CN114214423A

CN114214423A - Kit, device and method for diagnosis of lung cancer

Info

Publication number: CN114214423A
Application number: CN202210144702.1A
Authority: CN
Inventors: 赫捷; 高树庚; 高亦博; 郭威
Original assignee: Cancer Hospital and Institute of CAMS and PUMC
Current assignee: Cancer Hospital and Institute of CAMS and PUMC
Priority date: 2020-03-30
Filing date: 2020-05-11
Publication date: 2022-03-22
Anticipated expiration: 2040-05-11
Also published as: CN113774138A; CN111455057A; CN113930505A; CN114231638A; CN113774138B; CN114214420B; CN114214423B; CN114214419B; CN114214420A; CN114214422A; CN114231638B; CN114214419A; CN114214421A; CN113801936A; CN111455057B; CN113930505B; CN113801936B; CN114410794A; CN114410794B; CN114214421B

Abstract

The present invention discloses a kit, device and method for lung cancer diagnosis, wherein the kit includes primers and probes for detecting the combination of exosome long RNA markers and miRNA markers, and the long RNA markers include ARPC5, MBOAT2 , one or more of IL1B, the miRNA markers include miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, One or more of miR-4746-5p, miR-125a-5p. The exosome-based non-invasive early-stage lung cancer diagnosis method of the present invention has high sensitivity and high specificity in early-stage lung cancer, and provides important value for early-stage lung cancer diagnosis. It is of great help to the prevention and treatment of lung cancer in my country.

Description

Kit, device and method for lung cancer diagnosis

The invention relates to a kit, a device and a method for diagnosing lung cancer, which are divided applications, wherein the application number of a parent application is 202111109658.2, the application date is 2020 and 05 and 11 days.

Technical Field

The invention relates to the field of medical diagnosis, in particular to a diagnostic kit, a device and a method for early lung cancer.

Background

Lung cancer is one of the main cancer species in China and even in the world. According to the latest global cancer statistics in 2018, the incidence and mortality of lung cancer are the first in all cancer species. According to the Chinese tumor registration data of 2019, 390.2 ten thousand cancer cases are newly added in 2015 of China, and the death cases of cancer are about 233.8 ten thousand, wherein lung cancer is the main cause of cancer death in China. Therefore, in order to improve the diagnosis and treatment behaviors of lung cancer in China and improve the prognosis of lung cancer patients, early diagnosis becomes an important problem for diagnosis and treatment of lung cancer.

With the application of low dose helical CT, more and more imaging is shown with lung nodules (single lesions <3cm in the lung interstitium and no associated atelectasis or lymphadenopathy) being discovered. However, not all lung nodules are malignant, and identification of benign and malignant lung nodules has been a difficult point in thoracic surgical clinical diagnosis and treatment. At present, noninvasive detection means such as plasma circulating tumor cells and circulating tumor free DNA are adopted, but the detection sensitivity in early lung cancer diagnosis is not high; therefore, there is a need to develop a highly sensitive method for noninvasive early detection of lung cancer.

Disclosure of Invention

The invention provides an exosome-based reagent, an exosome-based device and an exosome-based method for non-invasive early lung cancer diagnosis.

The invention provides a kit for lung cancer diagnosis, which comprises primers and probes for detecting a long RNA marker of an exosome and miRNA marker combination, wherein the long RNA marker comprises one or more of ARPC5, MBOAT2 and IL1B, and the miRNA marker comprises one or more of miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p and miR-125a-5 p.

Preferably, the marker is a combination of miR-450b-5p, let-7f-2-3p and ARPC 5.

Preferably, the marker is a combination of miR-106b-3P, miR-30e-5p and MBOAT 2.

Preferably, the marker is a combination of miR-106b-3P, miR-30e-5p, miR-3615, miR-885-5p and ARPC 5.

Preferably, the marker is a combination of miR-106b-3P, miR-125a-5p, miR-3615, miR-450b-5p and IL 1B. .

Preferably, the source of exosomes comprises one or more of blood, saliva and sputum.

