CN114480612A - Application of exosome miR-654-5p in bronchoalveolar lavage fluid - Google Patents
Application of exosome miR-654-5p in bronchoalveolar lavage fluid Download PDFInfo
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Abstract
The invention discloses application of exosome miR-654-5p in bronchoalveolar lavage fluid. The invention utilizes RT-qPCR method, combines software to carry out multiple analysis, utilizes exosome miR-654-5p in bronchoalveolar lavage fluid, and can improve the reliability and accuracy of severe pneumonia patients: the content of the bronchoalveolar lavage fluid is most directly influenced by pathological and physiological changes of lung tissues, and the exosome in the bronchoalveolar lavage fluid is adopted, so that the specificity is high; the RT-qPCR detection method can efficiently identify severe pneumonia patients and has high sensitivity; the expression level of miR-654-5p is detected by adopting the modern molecular biology front-end technology, so that the method is efficient and rapid; the primers are specifically combined with the target sequence, so that the accuracy of the RT-qPCR amplification result is improved, severe pneumonia patients can be reliably detected, and the accuracy is high.
Description
Technical Field
The invention relates to the technical field of molecular detection of severe pneumonia, and particularly relates to application of exosome miR-654-5p in bronchoalveolar lavage fluid.
Background
Severe pneumonia is a serious and even fatal severe infectious disease, and infection originates in the lung and can rapidly progress to cause respiratory failure. The disease is a rapidly progressive disease, and clinically severe pneumonia and complicated systemic inflammatory response syndrome, multi-organ functional failure and the like seriously threaten the life of patients. Severe pneumonia is still not defined universally, and patients who need to stay in ICU can be considered as severe pneumonia. It is currently believed that severe pneumonia can be called if patients with pneumonia have severe conditions that require ventilatory support (acute respiratory failure, severe gas exchange dysfunction with hypercapnia or persistent hypoxemia), circulatory support (hemodynamic dysfunction, peripheral hypoperfusion) and intensive care therapy (sepsis due to pneumonia or other organ dysfunction due to underlying disease).
At present, whether a patient has severe pneumonia or not can be comprehensively judged by aspects such as mental state, physical examination, arterial qi and blood analysis examination, blood routine examination, imaging examination and the like of the patient. Many non-specific indicators may be associated with the severity of severe pneumonia, such as C-reactive protein (CRP), Procalcitonin (PCT), etc. However, these indexes have poor specificity and cannot accurately reflect the severity of severe pneumonia.
Exosomes are extracellular vesicles of about 50-150nm in size, surrounded by a phospholipid bilayer, and can be produced by a variety of cells, such as epithelial cells, macrophages, and endothelial cells, and are present in a variety of biological fluids, such as blood, sputum, urine, bronchoalveolar lavage fluid, pleural fluid, ascites, and synovial fluid. The exosome surface has abundant specific markers such as leukocyte differentiation antigens CD9, CD63, CD81, heat shock 70 protein, tumor sensitive gene 101, ALG-2 interactive protein X and major histocompatibility complex MHC I and II molecules. Pathological factors such as hypoxia, inflammation and the like can cause the change of components such as RNA, protein and the like in exosome, thereby affecting stress response and immune response. Micro ribonucleic acid (miRNA) widely exists in body fluid and cells, is related to cell proliferation, differentiation and disease occurrence and development, and a phospholipid bilayer of an exosome may have a protective effect on the miRNA, so that the miRNA is not easily degraded, so that the miRNA is stable and plays a biological role, and the miRNA is more stable and not easily degraded compared with the miRNA in a free form (Fan Yuxin, research on the influence and mechanism of the exosome from slow-block pulmonary alveolar macrophage on the proliferation capacity of airway epithelial cells [ D ] research on the mechanism of the miRNA 2020, China civil Release military medical university).
The prior art discloses an early lung adenocarcinoma specific exosome miRNA and application thereof, wherein the miRNA is suitable for auxiliary screening of early tumors and wins treatment opportunity for lung adenocarcinoma patients. However, no exosome miRNA detection method for severe pneumonia exists at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides application of an exosome miR-654-5p in preparation of a severe pneumonia detection product. The invention realizes the detection of quickly and specifically identifying severe pneumonia patients based on the detection of the expression level of exosome miR-654-5p in bronchoalveolar lavage fluid.
The first purpose of the invention is to provide the application of the exosome miR-654-5p in the preparation of a severe pneumonia detection product.
The second purpose of the invention is to provide a detection product for diagnosing severe pneumonia by using the expression level of exosome miR-654-5p in bronchoalveolar lavage fluid.
The third purpose of the invention is to provide a detection primer of exosome miR-654-5p in bronchoalveolar lavage fluid for preparing a detection product for severe pneumonia.
