Including the application of exosomal miR-106b-3p, let-7a-3p, etc. in the diagnosis of lung cancer

Abstract

The present invention discloses the use of exosome miR-106b-3p, let-7a-3p, etc. in the diagnosis of lung cancer, wherein the kit includes primers and probes for detecting exosome miRNA markers, and the exosome miRNA markers include let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, m One or more of iR-29a-3p, miR-30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-5p, miR-7706, and miR-885-5p. The present invention provides a non-invasive exosome-based lung cancer diagnosis method with high sensitivity and specificity in lung cancer, providing important value for early diagnosis and recurrence monitoring of lung cancer. This method is of great help in the prevention and treatment of lung cancer in my country.

Description

[0001] This application is a divisional application, the divisional application number is 202111059955.0, the application date is May 11, 2020, and the name of the invention is “Kits, devices and methods for diagnosing lung cancer”. Technical Field

[0002] The present invention relates to the field of medical diagnosis, and in particular to a diagnostic kit, device and method for early lung cancer. Background Art

[0003] With the application of low-dose spiral CT, an increasing number of imaging manifestations of pulmonary nodules (single lesions <3 cm within the pulmonary interstitium, without associated atelectasis or lymphadenopathy) have been discovered. However, not all pulmonary nodules are malignant, and differentiating between benign and malignant pulmonary nodules has always been a difficult point in the clinical diagnosis and treatment of thoracic surgery. Currently, non-invasive detection methods such as plasma circulating tumor cells and circulating tumor free DNA are also available, but their sensitivity in diagnosing early lung cancer is not high; therefore, there is an urgent need to develop a highly sensitive non-invasive method for early lung cancer detection. Summary of the Invention

[0004] The present invention provides a reagent, device and method for non-invasive early-stage lung cancer diagnosis based on exosomes.

[0005] On the one hand, the present invention provides a kit for diagnosing lung cancer, comprising primers and probes for detecting exosomal miRNA markers, wherein the exosomal miRNA markers include let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR -30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-5p, miR-7706, miR-885-5p or more.

[0006] Preferably, the exosomal miRNA marker is one or a combination of miR-3615, miR-502-3p, miR-450b-5p, miR-4746-5p, miR-10a-5p, miR-106b-3p, miR-125a-5p, and miR-885-5p.

[0007] Preferably, the exosomal miRNA marker is a combination of miR-106-3p, miR-125a-5p, and miR-3615.

[0008] Preferably, the exosomal miRNA marker is a combination of miR-106b-3p, miR-3615, and miR-450b-5p.

[0009] Preferably, the exosomal miRNA marker is a combination of miR-106b-3p, miR-125a-5p, miR-3615, miR-450b-5p, and miR-885-5p.

[0010] Preferably, the exosomal miRNA marker is a combination of miR-106b-3p, miR-10a-3p, miR-125a-5p, miR-3615, and miR-450b-5p.

[0011] Preferably, the source of exosomes includes one or more of blood, saliva and sputum.

[0012] Preferably, the primers and probes include:

[0013] Reverse transcription primers, PCR primers, and probes for detecting let-7a-3p: the let-7a-3p reverse transcription primer has the nucleotide sequence shown in SEQ ID NO: 1, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 2, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 3;

[0014] Reverse transcription primers, PCR primers, and probes for detecting let-7f-2: the let-7f-2 reverse transcription primer has the nucleotide sequence shown in SEQ ID NO: 4, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 5, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 6;

[0015] Reverse transcription primers, PCR primers, and probes for detecting miR-106b-3p: the reverse transcription primer for miR-106b-3p has the nucleotide sequence shown in SEQ ID NO: 7, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 8, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 9;

[0016] Reverse transcription primers, PCR primers, and probes for detecting miR-10a-3p: the reverse transcription primer for miR-10a-3p has the nucleotide sequence shown in SEQ ID NO: 10, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 11, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 12;

[0017] Reverse transcription primers, PCR primers, and probes for detecting miR-10a-5p: the reverse transcription primer for miR-10a-5p has the nucleotide sequence shown in SEQ ID NO: 13, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 14, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 15;

[0018] Reverse transcription primers, PCR primers, and probes for detecting miR-125a-5p: the reverse transcription primer for miR-125a-5p has the nucleotide sequence shown in SEQ ID NO: 16, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 17, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 18;

[0019] Reverse transcription primers, PCR primers, and probes for detecting miR-1294: the reverse transcription primer for miR-1294 has the nucleotide sequence set forth in SEQ ID NO: 19, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 20, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 21;

[0020] Reverse transcription primers, PCR primers, and probes for detecting miR-19a-3p: the reverse transcription primer for miR-19a-3p has the nucleotide sequence set forth in SEQ ID NO: 22, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 23, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 24;

[0021] Reverse transcription primers, PCR primers, and probes for detecting miR-22-3p: the reverse transcription primer for miR-22-3p has the nucleotide sequence shown in SEQ ID NO: 25, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 26, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 27;

[0022] Reverse transcription primers, PCR primers, and probes for detecting miR-29a-3p: the reverse transcription primer for miR-29a-3p has the nucleotide sequence shown in SEQ ID NO: 28, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 29, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 30;

[0023] Reverse transcription primers, PCR primers, and probes for detecting miR-30e-5p: the reverse transcription primer for miR-30e-5p has the nucleotide sequence set forth in SEQ ID NO: 31, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 32, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 33;

