CN111455057A - Kit, device and method for lung cancer diagnosis - Google Patents

Abstract

The invention discloses a kit, a device and a method for diagnosing lung cancer, wherein the kit comprises a primer and a probe for detecting the combination of an exosome long RNA marker and a miRNA marker, the long RNA marker comprises one or more of ARPC5, MBOAT2 and I L1B, 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.

Description

Kit, device and method for diagnosis of lung cancer

technical field

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

Background technique

Lung cancer is one of the major cancers in my country and even in the world. According to the latest global cancer statistics in 2018, lung cancer ranks first in morbidity and mortality among all cancer types. According to the 2019 China Cancer Registry data, there were 3.902 million new cancer cases in China in 2015, and about 2.338 million cancer deaths, among which lung cancer is the main cause of cancer death in China. Therefore, in order to improve the behavior of lung cancer diagnosis and treatment in my country and improve the prognosis of lung cancer patients, early diagnosis has become an important issue in the diagnosis and treatment of lung cancer.

With the use of low-dose helical CT, an increasing number of radiographic findings of pulmonary nodules (single lesions <3 cm in the interstitium without associated atelectasis or lymphadenopathy) have been identified. However, not all pulmonary nodules are malignant, and the identification of benign and malignant pulmonary nodules has always been a difficult point in the clinical diagnosis and treatment of thoracic surgery. At present, there are also non-invasive detection methods using plasma circulating tumor cells, circulating tumor cell-free DNA, etc., but their detection sensitivity in the diagnosis of early lung cancer is not high. Therefore, it is urgent to develop a high-sensitivity non-invasive early detection method for lung cancer.

SUMMARY OF THE INVENTION

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

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

Preferably, the marker is a combination of miR-450b-5p, let-7f-2-3p and ARPC5.

Preferably, the marker is a combination of miR-106b-3P, miR-30e-5p and MBOAT2.

Preferably, the marker is a combination of five of miR-106b-3P, miR-30e-5p, miR-3615, miR-885-5p and ARPC5.

Preferably, the marker is a combination of miR-106b-3P, miR-125a-5p, miR-3615, miR-450b-5p and IL1B. .

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

Preferably, the primers and probes include:

Primers and probes for detecting internal reference ACTB: the upstream primer of the RNA is shown in nucleotide sequence of SEQ ID NO: 1, the downstream primer is shown in nucleotide sequence of SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3. the nucleotide sequence shown;

Primers and probes for detecting ARPC5: the upstream primer of the RNA is shown in the nucleotide sequence shown in SEQ ID NO: 4, the downstream primer is shown in the nucleotide sequence shown in SEQ ID NO: 5, and the probe is shown in SEQ ID NO: 6 nucleotide sequence;

Primers and probes for detecting IL1B: the upstream primer of the RNA is shown in the nucleotide sequence shown in SEQ ID NO: 7, the downstream primer is shown in the nucleotide sequence shown in SEQ ID NO: 8, and the probe is shown in SEQ ID NO: 9 nucleotide sequence;

Primers and probes for detecting MBOAT2: the upstream primer of the RNA is shown in the nucleotide sequence shown in SEQ ID NO: 10, the downstream primer is shown in the nucleotide sequence shown in SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12 nucleotide sequence;

Reverse transcription primers, PCR primers and probes for the detection of let-7f-2: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 13, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 14 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 15;

Reverse transcription primers, PCR primers and probes for detecting miR-106b-3p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 16, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 17 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 18;

Reverse transcription primers, PCR primers and probes for detecting miR-125a-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 19, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 20 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 21;

Reverse transcription primers, PCR primers and probes for detecting miR-30e-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 22, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 23 sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 24;

Reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 25, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 26, The downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 27;

Reverse transcription primers, PCR primers and probes for detecting miR-450b-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 28, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 29 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 30;

Reverse transcription primers, PCR primers and probes for detecting miR-4746-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 31, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 32 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 33;

Reverse transcription primers, PCR primers and probes for detecting miR-885-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 34, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 35 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 36;

Reverse transcription primers, PCR primers and probes for detecting internal reference U6: the reverse transcription primers of the U6 are shown in the nucleotide sequence shown in SEQ ID NO: 39, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 37, The downstream primer is the nucleotide sequence shown in SEQ ID NO: 38, and the probe is the nucleotide sequence shown in SEQ ID NO: 40.

