How to detect coronavirus infection

Abstract

The purpose of the present invention is to provide a method for identifying a subject who is or may be suffering from COVID-19. The present invention provides a method for identifying whether or not a mammal subject is or may be suffering from COVID-19, the method comprising detecting the level of at least one modified nucleoside selected from the group consisting of 6-threonylcarbamoyladenosine (t6A) and 2-thiomethyl,6-threonylcarbamoyladenosine (ms2t6A) in a sample derived from the subject.

Description

[Technical field]

[0001] The present invention relates to a method for detecting a subject at risk of or suffering from COVID-19. More specifically, the present invention relates to a method for detecting a subject at risk of or suffering from COVID-19 by using modified nucleosides as targets. [Background technology]

[0002] The pneumonia of unknown cause reported in Wuhan, Hubei Province, People's Republic of China in December 2019 is an infectious disease caused by a novel pathogenic virus called SARS-CoV2 and has been named COVID-19. Since then, the infection has spread worldwide, causing many deaths and having a huge impact on social and economic activities. Currently, PCR and antigen tests are used to diagnose the disease. However, because antigen tests have low accuracy, PCR tests are used in most cases for definitive diagnosis. PCR tests are highly accurate, but because nasopharyngeal swabs, sputum, or saliva are used as samples, there is a risk of infection for medical workers and laboratory technicians, and the workers are under mental and physical strain. In addition, it takes a long time to get the test results, and currently pretreatment is often done by hand, which is cumbersome. In addition, the current test method cannot determine the severity or effectiveness of treatment, making it difficult to evaluate the patient's condition in the clinical setting. Therefore, a COVID-19 test method that can more easily evaluate the disease is desired.

[0003] The inventors have previously clarified and reported the physiological significance of chemical modifications in ribonucleic acid (RNA) in mammals. In the process, they discovered that once modified, nucleic acids are broken down into nucleosides and excreted outside the cells, and established a method for comprehensively analyzing modified nucleosides in blood and urine (Non-Patent Document 1). It is known that some of these modified nucleosides are common to all biological species, while others are specific to each biological species.

[0004] tRNA is an adaptor molecule that converts genetic information written in DNA, which consists of four bases, into the amino acid sequence of a protein. tRNA is modified after various post-transcriptional modifications, which are necessary not only for the folding and stability of tRNA but also for the accurate and efficient decoding of the genetic code.

[0005] N 6 -Threonylcarbamoyl adenosine (t 6 A) is a derivative of adenosine and has a chemical structure in which threonine is bound to the N6 position via a carbonyl group. 6 A is a modified base present at position 37 of tRNA that decodes the ANN codon, and is a modified nucleoside that is conserved in almost all organisms and is essential for the growth of many organisms. 6 A is known to play important roles in various steps of protein synthesis, including aminoacylation of tRNA, translocation reaction, accurate codon recognition, and maintenance of reading frame.

[0006] 2-Thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) is t 6 The chemical structure of A is that the 2-position of the adenine is thiomethylated. 2 t 6 A is a modified base at position 37 of tRNA with the anticodon UUU, and is transduced by the methylthiotransferase Cdkal1. 6 The present inventors have reported that it is biosynthesized from A (Non-Patent Document 2).

[0007] Modified nucleosides have also been reported to be associated with diseases. 2 t 6 The present inventors have reported that A is associated with type 2 diabetes (Non-Patent Document 2). However, there have been no reports of any association between these modified nucleosides and infectious viruses, including COVID-19. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ogawa et al., Molecular Cell 81, 659-674, 2021 [Non-Patent Document 2] Wei et al., J Clin Invest. 2011; 121(9):3598-3608 Summary of the Invention [Problem to be solved by the invention]

[0009] In one aspect, the present invention aims to provide a method for detecting a subject at risk of or suffering from COVID-19. In another aspect, the present invention aims to provide a method for predicting the severity of a patient suffering from COVID-19. [Means for solving the problem]

[0010] The inventors comprehensively analyzed modified nucleosides in the blood and urine of COVID-19 patients and healthy individuals using a mass spectrometer. As a result, 6-threonylcarbamoyl adenosine (t 6 A) and 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 The inventors further found that the amount of t in the plasma and urine of various fever patients was significantly higher in COVID-19 patients, and completed the present invention. 6 A and ms 2 t 6 The inventors analyzed the amount of modified nucleosides in the patients with COVID-19 and found that these modified nucleosides were specifically higher in the patients than in other patients with fever. 6 A and ms 2 t 6 We found that the amount of A correlated with subsequent changes in the patient's condition.

