KR20220110152A - Method for diagnosing of infectious respiratoy diseases using extracellular vesicles - Google Patents

Abstract

The present invention provides a method for diagnosing an infectious respiratory disease using extracellular vesicles, which comprises the steps of: separating a supernatant from a biological sample isolated from a subject; incubating a mixed solution that is formed by mixing the separated supernatant with a first solution; separating extracellular vesicles from the mixed solution; extracting a biomarker from the extracellular vesicles; measuring an expression level of the extracted biomarker; and determining, when the measured expression level of the biomarker is above a predetermined level, a respiratory disease. According to the present invention, the method is capable of diagnosing pathogens more accurately and rapidly.

Description

Method for diagnosing infectious respiratory diseases through extracellular vesicles

The present invention relates to a method for diagnosing infectious respiratory diseases through extracellular vesicles, and more particularly, to a method for isolating extracellular vesicles within a short time and diagnosing pathogens of respiratory diseases through the isolated extracellular vesicles.

Respiratory diseases caused by viral and bacterial infections are highly morbid, accounting for about half of all infectious diseases. Infectious respiratory diseases are usually transmitted by direct contact, and epidemics of infectious respiratory diseases can occur simultaneously in local or global regions.

Since these infectious respiratory diseases usually have similar clinical symptoms in the early stages of infection, it is difficult to clearly identify the causative agent only by examination and chest X-ray examination according to the clinical symptoms performed in actual point-of-care. Since symptoms, prescriptions, and prognosis according to each virus and bacteria are very diverse, the most important factor in the management of patients with infectious respiratory diseases may be early detection and identification of pathogens. In addition, the causative agents of infectious respiratory diseases may have resistance to antibiotics. Accordingly, the screening of pathogens causing respiratory diseases induces prescription of an effective antibiotic type and administration period, and more effectively reduces the antibiotic resistance rate, thereby preventing side effects due to the use of antibiotics.

Meanwhile, the diagnosis of the current causative pathogen is performed by laboratory techniques such as serological methods, pathogen antigen detection, pathogen culture, pathogen nucleic acid detection, and the like. Among these, the diagnostic method with the highest accuracy is the pathogen culture method. However, the pathogen culture method takes a long time of 7 days or more, which may miss an important treatment treatment period. Accordingly, a method using pathogen nucleic acid detection, that is, real-time polymerase chain reaction (PCR), is widely used, but the accuracy is lower than that of the pathogen culture method. Accordingly, there is a need for a method capable of rapidly and accurately detecting pathogens compared to conventional methods.

The description underlying the invention has been prepared to facilitate understanding of the invention. It should not be construed as an admission that the matters described in the background technology of the invention exist as prior art.

As a method for solving the above problems, many molecular biological methods capable of directly detecting pathogens, ie, viruses and bacteria, from a specimen have been introduced. The most commonly used molecular biological method is nucleic acid amplification (NAA), which amplifies specific RNA and DNA regions of pathogens using polymerase chain reaction. However, since the sensitivity and specificity of commercialized products have large deviations from actual clinical specimens, the development of a method showing higher accuracy in early diagnosis of infectious diseases and decision of treatment policy is required.

Meanwhile, nano-sized extracellular vesicles (EVs) exist in body fluid. The extracellular vesicle is secreted from the cell membrane and plays a very important function in cell-to-cell communication, and contains biologically active substances such as DNA, RNA, and proteins. More specifically, a substance derived from a pathogen, such as lipopolysaccharide (LPS) or peptidoglycan, can induce the production of inflammatory cytokines in immune cells and epidermal cells, and the extracellular vesicles are these hosts. Contains proteins that can regulate the inflammatory response of In addition, extracellular vesicles derived from pathogens are involved in antibiotic resistance, elimination of competing bacteria, virulence factor delivery, and host cell regulation. Furthermore, abnormal cells such as cells infected with pathogens and cancer cells secrete much larger amounts of extracellular vesicles than normal cells. The extracellular vesicles are surrounded by a double lipid membrane, so that major biological materials such as nucleic acids or proteins, such as DNA and RNA, present therein can be stably preserved.

Accordingly, the inventors of the present invention found that the accuracy of diagnosis for infectious respiratory diseases, that is, sensitivity and It was recognized that specificity could be further improved. In addition, since the extracellular vesicles are separable from body fluids, samples can be obtained non-invasively and used for disease diagnosis.

Furthermore, the inventors of the present invention have found that by using a high concentration of the polymer for precipitation, it is possible to isolate the extracellular vesicles within a short time with higher purity than in the prior art.

Accordingly, the inventors of the present invention sought to provide a more accurate diagnostic method for respiratory diseases by isolating extracellular vesicles from a biological sample isolated from an individual and using the separated extracellular vesicles.

The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the problems as described above, according to an embodiment of the present invention, separating the supernatant from the biological sample separated from the subject, culturing the mixed solution formed by mixing the separated supernatant and the first solution (incubation) step, separating extra cellular vesicles from the mixture, extracting the biomarker from the extracellular vesicle, measuring the expression level of the extracted biomarker, the measured expression level of the biomarker Provided is a method for diagnosing an infectious respiratory disease through the extracellular vesicles, comprising the step of determining a respiratory disease when the level is higher than a predetermined level.

At this time, according to a feature of the present invention, the first solution is at least one of the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (polyvinyl alcohol), and pyrrole. It may include one, but is not limited thereto. In addition, the aforementioned polyethylene glycol may have a molecular weight of about 8000 MW. The compound included in the first solution may be selected from, for example, a molecular weight of 5000 MW or more.

According to another feature of the present invention, the extracellular vesicles are exosomes, ectosomes, external vesicles (exovesicle) microvesicles, microparticles, apoptotic bodies (apoptotic body) , a membrane particle, a membrane vesicle, an exosome-like vesicle, and at least one of the group consisting of an ectosome-like vesicle, but is not limited thereto. In addition, the aforementioned extracellular vesicles may have a size of 100 nm or less.

