 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 accompanying drawings can be obtained based on these drawings.

[0036] Figure 1 : Flowchart of the diagnostic non-small cell lung cancer kit provided by the present invention;

[0037] Figure 2 : AUC curve analysis of SFTPA1 and SFTPA2 genes;

[0038] Figure 3 : Pan-cancer analysis of SFTPA1 and SFTPA2 genes; Figure 3 In b, the light blue bar graph represents the expression level of the SFTPA1 gene in cancer patients, and the light gray bar graph represents the expression level of the SFTPA1 gene in healthy people; Figure 3 In c, the light red bar graph represents the expression level of the SFTPA2 gene in cancer patients, and the light gray bar graph represents the expression level of the SFTPA2 gene in healthy people;

[0039] Figure 4 : Prognostic survival curves of SFTPA1 and SFTPA2 genes; Figure 4 a shows the survival curves of the overall survival (OS) and recurrence-free survival (RFS) of patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC); Figure 4 b shows the expression of SFTPA1 and SFTPA2 genes in different types of tumors (LUAD and LUSC) at different tumor stages (Stage Ⅰ - Ⅳ);

[0040] Figure 5 : Expression of SFTPA1 and SFTPA2 genes in different populations;

[0041] Figure 6 : Drug sensitivity analysis of SFTPA1 and SFTPA2 genes; GGO is ground-glass nodule, SN is solid nodule; MIA refers to minimally invasive adenocarcinoma, AIS refers to adenocarcinoma in situ, AH refers to atypical hyperplasia, BT is benign tumor, ADC refers to adenocarcinoma, SCC is squamous cell carcinoma, PDC is poorly differentiated carcinoma, LLL is left lower lobe of the lung, LUL is left upper lobe of the lung, RLL is right lower lobe of the lung, RML is right middle lobe of the lung, RUL is right upper lobe of the lung. Detailed implementation manners

[0042] The present invention will be further described in detail below with reference to the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0043] Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0044] Example 1: Preparation method of a kit for diagnosing early non-small cell lung cancer

[0045] (1) Design and preparation of probes: Obtain the full sequences of SFTPA1 (SEQ ID NO: 1, Gene ID: 653509) and SFTPA2 (SEQ ID NO: 2, Gene ID: 653510) genes using the GenBank website (https: / / www.ncbi.nlm.nih.gov / genbank / ); analyze the SFTPAs gene sequences through BLAST alignment and Primer3 respectively to determine their specific regions, that is, probes or PCR primers targeting these regions can accurately reflect the gene expression, so as to distinguish normal tissues from non-small cell lung cancer tissues based on the expression differences of SFTPAs genes; according to the specific regions of the genes, design oligonucleotide probes of 18 - 25 bp. The probe sequences should avoid forming secondary structures (hairpin structures) or self-complementarity, and ensure that their T m value (melting temperature) is between 50°C and 65°C to ensure the binding efficiency and specificity in the amplification reaction. The designed probe sequences are shown in SEQ ID NO: 3 - SEQ ID NO: 6 in the sequence listing (see Table 1); select a suitable fluorescent group (FAM group is preferred in this embodiment) to label the 5' end of the probe, and a quencher (TAMRA or BHQ1) (TAMRA is preferred in this embodiment) is labeled at the 3' end to ensure that the fluorescent signal is released and detected when the probe binds to the target sequence; use a DNA synthesizer ABI 3900 DNA synthesizer (Thermo Fisher Scientific, USA) to synthesize the designed probe sequences, and then purify the probes by high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity II high-performance liquid chromatography (HPLC) system (Agilent, USA) or AKTA Pure 25 protein purification system (Cytiva, Sweden) or polyacrylamide gel electrophoresis (PAGE) to remove incompletely synthesized products and other impurities to ensure the high purity and high specificity of the probes.