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

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

The invention provides a non-invasive early lung cancer diagnosis method based on exosomes, which has high sensitivity and high specificity in early lung cancer, and provides important value for the early diagnosis of lung cancer. It is of great help to the prevention and treatment of lung cancer in my country. Further, the combination with the highest sensitivity can achieve 100% sensitivity, and the combination with the highest specificity can achieve 96.67% specificity, which has better performance.

Description of drawings

Figure 1 is the ROC curve of the combination of miR-450b-5p+let-7f-2-3p+ARPC5 to detect lung cancer.

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

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

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

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

Figure 6 is the ROC curve of the combination of miR-106b-3P+miR-125a-5p+miR-3615+miR-450b-5p+IL1B to detect lung cancer.

Detailed ways

Extracellular vesicles (Extracellular Vesicles, EVs; the following vesicles all represent extracellular vesicles) refers to vesicle-like bodies with a double-membrane structure shed from the cell membrane or secreted by cells, ranging in diameter from 30-1000nm , Extracellular vesicles are mainly composed of microvesicles (MicroVesicles, MVs) and exosomes (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 are of great significance in the diagnosis of diseases, especially the exosomes in them.

Exosomes are small membranous vesicles with a diameter of 30-150 nm that are secreted into the extracellular environment after fusion of intracellular multivesicular bodies with cell membranes. It plays an important role in apoptosis, inflammatory response, tumor development and metastasis. It is widely distributed in body fluids, including blood, saliva, urine, breast milk, pleural and ascites, etc. It contains various inclusions such as DNA, RNA and protein, and can be used as a non-invasive diagnostic marker for various diseases such as tumors. miRNAs are the most abundant nucleic acid components in exosomes, so exosomal miRNAs have the potential for early diagnosis of lung cancer.

The kit, device and method provided by the present invention use the combination of long RNA and miRNA, which are found to be significantly differentially expressed in exosomes of patients with early-stage lung cancer through experimental research, as a marker for diagnosing early-stage lung cancer.

In an embodiment, the combination of markers for diagnosing lung cancer comprises at least one long RNA and one miRNA. The long RNAs include: ARPC5, MBOAT2, and IL1B. MiRNAs include: miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p, miR-125a-5p.

In some preferred embodiments, 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-885-5p+ARPC5 and miR-106b-3P+miR-125a-5p+miR-3615+miR-450b-5p+IL1B. The above combination can provide a better basis for the early diagnosis of lung cancer and predict the risk of the disease.

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

Primers and probes for detecting the internal reference ACTB: the upstream primer of the RNA is specifically the nucleotide sequence shown in SEQ ID NO: 1, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 2, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 2 The nucleotide sequence shown in No. 3;

Primers and probes for detecting ARPC5: the upstream primer of the RNA is specifically the nucleotide sequence shown in SEQ ID NO: 4, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 5, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 4 The nucleotide sequence shown in 6;

Primers and probes for detecting IL1B: the upstream primer of the RNA is specifically the nucleotide sequence shown in SEQ ID NO: 7, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 8, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 8 The nucleotide sequence shown in 9;

Primers and probes for detecting MBOAT2: the upstream primer of the RNA is specifically the nucleotide sequence shown in SEQ ID NO: 10, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 11, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 11 The nucleotide sequence shown in 12;

Reverse transcription primers, PCR primers and probes for the detection of let-7f-2: the reverse transcription primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 13, and the PCR upstream primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 14. The nucleotide sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 15;

Reverse transcription primers, PCR primers and probes for detecting miR-106b-3p: the reverse transcription primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 16, and the PCR upstream primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 17. The nucleotide sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 18;

Reverse transcription primers, PCR primers and probes for detecting miR-125a-5p: the reverse transcription primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 19, and the PCR upstream primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 20. The nucleotide sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 21;

Reverse transcription primers, PCR primers and probes for detecting miR-30e-5p: the reverse transcription primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 22, and the PCR upstream primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 23. The nucleotide sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 24;

Reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primers are specifically the nucleotide sequence shown in SEQ ID NO: 25, and the PCR upstream primers are specifically the nucleotide sequence shown in SEQ ID NO: 26 Sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 27;

Reverse transcription primers, PCR primers and probes for detecting miR-450b-5p: the reverse transcription primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 28, and the PCR upstream primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 29. The nucleotide sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 30;

Reverse transcription primers, PCR primers and probes for detecting miR-4746-5p: the reverse transcription primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 31, and the PCR upstream primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 32. The nucleotide sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 33;