According to another feature of the present invention, the biological sample may include at least one of the group consisting of blood, urine, pleural fluid, feces, sputum, abdominal fluid, breast milk, cerebrospinal fluid, saliva, lymph, and bronchoalveolar lavage fluid, but is limited thereto. It is not, and may include all of various bodily fluids that can be collected in the body.

According to another feature of the present invention, the amount of the biological sample, 150 to 350 μl may be.

According to another feature of the present invention, after the step of isolating the extracellular vesicles, the step of detecting the respiratory virus and the detected respiratory virus amplification curve is expressed or detected below a predetermined ct (threshold cycle) value. When the droplet expression is higher than or equal to a predetermined level, a method for diagnosing infectious respiratory diseases through the extracellular vesicles may be provided, further comprising the step of determining a viral respiratory disease caused by a respiratory virus.

In this case, the step of determining the viral respiratory disease is a case in which the amplification curve of the detected respiratory virus is expressed below a predetermined ct value, and the predetermined ct value may be 27, but is not limited thereto. It can be set in various ways depending on the condition, the detection kit used, the measuring device, and the type of virus.

In addition, the step of determining the viral respiratory disease is when the detected droplet expression of the respiratory virus is greater than or equal to a predetermined level, and the predetermined level value of droplet expression may be 1, but is not limited thereto, and each sample It can be set in various ways depending on the status of the virus and the type of virus.

At this time, the respiratory virus is respiratory syncytial virus A (RSV A), respiratory syncytial virus B, influenza A (influenza A), influenza B, parainfluenza 1 (parainfluenza 1), parainfluenza 2, parainfluenza 3, rhinovirus A (rhino A), metapneumovirus (metapneumovirus), coronavirus 229E (corona virus 229E), coronavirus OC43, coronavirus NL63 and may contain at least one of the group consisting of bocavirus, but , but is not limited thereto, and may include both DNA and RNA detectable respiratory viruses.

According to another feature of the present invention, the biomarker may be IS6110, and IS6110 is a biomarker with an increased copy number specifically in Mycobacterium tuberculosis.

Accordingly, according to another feature of the present invention, respiratory diseases are Mycobacterium tuberculosis (M), M. bovis, M. africanum, Caneti tuberculosis (M. canetii), and micro It may be a bacterial respiratory disease caused by M. microti, but is not limited thereto, and may include various diseases that can be detected through the expression of a specific IS6110 gene.

According to another feature of the present invention, after extracting the biomarker from the extracellular vesicle, measuring the expression level of the biomarker by rtPCR, and when the measured amplification curve of the biomarker is expressed at less than 27 ct value , There is provided a method for diagnosing an infectious respiratory disease through the extracellular vesicles, further comprising the step of determining a respiratory disease. In this case, the ct value of the amplification curve may be 27, but is not limited thereto, and may be variously set according to the state of each sample, the detection kit used, the measuring device, and the type of the biomarker.

Such biomarker measurement may have higher accuracy, ie, sensitivity, than other detection methods in ddPCR. Accordingly, according to another feature of the present invention, the predetermined level may be 1 copy/reaction (ml) in the case of ddPCR.

According to another feature of the present invention, the step of measuring the expression level of the biomarker includes a microarray analysis method, polymerase chain reaction (PCR), real-time reverse transcription polymerase chain reaction (through fluorescence, chemiluminescence, or electrical signal detection) rtPCR), digital microdroplet PCR (ddPCR), solid-state nanopore detection, RNA switch activation, Northern blot, or gene expression sequencing analysis (SAGE). No, it may include all measurement methods that can be measured through nucleic acids.

According to another feature of the present invention, the step of separating the supernatant is performed by centrifugation at 500 to 4000 g for 5 to 30 minutes, and the culturing step is performed at 2 to 5 ° for 1 to 3 hours, The step of isolating the extracellular vesicles may be performed by centrifugation at 1000 to 2000 g for 10 to 40 minutes.

According to another feature of the present invention, the first solution, with respect to the total volume of the mixed solution, may be 7 to 10% (w / v), but is not limited thereto, and may have various concentrations depending on the type and state of the sample. can be used

According to an embodiment of the present invention, the steps of isolating the extracellular vesicles from the biological sample isolated from the subject, extracting the biomarkers from the extracellular vesicles, measuring the expression level of the extracted biomarkers by ddPCR, and measuring Provided is a method for diagnosing infectious respiratory diseases through extracellular vesicles, comprising determining a respiratory disease when the expression level of the biomarker is greater than or equal to 1 copy/reaction (ml).

According to a feature of the present invention, the extracellular vesicles are exosomes, ectosomes, external vesicles, microvesicles, microparticles, apoptotic bodies, It may include, but is not limited to, at least one of the group consisting of a membrane particle, a membrane vesicle, an exosome-like vesicle, and an ectosome-like vesicle. In addition, the aforementioned extracellular vesicles may have a size of 100 nm or less.

According to another feature of the present invention, respiratory diseases include Mycobacterium tuberculosis (M), M. bovis, M. africanum, M. canetii, and Mycobacterium tuberculosis (M. canetii). M. microti) may be a bacterial respiratory disease, and the above-mentioned bacteria may have specifically amplified IS6110 gene.

Accordingly, according to another feature of the present invention, the biomarker may be IS6110, but is not limited thereto.

According to another feature of the present invention, after the step of isolating the extracellular vesicles, the step of detecting the respiratory virus by ddPCR, and when the copy/reaction (ml) value of the detected respiratory virus is 1 or more, the virus caused by the respiratory virus A method for diagnosing an infectious respiratory disease through the extracellular vesicles may be provided, further comprising determining as a sexual respiratory disease.