Preferably, the primers and probes comprise:

primers and probes for detection of internal reference ACTB: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 1, the downstream primer is a nucleotide sequence shown as a sequence number 2, and the probe is a nucleotide sequence shown as a sequence number 3;

primers and probes for detection of ARPC 5: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 4, the downstream primer is a nucleotide sequence shown as a sequence number 5, and the probe is a nucleotide sequence shown as a sequence number 6;

primers and probes for detection of IL 1B: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 7, the downstream primer is a nucleotide sequence shown as a sequence number 8, and the probe is a nucleotide sequence shown as a sequence number 9;

primers and probes for detection of MBOAT 2: the upstream primer of the RNA is a nucleotide sequence shown as a sequence number 10, the downstream primer is a nucleotide sequence shown as a sequence number 11, and the probe is a nucleotide sequence shown as a sequence number 12;

reverse transcription primers, PCR primers and probes for detecting let-7 f-2: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 13, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 14, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 15;

reverse transcription primers, PCR primers and probes for detecting miR-106b-3 p: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 16, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 17, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 18;

reverse transcription primers, PCR primers and probes for detecting miR-125a-5 p: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 19, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 20, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 21;

reverse transcription primers, PCR primers and probes for detecting miR-30e-5 p: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 22, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 23, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 24;

reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 25, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 26, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 27;

reverse transcription primers, PCR primers and probes for detecting miR-450b-5 p: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 28, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 29, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 30;

reverse transcription primers, PCR primers and probes for detecting miR-4746-5 p: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 31, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 32, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 33;

reverse transcription primers, PCR primers and probes for detecting miR-885-5 p: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 34, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 35, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 36;

reverse transcription primers, PCR primers and probes for detection of internal reference U6: the reverse transcription primer of U6 has a nucleotide sequence shown as sequence number 39, the PCR upstream primer has a nucleotide sequence shown as sequence number 37, the PCR downstream primer has a nucleotide sequence shown as sequence number 38, and the probe has a nucleotide sequence shown as sequence number 40.

In another aspect, the invention provides a device for lung cancer diagnosis, comprising primers and probes for detecting a combination of long RNA markers of exosomes, including one or more of ARPC5, MBOAT2, IL1B, and miRNA markers, including one or more of miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p, miR-125a-5 p.

In another aspect, the invention provides a method for lung cancer diagnosis comprising detecting the specificity of a combination of exosome long RNA markers comprising one or more of ARPC5, MBOAT2, IL1B and miRNA markers comprising one or more of miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p, miR-125a-5 p.

The invention provides a noninvasive early lung cancer diagnosis method based on exosome, which has high sensitivity and high specificity in early lung cancer and provides important value for early diagnosis of lung cancer. It is helpful for preventing and treating lung cancer in our country. Furthermore, the combination with the highest sensitivity can achieve the sensitivity of 100%, and the combination with the highest specificity can achieve the specificity of 96.67%, so that the combination has better performance.

Drawings

FIG. 1 is a ROC curve of the combination of miR-450b-5p + let-7f-2-3p + ARPC5 for detecting lung cancer.

FIG. 2 is a ROC curve of the combination of miR-106b-3P + miR-30e-5P + MBOAT2 for detecting lung cancer.

FIG. 3 is a ROC curve for the combination of miR-106b-3P + miR-30e-5P + miR-450b-5P + miR-885-5P + ARPC5 to detect lung cancer.

FIG. 4 is a ROC curve for the combination of miR-106b-3P + miR-30e-5P + miR-4746-5P + miR-885-5P + ARPC5 to detect lung cancer.

FIG. 5 is a ROC curve of the combination of miR-106b-3P + miR-30e-5P + miR-3615+ miR-885-5P + ARPC5 for detecting lung cancer.

FIG. 6 is a ROC curve of miR-106b-3P + miR-125a-5P + miR-3615+ miR-450b-5P + IL1B combination for detecting lung cancer.

Detailed Description

The extracellular vesicles (EVs; hereinafter vesicles are all referred to as extracellular vesicles) refer to vesicular bodies with a double-layer membrane structure, which are shed from cell membranes or secreted by cells, and have diameters of 30-1000nm, and mainly comprise MicroVesicles (MVs) and exosomes (exosomes), and the MicroVesicles are vesicles shed from cell membranes after cells are activated or damaged. Extracellular vesicles are of great interest in disease diagnosis, particularly exosomes, due to their unique biological characteristics.