[0046] (2) Design and preparation of PCR primers: According to the upstream and downstream sequences of the recognition regions of the SFTPA1 and SFTPA2 gene probes respectively, the corresponding PCR primers were designed. The primer length is generally 18 - 22 bp, and its Tm value is similar to that of the probe, usually between 50°C and 65°C. It should be noted that the targeted region of the designed primer should avoid overlapping with the probe, and it is ensured that no complementary structure is formed inside or non-specific binding occurs with other primers. Then, the designed primer sequences were verified through the OligoAnalyzer online tool (https: / / www.idtdna.com / pages / tools / oligoanalyzer), that is, the secondary structure, self-complementarity, amplification efficiency, and specificity of the primers were detected. The designed PCR primer sequences are shown in Sequence Listing SEQ ID NO:7 - SEQ ID NO:10 (see Table 1); the primers were synthesized by a DNA synthesizer, and after synthesis, they were purified by high-performance liquid chromatography (HPLC) or PAGE to remove impurities; after purification, quality detection was carried out, that is, the molecular weight of the primers was verified by mass spectrometry analysis (MALDI-TOF or LC-MS), and the concentration and purity of the primers were evaluated by combining ultraviolet absorption detection (at a wavelength of 260 nm), and at the same time, the accuracy of the primer length was verified by capillary electrophoresis (CE) analysis to ensure the high purity and high quality of the primers as well as the accuracy of the primer length and sequence.

[0047] Table 1 Probe and primer sequences involved in the non-small cell lung cancer diagnostic kit

[0048] Primer Name Primer Sequence Sequence Number SFTPA1 - Probe - F 5’ - [FAM] - ACCTGGGAGCCTTGCAGT - [TAMRA] - 3’ SEQ ID NO 3 SFTPA1 - Probe - R 5’ - [FAM] - TGCAGTCCGTGTTGGACA - [TAMRA] - 3’ SEQ ID NO 4 SFTPA2 - Probe - F 5’ - [FAM] - CAGCAGGAGCCTGTGGAA - [TAMRA] - 3’ SEQ ID NO 5 SFTPA2 - Probe - R 5’ - [FAM] - TGGGAGTGTCCTGTCAGA - [TAMRA] - 3’ SEQ ID NO 6 SFTPA1 - Primer - F 5’ - TGGAGGACGGTGAGGTTCA - 3’ SEQ ID NO 7 SFTPA1 - Primer - R 5’ - CGTCTGCTGGCAGTACCT - 3’ SEQ ID NO 8 SFTPA2 - Primer - F 5’ - TGCAGAGGTCGTGTGGAAC - 3’ SEQ ID NO 9 SFTPA2 - Primer - R 5’ - CTGTGAGGTCGTGGAGGAT - 3’ SEQ ID NO 10

[0049] (3) Optimization of the reaction system: That is, to determine the optimal working concentrations of the various components of the reaction system (Taq DNA polymerase (Takara ExTaq DNA Polymerase, Takara Bio Inc.), dNTPs (dNTP Mix, Thermo Fisher Scientific Inc.), MgCl2 (10 mM MgCl2 Solution, Invitrogen Inc.), reaction buffer (10×Ex Taq Buffer, Takara Bio Inc.), probe and PCR primers (see Table 1 for details)) to achieve the best amplification effect and detection sensitivity; usually in qPCR or dPCR, the MgCl2 concentration is in the range of 1.5 - 3.0 mM (2.5 mM is preferred in this example); by adjusting the concentrations of the probe (SEQ ID NO: 3 - SEQ ID NO: 6) and primers (SEQ ID NO: 7 - SEQ ID NO: 10), to ensure the efficient generation of amplification products and the maximization of fluorescence signals. Generally, the concentration of the probe is between 100 nM and 250 nM, and the primer concentration is between 200 nM and 500 nM. In this example, the optimal concentrations of the probe and primers were verified by experiments to be 200 nM and 400 nM respectively; to ensure the specificity and efficiency of the reaction, the thermal cycling conditions of PCR need to be optimized, including the pre-denaturation temperature (usually 95°C), primer annealing temperature (between 50°C and 65°C), and amplification time. In this example, the optimal pre-denaturation temperature, annealing temperature, and amplification time were verified by experiments to be 95°C, 60°C, and 30 s respectively. In summary, the best PCR reaction system (total volume of 20 μL) provided in this example contains 2 μL of the sample to be detected, 10 μL of 2× Taq DNA polymerase, 0.4 μL of 10 mM dNTPs, 1.5 μL of 25 mM MgCl2, 2 μL of reaction buffer (containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 0.1% Triton X-100), 0.4 μL of 200 nM probe (SEQ ID NO: 3 - SEQ ID NO: 6), and 0.8 μL of 400 nM PCR primers (SEQ ID NO: 7 - SEQ ID NO: 10), and the system was made up to 20 μL with ddH2O; the best PCR reaction program is: Initial denaturation: 95°C, 1 minute. Cycle reaction (20 cycles in total): Denaturation at 95°C, 15 seconds; Annealing at 55°C, 15 seconds; Extension at 60°C, 15 seconds. After completion: The sample temperature is maintained at 12°C and enters an infinite-time incubation state.