At this time, the respiratory virus is respiratory syncytial virus A (RSV A), respiratory syncytial virus B, influenza A (influenza A), influenza B, parainfluenza 1 (parainfluenza 1), parainfluenza 2, para may contain at least one of the group consisting of influenza 3, rhino A, metapneumovirus, corona virus 229E, coronavirus OC43, coronavirus NL63 and bocavirus have.

Hereinafter, the present invention will be described in more detail through examples. However, since these examples are only for illustrative purposes of the present invention, the scope of the present invention should not be construed as being limited by these examples.

The present invention has the effect of more accurately and rapidly diagnosing respiratory diseases through the extracellular vesicles.

More specifically, the size of the small extracellular vesicles is increased through the precipitation polymer of the present invention, and thus the extracellular vesicles can be separated even in a general centrifuge. Furthermore, since the present invention can separate extracellular vesicles of a certain size, the accuracy of the analysis, ie, reliability, can be high.

Furthermore, as the extracellular vesicles contain more genetic material and metabolites, the sensitivity may be improved, which may be more accurate for the diagnosis of pathogens than general fluid samples. Furthermore, in the present invention, by using the extracellular vesicles, the sensitivity is improved, and even a very low concentration of pathogen markers present in a sample can be accurately detected.

In addition, since the extracellular vesicles can be isolated from body fluids, pathogens can be non-invasively diagnosed.

Accordingly, through the present invention, it is possible to accurately diagnose an infection at an early stage, and furthermore, it is possible to confirm the drug resistance of the pathogen and confirm the therapeutic effect through the monitoring of the drug treatment.

The effect according to the present invention is not limited by the contents exemplified above, and more various effects are included in the present specification.

1 exemplarily illustrates the procedure of a method for diagnosing infectious respiratory diseases through extracellular vesicles according to an embodiment of the present invention.
Figure 2a shows the diagnosis result of Mycobacterium tuberculosis according to the extracellular vesicle separation method in the infectious respiratory disease diagnosis method through the extracellular vesicles according to an embodiment of the present invention.
Figure 2b shows the ddPCR results for Examples 1 and 2 in Figure 2a.
3a to 3c show the results of the nanoparticle characterization for Examples 1 and 2 in FIG. 1 .
Figure 4 shows the respiratory virus diagnosis results according to the extracellular vesicle separation method in the infectious respiratory disease diagnosis method through the extracellular vesicles according to an embodiment of the present invention.
5 shows the analysis performance test results according to the analysis method in Mycobacterium tuberculosis.
6 shows the analysis performance test results according to the culture results of Mycobacterium tuberculosis.
7 shows the diagnostic test results in the qPCR and ddPCR diagnostic methods according to the extracellular vesicle separation method from a low concentration sample.
Figures 8a to 8b shows the number of copies of Mycobacterium tuberculosis (log ₁₀ copies/ml) in the Mycobacterium tuberculosis positive sample according to whether extracellular vesicles are separated.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be embodied in various different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the art to which the present invention pertains It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

As used herein, the term “diagnosis” means to identify or characterize a pathological condition. For the purpose of the present invention, it may mean to identify the onset of a respiratory disease and the pathogen causing the onset, and further, it may include determining the development and alleviation of respiratory diseases by checking the presence or absence and level of expression of biomarkers. can

Hereinafter, with reference to FIG. 1, the procedure of the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention will be described in detail.

1 exemplarily illustrates the procedure of a method for diagnosing infectious respiratory diseases through extracellular vesicles according to an embodiment of the present invention.

Referring to FIG. 1 , the method for diagnosing a disease through the extracellular vesicles according to an embodiment of the present invention includes the steps of separating the supernatant from the biological sample isolated from the subject (S110), the separated supernatant and the first solution Culturing the mixed solution formed by mixing (S120), separating the extracellular vesicles from the mixed solution (S130), extracting the biomarker from the extracellular ER (S140), measuring the expression level of the extracted biomarker ( S150), when the measured expression level of the biomarker is greater than or equal to a predetermined level, determining a respiratory disease (S160).

First, in the step of separating the supernatant from the biological sample isolated from the subject (S110), centrifugation may be performed at 500 to 4000 g for 5 to 30 minutes. Accordingly, cell debris, waste products and large particles are precipitated in the form of pellets, which can be easily removed. In this case, the biological sample isolated from the subject may include, but is not limited to, blood, urine, pleural fluid, feces, sputum, abdominal fluid, breast milk, cerebrospinal fluid, saliva, lymph, and bronchoalveolar lavage fluid, and may be separated from the subject. It can contain all of the various bodily fluids present. In addition, the biological sample may include solutions of various biological origins, such as animals, plants, fungi, and algae, and may be cultured or cultured under conditions that release extracellular vesicles, extracellular vesicles, or secrete extracellular vesicles. can be incubated.

In addition, the amount of the biological sample used may be 350 μl or less, preferably 200 μl or less, but is not limited thereto, and samples of various capacities may be used depending on the type and state of the sample.

Next, in the step of culturing the mixed solution formed by mixing the separated supernatant and the first solution (S120), the first solution may be 7 to 10% (w/v) based on the total volume of the mixed solution, but limited thereto it's not going to be More specifically, in one embodiment of the present invention, the mixed solution is mixed by adding 3 or more of the supernatant from which the pellets are removed to the first solution 1 of 4X (4 times concentrated), and this is 1 to 1 at 2 to 5 ˚C Incubated for 3 hours.

However, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention is not limited to the mixing ratio of the first solution with the above-mentioned concentration. More specifically, in the step of forming the mixed solution ( S120 ), the final concentration when the first solution is mixed with the supernatant may be important. Accordingly, in the step of culturing the mixed solution (S120), 1X or more of various first solutions may be used, and the concentration of the first solution when mixed may be 7 to 10% (w/v) with respect to the total volume of the mixed solution. have.

During culturing, the extracellular vesicles in the mixed solution are combined with a polymer for precipitation contained in the first solution and increase in size (molecular weight increase), thereby increasing the precipitation rate of the extracellular vesicles.