[0024] Reverse transcription primers, PCR primers, and probes for detecting miR-3158-3p: the reverse transcription primer for miR-3158-3p has the nucleotide sequence set forth in SEQ ID NO: 34, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 35, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 36;

[0025] Reverse transcription primers, PCR primers, and probes for detecting miR-330-5p: the reverse transcription primer for miR-330-5p has the nucleotide sequence set forth in SEQ ID NO: 37, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 38, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 39;

[0026] Reverse transcription primers, PCR primers, and probes for detecting miR-3605-3p: the reverse transcription primer for miR-3605-3p has the nucleotide sequence set forth in SEQ ID NO: 40, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 41, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 42;

[0027] Reverse transcription primers, PCR primers, and probes for detecting miR-3615: the reverse transcription primer for miR-3615 has the nucleotide sequence set forth in SEQ ID NO: 43, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 44, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 45;

[0028] Reverse transcription primers, PCR primers, and probes for detecting miR-378h: the reverse transcription primer for miR-378h has the nucleotide sequence set forth in SEQ ID NO: 46, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 47, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 48;

[0029] Reverse transcription primers, PCR primers, and probes for detecting miR-425-3p: the reverse transcription primer for miR-425-3p has the nucleotide sequence set forth in SEQ ID NO: 49, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 50, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 51;

[0030] Reverse transcription primers, PCR primers, and probes for detecting miR-450b-5p: the reverse transcription primer for miR-450b-5p has the nucleotide sequence set forth in SEQ ID NO: 52, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 53, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 54;

[0031] Reverse transcription primers, PCR primers, and probes for detecting miR-4746-5p: the reverse transcription primer for miR-4746-5p has the nucleotide sequence set forth in SEQ ID NO: 55, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 56, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 57;

[0032] Reverse transcription primers, PCR primers, and probes for detecting miR-483-3p: the reverse transcription primer for miR-483-3p has the nucleotide sequence set forth in SEQ ID NO: 58, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 59, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 60;

[0033] Reverse transcription primers, PCR primers, and probes for detecting miR-502-3p: the reverse transcription primer for miR-502-3p has the nucleotide sequence set forth in SEQ ID NO: 61, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 62, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 63;

[0034] Reverse transcription primers, PCR primers, and probes for detecting miR-550a-5p: the reverse transcription primer for miR-550a-5p has the nucleotide sequence set forth in SEQ ID NO: 64, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 65, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 66;

[0035] Reverse transcription primers, PCR primers, and probes for detecting miR-651-5p: the reverse transcription primer for miR-651-5p has the nucleotide sequence set forth in SEQ ID NO: 67, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 68, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 69;

[0036] Reverse transcription primers, PCR primers, and probes for detecting miR-7706: the reverse transcription primer for miR-7706 has the nucleotide sequence set forth in SEQ ID NO: 70, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 71, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 72;

[0037] Reverse transcription primers, PCR primers and probes for detecting miR-885-5p: the reverse transcription primer of miR-885-5p has a nucleotide sequence as shown in sequence number 73, the upstream PCR primer has a nucleotide sequence as shown in sequence number 74, the downstream primer has a nucleotide sequence as shown in sequence number 80, and the probe has a nucleotide sequence as shown in sequence number 75.

[0038] Another aspect of the present invention provides a device for diagnosing lung cancer, comprising a reagent for detecting exosomal miRNA markers, wherein the exosomal miRNA markers include let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR-3 one or more of miR-0e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-5p, miR-7706, and miR-885-5p.

[0039] Another aspect of the present invention provides a method for diagnosing lung cancer, comprising detecting the specificity of exosomal miRNA markers, wherein the exosomal miRNA markers include let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR- one or more of miR-30e-5p, miR-3158-3p, miR-330-5p, miR-3605-3p, miR-3615, miR-378h, miR-425-3p, miR-450b-5p, miR-4746-5p, miR-483-3p, miR-502-3p, miR-550a-5p, miR-651-5p, miR-7706, and miR-885-5p.

[0040] This invention provides a noninvasive exosome-based lung cancer diagnosis method with high sensitivity and specificity, providing valuable insights into early diagnosis and recurrence monitoring. This approach significantly contributes to the prevention and treatment of lung cancer in my country. Furthermore, five of these miRNA markers (with a combined AUC of up to 0.951, a negative predictive value of 90.32%, a sensitivity of 90.00%, and a specificity of 93.33%) demonstrated exceptional diagnostic performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the result of exosome electron microscopy identification.

[0042] Figure 2 This is the ROC curve of miR-3615 alone for detecting lung cancer.

[0043] Figure 3 This is the ROC curve of miR-502-3p alone for detecting lung cancer.

[0044] Figure 4 This is the ROC curve of miR-450b-5p alone for detecting lung cancer.

[0045] Figure 5 This is the ROC curve of miR-4746-5p alone for detecting lung cancer.

[0046] Figure 6 This is the ROC curve of miR-10a-5p alone for detecting lung cancer.

[0047] Figure 7 This is the ROC curve of the miR-106-3p+miR-125a-5p+miR-3615 combination for detecting lung cancer.

[0048] Figure 8 This is the ROC curve of the miR-106b-3p+miR-3615+miR-450b-5p combination for detecting lung cancer.