The exosome is a membrane vesicle with the particle size of 30-150 nm secreted into the extracellular environment after an intracellular multivesicular body and a cell membrane are fused, is an important medium for intercellular information transfer, and plays an important role in antigen presentation, apoptosis, inflammatory reaction, tumorigenesis development and metastasis processes. It is widely distributed in body fluid, including blood, saliva, urine, milk, hydrothorax and ascites, etc.; contains various inclusion substances such as DNA, RNA, protein and the like, and can be used as noninvasive diagnosis markers of various diseases such as tumors and the like. And miRNA is the most abundant nucleic acid component in exosome, so exosome miRNA has the potential of being used for early diagnosis of lung cancer.

According to the kit, the device and the method provided by the invention, the combination of long RNA and miRNA which show significant differential expression in exosome of a patient with early lung cancer is found through experimental research to be used as a marker for diagnosing early lung cancer.

In embodiments, the marker combination for diagnosing lung cancer comprises at least one long RNA and one miRNA. Wherein the long RNA comprises: ARPC5, MBOAT2, IL 1B. The MiRNA includes: miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p and miR-125a-5 p.

In some preferred embodiments, wherein the preferred combination is: miR-450b-5P + let-7f-2-3P + ARPC5, miR-106b-3P + miR-30e-5P + MBOAT2, miR-106b-3P + miR-30e-5P + miR-450b-5P + miR-885-5P + ARPC5miR-106b-3P + miR-30e-5P + miR-4746-5P + miR-885-5P + ARPC5, miR-106b-3P + miR-30e-5P + miR-3615+ miR-885P + ARPC5 and miR-106b-3P + miR-125a-5P + miR-3615+ miR-450b-5P + IL 1B. The combination can provide better basis for early diagnosis of lung cancer and indicate the risk of diseases.

In addition, the kit for lung cancer diagnosis of the present invention includes primers and probes for detecting the above exosome-long RNA marker. Primers and probes for detecting exosome markers include:

primers and probes for detection of internal reference ACTB: the upstream primer of the RNA is specifically a nucleotide sequence shown as a sequence number 1, the downstream primer is specifically a nucleotide sequence shown as a sequence number 2, and the probe is specifically a nucleotide sequence shown as a sequence number 3;

primers and probes for detection of ARPC 5: the upstream primer of the RNA is specifically a nucleotide sequence shown as a sequence number 4, the downstream primer is specifically a nucleotide sequence shown as a sequence number 5, and the probe is specifically a nucleotide sequence shown as a sequence number 6;

primers and probes for detection of IL 1B: the upstream primer of the RNA is specifically a nucleotide sequence shown as a sequence number 7, the downstream primer is specifically a nucleotide sequence shown as a sequence number 8, and the probe is specifically a nucleotide sequence shown as a sequence number 9;

primers and probes for detection of MBOAT 2: the upstream primer of the RNA is specifically a nucleotide sequence shown as a sequence number 10, the downstream primer is specifically a nucleotide sequence shown as a sequence number 11, and the probe is specifically a nucleotide sequence shown as a sequence number 12;

reverse transcription primers, PCR primers and probes for detecting let-7 f-2: the reverse transcription primer is specifically a nucleotide sequence shown as a sequence number 13, the PCR upstream primer is specifically a nucleotide sequence shown as a sequence number 14, the downstream primer is specifically a nucleotide sequence shown as a sequence number 41, and the probe is specifically a nucleotide sequence shown as a sequence number 15;

reverse transcription primers, PCR primers and probes for detecting miR-106b-3 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 16, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 17, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 18;

reverse transcription primers, PCR primers and probes for detecting miR-125a-5 p: the reverse transcription primer is specifically a nucleotide sequence shown as a sequence number 19, the PCR upstream primer is specifically a nucleotide sequence shown as a sequence number 20, the PCR downstream primer is specifically a nucleotide sequence shown as a sequence number 41, and the probe is specifically a nucleotide sequence shown as a sequence number 21;

reverse transcription primers, PCR primers and probes for detecting miR-30e-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 22, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 23, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 24;

reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 25, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 26, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 27;

reverse transcription primers, PCR primers and probes for detecting miR-450b-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 28, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 29, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 30;

reverse transcription primers, PCR primers and probes for detecting miR-4746-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown as a sequence number 31, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 32, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 33;