[0011] The present invention includes the following aspects. [1] A method for determining whether a mammalian subject is at risk of or has COVID-19, comprising detecting the presence of 6-threonylcarbamoyl adenosine (t 6 A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) detecting an amount of a modified nucleoside; and providing the detected amount of the modified nucleoside for said determination. [2] The method according to [1] above, further comprising a step of determining whether the subject is at risk of being infected with or is infected with COVID-19 by comparing the amount of the detected modified nucleoside with a predetermined reference value. [3] The method according to [1] above, further comprising a step of assessing the severity of the subject (degree of COVID-19 infection, e.g., mild, moderate (moderate I, moderate II), severe) based on the amount of the modified nucleoside detected. [4] The method according to [2] above, further comprising a step of predicting a subsequent change in the subject's pathological condition based on the amount of the modified nucleoside detected. [5] A method for evaluating the efficacy of a treatment in a mammal suffering from COVID-19, comprising: determining whether 6-threonylcarbamoyl adenosine (t 6 A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) detecting an amount of a modified nucleoside; and providing the detected amount of the modified nucleoside for said evaluation. [6] The method according to any one of the above [1] to [5], wherein the subject-derived sample is plasma, serum, or urine. [7] The method according to any one of the above [1] to [6], wherein the amount of the modified nucleoside is detected by mass spectrometry (preferably tandem mass spectrometry (MS / MS)). [8] The method according to the above [7], wherein the sample has been subjected to deproteinization and desalting treatment. [9] The method according to [7] or [8] above, wherein the sample is plasma or serum, and the step of detecting the amount of the modified nucleoside includes a step of correcting the measurement result by the amount of adenosine in the plasma or serum.

[10] The method according to [7] or [8] above, wherein the sample is urine and the step of detecting the amount of the modified nucleoside includes a step of correcting the measurement result with the amount of at least one substance selected from the group consisting of creatinine, urea nitrogen, uric acid, adenosine, and 3-amino-3-carboxypropyluridine (acp3U) in the urine.

[11] The method according to any one of the above [1] to [6], wherein the amount of the modified nucleoside is detected by ELISA.

[12] The method according to any one of the above [1] to

[11] , wherein the subject is a human.

[13] The method according to

[12] above, wherein the subject is a patient with fever.

[14] A method for determining whether a mammalian subject is a subject for treatment of COVID-19, comprising: detecting 6-threonylcarbamoyl adenosine (t 6 A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) detecting an amount of a modified nucleoside; and providing the detected amount of the modified nucleoside for said determination.

[15] The method according to

[14] above, wherein the subject-derived sample is plasma, serum, or urine.

[16] The method according to

[14] or

[15] above, wherein the subject is a human.

[17] 6-Threonylcarbamoyl adenosine (t 6 A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) A marker for the progression or prediction of the progression of COVID-19.

[18] 6-Threonylcarbamoyladenosine (t 6A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) Use of a modified nucleoside as a marker for the severity or predictive marker of COVID-19.

[19] The use according to

[18] above, wherein the subject is a human and the sample is plasma, serum or urine.

[0012] The present invention also relates to a method for diagnosing whether or not a subject is infected with COVID-19 based on the results of a modified nucleoside detected by the method according to any one of the above [1] to

[13] . Thus, the present invention also relates to a diagnostic method comprising the steps of: (a) Detection of 6-threonylcarbamoyl adenosine (tAAD) in a patient-derived sample (preferably plasma, serum, or urine, more preferably plasma, serum, or urine that has been deproteinized and desalted). 6 A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) a modified nucleoside (preferably t 6 A and ms 2 t 6 Detecting the amount of A); and (b) diagnosing whether the patient is infected with COVID-19 based on the amount of modified nucleoside detected in step a. The amount of modified nucleoside in the above step (a) can be detected by mass spectrometry (preferably tandem mass spectrometry (MS / MS)) or ELISA. In the above step (a), when the sample is plasma or serum, more accurate detection can be achieved by correcting the amount of the detected modified nucleoside with the amount of adenosine in the plasma or serum. Furthermore, when the sample is urine, more accurate detection can be achieved by correcting the amount of the detected modified nucleoside with the amount of at least one substance selected from the group consisting of creatinine, urea nitrogen, uric acid, adenosine, and 3-amino-3-carboxypropyluridine (acp3U). Effect of the Invention

[0013] The methods of the present invention can determine whether a subject is at risk of or has COVID-19. [Brief description of the drawings]