[0050] (4) Sub-packaging of reagents: The above optimal PCR reaction system is sub-packaged into independent 1.5 mL enzyme-free EP tubes (Eppendorf). The vials are made of high-quality polypropylene (PP) material, with strong temperature resistance (-80 °C to 121 °C), no DNase / RNase contamination, good sealing, and transparency for easy observation, suitable for long-term storage and use of the PCR reaction system. Each vial contains the required Taq DNA polymerase, dNTPs, buffer, MgCl2, primers, and probes. At the same time, errors should be avoided during sub-packaging, that is, ensuring that the component ratios of the reaction units in each vial are consistent to ensure the accuracy of test results.

[0051] In some specific embodiments, in addition to the above PCR system, the kit further contains reagents required for RNA extraction and reverse transcription reagents. The RNA extraction system includes sample processing reagents (Quick-cfRNA TM Digestion Buffer 200 μL and Proteinase K 10 μL), binding reagents (Quick-cfRNA TM Binding Buffer 400 μL and 100% isopropanol 1.2 mL), and washing and recovery reagents (RNAPrep Buffer 600 μL, RNAWash Buffer 700 μL × 2, RNARecovery Buffer 15 μL, and DNase / RNase-Free water for elution). The total volume of the reverse transcription system is 20 μL, and the specific components include: template RNA 5 μL, Oligo(dT) or random primer (10 μM) 1 μL, dNTPs (10 mM) 1 μL, reverse transcriptase (such as Superscript IV) 1 μL, reverse transcription buffer (5×Buffer) 4 μL, RNase inhibitor (40 U / μL) 0.5 μL, MgCl2 (25 mM) 1.5 μL, and made up to 20 μL with ddH2O. The reagents suitable for extracting total RNA from blood samples (lysis buffer, washing buffer, elution buffer, etc.) are sub-packaged step by step to ensure that users can directly use the kit to extract high-quality RNA from blood samples; the reverse transcription system (transcriptase, reverse transcription buffer, and dNTPs) is sub-packaged according to the reaction requirements.

[0052] The sub-packaged reverse transcription reagents, RNA extraction reagents, and PCR reaction system are respectively packaged independently, and detailed operation instructions are provided, including the specific steps of sample processing, RNA extraction, reverse transcription, and qPCR or dPCR (see Example 2 for details). The kit needs to be stored at low temperature, usually at -20 °C.

[0053] Example 2: Method for using a kit for diagnosing early non-small cell lung cancer

[0054] The present invention provides a method for using the kit described in Example 1, and the specific steps are as follows( Figure 1 ):

[0055] Collect 5 - 10 mL of peripheral blood from the patient, and store the sample in an EDTA anticoagulation tube (Becton, Dickinson and Company, BD, USA). The sample should be processed within 2 hours or immediately frozen at -80 °C; Use the RNA extraction reagent provided in the kit to extract the total RNA of the sample: Add 200 μL of peripheral blood sample into a 1.5 mL enzyme-free EP tube, and add 200 μL of Quick-cfRNA TM Digestion Buffer and 10 μL of Proteinase K, and incubate at room temperature for 15 minutes to lyse the sample; Subsequently, add 400 μL of Quick-cfRNA TM Binding Buffer and 1.2 mL of 100% isopropanol, mix well, transfer to the RNA column, centrifuge at 12,000×g for 1 minute, and discard the filtrate; Add 600 μL of RNAPrep Buffer and 700 μL of RNA Wash Buffer in sequence, centrifuge at 12,000×g for 30 seconds, repeat the washing once, and then centrifuge the empty tube at 12,000×g for 2 minutes to remove residual ethanol; Finally, add 15 μL of RNA Recovery Buffer, let it stand at room temperature for 2 minutes, centrifuge at 12,000×g for 1 minute to elute and purify the RNA, and store the RNA on ice or at -80 °C.