reverse transcription primers, PCR primers and probes for detecting miR-885-5 p: the reverse transcription primer is specifically shown as a nucleotide sequence shown in a sequence number 34, the PCR upstream primer is specifically shown as a nucleotide sequence shown in a sequence number 35, the downstream primer is specifically shown as a nucleotide sequence shown in a sequence number 41, and the probe is specifically shown as a nucleotide sequence shown in a sequence number 36;

reverse transcription primers, PCR primers and probes for detection of internal reference U6: the reverse transcription primer of U6 is specifically shown as a nucleotide sequence shown as a sequence number 39, the PCR upstream primer is specifically shown as a nucleotide sequence shown as a sequence number 37, the downstream primer is specifically shown as a nucleotide sequence shown as a sequence number 38, and the probe is specifically shown as a nucleotide sequence shown as a sequence number 40. The nucleotide sequences of the primers and probes are shown in Table 1.

TABLE 1

Further, the source of exosomes includes one or more of blood, saliva, and sputum.

The kit, the device and the method are suitable for individuals, such as people at high risk of lung cancer, normal individuals and patients after lung cancer operation.

The technical solution of the present invention will be described in detail with reference to the following embodiments, which are a part of the embodiments of the present invention, but not all of them. Other embodiments, which can be derived by one of ordinary skill in the art from the embodiments of the present invention without creative efforts, are within the protection scope of the present invention.

In order to screen an exosome marker related to diagnosis of colon lung cancer, 50 cases of early lung cancer patients and 72 cases of controls respectively take blood of not less than 10ml and separate plasma, the blood is used for separating exosomes in the plasma by a classical ultracentrifugation method and extracting RNA, and the obtained RNA is respectively subjected to RNA library construction and sequencing. The data obtained were analyzed bioinformatically to compare differentially expressed RNA in early lung cancer patients and controls. These exosome-derived RNA-level markers can be used for early diagnosis of lung cancer.

Further directed to the method wherein the RNA marker is further analyzed by the following steps: (1) collecting body fluid samples (including blood, sputum and saliva) of individuals to be detected; (2) isolating exosomes in the body fluid; (3) detecting the expression level of target RNA by using a two-step method; (4) normalizing the expression level of the detection target RNA by using the reference gene; (5) substituting the normalized gene expression level into a judgment model to obtain an output value; (6) and judging whether the individual to be detected is lung cancer or not according to the output value of the model and the judgment threshold value.

The kit comprises a PCR primer, a probe and a standard substance for detecting the long RNA marker of the exosome and a two-step detection system of PCR.

The invention comprises selecting internal reference beta-Actin (ACTB) to carry out the quantification of long RNA, and taking U6 as the internal reference to carry out the quantification of miRNA. Wherein the expression level of the marker is calculated by using a quantitative formula 2 delta Ct according to the detection Ct value by using the quantification of the target RNA when the reference is selected. Having obtained the target RNA expression level, the ROC characteristic curve and AUC were used to assess the accuracy of lung cancer detection by single RNA or by combining multiple RNAs.

Example 1 screening of exosome mRNA and LncRNA markers associated with early lung cancer based on high throughput sequencing

In order to screen an exosome marker related to early lung cancer diagnosis, 50 cases of patients 72 and controls for early lung cancer diagnosis are respectively selected, blood is taken to be not less than 10ml, plasma is separated, exosome in the plasma is separated by a classical ultracentrifugation method, RNA is extracted by a qiagen miRNeasy mini kit, and the obtained RNA is subjected to micro-enucleation ribosome strand specific RNA library-building sequencing and small RNA library-building sequencing. The obtained data were analyzed by bioinformatics, and long RNAs and mirnas differentially expressed in early lung cancer patients and controls were compared to obtain long RNAs with significant differences as shown in table 2 below. These exosome-derived RNA-level markers can be used for early diagnosis of lung cancer.

