Applications of exosomal miR-106b-3p, miR-550-5p, etc. in the diagnosis of lung cancer

Abstract

The present invention discloses the application of exosomal miR-106b-3p, miR-550-5p, etc. in the diagnosis of lung cancer. The kit includes primers and probes for detecting exosomal miRNA markers, and the exosomal miRNA markers include one or more of 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. The present invention provides a non-invasive method for diagnosing lung cancer based on exosomes, which has high sensitivity and high specificity in lung cancer, provides important value for the early diagnosis and recurrence monitoring of lung cancer, and is of great help to the prevention and treatment of lung cancer in China.

Description

[0001] This application is a divisional application. The divisional application number is 202111059955.0, the application date is May 11, 2020, and the invention title is "Kit, Device and Method for Lung Cancer Diagnosis". Technical Field

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

[0003] With the application of low-dose spiral CT, more and more imaging findings of pulmonary nodules (single lesions within the pulmonary interstitium < 3 cm and without related atelectasis or lymphadenopathy) have been discovered. However, not all pulmonary nodules are malignant, and the differential diagnosis of 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 used, but their detection sensitivity in the diagnosis of early lung cancer is not high; therefore, there is an urgent need to develop a method for non-invasive early detection of lung cancer with high sensitivity. Summary of the Invention

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

[0005] On the one hand, the present invention provides a kit for lung cancer diagnosis, including primers and probes for detecting exosomal miRNA markers, and the exosomal miRNA markers include one or more of 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.

[0006] Preferably, the exosomal miRNA markers are 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, miR-885-5p.

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

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

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

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

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

[0012] Preferably, the primers and probes include:

[0013] Reverse transcription primer, PCR primer, and probe for detecting let-7a-3p: The reverse transcription primer for let-7a-3p 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 primer, PCR primer, and probe for detecting let-7f-2: The reverse transcription primer for let-7f-2 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 primer, PCR primer, and probe for detecting miR-106b-3p: The reverse transcription primer for miR-106b-3p has the nucleotide sequence shown in SEQ ID NO: 7, the PCR upstream 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 PCR upstream 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 PCR upstream 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 PCR upstream 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 shown in SEQ ID NO: 19, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 20, 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: 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 shown in SEQ ID NO: 22, the PCR upstream 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;

[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 PCR upstream 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 PCR upstream 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 of miR-30e-5p has the nucleotide sequence shown in SEQ ID NO: 31, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 32, 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: 33;

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

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

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

[0027] Reverse transcription primers, PCR primers and probes for detecting miR-3615: The reverse transcription primer of miR-3615 has the nucleotide sequence shown in SEQ ID NO: 43, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 44, 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: 45;

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

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

[0030] Reverse transcription primers, PCR primers and probes for detecting miR-450b-5p: The reverse transcription primer of 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;

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

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

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

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

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

[0036] Reverse transcription primers, PCR primers and probes for detecting miR-7706: The reverse transcription primer of miR-7706 has the nucleotide sequence shown in SEQ ID NO: 70, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 71, 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: 72;

[0037] Reverse transcription primers, PCR primers and probes for detecting miR-885-5p: The reverse transcription primer of miR-885-5p has the nucleotide sequence shown in SEQ ID NO: 73, the upstream PCR primer has the nucleotide sequence shown in SEQ ID NO: 74, 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: 75.

[0038] On the other hand, the present invention provides a device for diagnosing lung cancer, comprising reagents for detecting exosomal miRNA markers, and the exosomal miRNA markers include one or more of 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.

[0039] On the other hand, the present invention provides a method for diagnosing lung cancer, which includes detecting the specificity of exosomal miRNA markers, and the exosomal miRNA markers include one or more of 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.

[0040] The present invention provides a non-invasive method for diagnosing lung cancer based on exosomes, which has high sensitivity and high specificity in lung cancer, provides important value for the early diagnosis and recurrence monitoring of lung cancer, and is of great help to the prevention and treatment of lung cancer in China. Further, among them, 5 miRNA markers (the combined AUC can reach up to 0.951, and its negative predictive value is 90.32%, sensitivity is 90.00%, and specificity is 93.33%) have extremely excellent diagnostic performance. Brief Description of the Drawings

[0041] Figure 1 It is the result of electron microscopy identification of exosomes.

[0042] Figure 2 It is the ROC curve of miR-3615 for single detection of lung cancer.

[0043] Figure 3 It is the ROC curve of miR-502-3p for single detection of lung cancer.

[0044] Figure 4 It is the ROC curve of miR-450b-5p for single detection of lung cancer.