In this case, the first solution contains at least one of the group consisting of a polymer for precipitation, that is, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol, and pyrrole. may be, but is not limited thereto, and may include polyethylene glycol having a molecular weight of 5000 MW or more, about 8000 MW. The term “about” as used herein may be construed to include ±10% of the stated value. Furthermore, in one embodiment of the present invention, the first solution was used as a four-fold concentrated solution, but is not limited thereto, and various first solutions of 1X or more may be used.

Next, the step of separating the extracellular vesicles from the mixed solution (S130) may be performed by centrifugation at 1000 to 2000 g for 10 to 40 minutes, and the temperature at this time may be 2 to 6 ˚C. More specifically, extra cellular vesicles (EVs) may refer to vesicles produced in cells and secreted out of cells, exosomes, ectosomes, and exovesicle microvesicles. Included are microvesicles, microparticles, apoptotic bodies, membrane particles, membrane vesicles, exosome-like vesicles, and ectosome-like vesicles. These extracellular vesicles have a nano-level small size (100 nm or less) and a low density (1.13 to 1.19 g/ml), and a large centrifugal force is required due to the low density, so it is very difficult to separate them by a general centrifugation method. Accordingly, when centrifugation is used to separate the extracellular vesicles, an ultra-high-speed centrifuge having a high-speed rotation speed of 150,000 g or more is required. However, when the extracellular vesicles bind to the polymer for precipitation, the size increases and may be precipitated in a general centrifuge. That is, the extracellular vesicles with increased precipitation rate may have a high recovery rate even in a general centrifuge. Furthermore, the extracellular vesicles combined with the polymer for precipitation are not easily decomposed due to the formation of a protective film due to the polymer for precipitation, which may increase the half-life.

Accordingly, through the step of culturing the mixed solution formed by mixing the separated supernatant and the first solution (S120), a low-molecular extracellular vesicle is formed into a polymer capable of precipitating it, so that it can be easily separated in a general centrifuge.

Next, in the step of extracting the biomarker from the extracellular ER ( S140 ), nucleic acid, which is a genetic material in the extracellular ER, can be extracted, and the method of extracting the nucleic acid is to remove the cell wall and the cell membrane of the extracellular ER and extract only the nucleic acid All possible methods can be included.

In this case, the term "nucleic acid" as used herein means at least one nucleotide consisting of a sugar linked to an organic base (substituted pyrimidines such as cytosine, thymidine and uracil, or substituted purines such as adenine and guanine) can do. More specifically, the nucleic acid may include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), methylphosphonate nucleic acid, S-oligo, cDNA, miRNA and aptamer (aptamer), and the like, naturally occurring or artificial. may include all those synthesized with However, it may be preferably RNA and DNA, but is not limited thereto.

Next, in the step of measuring the expression level of the extracted biomarker (S150), the level measurement is a microarray analysis method through fluorescence, chemiluminescence, or electrical signal detection, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction ( RT-PCR), digital microdroplet PCR (ddPCR), solid-state nanopore detection, RNA switch activation, Northern blot, or sequencing of gene expression analysis (SAGE). it's not going to be However, a method capable of increasing the accuracy of the analysis may preferably be ddPCR.

Next, when the expression level of the measured biomarker is higher than or equal to a predetermined level, in the step of determining respiratory disease ( S160 ), respiratory disease is detected through, for example, IS6110, a target gene of a pathogen that can cause respiratory disease as a biomarker. can be diagnosed However, the biomarker is not limited thereto, and various target genes of pathogens that can cause respiratory diseases may be used. Furthermore, respiratory diseases that can be diagnosed through the aforementioned biomarker IS6110 include Mycobacterium tuberculosis (M), M. bovis, M. africanum, M. canetii, and bacterial respiratory diseases caused by M. microti.

More specifically, IS6110 is a useful target marker for the diagnosis of tuberculosis due to the large number of copies in mycobacterium tuberculosis. In most tuberculosis strain genes, there are more than 10 IS6110 copies, so the strain discrimination ability through the IS6110 gene can be very good. Accordingly, IS6110 may be used to diagnose a bacterial respiratory disease caused by tuberculosis, that is, Mycobacterium tuberculosis, in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention, but is not limited thereto, and in the tuberculosis strain A variety of specifically expressed markers can be used.

At this time, the threshold of expression level for diagnosing whether or not infection with Mycobacterium tuberculosis may be 1 copy/reaction (ml) in ddPCR, but is not limited thereto. Therefore, when the measured expression level of IS6110 is 1 copy/reaction (ml) or more, it can be determined as positive for tuberculosis caused by Mycobacterium tuberculosis. tuberculosis can be determined negatively. That is, ddPCR can detect even a small amount of DNA copy of about 1, so it can be measured more sensitively than other conventional measuring methods.

At this time, ddPCR is a method of dividing a predetermined amount of a sample into tens of thousands of droplets, amplifying the sample by PCR reaction, and then counting the target. Each split droplet is displayed as positive (1) or negative (0) depending on whether the target is amplified or not, and by calculating the target copy through Poisson distribution, it can be expressed as the number of copies per sample.

Meanwhile, a respiratory disease caused by Mycobacterium tuberculosis can be diagnosed through methods other than the above-described ddPCR. More specifically, in the method for infectious respiratory disease through the extracellular vesicles according to an embodiment of the present invention, after measuring the expression level of the extracted biomarker (S150), measuring the expression of the biomarker by rtPCR and measuring If the amplification curve of the biomarker is expressed below the 27 ct value, the method may further include determining a respiratory disease. In this case, the ct value of the amplification curve may be 27, but is not limited thereto, and may be variously set according to the state of each sample, the detection kit used, the measuring device, and the type of the biomarker.

Through the above process, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention is a more convenient way to isolate the extracellular vesicles in a short time, thereby diagnosing infectious respiratory diseases with improved accuracy. have.