TABLE 2

RNA type	ID	Pvalue	log2FC	regulated
					mRNA	MBOAT2	4.23E-05	1.023772461	up
mRNA	IL1B	0.000162055	1.050636789	up
					mRNA	ARPC5	0.000871259	1.066858402	up
miRNA	hsa-let-7f-2-3p	0.00027664	-0.446890593	down
					miRNA	hsa-miR-450b-5p	0.000293888	-0.402238286	down
miRNA	hsa-miR-106b-3p	0.001429894	0.189007463	up
					miRNA	hsa-miR-885-5p	0.001455856	-0.589409694	down
miRNA	hsa-miR-30e-5p	0.001592304	-0.329476279	down
					miRNA	hsa-miR-3615	0.00283095	0.419677035	up
miRNA	hsa-miR-4746-5p	0.003524579	0.315578167	up
					miRNA	hsa-miR-125a-5p	0.004298578	-0.492462437	down

Example 2 fluorescent quantitation PCR platform based miRNA detection system

1. miRNA reverse transcription reaction system

miRNA reverse transcription reagents, enzymes and oligdT were purchased from TAKARA, standards were synthesized from Shanghai Yinxie substrate, and primers with reversed specificity were synthesized by Suzhou Hongyu. A20 ul reverse transcription system was used, as shown in Table 3 below.

TABLE 3

2. PCR reaction system

The PCR reaction mixture was purchased from TAKARA, the upstream primer, the probe, i.e., the universal downstream primer, was synthesized by Honghong, Suzhou, and the fluorescent quantitative PCR instrument was ABI 7500. The PCR reaction system is shown in Table 4 below:

TABLE 4

The PCR procedure was 95 ℃ for 10min, (95 ℃ for 15s, 55 ℃ for 30s) for 15 cycles without fluorescence collection, and (95 ℃ for 15s, 55 ℃ for 30s) for 35 cycles with fluorescence collection.

Example 3 fluorescent quantitation-based PCR platform mRNA and LncRNA detection system

1. Reverse transcription of mRNA and LncRNA

PrimeScript from takara was used^TMRT reagent Kit (Perfect Real Time) and Premix Ex Taq^TM(Probe qPCR) kit for reverse transcription and qPCR detection.

Preparing a reverse transcription reaction system (reaction liquid is prepared on ice), then putting the reaction system into a PCR instrument for reaction under the reaction conditions of 37 ℃ for 60min, 85 ℃ for 5s and 12 ℃ for infinity, and adding 50ul DEPC-H after the reverse transcription is finished₂Diluting with O, taking 3ul as a template, and carrying out PCR reaction. The reverse transcription reaction system is shown in Table 5 below.

TABLE 5

2. mRNA and LncRNA qPCR

The qPCR reaction system was prepared as follows (reaction solution was prepared on ice) and a no template control was set as a negative control. Then, the mixture was put into a real-time fluorescence PCR apparatus (ABI7500) to perform amplification detection under the following reaction conditions. The qPCR reaction system is shown in table 6 below and the qPCR reaction conditions are shown in table 7 below.

TABLE 6

TABLE 7

Example 4 evaluation of Effect of Single-marker Lung cancer early diagnosis detection

1. Sample collection

10ml of blood of control samples of early stage (stage I and stage II) lung nodule lung cancer patients, benign lung nodule patients, healthy persons, etc. diagnosed in hospitals were collected and separated into plasma.

2. Exosome RNA extraction

Plasma exosome separation is carried out by ultracentrifugation or Exosuurur of Echobiotech (Beijing Enzekangtai), long RNA in exosome is extracted from the separated exosome by a Qiagen MiReasy mini kit, RNA concentration and quality are detected by Agilent2100, and the RNA concentration is recorded.

3. RNA two-step detection system

A two-step method detection system based on mRNA of a PCR platform in example 1 is adopted to detect plasma exosome mRNA and LncRNA of 30 patients with early lung cancer and 30 control samples (healthy people and benign nodules), the Ct value of target long RNA is detected, and the relative expression amount is calculated according to the Ct value and a relative quantitative formula.

4. Evaluation of exosome RNA single marker diagnosis early lung cancer performance

As shown in the following table, the Ct values of the target genes were detected for plasma exosomes of 30 and 30 control samples (healthy and benign lesions) of early stage lung cancer patients, wherein the long RNA was referenced to the internal reference β -Actin, and the miRNA was referenced to the internal reference U6; and obtaining the relative expression level of the RNA according to the Ct value. And calculating the fold change of the relative expression quantity of the combined marker by using a relative quantitative formula value so as to obtain the relative expression quantity of the RNA. And performing statistical analysis on the detection result by adopting an R language. The performance evaluation of the single marker is shown in the following table 8, wherein miR-450b-5p and miR-3615 have good sensitivity, and let-7f-2-3p has good specificity.