[0014] [Figure 1] The left diagram shows the structure of 6-threonylcarbamoyladenosine (t6A), and the right diagram shows the structure of 2-thiomethyl,6-threonylcarbamoyladenosine (ms2t6A). [Diagram 2] These are the results of an analysis of modified nucleosides in the RNA of ACE2-overexpressing HEK293 cells infected with SARS-CoV2. [Diagram 3] The left panel shows the amount of t6A in serum (after correction for serum adenosine), and the right panel shows the amount of ms2t6A in serum (after correction for serum adenosine). [Figure 4] The results show a comparison of urinary t6A levels (corrected for urinary acp3U) between COVID-19 patients and other patients with fever. [Diagram 5] The ROC curve of t6A amount corrected by acp3U is shown. [Figure 6] The results show a comparison of urinary ms2t6A levels (corrected for urinary acp3U) between COVID-19 patients and other patients with fever. [Figure 7] The ROC curve of ms2t6A amount corrected by acp3U is shown. [Figure 8] Comparison of detection of modified nucleic acids (t6A and ms2t6A) in COVID-19 infected patients and healthy subjects, without correction using an internal standard. Data represent mean ± SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001. [Figure 9]Comparison of subject samples for detection of modified nucleic acids (t6A and ms2t6A) in COVID-19 infected patients and healthy controls. Urine samples were acp3U corrected and serum samples were adenosine corrected. Data represent mean ± SEM, points represent individual subjects. ****, P<0.0001. [Figure 10] The results show a comparison of detection of modified nucleic acids (t6A and ms2t6A) in asymptomatic / mildly ill and severely ill COVID-19 patients. [Figure 11] The results show the detection of modified nucleic acid (ms2t6A) at the time of admission of COVID-19 infected patients and the subsequent changes in the pathological condition of the patients. Data are the mean ± SEM. * P < 0.0035. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will now be described in detail with reference to exemplary embodiments, but the present invention is not limited to the embodiments described below. Unless otherwise specified in the text, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In addition, any materials and methods equivalent or similar to those described in this specification can be used in the same manner in the practice of the present invention. In addition, all publications and patents cited in this specification in relation to the invention described in this specification are incorporated by way of example as a part of this specification, as a description of methods, materials, etc. that can be used in the present invention.

[0016] In this specification, the description of "A to B" indicating a numerical range means a numerical range including the endpoints A and B. The same applies to "from A to B."

[0017] COVID-19 is the name given to the pneumonia of unknown cause that broke out in December 2019 and caused a global pandemic. Meanwhile, SARS-CoV-2 is the name of the virus that causes it. In this specification, COVID-19 is used to refer to the disease, and SARS-CoV-2 is used to refer to the virus. Regardless of which name is used, if it is clear from the context whether it refers to the disease or the virus, it shall be interpreted as meaning that it is.

[0018] As used herein, a "subject" or "patient" refers to any mammal, including, but not limited to, humans, non-human primates, including non-human primates such as chimpanzees, other apes and monkey species, farm animals such as cows, sheep, pigs, goats and horses, domestic mammals such as dogs and cats, and small or laboratory animals, including rodents such as mice, rats and guinea pigs, and is preferably a human. A "subject" or "patient" also includes adults, infants and neonates.

[0019] In the present invention, the term "sample" refers to any sample derived from a subject that may contain modified nucleosides. Although not particularly limited, a body fluid sample derived from a subject is preferably used as the sample. A "body fluid sample" refers to any liquid sample that can be isolated from an individual's body, including, but not limited to, blood, plasma, serum, saliva, urine, tears, sweat, etc. Preferably, the body fluid is plasma, serum, or urine. In one embodiment, the sample used in the present invention is derived from a human.

[0020] In the present invention, "having COVID-19" means that a mammalian subject is infected with the causative virus SARS-CoV2 and can be determined to have COVID-19. In the present invention, "possibly having COVID-19" means that a mammalian subject is suspected of being infected with the causative virus SARS-CoV2.

[0021] A subject determined to be infected with or at risk of being infected with COVID-19 by the method of the present invention can be diagnosed as having COVID-19 by combining with other COVID-19 detection methods. Other detection methods include, but are not limited to, PCR tests and antigen tests, and preferably PCR tests.

[0022] In the present invention, a subject being a target for treatment of COVID-19 means that the subject to which the detection method of the present invention is performed can be determined to be infected with SARS-CoV2, the causative virus, suffering from COVID-19, and in need of treatment.

[0023] The severity of COVID-19 is classified into mild, moderate (moderate I, moderate II), and severe cases, for example, by referring to the "Guidelines for the Treatment of Novel Coronavirus Infections" issued by the Ministry of Health, Labor, and Welfare. Other examples include the WHO treatment guidelines, guidelines published by the National Institutes of Health in the United States, and guidelines published by other countries.

[0024] The modified nucleoside to be detected is 6-threonylcarbamoyl adenosine (t 6 A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms 2 t 6 A) t 6 A and / or ms 2 t 6 There are no particular limitations on the method for detecting A, so long as it can detect the modified nucleoside, but it is preferably detected by mass spectrometry or ELISA.