[0056] The extracted RNA was used for reverse transcription reaction. In a reaction system with a total volume of 20 μL, 5 μL of RNA sample, 1 μL of Oligo(dT) primer (10 μM), 1 μL of dNTPs (10 mM), 4 μL of 5× reverse transcription buffer, 0.5 μL of RNase inhibitor (40 U / μL), 1 μL of reverse transcriptase, 1.5 μL of MgCl2 (25 mM), and 6 μL of enzyme-free ddH2O were added. The reaction procedure was incubation at 42 °C for 60 minutes and termination of the reaction at 95 °C for 5 minutes. The synthesized cDNA was immediately placed on ice or stored at -20 °C for subsequent PCR detection. Using the specific primers and probes provided in the kit, the expression levels of SFTPA1 and SFTPA2 genes were detected by qPCR technology or dPCR technology (qPCR technology was preferably used in this example). That is, in the qPCR experiment, gene expression was monitored by the real-time accumulation of fluorescence signals, or in the dPCR experiment, the absolute copy number of the gene was calculated by counting the positive reaction units. By comparing the Ct values in qPCR or the gene copy numbers in dPCR, and by comparing the expression levels of SFTPA1 and SFTPA2 genes in the test subjects with those in healthy people and non-small cell lung cancer patients respectively, it was used to preliminarily diagnose whether the test subjects had non-small cell lung cancer. The PCR system and reaction procedure were the same as those described in Example 1.

[0057] In some specific embodiments, a positive control (sample of a confirmed non-small cell lung cancer patient) and a negative control (sample of a healthy person) were set up simultaneously in the detection experiment. By comparing the Ct values or gene copy numbers of the test sample with those of the positive / negative samples, it was determined whether the test subject had non-small cell lung cancer. That is, if the Ct value or gene copy number of the test sample was closer to that of the positive sample, the test result was positive; otherwise, it was negative.

[0058] In some other specific embodiments, qPCR was used for detection, and the determination criteria for the detection results were as follows: when the detection value of the biomolecular marker composition (i.e., the relative expression level of the gene, calculated by the 2-△△ct method) ≤ 0.40903, it was diagnosed that the test subject did not have non-small cell lung cancer; or when the detection value of the biomolecular marker composition ≥ 0.40903, it was diagnosed that the test subject had non-small cell lung cancer. To preliminarily diagnose whether the test subject had non-small cell lung cancer.

[0059] Example 3: Screening of molecular markers for diagnosing non-small cell lung cancer

[0060] To obtain molecular markers capable of specifically diagnosing non-small cell carcinoma, in this example, RNA sequencing was performed on blood samples from 45 non-small cell lung cancer patients and blood samples from 30 healthy controls. The differentially expressed genes between the two groups of samples were analyzed, and the top 3 genes with the largest fold change in differential expression (SFTPA1, SFTPA2, and KRT19) were selected. The expression levels of these 3 genes and their combinations in non-small cell lung cancer patients (n = 45) and normal individuals (n = 30) were detected by qPCR. The specific steps were the same as those described in Examples 1-2, and ROC curve analysis was performed on the detection results. The specific results are shown in Table 2 and Figure 2 , where the threshold is the relative expression level of the molecular marker detected by qPCR technology and calculated by the 2-△△ct method.

[0061] Table 2 ROC curve analysis of different genes and their combinations

[0062]

[0063]

[0064] The higher the sensitivity, specificity, and AUC value, the more accurately the detection of the corresponding molecular marker can diagnose whether the test subject has non-small cell carcinoma. The sensitivity of the SFTPA1 and SFTPA2 gene combination is 0.83, the specificity is 0.89, and the AUC value is 0.85 ( Figure 2 ), and these indicators are all superior to those of detecting a single molecular marker and the combination of two or three molecular markers, indicating that this combination can better distinguish between healthy people and non-small cell lung cancer patients and has good application prospects in early diagnosis, especially in controlling false positives during tumor screening.

[0065] To further verify the application of the SFTPA1 and SFTPA2 gene combination in diagnosing non-small cell lung cancer, in this example, the relative expression levels of different markers and their combinations were detected in another batch of samples different from the above (i.e., 20 clinically known non-small cell lung cancer patients and 20 non-non-small cell lung cancer patients). Samples with a relative expression level greater than the threshold were considered to have non-small cell lung cancer, and those with a lower level were considered normal individuals. The analysis results are shown in Table 3, where the accuracy rate = number of correct diagnoses / total number of diagnoses × 100%.