TABLE 8

Example 5 evaluation of Long RNA and miRNA in combination for early diagnosis of Lung cancer

1. Three marker combination performance evaluation

Relative expression levels of each long RNA were calculated according to the method described in example 3, and the three marker combinations were trained using logistic regression, and the combinations with AUC of 0.76 or higher for the three marker combinations were obtained as shown in table 9 below. The miR-450b-5p + let-7f-2-3p + ARPC5 combination has the optimal performance, the accuracy is 76.67%, and the AUC curves are respectively shown in figure 1; the miR-106b-3P + miR-30e-5P + MBOAT2 has better specificity (80%), and the AUC curve is shown in the following figure 1.

TABLE 9

2. Five marker combination performance evaluation

Relative expression levels of each long RNA were calculated according to the method described in example 3, and five marker combinations were trained using logistic regression, and the combinations having AUC of 0.85 or more of the five marker combinations were obtained as shown in table 10 below. Wherein two combinations of miR-106b-3P + miR-30e-5P + miR-450b-5P + miR-885-5P + ARPC5 and miR-106b-3P + miR-30e-5P + miR-4746-5P + miR-885-5P + ARPC5 have good sensitivity which is 93.33% and 100% respectively, and AUC curves are shown in figure 3 and figure 4; the miR-106b-3P + miR-30e-5P + miR-3615+ miR-885-5P + ARPC5 and miR-106b-3P + miR-125a-5P + miR-3615+ miR-450b-5P + IL1B have good specificity of 90% and 96.67% respectively, and AUC curves are shown in figure 5 and figure 6.

Watch 10

The data show that the exosome-based noninvasive early lung cancer diagnosis method has high sensitivity and high specificity in early lung cancer, and provides important value for early diagnosis of lung cancer. It is helpful for preventing and treating lung cancer in our country. The combination with the highest sensitivity can achieve the sensitivity of 100%, and the combination with the highest specificity can achieve the specificity of 96.67%, so that the method has better performance.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

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<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactcacag 50

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cgtccctgag accctttaac 20

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tcgcactgga tacgactcac aggt 24

<210> 22

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccttcca 50

<210> 23

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

accgctgtaa acatccttga c 21

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tcgcactgga tacgaccttc ca 22

<210> 25

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgagccg 50

<210> 26

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cctctctcgg ctcctcg 17

<210> 27

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tcgcactgga tacgacgagc cg 22

<210> 28

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactattca 50

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tccgcttttg caatatgttc c 21

<210> 30

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ttcgcactgg atacgactat tcagga 26

<210> 31

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactctgca 50

<210> 32

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ccggtcccag gagaacc 17

<210> 33

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

tcgcactgga tacgactctg ca 22

<210> 34

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagaggc 50

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

acgctccatt acactaccct 20

<210> 36

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

tcgcactgga tacgacagag gc 22

<210> 37

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ctcgcttcgg cagcaca 17

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

aacgcttcac gaatttgcgt 20

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aacgcttcac gaatttgcgt 20

<210> 40

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

agaagattag catggcccct gcgca 25

<210> 41

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

gtgcagggtc cgaggt 16

Claims

1. The application of primers and probes for detecting exosome long RNA markers and miRNA markers in preparation of lung cancer diagnostic kits is characterized in that the markers are a combination of miR-106b-3P, miR-30e-5p, miR-3615, miR-885-5p and IL 1B.

2. The use according to claim 1, wherein the source of exosomes comprises one or more of blood, saliva and sputum.

3. The use of claim 1, wherein the primers and probes comprise:

reverse transcription primers, PCR primers and probes for detecting miR-885-5 p: the reverse transcription primer is shown as a nucleotide sequence in a sequence number 34, the PCR upstream primer is shown as a nucleotide sequence in a sequence number 35, the PCR downstream primer is shown as a nucleotide sequence in a sequence number 41, and the probe is shown as a nucleotide sequence in a sequence number 36.

4. The application of primers and probes for detecting exosome long RNA markers and miRNA markers in preparation of a lung cancer diagnosis device is characterized in that the markers are a combination of miR-106b-3P, miR-30e-5p, miR-3615, miR-885-5p and IL 1B.