Reverse transcription primers, PCR primers and probes for detecting miR-885-5p: the reverse transcription primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 34, and the PCR upstream primers are specifically shown in the nucleotide sequence shown in SEQ ID NO: 35. The nucleotide sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 41, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 36;

The reverse transcription primers, PCR primers and probes used to detect the internal reference U6: the reverse transcription primer of the U6 is specifically the nucleotide sequence shown in SEQ ID NO: 39, and the PCR upstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 37 The sequence, the downstream primer is specifically the nucleotide sequence shown in SEQ ID NO: 38, and the probe is specifically the nucleotide sequence shown in SEQ ID NO: 40. The nucleotide sequences of 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 kits, devices and methods of the present invention are suitable for individuals with high risk of lung cancer, normal individuals and patients after lung cancer surgery.

The technical solutions of the present invention will be described in a complete and clear manner below with reference to examples of implementations. The described embodiments are part of the embodiments of the present invention, rather than all examples. Based on the embodiments of the present invention, other embodiments obtained by persons of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In order to screen the exosome markers related to the diagnosis of lung cancer, 50 patients with early-stage lung cancer and 72 controls were collected, and the blood was not less than 10ml, and the plasma was separated. RNA was extracted, and the obtained RNAs were respectively subjected to RNA library construction and sequencing. The obtained data were subjected to bioinformatics analysis to compare differentially expressed RNAs in early-stage lung cancer patients and controls. These exosome-derived RNA-level markers can be used for early diagnosis of lung cancer.

For further application analysis of RNA markers, the method is described as follows: (1) collecting body fluid samples (including blood, sputum and saliva) of the individual to be tested; (2) separating exosomes in body fluids; (3) ) using a two-step method to detect the expression level of the target RNA; (4) using the internal reference gene to normalize the expression level of the detected target RNA; (5) substituting the normalized gene expression level into the judgment model to obtain the output value; (6) ) according to the output value of the model and the judgment threshold whether the individual to be tested is lung cancer.

The kit includes PCR primers, probes, standards and a two-step PCR detection system for detecting exosomal long RNA markers.

The present invention includes selecting internal reference β-Actin (ACTB) to quantify long RNA, and using U6 as internal reference to perform miRNA quantification. The quantification of the target RNA when the reference is selected is based on the detected Ct value using the quantitative formula 2ΔΔCt to calculate the expression level of the marker. After obtaining the target RNA expression level, the ROC characteristic curve and AUC were used to evaluate the accuracy of single RNA or combined multiple RNAs to detect lung cancer.

Example 1 Screening of exosomal mRNA and LncRNA markers related to early lung cancer based on high-throughput sequencing

In order to screen exosome markers related to the diagnosis of early-stage lung cancer, 72 patients with early-stage lung cancer diagnosis and 50 controls were collected, and the blood was not less than 10ml and the plasma was separated. RNA was extracted by qiagen miRNeasy mini kit, and the obtained RNA was subjected to micro-deribosome strand-specific RNA library construction and sequencing and small RNA library construction and sequencing. The obtained data were analyzed by bioinformatics, and the differentially expressed long RNAs and miRNAs in the early stage lung cancer patients and controls were compared, and the significantly different long RNAs were obtained as shown in Table 2 below. These exosome-derived RNA-level markers can be used for early diagnosis of lung cancer.

Table 2

Example 2 miRNA detection system based on fluorescence quantitative PCR platform

1. miRNA reverse transcription reaction system

miRNA reverse transcription reagents, enzymes and oligdT were purchased from TAKARA, the standards were synthesized from Shanghai Yingweijieji, and the specific reverse primers were synthesized by Suzhou Hongxun. A 20ul 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 primers and probes, that is, the universal downstream primers, were synthesized by Suzhou Hongxun, and the fluorescence quantitative PCR instrument was ABI 7500. The PCR reaction system is shown in Table 4 below:

Table 4

The PCR program was 95°C for 10 min, (95°C for 15s; 55°C for 30s) for 15 cycles without collecting fluorescence, and (95°C for 15s; 55°C for 30s) for 35 cycles to collect fluorescence.

Example 3 mRNA and LncRNA detection system based on fluorescence quantitative PCR platform

1. mRNA and LncRNA reverse transcription

Reverse transcription and qPCR detection were performed using takara's PrimeScript ^™ RT reagent Kit (Perfect Real Time) and Premix Ex Taq ^™ (Probe qPCR) kit.