[0045] Figure 5 It is the ROC curve of miR-4746-5p for single detection of lung cancer.

[0046] Figure 6 It is the ROC curve of miR-10a-5p for single detection of lung cancer.

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

[0048] Figure 8 The ROC curve for the combined detection of lung cancer by miR-106b-3p + miR-3615 + miR-450b-5p.

[0049] Figure 9 The ROC curve for the combined detection of lung cancer by miR-106b-3p + miR-125a-5p + miR-3615 + miR-450b-5p + miR-885-5.

[0050] Figure 10 The ROC curve for the combined detection of lung cancer by miR-106b-3p + miR-10a-3p + miR-125a-5p + miR-3615 + miR-450b-5p.

[0051] Figure 11 The ROC curve for the combined detection of lung cancer by miR-106b-3P + miR-10a-5p + miR-125a-5p + miR-3615 + miR-450b-5p (using U6 as a reference). Detailed implementation manner

[0052] Extracellular Vesicles (EVs; hereinafter, vesicles all represent extracellular vesicles) refer to vesicular bodies with a double-membrane structure that are shed from the cell membrane or secreted by cells, with diameters ranging from 30 to 1000 nm. Extracellular vesicles mainly consist of MicroVesicles (MVs) and exosomes. Microvesicles are small vesicles shed from the cell membrane after cell activation or injury. Due to the unique biological characteristics of extracellular vesicles, they have important significance in disease diagnosis, especially exosomes among them.

[0053] Exosomes are membranous small vesicles with a diameter between 30 and 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 information transmission and play important roles in antigen presentation, apoptosis, inflammatory responses, tumorigenesis, development, and metastasis. They are widely distributed in body fluids, including blood, saliva, urine, milk, and pleural and peritoneal effusions, etc.; contain various inclusions such as DNA, RNA, and proteins, and can be used as non-invasive diagnostic markers for various diseases such as tumors. miRNA is the most abundant nucleic acid component in exosomes, so exosomal miRNA has the potential for early diagnosis of lung cancer.

[0054] The kit, device, and method provided by the present invention use one or more of the miRNAs that show significant differential expression in the exosomes of patients with early lung cancer found through experimental research as markers for diagnosing early lung cancer.

[0055] The 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-3158-3p, 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.

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

[0057] In addition, the kit for diagnosing lung cancer 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 primer, PCR primer and probe for detecting let-7a-3p: The reverse transcription primer of let-7a-3p has the nucleotide sequence shown in SEQ ID NO: 1, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 2, the downstream primer specifically 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 primer, PCR primer and probe for detecting let-7f-2: The reverse transcription primer of let-7f-2 has the nucleotide sequence shown in SEQ ID NO: 4, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 5, the downstream primer specifically 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 of miR-106b-3p has the nucleotide sequence shown in SEQ ID NO: 7, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 8, the downstream primer specifically 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 of miR-10a-3p has the nucleotide sequence shown in SEQ ID NO: 10, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 11, the downstream primer specifically 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 of miR-10a-5p has the nucleotide sequence shown in SEQ ID NO: 13, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 14, the downstream primer specifically 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 of miR-125a-5p has the nucleotide sequence shown in SEQ ID NO: 16, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 17, the downstream primer specifically 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 of miR-1294 has the nucleotide sequence shown in SEQ ID NO: 19, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 20, the downstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 21;

[0065] Reverse transcription primers, PCR primers and probes for detecting miR-19a-3p: The reverse transcription primer of miR-19a-3p has the nucleotide sequence shown in SEQ ID NO: 22, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 23, the downstream primer specifically 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 of miR-22-3p has the nucleotide sequence shown in SEQ ID NO: 25, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 26, the downstream primer specifically 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 of miR-29a-3p has the nucleotide sequence shown in SEQ ID NO: 28, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 29, the downstream primer specifically 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 of miR-30e-5p has the nucleotide sequence shown in SEQ ID NO: 31, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 32, the downstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 33;

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

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

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

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

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

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

[0075] Reverse transcription primers, PCR primers and probes for detecting miR-450b-5p: The reverse transcription primer of miR-450b-5p has the nucleotide sequence shown in SEQ ID NO: 52, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 53, the downstream primer specifically 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 of miR-4746-5p has the nucleotide sequence shown in SEQ ID NO: 55, the PCR upstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 56, the downstream primer specifically has the nucleotide sequence shown in SEQ ID NO: 80, and the probe has the nucleotide sequence shown in SEQ ID NO: 57;

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

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

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

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

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

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

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

[0084] Table 1

[0085]

[0086]

[0087]

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

[0089] The kits, devices, and methods of the present invention are applicable to individuals who are at high risk of lung cancer, normal individuals, and patients after lung cancer surgery.