[0049] Figure 9 This is the ROC curve of the miR-106b-3p+miR-125a-5p+miR-3615+miR-450b-5p+miR-885-5 combination for detecting lung cancer.

[0050] Figure 10 This is the ROC curve of the miR-106b-3p+miR-10a-3p+miR-125a-5p+miR-3615+miR-450b-5p combination for detecting lung cancer.

[0051] Figure 11 This is the ROC curve of the miR-106b-3P+miR-10a-5p+miR-125a-5p+miR-3615+miR-450b-5p combination for detecting lung cancer (U6 as a reference). DETAILED DESCRIPTION

[0052] Extracellular vesicles (EVs) are small, double-membrane vesicles that are shed from the cell membrane or secreted by cells. Their diameters range from 30 to 1000 nm. EVs primarily consist of microvesicles (MVs) and exosomes. MVs are small vesicles that shed from the cell membrane following cell activation or damage. Due to their unique biological characteristics, EVs, particularly exosomes, are of great significance in disease diagnosis.

[0053] Exosomes are small membrane-bound vesicles ranging in size from 30 to 150 nm that are secreted into the extracellular environment after the fusion of intracellular multivesicular bodies with the cell membrane. They are important mediators of intercellular communication and play a crucial role in antigen presentation, apoptosis, inflammatory responses, tumor development, and metastasis. They are widely distributed in body fluids, including blood, saliva, urine, breast milk, and pleural and ascites fluids. They contain a variety of substances, including DNA, RNA, and proteins, and can serve as noninvasive diagnostic markers for various diseases, including tumors. MiRNAs are the most abundant nucleic acid component in exosomes, and therefore exosomal miRNAs have the potential to be used for the early diagnosis of lung cancer.

[0054] The kit, device and method provided by the present invention use one or more miRNAs found through experimental studies to be significantly differentially expressed in the exosomes of patients with early-stage lung cancer as markers for diagnosing early-stage lung cancer.

[0055] Significantly differentially expressed miRNAs include: let-7a-3p, let-7f-2, miR-106b-3p, miR-10a-3p, miR-10a-5p, miR-125a-5p, miR-1294, miR-19a-3p, miR-22-3p, miR-29a-3p, miR-30e-5p, miR-3158-3 p,miR-330-5p,miR-3605-3p,miR-3615,miR-378h,miR-425-3p,miR-450b-5p,miR-4 746-5p,miR-483-3p,miR-502-3p,miR-550a-5p,miR-651-5p,miR-7706,miR-885-5p.

[0056] In some preferred embodiments, significantly differentially expressed miRNA markers are used in combination, with preferred combinations including one or more of miR-3615, miR-502-3p, miR-450b-5p, miR-4746-5p, miR-10a-5p, miR-106b-3p, miR-125a-5p, and miR-885-5p. Such combinations can provide a better basis for early diagnosis of lung cancer and predict disease risk.

[0057] In addition, the kit for lung cancer diagnosis of the present invention includes primers and probes for detecting the above-mentioned exosomal miRNA markers. The primers and probes for detecting exosomal miRNA markers include:

[0058] Reverse transcription primers, PCR primers, and probes for detecting let-7a-3p: the let-7a-3p reverse transcription primer has the nucleotide sequence shown in SEQ ID NO: 1, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 2, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 3;

[0059] Reverse transcription primers, PCR primers, and probes for detecting let-7f-2: the let-7f-2 reverse transcription primer has the nucleotide sequence shown in SEQ ID NO: 4, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 5, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 6;

[0060] Reverse transcription primers, PCR primers, and probes for detecting miR-106b-3p: the reverse transcription primer for miR-106b-3p has the nucleotide sequence shown in SEQ ID NO: 7, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 8, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 9;

[0061] Reverse transcription primers, PCR primers, and probes for detecting miR-10a-3p: the reverse transcription primer for miR-10a-3p has the nucleotide sequence shown in SEQ ID NO: 10, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 11, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 12;

[0062] Reverse transcription primers, PCR primers, and probes for detecting miR-10a-5p: the reverse transcription primer for miR-10a-5p has the nucleotide sequence shown in SEQ ID NO: 13, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 14, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 15;

[0063] Reverse transcription primers, PCR primers, and probes for detecting miR-125a-5p: the reverse transcription primer for miR-125a-5p has the nucleotide sequence shown in SEQ ID NO: 16, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 17, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 18;

[0064] Reverse transcription primers, PCR primers, and probes for detecting miR-1294: the reverse transcription primer for miR-1294 has the nucleotide sequence set forth in SEQ ID NO: 19, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 20, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 21;

[0065] Reverse transcription primers, PCR primers, and probes for detecting miR-19a-3p: the reverse transcription primer for miR-19a-3p has the nucleotide sequence shown in SEQ ID NO: 22, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 23, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 24;

[0066] Reverse transcription primers, PCR primers, and probes for detecting miR-22-3p: the reverse transcription primer for miR-22-3p has the nucleotide sequence shown in SEQ ID NO: 25, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 26, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 27;

[0067] Reverse transcription primers, PCR primers, and probes for detecting miR-29a-3p: the reverse transcription primer for miR-29a-3p has the nucleotide sequence shown in SEQ ID NO: 28, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 29, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 30;