Prepare a reverse transcription reaction system according to the following components (the reaction solution is prepared on ice), and then put it into the PCR machine for the reaction. The reaction conditions are 37 °C for 60 min, 85 °C for 5 s, 12 °C ∞, and add 50ul after reverse transcription. Dilute with DEPC-H ₂ O, take 3ul as template, and carry 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 according to the following components (the reaction solution was prepared on ice), and the no-template control was set as a negative control. Then put it into a real-time fluorescent PCR instrument (ABI7500) for amplification detection according to 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 the effect of single-marker early diagnosis and detection of lung cancer

1. Sample collection

10ml of blood was collected from patients with early stage (I and II) lung nodule lung cancer diagnosed by the hospital, patients with benign lung nodules, healthy people and other control samples, and separated into plasma.

2. Exosomal RNA extraction

Plasma exosomes were isolated by ultracentrifugation or Exosupur from Echobiotech (Beijing Enzekangtai). The isolated exosomes were extracted with Qiagen miReasy mini kit to extract long RNA from exosomes, and Agilent 2100 was used to detect RNA concentration and quality. , and record the RNA concentration.

3. RNA two-step detection system

Using the two-step detection system based on PCR platform mRNA in Example 1, the plasma exosomal mRNA and LncRNA of 30 early-stage lung cancer patients and 30 control samples (healthy and benign nodules) were detected, and the target long RNA was detected. The relative expression level was calculated according to the Ct value and relative quantitative formula.

4. Evaluation of the performance of exosomal RNA single marker in the diagnosis of early lung cancer

As shown in the table below, the plasma exosomes of 30 early-stage lung cancer patients and 30 control samples (healthy people and benign lesions) were used to detect the Ct value of the target gene, in which the long RNA was used as the reference, β-Actin was used as the reference, and the miRNA was used as the reference. U6 is the reference; according to the Ct value, the relative expression level of RNA is obtained. Using the relative quantitative formula value, the fold change of the relative expression of the combined markers was calculated, and then the relative expression of RNA was obtained. Statistical analysis was performed using R language for the detection results. The performance evaluation of individual markers is shown in Table 8 below, in which miR-450b-5p and miR-3615 have good sensitivity, and let-7f-2-3p has good specificity.

Table 8

Example 5 Evaluation of early diagnosis effect of long RNA and miRNA combination in lung cancer

1. Performance evaluation of three-marker combination

The relative expression level of each long RNA was calculated according to the method in Example 3, and the three marker combinations were trained by logistic regression. Among them, the combination of miR-450b-5p+let-7f-2-3p+ARPC5 has the best performance, with an accuracy of 76.67%, and the AUC curves are shown in Figure 1; miR-106b-3P+miR-30e-5p+ MBOAT2 has good specificity (80%), and its AUC curve is shown in Figure 1 below.

Table 9

2. Performance evaluation of five-marker combination

The relative expression level of each long RNA was calculated according to the method in Example 3, and the five marker combinations were trained by logistic regression. Among them 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 and other two combinations have good sensitivity, which are 93.33% and 100%, respectively, and their AUC curves are shown in Figure 3 and Figure 4; 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 had good specificities of 90% and 96.67%, respectively, and their AUC curves were shown in Figure 5 and Figure 6 .

Table 10

It can be seen from the above data that the exosome-based non-invasive early lung cancer diagnosis method of the present invention has high sensitivity and high specificity in early lung cancer, and provides important value for the early diagnosis of lung cancer. It is of great help to the prevention and treatment of lung cancer in my country. Among them, the combination with the highest sensitivity can achieve 100% sensitivity, and the combination with the highest specificity can achieve 96.67% specificity, which has better performance.

The above-disclosed preferred embodiments of the present invention are provided only to help illustrate the present invention. The preferred embodiments do not exhaust all the details, nor do they limit the invention to only the described embodiments. Obviously, many modifications and variations are possible in light of the content of this specification. The present specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is to be limited only by the claims and their full scope and equivalents.

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<213> Artificial Sequence

<400> 30

ttcgcactgg atacgactat tcagga 26

<210> 31

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 31

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactctgca 50

<210> 32

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 32

ccggtcccag gagaacc 17

<210> 33

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 33

tcgcactgga tacgactctg ca 22

<210> 34

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 34

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagaggc 50

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 35

acgctccatt acactaccct 20

<210> 36

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 36

tcgcactgga tacgacagag gc 22

<210> 37

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 37

ctcgcttcgg cagcaca 17

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 38

aacgcttcac gaatttgcgt 20

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 39

aacgcttcac gaatttgcgt 20

<210> 40

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 40

agaagattag catggcccct gcgca 25

<210> 41

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 41

gtgcagggtc cgaggt 16

Claims (9)