The fourth purpose of the invention is to provide a detection kit for severe pneumonia.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention extracts exosome in a sample to be detected by an ultracentrifugation method, enriches exosome RNA, and synthesizes cDNA of the exosome RNA by reverse transcription by referring to miRNAFirstStrand Synthesis operating instructions of Takara company. Designing a specific primer hsa-miR-654-5p of an exosome miR-654-5p, preparing an RT-qPCR reaction system, and carrying out 2-ΔΔCTThe method calculates the expression level of miR-654-5p corresponding to each sample, and determines whether the sample is from a severe pneumonia patient.
Accordingly, the invention claims the following:
an application of exosome miR-654-5p in bronchoalveolar lavage fluid, wherein the exosome miR-654-5p is used for preparing a detection product for severe pneumonia.
Preferably, the nucleotide sequence of the exosome miR-654-5p is shown as SEQ ID NO 1.
An application of a product for detecting the expression level of exosome miR-654-5p in bronchoalveolar lavage fluid in the preparation of a product for detecting severe pneumonia diagnosis.
Preferably, the nucleotide sequence of the exosome miR-654-5p is shown as SEQ ID NO 1.
An application of a detection primer of exosome miR-654-5p in bronchoalveolar lavage fluid in preparation of a detection product for severe pneumonia.
Preferably, the nucleotide sequence of the exosome miR-654-5p is shown as SEQ ID NO 1.
Preferably, the nucleotide sequence of the detection primer is shown as SEQ ID NO. 2.
More preferably, the product is a detection reagent and/or a detection kit.
The invention also claims a detection kit for severe pneumonia, which contains an expression level detection reagent of the exosome miR-654-5p in claim 1.
Preferably, the expression level detection reagent is a detection primer with a nucleotide sequence shown as SEQ ID NO. 2.
Preferably, the detection kit further comprises reagents for RT-qPCR.
Preferably, the reagent for RT-qPCR is miRNA first Strand Synthesis kit and kit of Takara corporation
Premix Ex TaqTM
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the RT-qPCR method is utilized, the software is combined for multiple analysis, and the exosome miR-654-5p in bronchoalveolar lavage fluid is utilized, so that the reliability and accuracy of detecting severe pneumonia patients can be improved;
(1) the content of the bronchoalveolar lavage fluid is most directly influenced by pathological and physiological changes of lung tissues, and the exosome in the bronchoalveolar lavage fluid is adopted, so that the specificity is high; (2) the RT-qPCR detection method can efficiently identify severe pneumonia patients and has high sensitivity; (3) the expression level of miR-654-5p is detected by adopting the modern molecular biology front-end technology, so that the method is efficient and rapid; (4) the primers are specifically combined with the target sequence, so that the accuracy of the RT-qPCR amplification result is improved, severe pneumonia patients can be reliably detected, and the accuracy is high.
Drawings
FIG. 1 is a flow chart of a method for detecting an expression level of exosome miR-654-5p in bronchoalveolar lavage fluid by an RT-qPCR method.
FIG. 2 Receiver Operating Characteristic (ROC) curves for diagnosing severe pneumonia with miR-654-5p expression in exosomes in bronchoalveolar lavage fluid.
FIG. 3 comparison of the diagnostic effect of exosome miR-654-5p in BALF with clinical common serum biomarker hsCRP, PCT level.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 primer design
1. Experimental method for obtaining RT-qPCR specific primer
And logging in a website http:// www.mirbase.org/index.shtml to obtain miRBase: > hsa-miR-654-5p MIMAT0003330, and obtaining the primer with high sensitivity, good specificity and high amplification efficiency, wherein the nucleotide sequence of the primer is Mature sequence hsa-miR-654-5p shown in SEQ ID NO: 1.
Mature sequence of exosome miR-654-5p (SEQ ID NO: 1):
5’-UGGUGGGCCGCAGAACAUGUGC-3’；
2. obtaining the experimental result of RT-qPCR specific primers
From the RT-qPCR specific Primer combinations determined in the above experiments, U6 Forward Primer, U6 Reverse Primer and mRQ 3' Primer were obtained using miRNA first Strand Synthesis kit from Takara, Inc., respectively.
And a specific primer of the exosome miR-654-5p (SEQ ID NO: 2):
5’-TGGTGGGCCGCAGAACATG-3’；
example 2 construction of a kit for detecting Severe pneumonia
One, component
(1) The Primer combination obtained in example 1 (U6 Forward Primer, U6 Reverse Primer and mRQ3 ' Primer, and specific primers for exosome miR-654-5p (SEQ ID NO: 2): 5'-TGGTGGGCCGCAGAACATG-3' were obtained using miRNA first Strand Synthesis kit of Takara corporation, respectively);
(2)ddH2O；
(3)
Premix Ex TaqTM；
(4) the miRNA first Strand Synthesis kit of Takara corporation.