[0066] Table 3 Prediction results of different markers

[0067]

[0068] As can be seen from Table 3, the diagnostic accuracy based on the combination of SFTPA1 and SFTPA2 genes is the highest, and this result is consistent with the result of ROC curve analysis. All in all, the above analysis results prove that the combination of SFTPA1 and SFTPA2 genes is the best biomarker combination for diagnosing non-small cell lung cancer. Therefore, this combination will be the focus of subsequent analysis.

[0069] Next, in this example, pan-cancer analysis of SFTPA1 and SFTPA2 genes was performed through the GEPIA2.0 (http: / / gepia2.cancer-pku.cn / ) and TIMER2.0 (http: / / timer.cistrome.org / ) databases, and the data was sourced from the TCGA and GTEx databases. In GEPIA2.0, through the Expression DIY function, the Box Plot mode was selected to compare the expression differences of SFTPA1 and SFTPA2 in different cancers and normal tissues, and the chart was exported. In TIMER2.0, using the Gene DE module, the SFTPA1 and SFTPA2 genes were input, and the system automatically generated the expression difference results of these two genes between multiple cancer types and normal tissues. Statistical data such as expression levels, p-values, and fold changes were extracted, and the corresponding visualization charts were generated. The above analysis provides reliable data support for the diagnostic value of SFTPA1 and SFTPA2 genes as potential biomarkers for non-small cell lung cancer. The results show ( Figure 3) Compared with normal healthy individuals, the expression levels of SFTPA1 and SFTPA2 genes in patients with lung adenocarcinoma (LUAD) and non-small cell lung cancer (LUSC) are significantly reduced. Among them, the reduction degree in patients with non-small cell lung cancer (LUSC) is greater. This result indicates that SFTPA1 and SFTPA2 genes can be used as potential molecular markers for diagnosing lung adenocarcinoma and non-small cell lung cancer (lung squamous cell carcinoma), and can be used to distinguish healthy individuals from patients with the above two types of lung cancer, especially for the early screening of non-small cell lung cancer. Through the analysis of the expression differences of SFTPA1 and SFTPA2 genes in this patent, the results show that there are significant differences in these two genes between healthy individuals and lung cancer patients (especially non-small cell lung cancer patients), and certain diagnostic specificity and sensitivity are also shown in early lung cancer. For example, through the pan-cancer analysis of the TIMER2.0 and GEPIA 2.0 databases, the expression levels of SFTPA1 and SFTPA2 in non-small cell lung cancer tissues are significantly lower than those in normal tissues, and the statistical results (p<0.05) support their potential as lung cancer diagnostic markers. In addition, combined with the ROC curve analysis, the sensitivity of the combined detection of SFTPA1 and SFTPA2 genes at the optimal diagnostic threshold is 62.5%, and the specificity is 78.57%, further proving that the detection based on these two genes can effectively distinguish healthy individuals from lung cancer patients, providing important data support and theoretical basis for the early screening of non-small cell lung cancer, and laying a foundation for the subsequent design of an early diagnostic kit based on the expression differences of SFTPA1 and SFTPA2 genes.

[0070] Example 4: Screening of prognostic molecular markers for non-small cell lung cancer

[0071] In order to obtain molecular markers that can specifically predict the prognosis of non-small cell carcinoma, in this example, RNA sequencing was performed on the blood samples of 25 patients with good prognosis of non-small cell lung cancer (that is, the spread of cancer cells was effectively controlled, the patients recovered and there were no signs of recurrence) and 15 patients with poor prognosis (that is, the cancer cells spread to other organs, the cancer recurred or the patients died). The differentially expressed genes between these two groups of samples were analyzed, and the top 3 genes with the largest fold change in differential expression (SFTPA1, SFTPA2, and TP53) were selected. The expression levels of these 3 genes and their combinations in patients with good prognosis (n = 10) and poor prognosis (n = 15) of non-small cell lung cancer were detected by qPCR. The specific steps were the same as those described in Examples 1-2; then all the detection results were analyzed by the ROC curve. The specific results are shown in Table 4, where the threshold is the relative expression level of the molecular marker calculated by the 2-△△ct method using qPCR technology.