1. a test kit for lung cancer diagnosis, is characterized in that, comprises the primer and the probe that detects exosome long RNA marker and miRNA marker combination, and described long RNA marker comprises ARPC5, MBOAT2, IL1B in primer and probe. One or more, the miRNA markers include miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746- One or more of 5p, miR-125a-5p. 2 . The kit according to claim 1 , wherein the marker is a combination of miR-450b-5p, let-7f-2-3p and ARPC5. 3 . 3. The kit according to claim 1, wherein the marker is a combination of miR-106b-3P, miR-30e-5p and MBOAT2. 4. The kit according to claim 1, wherein the marker is a combination of miR-106b-3P, miR-30e-5p, miR-3615, miR-885-5p and ARPC5. 5. The kit according to claim 1, wherein the marker is a combination of miR-106b-3P, miR-125a-5p, miR-3615, miR-450b-5p and IL1B. 6. The kit according to claim 1, wherein the source of exosomes comprises one or more of blood, saliva and sputum. 7. kit according to claim 1, is characterized in that, described primer and probe comprise: Primers and probes for detecting internal reference ACTB: the upstream primer of the RNA is shown in nucleotide sequence of SEQ ID NO: 1, the downstream primer is shown in nucleotide sequence of SEQ ID NO: 2, and the probe is shown in SEQ ID NO: 3. the nucleotide sequence shown; Primers and probes for detecting ARPC5: the upstream primer of the RNA is shown in the nucleotide sequence shown in SEQ ID NO: 4, the downstream primer is shown in the nucleotide sequence shown in SEQ ID NO: 5, and the probe is shown in SEQ ID NO: 6 nucleotide sequence; Primers and probes for detecting IL1B: the upstream primer of the RNA is shown in the nucleotide sequence shown in SEQ ID NO: 7, the downstream primer is shown in the nucleotide sequence shown in SEQ ID NO: 8, and the probe is shown in SEQ ID NO: 9 nucleotide sequence; Primers and probes for detecting MBOAT2: the upstream primer of the RNA is shown in the nucleotide sequence shown in SEQ ID NO: 10, the downstream primer is shown in the nucleotide sequence shown in SEQ ID NO: 11, and the probe is shown in SEQ ID NO: 12 nucleotide sequence; Reverse transcription primers, PCR primers and probes for the detection of let-7f-2: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 13, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 14 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 15; Reverse transcription primers, PCR primers and probes for detecting miR-106b-3p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 16, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 17 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 18; Reverse transcription primers, PCR primers and probes for detecting miR-125a-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 19, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 20 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 21; Reverse transcription primers, PCR primers and probes for detecting miR-30e-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 22, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 23 sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 24; Reverse transcription primers, PCR primers and probes for detecting miR-3615: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 25, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 26, The downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 27; Reverse transcription primers, PCR primers and probes for detecting miR-450b-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 28, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 29 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 30; Reverse transcription primers, PCR primers and probes for detecting miR-4746-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 31, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 32 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 33; Reverse transcription primers, PCR primers and probes for detecting miR-885-5p: the reverse transcription primers are shown in the nucleotide sequence shown in SEQ ID NO: 34, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 35 Sequence, the downstream primer is the nucleotide sequence shown in SEQ ID NO: 41, and the probe is the nucleotide sequence shown in SEQ ID NO: 36; Reverse transcription primers, PCR primers and probes for detecting internal reference U6: the reverse transcription primers of the U6 are shown in the nucleotide sequence shown in SEQ ID NO: 39, and the PCR upstream primers are shown in the nucleotide sequence shown in SEQ ID NO: 37, The downstream primer is the nucleotide sequence shown in SEQ ID NO: 38, and the probe is the nucleotide sequence shown in SEQ ID NO: 40. 8. A device for lung cancer diagnosis, characterized in that it includes primers and probes that detect the combination of exosome long RNA markers and miRNA markers, and the long RNA markers include one of ARPC5, MBOAT2, and IL1B. One or more, the miRNA markers include miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p , one or more of miR-125a-5p. 9. A method for lung cancer diagnosis, comprising detecting the specificity of an exosome long RNA marker and a miRNA marker combination, the long RNA marker comprising one of ARPC5, MBOAT2, IL1B or Various, the miRNA markers include miR-450b-5p, miR-let-7f, miR-3615, miR-885-5p, miR-106b-3p, miR-30e-5p, miR-4746-5p, miR One or more of -125a-5p.

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