Confirmation of detectability in Mycobacterium tuberculosis

Referring to FIG. 2A, the results of the diagnosis of Mycobacterium tuberculosis according to the extracellular vesicle isolation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention are shown. In Comparative Example 1, a sample amount of 1 mL was used, the extracellular vesicles were isolated using Exoquick, an extracellular vesicle separation kit, and IS6110 copy number (copy/ml), a marker for Mycobacterium tuberculosis, was measured. In Comparative Example 2, a mixture containing dextran, not the first solution, was used in the process of the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention, and by the above-described method The extravesicular vesicles were isolated and the IS6110 copy number in the tuberculosis group was measured.

Dextran is a first solution used in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention, that is, a polymer material having a molecular weight 10 times higher than that of PEG, and the precipitation rate through the first solution That is, it was used as a control to confirm the yield of extracellular vesicles.

In addition, in Example 1, a sample amount of 300 μL was used, and in Example 1, the extracellular vesicles were isolated by the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention, and IS6110 replication in Mycobacterium tuberculosis. The number was measured. Furthermore, each copy number was determined using ddPCR.

In addition, in the method using rtPCR, a sample amount of 1 mL was used, respectively, and the extracellular vesicle separation method was not performed.

First, Sample 1 shows that both rtPCR and Mycobacterium tuberculosis culture test results are negative (Neg), and there is no Mycobacterium tuberculosis at all in Comparative Example 1, Comparative Example 2 and Example 1. That is, the negative determination for the conjunctivitis is shown to be effective in both Comparative Examples and Examples.

Sample 2 shows that both rtPCR and Mycobacterium tuberculosis culture test results are positive (Pos). Furthermore, the number of copies for Mycobacterium tuberculosis in ddPCR is shown to be similar in Comparative Example 1 and Example 1 at 2317 copies/ml and 1885 copies/ml, respectively, and Comparative Example 2 is the lowest at 170 copies/ml. That is, the diagnostic method of the present invention using a sample amount of 300 μL appears to have similar detection power to Comparative Example 1 using the conventional extracellular vesicle separation method using a sample amount of 1 mL, and even with a smaller sample amount than the conventional method DNA content can be measured. Furthermore, in the polymer for precipitation, it appears that the first solution used in Example 1, that is, PEG, has a higher detection power than the dextran used in Comparative Example 2. As a result, the diagnostic method of the present invention showed utility in target detection than the conventional extracellular vesicle separation method.

Sample 3 shows that both rtPCR and Mycobacterium tuberculosis culture test results are positive (Pos). Furthermore, the number of copies for Mycobacterium tuberculosis in ddPCR is shown to be similar in Comparative Example 1 and Example 1 at 8088 copies/ml and 7759 copies/ml, respectively, and Comparative Example 2 is shown to be the lowest at 1249 copies/ml. That is, as in Sample 2, Example 1 is shown to have similar detection power to Comparative Example 1, which is a conventional method, even with a small amount of sample. Furthermore, Comparative Example 2, in which a mixture containing dextran was used, showed lower detection power than Example 1.

Furthermore, referring to FIG. 2B , ddPCR results for Example 1 and Comparative Example 2 in FIG. 2A are shown. In this case, the x-axis means each sample and the amount of the sample in the ddPCR well, and the y-axis means the fluorescence amplitude for each droplet. Furthermore, the horizontal line means a threshold value that separates positive and negative. At this time, the threshold value is shown as 3045, but is not limited thereto, and may vary for each experimental batch.

Sample 1, in which both rtPCR and Mycobacterium tuberculosis culture results were negative, showed no fluorescence expression in Example 1 and Comparative Example 2. This may mean that both the methods of Example 1 and Comparative Example 2 can accurately diagnose negative for Mycobacterium tuberculosis.

Sample 2, which was positive for both rtPCR and Mycobacterium tuberculosis culture results, exhibits fluorescence expression between 7000 and 9000 in Example 1 and Comparative Example 2. However, the number of expressed droplets appears to be greater in Example 1 than in Comparative Example 2. That is, in the separation of extracellular vesicles, the method in Example 1 using PEG, the first solution of the present invention, can mean to have a higher yield than the method in Comparative Example 2 using a mixture containing dextran. have.

Therefore, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention has a higher yield in the separation of the extracellular vesicles, and is expressed above 3045, which is a threshold value for separating positive and negative, to Mycobacterium tuberculosis. It may mean that a positive diagnosis can be made accurately.

Sample 3 showing both rtPCR and Mycobacterium tuberculosis culture results showed that the fluorescence expression of Example 1 and Comparative Example 2 was between 6000 and 10000. However, as in Sample 2, the number of expressed droplets is greater in Example 1 than in Comparative Example 2, which may mean that the method in Example 1 has a higher yield than the method in Comparative Example 2. .

Accordingly, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention has a higher yield in the separation of the extracellular vesicles, and is expressed above 3045, which is the threshold for separating positive and negative, for Mycobacterium tuberculosis. It can mean that a positive diagnosis can be accurately made. As a result, Example 1, in which the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention is used, has a higher yield, and an accurate negative or positive diagnosis for Mycobacterium tuberculosis can be made based on the threshold. .

Accordingly, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention can diagnose Mycobacterium tuberculosis even in 200 μL, which is less than 1 ml used in the conventional method.

Referring to FIGS. 3A to 3C , the nanoparticle characterization results for Example 1 and Comparative Example 2 are shown. At this time, the nanoparticle characteristic analysis is measured twice each using nanosight, so that the size distribution of the nanoparticles can be confirmed.

First, FIG. 3a shows the results of analysis of the nanoparticle characteristics in Sample 1 for Example 1 and Comparative Example 2. As shown in FIG. Referring to (a) of FIG. 3A , the nanoparticles in Example 1 are shown to have a size of 1 to 400 nm, and the nanoparticles having a size of 100 nm appear to be the most.