[0068] Reverse transcription primers, PCR primers, and probes for detecting miR-30e-5p: the reverse transcription primer for miR-30e-5p has the nucleotide sequence set forth in SEQ ID NO: 31, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 32, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 33;

[0069] Reverse transcription primers, PCR primers, and probes for detecting miR-3158-3p: the reverse transcription primer for miR-3158-3p has the nucleotide sequence set forth in SEQ ID NO: 34, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 35, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 36;

[0070] Reverse transcription primers, PCR primers, and probes for detecting miR-330-5p: the reverse transcription primer for miR-330-5p has the nucleotide sequence set forth in SEQ ID NO: 37, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 38, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 39;

[0071] Reverse transcription primers, PCR primers, and probes for detecting miR-3605-3p: the reverse transcription primer for miR-3605-3p has the nucleotide sequence set forth in SEQ ID NO: 40, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 41, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 42;

[0072] Reverse transcription primers, PCR primers, and probes for detecting miR-3615: the reverse transcription primer for miR-3615 has the nucleotide sequence set forth in SEQ ID NO: 43, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 44, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 45;

[0073] Reverse transcription primers, PCR primers, and probes for detecting miR-378h: the reverse transcription primer for miR-378h has the nucleotide sequence set forth in SEQ ID NO: 46, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 47, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 48;

[0074] Reverse transcription primers, PCR primers, and probes for detecting miR-425-3p: the reverse transcription primer for miR-425-3p has the nucleotide sequence set forth in SEQ ID NO: 49, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 50, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 51;

[0075] Reverse transcription primers, PCR primers, and probes for detecting miR-450b-5p: the reverse transcription primer for miR-450b-5p has the nucleotide sequence shown in SEQ ID NO: 52, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 53, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 54;

[0076] Reverse transcription primers, PCR primers, and probes for detecting miR-4746-5p: the reverse transcription primer for miR-4746-5p has the nucleotide sequence set forth in SEQ ID NO: 55, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 56, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 57;

[0077] Reverse transcription primers, PCR primers, and probes for detecting miR-483-3p: the reverse transcription primer for miR-483-3p has the nucleotide sequence set forth in SEQ ID NO: 58, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 59, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 60;

[0078] Reverse transcription primers, PCR primers, and probes for detecting miR-502-3p: the reverse transcription primer for miR-502-3p has the nucleotide sequence set forth in SEQ ID NO: 61, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 62, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 63;

[0079] Reverse transcription primers, PCR primers, and probes for detecting miR-550a-5p: the reverse transcription primer for miR-550a-5p has the nucleotide sequence set forth in SEQ ID NO: 64, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 65, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 66;

[0080] Reverse transcription primers, PCR primers, and probes for detecting miR-651-5p: the reverse transcription primer for miR-651-5p has the nucleotide sequence set forth in SEQ ID NO: 67, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 68, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 69;

[0081] Reverse transcription primers, PCR primers, and probes for detecting miR-7706: the reverse transcription primer for miR-7706 has the nucleotide sequence set forth in SEQ ID NO: 70, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 71, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 72;

[0082] Reverse transcription primers, PCR primers, and probes for detecting miR-885-5p: the reverse transcription primer for miR-885-5p has the nucleotide sequence set forth in SEQ ID NO: 73, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 74, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 75;

[0083] Reverse transcription primers, PCR primers, and probes for detecting the internal reference U6: The U6 reverse transcription primer has the nucleotide sequence set forth in SEQ ID NO: 78, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 76, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 78, and the probe has the nucleotide sequence set forth in SEQ ID NO: 79. The nucleotide sequences of the primers and probe are shown in Table 1.

[0084] Table 1

[0085]

[0086]

[0087]

[0088] Furthermore, the source of exosomes includes one or more of blood, saliva and sputum.

[0089] The kit, device and method of the present invention are applicable to individuals who may be people at high risk of lung cancer, normal individuals and patients after lung cancer surgery.

[0090] The following is a complete and clear description of the technical solution of the present invention in conjunction with implementation examples. The described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by ordinary technicians in this field without making creative efforts are all within the scope of protection of the present invention.

[0091] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0092] To identify exosome biomarkers relevant to the diagnosis of colorectal cancer, at least 10 ml of blood was collected from 50 patients with early-stage lung cancer and 72 controls. Plasma was then separated using a classical ultracentrifugation method to isolate exosomes from the plasma and extract RNA. The resulting RNA was then sequenced and used to construct RNA libraries. The resulting data were then analyzed using bioinformatics to compare differentially expressed RNAs between patients with early-stage lung cancer and controls. These exosome-derived mRNA-level biomarkers could be used for early diagnosis of lung cancer.

[0093] Further application analysis of the RNA markers is carried out, and the method is as follows: (1) collecting body fluid samples (including blood, sputum and saliva) from the individual to be tested; (2) isolating exosomes in the body fluids; (3) extracting exosome RNA using the spkin external reference cel-miR-39; (4) using a two-step method to detect the expression level of the target RNA; (5) using the external reference gene to normalize the expression level of the detected target RNA; (6) substituting the normalized gene expression level into the judgment model to obtain the output value; (7) judging whether the individual to be tested has lung cancer based on the output value of the model and the judgment threshold.

[0094] The kit includes PCR primers, probes, standards and a two-step detection system of reverse PCR for detecting exosome RmiNA markers.