Second, use method
FIG. 1 shows a method for using the kit of the present invention, which comprises the following steps:
1. and (3) extracting exosomes in the bronchoalveolar lavage fluid to be detected.
Step 1: bronchoalveolar lavage fluid was collected, centrifuged using a centrifugal force of 300 × g for 10min, and then the remaining cells were removed to collect the supernatant.
Step 2: and (2) transferring the supernatant obtained in the step (1) into a new centrifuge tube, centrifuging by adopting the parameters of 14000 Xg of centrifugal force and 30min of centrifugal time, then removing cells, bacteria, apoptotic bodies and the like, and collecting the supernatant.
And step 3: transferring the supernatant obtained in the step 2 to an ultracentrifuge tube, ultracentrifuging by adopting the parameters of centrifugal force of 100000 Xg and centrifugation time of 90min, discarding the supernatant, adding sterile PBS to resuspend and precipitate to obtain an exosome, and storing at-80 ℃ for later use or immediately using.
2. Extraction of exosome RNA
Step 1: extracting an exosome sample, adding Trizol into the sample, and supplementing 1 ml; keeping the temperature at 15-30 ℃, and standing for 5 min;
step 2: taking out the sample in the step 1, adding 200 mu L of chloroform, carrying out vortex oscillation and uniform mixing for 15s, and standing for 5min at room temperature; ultracentrifugation is carried out at 4 ℃ by adopting the parameters of centrifugal force of 12000 Xg and centrifugation time of 15 min; centrifuging, separating into 3 layers, and collecting the upper colorless transparent RNA layer for later use;
and step 3: collecting the supernatant in the step 2, transferring the supernatant into a new RNA enzyme-free EP tube, and adding isopropanol to precipitate RNA molecules, wherein the volume of the isopropanol is the same as that of the supernatant in the step 2; keeping the temperature at 15-30 ℃, and standing for 5 min; ultracentrifugation is carried out at 4 ℃ by adopting the parameters of centrifugal force of 12000 Xg and centrifugation time of 10min, and colloidal sediment appears at the bottom of a centrifugal tube;
and 4, step 4: discarding the supernatant in the step 3, adding 1mL of 75% ethanol into the colloidal precipitate at the bottom of the centrifuge tube, performing ultracentrifugation at 4 ℃ by adopting the parameters of centrifugal force of 12000 Xg and centrifugal time of 5min after vortex oscillation, and after two times of washing, inverting the EP tube on the filter paper until the ethanol is completely volatilized;
and 5: adding 15 mu L of DEPC water prepared by Biyuntian biology company into the sample obtained in the step 4, dissolving RNA precipitate, blowing and beating the precipitate by using a pipette, keeping the temperature at 55-60 ℃, and standing for 10min to promote RNA dissolution;
step 6: RNA quality and concentration were determined using a NanoDprop2000 ultraspectrophotometer.
3. Synthesis of cDNA
The extracted exosome RNA was reverse transcribed to synthesize the desired cDNA, referring to the miRNA first Strand Synthesis protocol of Takara.
4. Reagents and specific operating parameters required by RT-qPCR experiment
(1) Reagent: 0.5. mu.L of cDNA, 12.5. mu.L
Premix Ex TaqTM0.5. mu.L of 10. mu.M forward primer, 0.5. mu.L of 10. mu.M forward primer11. mu.L ddH and2O。
(2) and (3) amplification procedure: denaturation at 95 ℃ for 10 s; qPCR reaction at 95 ℃ for 5s, 60 ℃ for 20s, x 40 cycles; the dissolution curves were 95 ℃ for 60s, 55 ℃ for 30s, 95 ℃ for 30 s.
5. Analysis of amplification products
Setting three multiple holes for each sample, reading CT value of each sample, and passing through 2-ΔΔCTCalculating the expression level of miR-654-5p corresponding to each sample.
6. Statistical analysis, selection of diagnostic threshold
Analysis and processing through 2 using MedCalc 19.6.0 statistical analysis software-ΔΔCTAnd calculating the relative expression quantity of the obtained miR-654-5p, and determining 0.841 as the optimal diagnostic standard. Namely, when the examined person is a severe pneumonia patient and needs to be closely monitored or examined by imaging, the relative expression quantity of miR-654-5p is more than 0.841.
Example 3 analysis of the diagnostic Effect of recognizing Severe pneumonia by exosome miR-654-5p
First, experiment method
Bronchoalveolar lavage fluid samples were collected from 7 patients without respiratory infection and 28 patients with severe pneumonia, and the samples were examined using the kit of example 2.