[0072] Table 4 ROC curve analysis of different genes and their combinations

[0073] Molecular Marker Sensitivity Specificity AUC Value Threshold SFTPA1 0.36 0.40 0.42 0.12 SFTPA2 0.31 0.39 0.39 0.15 TP53 0.32 0.41 0.42 0.25 SFTPA1 and TP53 0.62 0.74 0.79 0.42 SFTPA1 and SFTPA2 0.70 0.85 0.88 0.25 SFTPA2 and TP53 0.61 0.70 0.78 0.38 SFTPA1, SFTPA2 and TP53 0.66 0.80 0.83 0.50

[0074] The higher the sensitivity, specificity, and AUC value are, the more accurately the corresponding molecular marker can predict the prognosis of non-small cell carcinoma patients. The sensitivity of the SFTPA1 and SFTPA2 gene combination is 0.70, the specificity is 0.85, and the AUC value is 0.88. These indicators are all better than those of detecting a single molecular marker and the combination of two or three molecular markers, indicating that this combination can most accurately predict the prognosis of non-small cell carcinoma patients.

[0075] To further verify the application of the SFTPA1 and SFTPA2 gene combination in predicting the prognosis of non-small cell lung cancer, this example detected the expression levels of different markers and their combinations in another batch of samples different from the above (i.e., 20 clinically known patients with good prognosis of non-small cell lung cancer and 20 patients with poor prognosis of non-small cell lung cancer). If the relative gene expression level of the detection object is less than the threshold, it is determined to have a good prognosis, and vice versa for a poor prognosis. The analysis results are shown in Table 5, where the accuracy rate = the number of correct diagnoses / the total number of diagnoses × 100%.

[0076] Table 5 Prediction results based on different markers

[0077]

[0078] As can be seen from Table 5, the prediction accuracy based on the SFTPA1 and SFTPA2 gene combination is the highest, and this result is consistent with the result of the ROC curve analysis. In other words, the above analysis results all prove that the SFTPA1 and SFTPA2 gene combination can most accurately predict the prognosis of non-small cell lung cancer, so this combination will be focused on in the follow-up analysis.

[0079] Next, the prognostic survival curves of the SFTPA1 and SFTPA2 genes in non-small cell lung cancer patients were further analyzed. The data used were from the Kaplan-Meier Plotter database (http: / / kmplot.com / analysis / ), which integrated public microarray data from the GEO and TCGA databases; according to the mRNA expression levels of SFTPA1 and SFTPA2 in the pan-cancer RNA sequence data, the survival curves of the overall survival (OS) and recurrence-free survival (RFS) of patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were respectively plotted ( Figure 4 a), and the expression levels of the SFTPA1 and SFTPA2 genes in different periods and different tumors were also analyzed ( Figure 4b). During the analysis, according to the gene expression levels, patients were divided into a high-expression group and a low-expression group, and the optimal cut-off value was automatically selected for grouping to ensure the accuracy and consistency of the analysis results; the statistical results were based on a significance criterion of p < 0.05 to clarify the relationship between the expression levels of the SFTPA1 and SFTPA2 genes and the prognosis of patients with non-small cell lung cancer.

[0080] From Figure 4 the OS (overall survival) curves of the SFTPA1 and SFTPA2 genes in a, it can be seen that the p-value < 0.05, indicating that there is a statistically significant difference between the high-expression group and the low-expression group of the SFTPA1 and SFTPA2 genes, and the HR values of both are approximately 0.7 (less than 1), suggesting that the high expression of the SFTPA1 or SFTPA2 gene is closely related to the reduction of the patient's death risk. That is, at the same time point, patients with high expression levels of the SFTPA1 or SFTPA2 gene have a higher survival rate. In addition, although the p-value did not reach statistical significance in the RFS (recurrence-free survival) analysis (possibly due to a small sample size), the HR values were all less than 1, suggesting that the high expression of the SFTPA1 and SFTPA2 genes can reduce the recurrence risk to a certain extent, and this result is consistent with the trend of the OS curve. The differences in the prognostic survival curves indicate that the expression levels of the SFTPA1 and SFTPA2 genes vary among different patient groups, and this difference may be related to the occurrence and development of tumors and the clinical outcomes of patients, and can also be used to evaluate the prognosis of patients to a certain extent based on the expression levels of the SFTPA1 and SFTPA2 genes.

[0081] From Figure 4 b, it can be seen that the expression levels of the SFTPA1 and SFTPA2 genes are not affected by tumor stage, meaning that these two genes can be used to evaluate the prognosis of tumors at different stages.