[0095] This involves quantifying the target RNA using either the external reference Cel-miR-39 or the internal reference U6. When using the reference, the target RNA is quantified using the quantitative formula 2ΔΔCt based on the detection Ct value. Once the target RNA expression level is determined, receiver operating characteristic curves and area under the curve (AUC) are used to assess the accuracy of single RNA or combined RNA detection for lung cancer.

[0096] Example 1 Screening of exosomal miRNA markers associated with early lung cancer based on high-throughput sequencing

[0097] To identify exosome biomarkers relevant to the diagnosis of early-stage lung cancer, at least 10 ml of blood was collected from 72 patients diagnosed with early-stage lung cancer and 50 controls. Plasma was separated and exosomes were isolated using classical ultracentrifugation. RNA was extracted using the Qiagen miRNeasy mini kit, and the resulting RNA was sequenced using a small RNA library. The resulting data were subjected to bioinformatics analysis to compare differentially expressed miRNAs between patients with early-stage lung cancer and controls. Significantly differentially expressed miRNAs are shown in Table 2 below. These exosome-derived RNA biomarkers can be used for the early diagnosis of lung cancer.

[0098] Table 2

[0099]

[0100]

[0101] Example 2 miRNA detection system based on fluorescent quantitative PCR platform

[0102] 1. miRNA reverse transcription reaction system

[0103] MiRNA reverse transcription reagents, enzymes, and oligodT were purchased from TAKARA, standards were synthesized by Shanghai Invitrogen, and specific reverse transcription primers were synthesized by Suzhou Hongxun. A 20 μl reverse transcription system was used, as shown in Table 3 below.

[0104] Table 3

[0105]

[0106] 2. PCR reaction system

[0107] The PCR reaction mixture was purchased from TAKARA. The upstream primer and probe (i.e., universal downstream primer) were synthesized by Suzhou Hongxun. The fluorescence quantitative PCR instrument was an ABI 7500. The PCR reaction system is shown in Table 4 below.

[0108] Table 4

[0109]

[0110] The PCR program was 95°C for 10 min, 15 cycles of (95°C for 15 s; 55°C for 30 s) without collecting fluorescence, and 35 cycles of (95°C for 15 s; 55°C for 30 s) with fluorescence collection.

[0111] Example 3 Evaluation of the effectiveness of single marker for early diagnosis of lung cancer using Cel-miR-39 as a reference

[0112] 1. Sample collection

[0113] 10 ml of blood was collected from patients with early-stage (stage I and II) lung cancer with pulmonary nodules confirmed by the hospital, patients with benign pulmonary nodules, healthy controls, and other control samples, and the blood was separated into plasma.

[0114] 2. Exosome RNA Extraction

[0115] Ultracentrifugation or Exosupur from Echobiotech (Beijing Enze Kangtai) was used to separate plasma exosomes (exosome electron microscopy results were as follows Figure 1 ), the separated exosomes were used to extract miRNA from the exosomes using the Qiagen miReasy minikit kit, and the RNA concentration and quality were detected using Agilent 2100, and the RNA concentration was recorded.

[0116] 3. RNA two-step detection system

[0117] The two-step PCR-based miRNA detection system in Example 1 was used to detect plasma exosomal miRNA in 30 patients with early lung cancer and 30 control samples (healthy people and benign nodules). The Ct value of the target miRNA was detected, and the relative expression level was calculated based on the Ct value and the relative quantitative formula.

[0118] 4. Evaluation of the performance of exosomal miRNA in diagnosing early lung cancer

[0119] (1) Performance evaluation of miR-3615 detection alone

[0120] like Figure 2As shown, the Ct values ​​of miR-3615 were detected in plasma exosomes from 30 patients with early-stage lung cancer and 30 controls (healthy individuals and benign lesions). Using the external reference Cel-miR-39, the Ct values ​​were used to determine the miRNA copy number. The relative quantification formula was used to calculate the fold change in relative expression of the combined markers, and then the relative RNA expression level was calculated. The results were analyzed using the R programming language, and the t-test showed a p-value of 0.00058 <= 0.05, indicating that exosomal miR-3615 was significantly associated with early-stage lung cancer. The AUC for miR-3615 alone for diagnosing early-stage lung cancer was 0.785, with a negative predictive value of 85%, a sensitivity of 90%, and a specificity of 56.57%, demonstrating its potential as a diagnostic biomarker.

[0121] (2) Performance evaluation of miR-502-3p detection alone

[0122] like Figure 3 As shown, the Ct values ​​of miR-502-3p were measured in plasma exosomes from 30 patients with early-stage lung cancer and 30 controls (healthy individuals and benign lesions). The Ct values ​​were used as a reference for miR-502-3p, and the miRNA copy number was calculated based on the Ct values. The relative quantification formula was used to calculate the fold change in relative expression of the combined markers, and the relative RNA expression was then calculated. The results were analyzed using the R programming language, and the p-value was 0.00139 < 0.05, indicating that exosomal miR-502-3p was significantly associated with early-stage lung cancer. The AUC for miR-502-3p alone for diagnosing early-stage lung cancer was 0.756, with a negative predictive value of 68.42%, a sensitivity of 60%, and a specificity of 86.67%, demonstrating its potential as a diagnostic biomarker.