Second, experimental results
FIG. 2 is a Receiver Operating Characteristic (ROC) curve of miR-654-5p expression in exosomes in bronchoalveolar lavage fluid for diagnosing severe pneumonia, which can be used for evaluating the diagnosis effect of exosomes miR-654-5p on severe pneumonia. The result shows that the area under the working characteristic curve (AUC) of a subject diagnosed with severe pneumonia by the expression level of exosome miR-654-5P in bronchoalveolar lavage fluid is 0.857, the 95% confidence interval (95% CI) is 0.688-0.955, the standard error is 0.067, and P is 0.001; when the cut-off value was 0.841, the sensitivity was 85.71% and the specificity was 100%.
Example 4 comparison of the test results with the diagnostic results of the common clinical biomarkers
First, experiment method
A peripheral venipuncture blood sampling method is used for obtaining a peripheral blood sample of a patient, and a Hitachi 600 type full-automatic biochemical analyzer is selected for detecting blood biochemical indexes Procalcitonin (PCT) and hypersensitive C reactive protein (hsCRP) of the patient.
The kit in example 2 is used for detecting the expression amount of exosome miR-654-5p in patient bronchoalveolar lavage fluid.
The diagnostic effect of the three biomarkers was compared using the ROC curve.
Second, experimental results
The results show that the area under the curve with the biomarker procalcitonin is 0.643, the standard error is 0.182 and the 95% confidence interval is 0.455-0.804. The area under the curve of the biomarker is hypersensitive C-reactive protein is 0.777, the standard error is 0.156, and the 95% confidence interval is 0.595-0.904. The area under the curve with the biomarker miR-654-5p is 0.857 standard error and 0.067, and the 95% confidence interval is 0.688-0.955. The area under the curve of the combined biomarker, the standard error and the 95% confidence interval is 0.756-0.982, and is 0.911 and 0.054.
The specific operation of the combination of the three is as follows:
medcalc software is adopted to realize the analysis of the working characteristic curve of the testee in the multi-index combined diagnosis.
Step 1: defining variables and establishing a table: as the first column (testa) is the value of procalcitonin; the second column (Testb) is the value of hypersensitive C-reactive protein; the third column (test) is the value of miR-654-5p, a numerical variable; disease is a disease state, 0 is disease-free, 1 is disease, i.e. severe pneumonia.
Step 2: importing data into Medcalc
And step 3: logistic regression
Clicking Statistics-Regression-Logistic Regression in a Logistic Regression dialog box, selecting Disease in a Dependent Variable box, and respectively selecting testa, testb and testc in the first, second and third lines in an Independent Variable box. In the Logistic Regression result dialog box, Save predicted probabilities are stored in the data set
And 4, step 4: at this time, an extra LOGREGR _ Pred1 is added in the data frame, namely the predicted probability values of testa, testb and testc generated by logistic, the diagnosis capability of testa, testb and testc is comprehensively reflected, and the working characteristic curve of the subject drawn by LOGREGR _ Pred1 is the joint diagnosis of the three.
FIG. 3 is a characteristic curve of the work of a subject with the diagnostic effect of exosome miR-654-5p and clinical common serum biomarkers of hypersensitivity C-reactive protein and procalcitonin level in bronchoalveolar lavage fluid.
As can be seen by combining the figure 3 and the experimental results, the exosome miR-654-5p in the bronchoalveolar lavage fluid has a high diagnosis effect, and the area value under the curve is 0.857, which is higher than the common clinical procalcitonin and hypersensitive C-reactive protein. The area under the curve of the multi-index combined diagnosis is 0.911, which is larger than that of other single-index detection.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
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Claims (10)
1. An application of exosome miR-654-5p in bronchoalveolar lavage fluid is characterized in that exosome miR-654-5p is used for preparing a detection product for severe pneumonia.
2. The use according to claim 1, wherein the nucleotide sequence of the exosome miR-654-5p is shown as SEQ ID NO. 1.
3. An application of a product for detecting the expression level of exosome miR-654-5p in bronchoalveolar lavage fluid in the preparation of a product for detecting severe pneumonia diagnosis.
4. The use according to claim 3, wherein the nucleotide sequence of the exosome miR-654-5p is shown as SEQ ID NO. 1.
5. An application of a detection primer of exosome miR-654-5p in bronchoalveolar lavage fluid in preparation of a detection product for severe pneumonia.
6. The use according to claim 5, wherein the nucleotide sequence of the exosome miR-654-5p is shown as SEQ ID NO. 1.
7. The use of claim 5, wherein the nucleotide sequence of the detection primer is shown as SEQ ID NO. 2.
8. Use according to any one of claims 1 to 7, wherein the product is a detection reagent and/or a detection kit.
9. A severe pneumonia detection kit, which is characterized by comprising an expression level detection reagent of the exosome miR-654-5p in claim 1.
10. The detection kit according to claim 9, wherein the expression level detection reagent is a detection primer having a nucleotide sequence shown in SEQ ID NO. 2.
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