[0090] The technical solutions of the present invention will be described completely and clearly below in conjunction with the embodiments. The described embodiments are some, but not all, of the embodiments 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.

[0091] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0092] To screen for exosomal markers related to the diagnosis of lung cancer, 50 early-stage lung cancer patients and 72 controls were recruited. At least 10 ml of blood was collected and plasma was separated. The exosomes in the plasma were isolated by the classical ultracentrifugation method and RNA was extracted. The obtained RNA was used for RNA library construction and sequencing respectively. The obtained data were subjected to bioinformatics analysis to compare the differentially expressed RNAs in early-stage lung cancer patients and controls. These mRNA-level markers from exosomes can be used for the early diagnosis of lung cancer.

[0093] Furthermore, the application analysis of the RNA markers is as follows: (1) Collect body fluid samples (including blood, sputum, and saliva) of the individual to be tested; (2) Isolate exosomes from the body fluid; (3) Extract exosomal RNA using spkin external reference cel-miR-39; (4) Detect the expression level of the target RNA using a two-step method; (5) Normalize the expression level of the detected target RNA using an external reference gene; (6) Substitute the normalized gene expression level into the determination model to obtain an output value; (7) Determine whether the individual to be tested has lung cancer based on the output value of the model and the determination threshold.

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

[0095] The method includes selecting an external reference Cel-miR-39 or an internal reference U6 for quantification of the target RNA. When the reference is selected, the quantification of the target RNA is calculated according to the detection Ct value using the quantitative formula 2ΔΔCt to calculate the expression level of the marker. After obtaining the expression level of the target RNA, the ROC characteristic curve and AUC are used to evaluate the accuracy of single RNA or combined multiple RNAs in detecting lung cancer.

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

[0097] In order to screen for exosome markers related to early lung cancer diagnosis, 72 patients with early lung cancer diagnosis and 50 controls were collected, and no less than 10 ml of blood was collected and plasma was separated. The exosomes in the plasma were separated by the classical ultracentrifugation method, and RNA was extracted using the qiagen miRNeasy mini kit. The obtained RNA was sequenced for small RNA library construction. The obtained data were analyzed by bioinformatics, and the differentially expressed miRNAs in early lung cancer patients and controls were compared. The significantly different miRNAs were shown in Table 2 below. These RNA-level markers from exosomes can be used for 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] The miRNA reverse transcription reagent, enzyme and oligodT were purchased from TAKARA, the standard was synthesized by Shanghai Invitrogen, and the specific reverse primer was synthesized by Suzhou Hongxun. A 20ul 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., the universal downstream primer, were synthesized by Suzhou Hongxun, and the fluorescence quantitative PCR instrument was ABI 7500. The PCR reaction system is shown in Table 4 below.

[0108] Table 4

[0109]

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

[0111] Evaluation of the detection effect of early lung cancer diagnosis using Cel-miR-39 as a reference single biomarker other than Example 3

[0112] 1. Sample collection

[0113] Collect 10 ml of blood from patients with early-stage (stage I and II) pulmonary nodule lung cancer, patients with benign pulmonary nodules, and healthy controls diagnosed by the hospital, and separate it into plasma.

[0114] 2. Extraction of exosomal RNA

[0115] Ultracentrifugation or Exosupur from Echobiotech (Beijing Enzekangtai) was used to isolate plasma exosomes (the results of exosome electron microscopy identification are as Figure 1 ). The miRNA in the isolated exosomes was extracted using the Qiagen miReasy mini kit, and the RNA concentration and quality were detected using an Agilent 2100, and the RNA concentration was recorded.

[0116] 3. Two-step RNA detection system

[0117] The two-step detection system for miRNA based on the PCR platform in Example 1 was used to detect the plasma exosomal miRNA of 30 early lung cancer patients 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 according to the Ct value and the relative quantification formula.

[0118] 4. Performance evaluation of exosomal miRNA in diagnosing early lung cancer

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

[0120] As Figure 2As shown, the Ct values of miR-3615 in the plasma exosomes of 30 early lung cancer patients and 30 control samples (healthy people and benign lesions) were detected. Using Cel-miR-39 as an external reference, the copy number of miRNA was obtained according to the Ct value. The relative expression level of the combined marker was calculated using the relative quantification formula value, and then the relative expression level of RNA was obtained. The t-test analysis of the test results using R language showed that pvalue = 0.00058 <= 0.05, indicating that exosomal miR-3615 was significantly associated with early lung cancer. The AUC of miR-3615 in the diagnosis of early lung cancer alone was 0.785, with a negative predictive value of 85%, a sensitivity of 90%, and a specificity of 56.57%, showing the potential of a diagnostic marker.