[0123] (3) Performance evaluation of miR-450b-5p detection alone

[0124] like Figure 4 As shown, the Ct values ​​of miR-450b-5p were measured in plasma exosomes from 30 patients with early-stage lung cancer and 30 controls (healthy individuals and benign lesions). Using the external reference Cel-miR-39, the Ct values ​​were used to determine the miRNA copy number. The relative quantification formula was used to calculate the fold change in relative expression of the combined markers, and the relative RNA expression was then calculated. The results were analyzed using the R programming language, using a t-test with a p value of 0.00199 < 0.05, indicating that exosomal miR-450b-5p was significantly associated with early-stage lung cancer. The AUC for miR-450b-5p alone for diagnosing early-stage lung cancer was 0.744, with a negative predictive value of 80%, a sensitivity of 86.67%, and a specificity of 53.33%, demonstrating its potential as a diagnostic biomarker.

[0125] (4) Performance evaluation of miR-4746-5p detection alone

[0126] like Figure 5 As shown, the Ct values ​​of miR-4746-5p were measured in plasma exosomes from 30 patients with early-stage lung cancer and 30 controls (healthy individuals and benign lesions). The Ct values ​​were used as a reference for miR-4746-5p copy number, using Cel-miR-39 as an external reference. The relative expression of the combined markers was calculated using the relative quantification formula to determine the relative RNA expression level. The results were analyzed using the R programming language, using a t-test. The p-value was 0.00183 < 0.05, indicating that exosomal miR-4746-5p was significantly associated with early-stage lung cancer. The AUC for miR-4746-5p alone for diagnosing early-stage lung cancer was 0.743, with a negative predictive value of 66.67%, a sensitivity of 60%, and a specificity of 80%, demonstrating its potential as a diagnostic biomarker.

[0127] (5) Performance evaluation of miR-10a-5p detection alone

[0128] like Figure 6 As shown, the Ct values ​​of miR-10a-5p were measured in plasma exosomes from 30 patients with early-stage lung cancer and 30 controls (healthy individuals and benign lesions). Using the external reference Cel-miR-39, the Ct values ​​were used to determine the miRNA copy number. The relative quantification formula was used to calculate the fold change in relative expression of the combined markers, and the relative RNA expression was then calculated. The results were analyzed using the R programming language, using a t-test. The p-value was 0.00183 < 0.05, indicating that exosomal miR-10a-5p was significantly associated with early-stage lung cancer. The area under the predictive analysis (AUC) for miR-10a-5p alone in the diagnosis of early-stage lung cancer was 0.73, with a negative predictive value of 77.27%, a sensitivity of 83.33%, and a specificity of 56.67%, demonstrating its potential as a diagnostic biomarker.

[0129] (6) Performance evaluation of other markers significantly associated with early lung cancer

[0130] The performance evaluation of other significantly correlated miRNAs is shown in Table 5 below.

[0131] Table 5

[0132]

[0133]

[0134] From the data shown in Table 5, it can be seen that the miRNAs described in the table all have the potential to be diagnostic markers.

[0135] Example 4 Evaluation of the effectiveness of multi-marker combination for early diagnosis of lung cancer using Cel-miR-39 as a reference

[0136] 1. Performance evaluation of three-marker combination

[0137] The relative expression level of each miRNA was calculated according to the method in Example 3, and the three marker combinations were trained using logistic regression. The three marker combinations with AUCs above 0.85 are shown in Table 6 below. Among them, the two combinations of miR-106-3p+miR-125a-5p+miR-3615 and miR-106b-3p+miR-3615+miR-450b-5p performed best, with AUCs of 0.887 and 0.881, respectively. Their AUC curves are shown in Table 6. Figure 7 and Figure 8 shown.

[0138] Table 6

[0139]

[0140]

[0141] 2. Performance evaluation of five-marker combination

[0142] The relative expression level of each miRNA was calculated according to the method in Example 3, and the five marker combinations were trained using logistic regression. The five marker combinations with AUCs above 0.93 are shown in Table 7 below. Among them, the two combinations of miR-106b-3p+miR-125a-5p+miR-3615+miR-450b-5p+miR-885-5p and miR-106b-3p+miR-10a-3p+miR-125a-5p+miR-3615+miR-450b-5p performed best, with AUCs of 0.951 and 0.948, respectively. Their AUC curves are shown in Table 7. Figure 9 and Figure 10 shown.

[0143] Table 7

[0144]

[0145]

[0146] Example 5 Evaluation of the effectiveness of a multi-marker combination for early diagnosis of lung cancer using internal reference U6

[0147] The relative expression of each miRNA was calculated using U6 as a reference according to the method in Example 3. The five marker combinations were trained using logistic regression. The five marker combinations with an AUC of more than 85 were shown in Table 8. Among them, the combination of miR-106b-3P+miR-10a-5p+miR-125a-5p+miR-3615+miR-450b-5p performed best with an AUC of 0.864. The AUC curves are shown in Table 8. Figure 11 shown.

[0148] Table 8

[0149]

[0150]

[0151] The above data demonstrate that the present invention's lung cancer detection method based on exosomal miRNA signatures can achieve non-invasive diagnosis of lung cancer, providing valuable insights into early diagnosis and recurrence monitoring, and significantly contributing to the prevention and treatment of lung cancer in my country. Five of the miRNA markers (with a combined AUC of up to 0.951, a negative predictive value of 90.32%, a sensitivity of 90.00%, and a specificity of 93.33%) demonstrated exceptional diagnostic performance.