[0082] Generally speaking, the SFTPA1 and SFTPA2 genes showed positive survival advantages in both the overall survival (OS) and recurrence-free survival (RFS) analyses. The overall survival rate and recurrence-free survival rate of patients in the high-expression group were significantly better than those in the low-expression group, with a p-value less than 0.05, showing statistical significance, and the HR value was approximately 0.7 (less than 1), further demonstrating that the high expression of the SFTPA1 and SFTPA2 genes is closely related to the reduction of the patient's death risk and recurrence risk. This result emphasizes the important role of the SFTPA1 and SFTPA2 genes in improving the prognosis of patients with non-small cell lung cancer and has high clinical application value. Combining the results of OS and RFS, the SFTPA1 and SFTPA2 genes, as potential biomarkers, not only perform outstandingly in improving the patient's survival rate but also show potential in the assessment of disease recurrence risk, and are worthy of further promotion and application in the early diagnosis and individualized treatment of non-small cell lung cancer.

[0083] Example 5: Expression of SFTPA1 and SFTPA2 Genes in Different Populations

[0084] In this example, the expression levels of the two genes were detected in clinically collected populations with different backgrounds (the detection method was the same as that described in Example 2). The specific results are shown in Table 6 (LLL is the left lower lung lobe, LUL is the left upper lung lobe, RLL is the right lower lung lobe, RML is the right middle lung lobe, RUL is the right upper lung lobe; MIA refers to minimally invasive adenocarcinoma, AIS refers to adenocarcinoma in situ, AH refers to atypical hyperplasia, BT is a benign tumor, ADC refers to adenocarcinoma, SCC is squamous cell carcinoma, PDC is poorly differentiated carcinoma, BLD refers to benign lung diseases, GGO is ground-glass nodule, and SN is solid nodule) and Figure 5 . Compared with the tumor-bearing population, the expression levels of SFTPAs genes in patients with benign lung diseases were significantly decreased. Therefore, SFTPAs genes can be used to distinguish tumor patients from those with benign lung diseases. On the other hand, age, gender, lesion location, pathological type, tumor stage, and type did not cause differences in the expression levels of SFTPAs genes. Therefore, different populations can diagnose non-small cell lung cancer or predict the prognosis of non-small cell lung cancer by detecting the expression levels of SFTPAs genes, that is, the kit provided in this example has universality.

[0085] Table 6 Expression Differences of SFTPAs Genes in Different Populations with Different Backgrounds

[0086]

[0087]

[0088] Example 6: Drug Sensitivity Analysis of SFTPA1 and SFTPA2 Genes

[0089] To study the correlation between genes SFTPA1 and SFTPA2 and different drugs, in this example, the gene expression data and drug sensitivity data of the NCI-60 cancer cell line were downloaded through the CellMiner database (https: / / discover.nci.nih.gov / cellminer / home.do); R software was used for data analysis. First, the correlation analysis was performed between the expression levels of SFTPA1 and SFTPA2 and the half-maximal inhibitory concentration (IC50) of different drugs to screen out 10 drugs that were significantly correlated with gene expression. The 10 drugs were commonly used clinically for targeted treatment of non-small cell lung cancer; the Spearman correlation analysis was used to evaluate the relationship between gene expression and drug sensitivity, and the correlation coefficient (R value) and p value were calculated to determine the correlation between high and low expression of SFTPA1 and SFTPA2 genes and drug sensitivity. A p value < 0.05 was considered to have statistical significance.

[0090] It can be seen from Figure 6 that 10 drugs basically do not affect the expression level of the SFTPA2 gene (R < 0.5); while the expression levels of the SFTPA1 gene increase with the increasing dosages of three drugs, namely Alectinb (a tyrosine kinase inhibitor targeting the EML4-ALK fusion gene), rifa (rifampicin, a rifamycin antibiotic that blocks RNA synthesis by inhibiting bacterial RNA polymerase and has broad-spectrum antibacterial effects), and LDK-378 (an anaplastic lymphoma tyrosine kinase inhibitor), showing a positive correlation (R > 0.6). This indicates that the three drugs can promote the expression of the SFTPA1 gene (the expression level of the SFTPA1 gene at the tissue level in non-small cell carcinoma patients is lower compared to normal people), thereby achieving specific therapeutic effects. Therefore, in addition to being used for the early diagnosis of non-small cell lung cancer, the detection method proposed in the present invention can also provide information basis for individualized treatment, helping doctors select more appropriate drug treatment regimens and further improving the clinical efficacy. At the same time, the above analysis also explores the clinical application value of SFTPA1 and SFTPA2 genes as potential predictors of different drug responses. Specifically, the SFTPA1 gene serves as a marker for predicting the responses to Alectinb, rifa, and LDK-378 drugs.