Furthermore, referring to (b) of FIG. 3A, the nanoparticles in Comparative Example 2 appear to have a size of 1 to 300 nm, and particles having a size of 100 nm are the most numerous, but 100 to 200 nm Nanoparticles with a size of

As a result, in Sample 1, Example 1 shows that only nanoparticles having the same size of 100 nm are mainly distributed, whereas in Comparative Example 2, various nanoparticles having a size of 0 to 200 nm are distributed. That is, Example 1 may mean that nanoparticles having a more uniform size than Comparative Example 2 can be separated.

Next, FIG. 3b shows the results of analysis of nanoparticle properties in Sample 2 for Example 1 and Comparative Example 2. FIG. Referring to (a) of FIG. 3B , the nanoparticles in Example 1 are shown to have a size of 1 to 350 nm, and the nanoparticles having a size of 100 nm to 200 nm are the largest.

Furthermore, referring to (b) of FIG. 3B , the nanoparticles in Comparative Example 2 are shown to have a size of 1 to 350 nm, and the nanoparticles having a size of 150 nm to 300 nm are the most numerous.

As a result, in Sample 2, Example 1 showed that nanoparticles having a size of 100 nm to 200 nm were mainly distributed, but Comparative Example 2 showed that nanoparticles having a relatively large size of 150 nm to 300 nm were mainly distributed. appears to be That is, Example 1 may mean that more nanoparticles having a smaller size than Comparative Example 2 can be separated.

Next, FIG. 3c shows the results of analysis of the nanoparticle characteristics in Sample 3 for Example 1 and Comparative Example 2. FIG. Referring to (a) of FIG. 3C , the nanoparticles in Example 1 are shown to have a size of 50 to 550 nm, and the nanoparticles having a size of 100 nm to 150 nm are the most numerous.

Furthermore, referring to (b) of FIG. 3C , the nanoparticles in Comparative Example 2 are shown to have a size of 1 to 300 nm, and the nanoparticles having a size of 150 nm to 200 nm are the most numerous.

That is, Example 1 in Sample 3 may mean that more nanoparticles having a smaller size than Comparative Example 2 can be separated, as in FIG. 3B described above.

In addition, as the nanoparticles in Comparative Example 2 have a large distribution in both 150 nm and 200 nm sizes, not a large distribution in a specific size such as 150 nm in Example 1, as in FIG. 1 may mean that nanoparticles of a more uniform size than Comparative Example 2 can be separated

Confirmation of detection power for respiratory viruses of the method for diagnosing infectious respiratory diseases through extracellular vesicles according to an embodiment of the present invention

4, the respiratory virus diagnosis result according to the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention is shown. In this case, Comparative Example 1 did not include the process of isolating the extracellular vesicles, but measurement was performed for respiratory viruses, and Comparative Example 2 is the process of isolating the extracellular vesicles using Exoquick, a conventional extracellular vesicle separation kit. and measurement was performed for respiratory viruses, Example 1 was performed by the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention. Furthermore, the amount of sample used in each method was the same as 500 μL, and the results were expressed as Ct values. The Ct value means the number of cycles at a point exceeding a threshold at which the amplified product can be detected.

First, the results of Sample 1 show that both Comparative Examples 1, 2 and Example 1 are influenza virus A (inf A), and the ct value of Example 1 is 18.02, indicating that Example 1 has the lowest ct value. That is, it appears that the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention has the highest expression level for influenza virus A.

Then, the result of Sample 2 shows that Comparative Examples 1, 2 and Example 1 are both rhinovirs (Rhino), and the ct value is 11.39, which is the lowest in Example 1. That is, it appears that the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention has the highest expression level for rhinovirus.

Then, the results of Sample 3 show that Comparative Examples 1, 2 and Example 1 are all negative for respiratory virus.

Then, the results of Sample 4 show that both Comparative Examples 1, 2 and Example 1 are metapneumovirus (MPV), and the ct value of Example 1 is 11.61, indicating that Example 1 has the lowest ct value. That is, it appears that the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention has the highest expression level for metapneumovirus.

Then, the results of Sample 5 indicate that both Comparative Examples 1, 2 and Example 1 are metapneumovirus (MPV), and the ct value of Comparative Example 2 is 4, indicating that it has the lowest ct value, Example 1 is 4.1, which is shown to have a similar ct value to Comparative Example 2. That is, the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention appears to have an expression level for metapneumovirus similar to the conventional extracellular vesicle separation method.

Then, the results of Sample 6 show that both Comparative Examples 1, 2 and Example 1 are influenza virus A (inf A), and the ct value is 17.41, showing that Example 1 has the lowest ct value. . That is, it appears that the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention has the highest expression level for influenza virus A.

On the other hand, Comparative Example 1 does not appear to be detected with respect to corona virus 229E (corona virus 229E). However, Comparative Examples 2 and 1, including the process of isolating the extracellular vesicles, were shown to be positive for corona virus 229E, and the ct values were similar to those of Comparative Examples 2 and 1, respectively, 26.397 and 26.7. appears to have a value. That is, the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention appears to have a similar expression level to the conventional extracellular vesicle separation method for coronavirus 229E.

Then, the result of Sample 7 shows that both Comparative Examples 1, 2 and Example 1 are corona virus NL63, and the ct value is 11.42, showing that Comparative Example 2 has the lowest ct value, Example 1 is 11.54, which is shown to have a similar ct value to Comparative Example 2. That is, the extracellular vesicle separation method in the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention has a similar expression level to the conventional extracellular vesicle separation method for coronavirus NL63.

As a result, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention is similar to or superior to the conventional method in not only Mycobacterium tuberculosis but also various respiratory viruses. Accordingly, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention can provide more improved sensitivity and specificity.