[0152] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents. Sequence Listing <110> Cancer Hospital of Chinese Academy of Medical Sciences <120> Including the application of exosomal miR-106b-3p, let-7a-3p, etc. in the diagnosis of lung cancer <130> 200001 <160> 80 <170> SIPOSequenceListing 1.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <400> 1 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgaaaga 50 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 cgcgccctat acaatctact g 21 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <400> 3 tcgcactgga tacgacgaaa gaca 24 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <400> 4 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacggaaag 50 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5 agcgcctata cagtctactg t 21 <210> 6 <211> twenty four <212> DNA <213> Artificial Sequence <400> 6 tcgcactgga tacgacggaa agac 24 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <400> 7 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgcagca 50 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <400> 8 accgcactgt gggtact 17 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 tcgcactgga tacgacgcag ca 22 <210> 10 <211> 50 <212> DNA <213> Artificial Sequence <400> 10 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactattcc 50 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <400> 11 acgcgcaaat tcgtatctag g 21 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <400> 12 ttcgcactgg atacgactat tcccc 25 <210> 13 <211> 50 <212> DNA <213> Artificial Sequence <400> 13 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccacaaa 50 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 cgctaccctg tagatccgaa 20 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 ttcgcactgg atacgaccac aaatt 25 <210> 16 <211> 50 <212> DNA <213> Artificial Sequence <400> 16 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactcacag 50 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 cgtccctgag accctttaac 20 <210> 18 <211> twenty four <212> DNA <213> Artificial Sequence <400> 18 tcgcactgga tacgactcac aggt 24 <210> 19 <211> 50 <212> DNA <213> Artificial Sequence <400> 19 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagacaa 50 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <400> 20 tgctgtgagg ttggcattg 19 <210> twenty one <211> twenty four <212> DNA <213> Artificial Sequence <400> twenty one ttcgcactgg atacgacaga caac 24 <210> twenty two <211> 50 <212> DNA <213> Artificial Sequence <400> twenty two gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactcagtt 50 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <400> twenty three cgctgtgcaa atctatgcaa 20 <210> twenty four <211> 25 <212> DNA <213> Artificial Sequence <400> twenty four tcgcactgga tacgactcag ttttg 25 <210> 25 <211> 50 <212> DNA <213> Artificial Sequence <400> 25 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacacagtt 50 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <400> 26 accaagctgc cagttgaag 19 <210> 27 <211> twenty four <212> DNA <213> Artificial Sequence <400> 27 tcgcactgga tacgacacag ttct 24 <210> 28 <211> 50 <212> DNA <213> Artificial Sequence <400> 28 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactaaccg 50 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 cccgtagcac catctgaaat 20 <210> 30 <211> twenty four <212> DNA <213> Artificial Sequence <400> 30 ttcgcactgg atacgactaa ccga 24 <210> 31 <211> 50 <212> DNA <213> Artificial Sequence <400> 31 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccttcca 50 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence <400> 32 accgctgtaa acatccttga c 21 <210> 33 <211> twenty two <212> DNA <213> Artificial Sequence <400> 33 tcgcactgga tacgaccttc ca 22 <210> 34 <211> 50 <212> DNA <213> Artificial Sequence <400> 34 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgtcctg 50 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <400> 35 acgaagggct tcctctctg 19 <210> 36 <211> twenty two <212> DNA <213> Artificial Sequence <400> 36 tcgcactgga tacgacgtcc tg 22 <210> 37 <211> 50 <212> DNA <213> Artificial Sequence <400> 37 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgcctaa 50 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <400> 38 actctctggg cctgtgtc 18 <210> 39 <211> twenty two <212> DNA <213> Artificial Sequence <400> 39 tcgcactgga tacgacgcct aa 22 <210> 40 <211> 50 <212> DNA <213> Artificial Sequence <400> 40 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacctagag 50 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <400> 41 agcctccgtg ttacctgtc 19 <210> 42 <211> twenty four <212> DNA <213> Artificial Sequence <400> 42 tcgcactgga tacgacctag agga 24 <210> 43 <211> 50 <212> DNA <213> Artificial Sequence <400> 43 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgagccg 50 <210> 44 <211> 17 <212> DNA <213> Artificial Sequence <400> 44 cctctctcgg ctcctcg 17 <210> 45 <211> twenty two <212> DNA <213> Artificial Sequence <400> 45 tcgcactgga tacgacgagc cg 22 <210> 46 <211> 50 <212> DNA <213> Artificial Sequence <400> 46 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacccatct 50 <210> 47 <211> 18 <212> DNA <213> Artificial Sequence <400> 47 gcgactggac ttggtgtc 18 <210> 48 <211> twenty three <212> DNA <213> Artificial Sequence <400> 48 tcgcactgga tacgacccat ctg 23 <210> 49 <211> 50 <212> DNA <213> Artificial Sequence <400> 49 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgggcgg 50 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <400> 50 agcatcggga atgtcgtgt 19 <210> 51 <211> twenty one <212> DNA <213> Artificial Sequence <400> 51 tcgcactgga tacgacgggc g 21 <210> 52 <211> 50 <212> DNA <213> Artificial Sequence <400> 52 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactattca 50 <210> 53 <211> twenty one <212> DNA <213> Artificial Sequence <400> 53 tccgcttttg caatatgttc c 21 <210> 54 <211> 26 <212> DNA <213> Artificial Sequence <400> 54 ttcgcactgg atacgactat tcagga 26 <210> 55 <211> 50 <212> DNA <213> Artificial Sequence <400> 55 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactctgca 50 <210> 56 <211> 17 <212> DNA <213> Artificial Sequence <400> 56 ccggtcccag gagaacc 17 <210> 57 <211> twenty two <212> DNA <213> Artificial Sequence <400> 57 tcgcactgga tacgactctg ca 22 <210> 58 <211> 50 <212> DNA <213> Artificial Sequence <400> 58 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacaagacg 50 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <400> 59 cgctcactcc tctcctcc 18 <210> 60 <211> twenty three <212> DNA <213> Artificial Sequence <400> 60 tcgcactgga tacgacaaga cgg 23 <210> 61 <211> 50 <212> DNA <213> Artificial Sequence <400> 61 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactgaatc 50 <210> 62 <211> 18 <212> DNA <213> Artificial Sequence <400> 62 acaatgcacc tgggcaag 18 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <400> 63 ttcgcactgg atacgactga atcct 25 <210> 64 <211> 50 <212> DNA <213> Artificial Sequence <400> 64 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgggctc 50 <210> 65 <211> 19 <212> DNA <213> Artificial Sequence <400> 65 cgagtgcctg agggagtaa 19 <210> 66 <211> twenty two <212> DNA <213> Artificial Sequence <400> 66 tcgcactgga tacgacgggc tc 22 <210> 67 <211> 50 <212> DNA <213> Artificial Sequence <400> 67 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccaaaag 50 <210> 68 <211> twenty one <212> DNA <213> Artificial Sequence <400> 68 cgcgctttag gataagcttg a 21 <210> 69 <211> 25 <212> DNA <213> Artificial Sequence <400> 69 ttcgcactgg atacgaccaa aagtc 25 <210> 70 <211> 50 <212> DNA <213> Artificial Sequence <400> 70 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactctcgg 50 <210> 71 <211> 17 <212> DNA <213> Artificial Sequence <400> 71 tgaagcgcct gtgctct 17 <210> 72 <211> twenty three <212> DNA <213> Artificial Sequence <400> 72 tcgcactgga tacgactctc ggc 23 <210> 73 <211> 50 <212> DNA <213> Artificial Sequence <400> 73 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagaggc 50 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <400> 74 acgctccatt acactaccct 20 <210> 75 <211> twenty two <212> DNA <213> Artificial Sequence <400> 75 tcgcactgga tacgacagag gc 22 <210> 76 <211> 17 <212> DNA <213> Artificial Sequence <400> 76 ctcgcttcgg cagcaca 17 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <400> 77 aacgcttcac gaatttgcgt 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <400> 78 aacgcttcac gaatttgcgt 20 <210> 79 <211> 25 <212> DNA <213> Artificial Sequence <400> 79 agaagattag catggcccct gcgca 25 <210> 80 <211> 16 <212> DNA <213> Artificial Sequence <400> 80 gtgcagggtc cgaggt 16