[0091] Example 7: Optimization of the means for detecting the expression levels of SFTPA1 and SFTPA2 genes

[0092] In this example, the expression levels of SFTPA1 and SFTPA2 genes were detected by qPCR technology and dPCR technology respectively, using the kit described in Example 1, and the operation method was the same as that described in Example 2.

[0093] The qPCR detection results showed that there were significant differences in the expression levels of SFTPA1 and SFTPA2 genes between the tumor group (n = 41) and the control group (n = 31). Using GAPDH as the internal reference gene, the relative expression level (T / R value) in the tumor group was 0.2145 ± 0.2072, which was significantly higher than that in the control group of 0.0834 ± 0.0831 (p < 0.01, Mann-Whitney U test). At the same time, the dPCR detection results showed that the copy numbers of SFTPA1 and SFTPA2 in the tumor group were higher than those in the control group, and the difference was statistically significant (p < 0.05). Combining the detection results of qPCR and dPCR technologies, the kit provided by the present invention is preferably used in combination with qPCR technology, which can achieve sensitive detection of the expression levels of SFTPA1 and SFTPA2 genes, providing important technical support for the early screening and diagnosis of non-small cell lung cancer.

[0094] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention, and any reference signs in the claims should not be regarded as limiting the claims involved.

Claims

1. A use of a biomolecular marker composition for preparing a reagent for determining whether a patient has non-small cell lung cancer, characterized in that: The biomolecule marker composition comprises SFTPA1 and SFTPA2 genes, and the SFTPA1 gene and SFTPA2 gene respectively contain the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence list.

2. A use of a biomolecular marker composition for preparing a reagent for evaluating the therapeutic effect of non-small cell lung cancer, characterized in that: The primers and / or probes specifically target the SFTPA1 or SFTPA2 gene, and the SFTPA1 gene and SFTPA2 gene contain the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing, respectively.

3. A use of a primer or / and a probe for preparing a reagent for determining whether a patient has non-small cell lung cancer, characterized in that: The primers and / or probes specifically target the SFTPA1 or SFTPA2 gene, and the SFTPA1 gene and SFTPA2 gene contain the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing, respectively.

4. A kit for diagnosing non-small cell lung cancer, characterized in that: The kit comprises primers and / or probes specifically targeting SFTPA1 or SFTPA2 gene, wherein the SFTPA1 gene and SFTPA2 gene respectively contain the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence list.

5. The kit according to claim 4, characterized in that The probe targeting the SFTPA1 gene comprises the sequences shown in SEQ ID NOs: 3-4 in the sequence listing; the primers targeting the SFTPA1 gene comprise the sequences shown in SEQ ID NOs: 7-8 in the sequence listing.

6. The kit according to claim 4, characterized in that The probe targeting the SFTPA2 gene comprises the sequences shown in SEQ ID NOs: 5-6 in the sequence listing; the primers targeting the SFTPA2 gene comprise the sequences shown in SEQ ID NOs: 9-10 in the sequence listing.

7. The kit according to claim 4, characterized in that The kit also contains DNA polymerase, dNTPs, MgCl2, and reaction buffer; the kit also contains reagents required for RNA extraction and reverse transcription, wherein the reagents required for RNA extraction include lysis solution, cleaning solution, eluent, etc.; the reagents required for reverse transcription include reverse transcriptase, reverse transcription buffer, and dNTPs.

8. A system for diagnosing early-stage non-small cell lung cancer, characterized in that: The system comprises a data analysis module; the data analysis module is used to analyze the detection value of the biomolecule marker composition in the detection sample, the biomolecule marker composition comprises SFTPA1 and SFTPA2 genes, and the SFTPA1 gene and SFTPA2 gene respectively contain the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence list.

9. The system according to claim 8, characterized in that The data analysis module compares the detection value of the biomolecule marker composition in the test sample with the normal value and / or abnormal value.

10. The system according to claim 9, characterized in that The system uses fluorescent real-time PCR or digital PCR to detect the value of the biomolecule marker composition.

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