In addition, the method for diagnosing infectious respiratory diseases through the extracellular vesicles according to an embodiment of the present invention can detect the above-mentioned pathogen based on the ct value of 27 in rtPCR when measuring Mycobacterium tuberculosis and respiratory viruses. More specifically, according to the above results, influenza virus A, rhinovirus, metapneumovirus, coronavirus 229E, and coronavirus NL63 appear to have ct values of 19, 13, 12, 5, 27 and 13 or less, respectively. Accordingly, when the amplification curve is expressed based on a range capable of detecting all of the aforementioned pathogens, that is, based on a value of 27 ct, it can be determined as positive for respiratory diseases caused by the aforementioned pathogens. However, the ct value is not limited thereto, and may be set in various ways according to the state of each sample, the detection kit used, the measuring device, and the type of biomarker.

Confirmation of analysis performance of the method for diagnosing infectious respiratory diseases through extracellular vesicles according to an embodiment of the present invention

Referring to FIG. 5 , the analysis performance test results according to the analysis method in Mycobacterium tuberculosis are shown. At this time. Sensitivity refers to the probability of judging positive for a positive individual, specificity refers to the probability of judging negative for a negative individual, and accuracy refers to the probability of judging negative for a negative individual. It may mean a probability of determining a true value.

First, the results of the Acid-Fast Bacillus Smear (AFB smear) test showed that 15 positive (Pos) and 29 negative (Neg) among pulmonary tuberculosis patients were found, and among non-tuberculosis patients, 2 positive and 144 negative. appears to be a name. Accordingly, the sensitivity is 34.1%, the specificity is 98.6%, and the accuracy is 83.7%.

Then, the qPCR results measured without performing the extracellular vesicle separation method showed that 21 patients were positive (Pos) and 23 were negative (Neg) among pulmonary tuberculosis patients, and 0 positive and 146 non-tuberculosis patients were negative. appears to be a name. Accordingly, the sensitivity is 47.7%, the specificity is 100%, and the accuracy is 87.9%.

Then, the extracellular vesicle isolation method was performed and the measured qPCR results showed that 24 positive (Pos) and 20 negative (Neg) patients were among pulmonary tuberculosis patients, and 0 positive and 146 non-tuberculosis patients were negative. appears to be Accordingly, the sensitivity is 54.6%, the specificity is 100%, and the accuracy is 89.5%.

Then, the ddPCR results measured without performing the extracellular vesicle separation method showed that 27 positive (Pos) and 17 negative (Neg) patients among pulmonary tuberculosis patients were positive and 146 were negative among non-tuberculosis patients. appears to be a name. Accordingly, the sensitivity is 61.4%, the specificity is 100%, and the accuracy is 91.1%.

Then, the extracellular vesicle separation method was performed and the measured ddPCR results showed that 33 positive (Pos) and 11 negative (Neg) patients were among pulmonary tuberculosis patients, and among non-tuberculosis patients, 0 positive and 146 negative patients were appears to be Accordingly, the sensitivity is 75.0%, the specificity is 100%, and the accuracy is 94.2%.

Then, the results of the Mycobacterium tuberculosis culture test showed that 39 positive (Pos) and negative (Neg) patients were among pulmonary tuberculosis patients, and 0 positive and 146 negative among non-tuberculosis patients. Accordingly, the sensitivity is 88.6%, the specificity is 100%, and the accuracy is 97.4%.

As a result, it appears that the method of performing the extracellular vesicle isolation method and measuring using ddPCR is most similar to the Mycobacterium tuberculosis culture test with the highest sensitivity and purification.

Referring to FIG. 6 , the analysis performance test results according to the culture results of Mycobacterium tuberculosis are shown.

First, in a population including both positive and negative (All) in the smear test, the sensitivity result of qPCR measured without performing the extracellular vesicle separation method is 46.2%, and the qPCR measured after performing the extracellular vesicle separation method The sensitivity result is 53.9%. That is, the qPCR diagnostic method through the extracellular vesicle isolation can provide improved sensitivity than the qPCR diagnosis that does not include the process of separating the extracellular vesicles.

Similarly, the sensitivity result of ddPCR measured without performing the extracellular vesicle separation method in the population including both positive and negative (All) in the smear test was shown to be 61.5%, and the extracellular vesicle separation method was performed and measured The sensitivity result of ddPCR appears to be 76.9%. That is, similar to the above-described qPCR results, the ddPCR method through extracellular vesicle separation can provide improved sensitivity than ddPCR, which does not include a process for separating extracellular vesicles.

Furthermore, the sensitivity of ddPCR measured by performing the extracellular vesicle isolation method was 76.9%, which is higher than that of qPCR 53.9%. That is, it may mean that the diagnostic method using ddPCR has improved sensitivity, ie, improved diagnostic power, compared to the diagnostic method using qPCR. Accordingly, the method for diagnosing infectious respiratory diseases through the extracellular vesicle separation method according to an embodiment of the present invention may preferably be a method using ddPCR, but is not limited thereto, and the sensitivity in various measurement methods can be improved. have.

Furthermore, in the smear-positive population (n=17), the sensitivity result of qPCR measured without performing the extracellular vesicle isolation method was found to be 85.7%, and the sensitivity result of qPCR measured with the extracellular vesicle isolation method performed was It is found to be 92.9%. In addition, the sensitivity result of ddPCR measured without performing the extracellular vesicle separation method in the smear positive population is 92.9%, and the sensitivity result of ddPCR measured after performing the extracellular vesicle separation method is 100%. That is, as in the group including both positive and negative in the smear test, it is possible to provide more improved sensitivity in each qPCR and ddPCR diagnostic method through extracellular vesicle separation, and the diagnostic method through ddPCR is the diagnostic method through qPCR. It appears to have improved sensitivity.