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

[0122] As Figure 3 shown, the Ct values of miR-502-3p in the plasma exosomes of 30 early lung cancer patients and 30 control samples (healthy people and benign lesions) were detected. Using Cel-miR-39 as an external reference, the copy number of miRNA was obtained according to the Ct value. The relative expression level of the combined marker was calculated using the relative quantification formula value, and then the relative expression level of RNA was obtained. The t-test analysis of the test results using R language showed that pvalue = 0.00139 <= 0.05, indicating that exosomal miR-502-3p was significantly associated with early lung cancer. The AUC of miR-502-3p in the diagnosis of early lung cancer alone was 0.756, with a negative predictive value of 68.42%, a sensitivity of 60%, and a specificity of 86.67%, showing the potential of a diagnostic marker.

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

[0124] As Figure 4 shown, the Ct values of miR-450b-5p in the plasma exosomes of 30 early lung cancer patients and 30 control samples (healthy people and benign lesions) were detected. Using Cel-miR-39 as an external reference, the copy number of miRNA was obtained according to the Ct value. The relative expression level of the combined marker was calculated using the relative quantification formula value, and then the relative expression level of RNA was obtained. The t-test analysis of the test results using R language showed that pvalue = 0.00199 <= 0.05, indicating that exosomal miR-450b-5p was significantly associated with early lung cancer. The AUC of miR-450b-5p in the diagnosis of early lung cancer alone was 0.744, with a negative predictive value of 80%, a sensitivity of 86.67%, and a specificity of 53.33%, showing the potential of a diagnostic marker.

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

[0126] As Figure 5 shown, the Ct values of miR-4746-5p were detected in the plasma exosomes of 30 early lung cancer patients and 30 control samples (healthy people and benign lesions). Using Cel-miR-39 as an external reference, the copy number of miRNA was obtained according to the Ct value. Using the relative quantitative formula value, the fold change of the relative expression level of the combined marker was calculated, and then the relative expression level of RNA was obtained. The t-test analysis was performed on the test results using R language, and pvalue = 0.00183 <= 0.05, indicating that exosomal miR-4746-5p was significantly correlated with early lung cancer. The AUC of miR-4746-5p for the diagnosis of early lung cancer alone was 0.743, its negative predictive value was 66.67%, sensitivity was 60%, and specificity was 80%, showing the potential of a diagnostic marker.

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

[0128] As Figure 6 shown, the Ct values of miR-10a-5p were detected in the plasma exosomes of 30 early lung cancer patients and 30 control samples (healthy people and benign lesions). Using Cel-miR-39 as an external reference, the copy number of miRNA was obtained according to the Ct value. Using the relative quantitative formula value, the fold change of the relative expression level of the combined marker was calculated, and then the relative expression level of RNA was obtained. The t-test analysis was performed on the test results using R language, and pvalue = 0.00183 <= 0.05, indicating that exosomal miR-10a-5p was significantly correlated with early lung cancer. The AUC of miR-10a-5p for the diagnosis of early lung cancer alone was 0.73, its negative predictive value was 77.27%, sensitivity was 83.33%, and specificity was 56.67%, showing the potential of a diagnostic marker.

[0129] (6) Performance evaluation of other markers significantly correlated 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] It can be seen from the data shown in Table 5 that the miRNAs described in the table all have the potential of diagnostic markers.

[0135] Evaluation of the Detection Effect of Multi-Marker Combinations for Early Diagnosis of Lung Cancer with Cel-miR-39 as a Reference Except for Example 4

[0136] 1. Performance Evaluation of Three-Marker Combinations

[0137] Calculate the relative expression levels of each miRNA according to the method in Example 3, and use logistic regression to train the three-marker combinations. The combinations with an AUC of more than 0.85 for the three-marker combinations are shown in Table 6 below. Among them, the two combinations miR-106-3p + miR-125a-5p + miR-3615 and miR-106b-3p + miR-3615 + miR-450b-5p have the best performance, with AUC values of 0.887 and 0.881 respectively. Their AUC curves are shown in Figure 7 and Figure 8 respectively.