Claims

1. Use of primers and probes for detecting plasma exosome miRNA markers in the preparation of a lung cancer diagnostic kit, characterized in that: The exosomal miRNA marker is a combination of miR-106b-3p, miR-125a-5p, miR-3615, miR-450b-5p, and let-7a-3p.

2. The use according to claim 1, characterized in that The primers and probes include: Reverse transcription primers, PCR primers, and probes for detecting let-7a-3p: the let-7a-3p reverse transcription primer has the nucleotide sequence shown in SEQ ID NO: 1, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 2, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 3; Reverse transcription primers, PCR primers, and probes for detecting miR-106b-3p: the reverse transcription primer for miR-106b-3p has the nucleotide sequence shown in SEQ ID NO: 7, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 8, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 9; Reverse transcription primers, PCR primers, and probes for detecting miR-125a-5p: the reverse transcription primer for miR-125a-5p has the nucleotide sequence shown in SEQ ID NO: 16, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 17, the downstream primer has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 18; Reverse transcription primers, PCR primers, and probes for detecting miR-3615: the reverse transcription primer for miR-3615 has the nucleotide sequence set forth in SEQ ID NO: 43, the upstream PCR primer has the nucleotide sequence set forth in SEQ ID NO: 44, the downstream primer has the nucleotide sequence set forth in SEQ ID NO: 80, and the probe has the nucleotide sequence set forth in SEQ ID NO: 45; Reverse transcription primers, PCR primers and probes for detecting miR-450b-5p: the reverse transcription primer of miR-450b-5p has a nucleotide sequence as shown in sequence number 52, the upstream PCR primer has a nucleotide sequence as shown in sequence number 53, the downstream primer has a nucleotide sequence as shown in sequence number 80, and the probe has a nucleotide sequence as shown in sequence number 54.

3. Use of primers and probes for detecting plasma exosome miRNA markers in the preparation of a device for lung cancer diagnosis, characterized in that: The exosomal miRNA marker is a combination of miR-106b-3p, miR-125a-5p, miR-3615, miR-450b-5p, and let-7a-3p.

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