Furthermore, in the smear negative population (n = 173), the sensitivity result of qPCR measured without performing the extracellular vesicle isolation method was found to be 24.0%, and the sensitivity result of qPCR measured with the extracellular vesicle isolation method performed was It appears to be 32.0%. In addition, the sensitivity result of ddPCR measured without performing the extracellular vesicle separation method in the smear positive population is 44.0%, and the sensitivity result of ddPCR measured after performing the extracellular vesicle separation method is 64.0%. That is, as in the group including both positive and negative in the smear test, it is possible to provide more improved sensitivity in each qPCR and ddPCR diagnostic method through extracellular vesicle separation, and the diagnostic method through ddPCR is the diagnostic method through qPCR. It appears to have improved sensitivity.

Referring to FIG. 7 , the diagnostic test results in the qPCR and ddPCR diagnostic methods according to the extracellular vesicle separation method from a low concentration sample are shown.

First, as a result of the culture of all samples, it appears that the Mycobacterium tuberculosis culture is positive.

Then, performing the extracellular vesicle separation method and the measured ddPCR results all appear to be 100% positive, and the ddPCR results measured without performing the extracellular vesicle separation method are 53.3% positive.

On the other hand, 46.7% of the qPCR results measured after performing the extracellular vesicle isolation method are shown to be positive, and 26.7% of the qPCR results measured without performing the extracellular vesicle isolation method are shown to be positive.

That is, even in a low concentration sample, the method of performing a diagnostic test through extracellular vesicle separation and ddPCR appears to have the highest diagnostic accuracy. Accordingly, the method for diagnosing infectious respiratory diseases through the extracellular vesicle separation method according to an embodiment of the present invention can diagnose even a very low concentration of pathogens present in a sample with high sensitivity.

Referring to Figures 8a to 8b, the number of copies of Mycobacterium tuberculosis (log ₁₀ copies/ml) in the Mycobacterium tuberculosis positive sample according to whether extracellular vesicles are isolated or not is shown. At this time, the copy number of Mycobacterium tuberculosis was measured by targeting the IS6100 gene in Mycobacterium tuberculosis, and the copy number was measured using ddPCR.

First, referring to FIG. 8A , a scatter plot result of the number of copies of Mycobacterium tuberculosis in a clinical sample according to whether or not extracellular vesicles are separated is shown. The correlation coefficient between clinical samples according to the separation of the extracellular vesicles is 0.827, indicating that the correlation between the two measurement variables is 0.8 or more (correlated).

Then, referring to FIG. 8B , the results of Bland-Altman Plot are shown in the number of copies of Mycobacterium tuberculosis in clinical samples according to whether or not extracellular vesicles are isolated. At this time, the Bland-Altonman diagram is a graph to find out the difference between the measured value and the estimated value measured by the two measurement methods. difference by as much as 95%, the limits of agreement (LOA).

More specifically, it appears that the average difference between clinical samples depending on whether extracellular vesicles are isolated is 0.2 ± 0.94 log ₁₀ copies/ml, and the 95% LOA upper and lower limits of 0.2 ± 0.94 log ₁₀ copies/ml are -1.7 and 2.0. In addition, it appears that the difference between the two clinical samples increases as the value increases.

As a result, the degree of agreement between the two clinical samples is similar from 0 to 5 log ₁₀ copies/ml, but from more than 5 log ₁₀ copies/ml, the diagnostic difference between the two clinical samples is large. Accordingly, in the results in FIGS. 5 to 7 described above, as the sample including the separation process of the extracellular vesicles has more improved sensitivity, infectious respiratory diseases through the extracellular vesicles separation method according to an embodiment of the present invention The diagnostic method can make an accurate diagnosis with reduced experimental error.

Although the embodiments of the present invention have been described in more detail with reference to the accompanying drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be made within the scope without departing from the technical spirit of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.

Claims (6)

separating the supernatant from the biological sample isolated from the subject;
A mixture formed by mixing the separated supernatant and a first solution containing a low molecular weight polyethylene glycol (PEG) having a molecular weight of 5000 to 8500 MW is cultured at 2 to 5° for 1 to 3 hours (incubation) step;
separating extra cellular vesicles from the cultured mixture;
extracting exo DNA derived from the extracellular ER as a biomarker from the extracellular ER;
measuring the expression level of exo DNA derived from the extracted extracellular vesicles by ddPCR; and
When the measured expression level of exo DNA derived from the extracellular vesicle is above a predetermined level, determining a respiratory disease,
The respiratory disease is bacteria caused by Mycobacterium tuberculosis (M), Mycobacterium bovis, M. africanum, M. canetii, and M. microti. sexual respiratory disease,
A method for diagnosing infectious respiratory diseases through the extracellular vesicles, characterized in that the recovery rate of the extracellular vesicles is increased by using the polyethylene glycol (PEG), which is a polymer material having a low molecular weight. The method of claim 1,
The extracellular vesicles,
exosomes, ectosomes, exovesicles, microvesicles, microparticles, apoptotic bodies, membrane particles, membrane vesicles, exosome-like vesicles, and A method for diagnosing infectious respiratory diseases through extracellular vesicles, comprising at least one of the group consisting of ectosome-like vesicles. The method of claim 1,
The biological sample,
A method for diagnosing infectious respiratory diseases through extracellular vesicles, comprising at least one of the group consisting of blood, urine, pleural fluid, feces, sputum, intraperitoneal fluid, breast milk, cerebrospinal fluid, saliva, lymph and bronchoalveolar lavage fluid. The method of claim 1,
Separating the supernatant is
A method for diagnosing infectious respiratory diseases through extracellular vesicles, performed by centrifugation at 500 to 4000 g for 5 to 30 minutes. The method of claim 1,
The first solution is
Based on the total volume of the mixed solution, 7 to 10% (w / v), Infectious respiratory disease diagnosis method through the extracellular vesicles. The method of claim 1,
The step of isolating the extracellular vesicles,
A method for diagnosing infectious respiratory diseases through extracellular vesicles, performed by centrifugation at 1000 to 2000 g for 10 to 40 minutes.

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