[0138] Table 6

[0139]

[0140]

[0141] 2. Performance Evaluation of Five-Marker Combinations

[0142] Calculate the relative expression levels of each miRNA according to the method in Example 3, and use logistic regression to train the five-marker combinations. The combinations with an AUC of more than 0.93 for the five-marker combinations are shown in Table 7 below. Among them, the two combinations 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 have the best performance, with AUC values of 0.951 and 0.948 respectively. Their AUC curves are shown in Figure 9 and Figure 10 respectively.

[0143] Table 7

[0144]

[0145]

[0146] Evaluation of the Detection Effect of Multi-Marker Combinations for Early Diagnosis of Lung Cancer with U6 as a Reference within Example 5

[0147] Calculate the relative expression level of each miRNA with U6 as the reference according to the method in Example 3, and use logistic regression to train the five biomarker combinations. The combinations with an AUC of more than 85 for the five-biomarker combination are shown in Table 8 below. Among them, the combination of mmiR-106b-3P + miR-10a-5p + miR-125a-5p + miR-3615 + miR-450b-5p has the best performance, with an AUC of 0.864. Its AUC curve is respectively as Figure 11 shown.

[0148] Table 8

[0149]

[0150]

[0151] It can be seen from the above data that the lung cancer detection method based on exosomal miRNA markers of the present invention can achieve non-invasive diagnosis of lung cancer, provides important value for the early diagnosis and recurrence monitoring of lung cancer, and is of great help to the prevention and treatment of lung cancer in China. Among them, 5 miRNA markers (the combined AUC can reach up to 0.951, with a negative predictive value of 90.32%, a sensitivity of 90.00%, and a specificity of 93.33%) have extremely excellent diagnostic performance.

[0152] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principle and practical application of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents. Sequence Listing <110> Cancer Hospital, Chinese Academy of Medical Sciences <120> Application of Exosomal miR-106b-3p, miR-550-5p, 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> 21 <212> DNA <213> Artificial Sequence <400> 2 cgcgccctat acaatctact g 21 <210> 3 <211> 24 <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> 21 <212> DNA <213> Artificial Sequence <400> 5 agcgcctata cagtctactg t 21 <210> 6 <211> 24 <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> 22 <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> 21 <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> 24 <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> 21 <211> 24 <212> DNA <213> Artificial Sequence <400> 21 ttcgcactgg atacgacaga caac 24 <210> 22 <211> 50 <212> DNA <213> Artificial Sequence <400> 22 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactcagtt 50 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <400> 23 cgctgtgcaa atctatgcaa 20 <210> 24 <211> 25 <212> DNA <213> Artificial Sequence <400> 24 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> 24 <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> 24 <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> 21 <212> DNA <213> Artificial Sequence <400> 32 accgctgtaa acatccttga c 21 <210> 33 <211> 22 <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> 22 <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> 22 <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> 24 <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> 22 <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> 23 <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> 21 <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> 21 <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> 22 <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> 23 <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> 22 <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> 21 <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> 23 <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> 22 <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 a primer and a probe for detecting plasma exosomal miRNA markers in the preparation of a lung cancer diagnostic kit, characterized in that, The exosomal miRNA markers are a combination of miR-106b-3p, miR-125a-5p, miR-3615, miR-450b-5p, and miR-550-5p.

2. The application according to claim 1, wherein The primers and probes include: Reverse transcription primer, PCR primers, and probe for detecting miR-106b-3p: The reverse transcription primer for miR-106b-3p has the nucleotide sequence shown in SEQ ID NO: 7, the PCR upstream 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 primer, PCR primers, and probe for detecting miR-125a-5p: The reverse transcription primer for miR-125a-5p has the nucleotide sequence shown in SEQ ID NO: 16, the PCR upstream 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 primer, PCR primers, and probe for detecting miR-3615: The reverse transcription primer for miR-3615 has the nucleotide sequence shown in SEQ ID NO: 43, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 44, 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: 45; Reverse transcription primer, PCR primers, and probe for detecting miR-450b-5p: The reverse transcription primer for miR-450b-5p has the nucleotide sequence shown in SEQ ID NO: 52, the PCR upstream 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; Reverse transcription primer, PCR primers, and probe for detecting miR-550a-5p: The reverse transcription primer for miR-550a-5p has the nucleotide sequence shown in SEQ ID NO: 64, the PCR upstream primer has the nucleotide sequence shown in SEQ ID NO: 65, 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:

66.

3. Use of a primer and a probe for detecting miRNA markers in plasma exosomes in the preparation of a device for diagnosing lung cancer, characterized in that, The exosomal miRNA markers are a combination of miR-106b-3p, miR-125a-5p, miR-3615, miR-450b-5p, and miR-550-5p.

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