[0025] "Mass spectrometry" or "MS" is an analytical technique for identifying compounds by their mass. It involves applying high voltage or other energy to the sample to be analyzed to ionize it, then filtering, detecting and / or measuring the ions based on their mass-to-charge ratio (m / z). There are many types of mass spectrometers, depending on the method of ionization and detection of the sample. 6 A and / or ms 2t 6 There are no particular limitations on the type of mass spectrometer that can be used as long as it can be used to detect A. Various mass spectrometers, such as MS and its improved versions, TOF-MS and MALDI-TOF-MS, are commercially available and can be used appropriately in the present invention.

[0026] When mass spectrometry is used, detection can be achieved using a single mass spectrometer, but it is preferable to use a tandem mass spectrometer (tandem MS / MS) in which two mass spectrometers are connected in tandem. Tandem MS / MS is an instrument in which two mass spectrometers (MS) are connected in series with a collision activation chamber between them. First, a sample is ionized in the first MS, and then only ions with a specific mass number are selected and introduced into the collision activation chamber where they are collided with an inert gas such as Xe (xenon). Secondary ions (product ions) generated from the ions selected by the first MS are then detected by the second MS.

[0027] Ionization methods include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electron ionization (EI), fast electron impact (FAB) / liquid secondary ionization (LSIMS), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, and particle beam ionization. Those skilled in the art can select an appropriate ionization method based on the analyte to be measured, the type of sample, the type of detector, the selection of positive ion mode or negative ion mode, and the like. 6 A and / or ms 2 t 6 As long as A can be detected, there is no particular limitation, and the above-mentioned methods can be used appropriately.

[0028] Tandem MS / MS is usually performed using selected reaction monitoring (SRM). Selective reaction monitoring refers to operating a mass spectrometer to continuously detect only the signal amount of a specific product ion generated from the analyte in a multi-stage mass analysis of two or more stages, instead of acquiring a product ion spectrum. In SRM, tandem mass analysis can be spatial or temporal.

[0029] When mass spectrometry is used, it is preferable to couple liquid chromatography (LC) or GC (gas chromatography) before a mass spectrometer (MS or MS / MS), more preferably to couple LC, and to separate a sample by LC or GC, and then introduce the sample into the mass spectrometer for analysis. By coupling LC or GC before a mass spectrometer, for example, even when a blood sample or a urine sample is used, analysis can be performed satisfactorily.

[0030] Types of chromatography that can be used for LC include partition chromatography, normal phase liquid chromatography (NPLC), displacement chromatography, reversed phase liquid chromatography (RPLC), size exclusion chromatography, ion exchange chromatography, affinity chromatography, and the like.

[0031] Generally, a mass spectrometer consists of a sample introduction section, an ionization section (ion source), a mass separation section (analyzer), a detection section (detector), a vacuum exhaust section (vacuum pump), an instrument control section, and a data processing section (data system).

[0032] Examples of the analyzer used in the mass spectrometer include a triple quadrupole analyzer, an ion trap analyzer, and a time-of-flight analyzer, with a triple quadrupole analyzer or a quadrupole time-of-flight (QTOF) analyzer being preferred. In view of the fact that many commercially available instrument platforms that are advantageous for SRM assays use triple quadrupole analyzers, it is more preferred to perform tandem MS / MS using a triple quadrupole analyzer in the detection method of the present invention. As generally understood by those skilled in the art, the term "triple quadrupole" as used herein includes not only quadrupoles, but also cases in which a multipole or stacked electrode is used instead of a quadrupole. In addition, in the detection of the present invention, mass spectrometry may be performed in either negative ion mode or positive ion mode.

[0033] In the method of the present invention, t 6 A and / or ms 2 t 6 For detection of A, a triple quadrupole LC / MS / MS is preferably used, in which an LC is coupled before a mass spectrometer.

[0034] In the method of the present invention, mass spectrometry is used to determine t 6 A and / or ms 2 t 6 When detecting A, it is preferable to preliminarily deproteinize and / or desalt the sample. These pretreatments allow for accurate and sensitive detection of the target substance.

[0035] Methods for deproteinization generally include insolubilization by denaturation of proteins (addition of acids such as perchloric acid, trichloroacetic acid, metaphosphoric acid, etc., addition of water-miscible organic solvents such as acetone, acetonitrile, methanol, ethanol, etc., heating and cooling), and physical removal (ultrafiltration using membrane filters (centrifugal filtration devices, etc.), dialysis using dialysis tubes, ultracentrifugation), etc. Deproteinization can also be performed by using permeation-limiting packing materials such as inner-phase packing materials, hybrid-type packing materials, and hydrophilic polymer packing materials. 6 A and / or ms2 t 6 As long as the detection of A is not hindered, the method is not limited, but an example of a preferred method of deproteinization is deproteinization using an insolubilization method by protein denaturation using a water-miscible organic solvent, for example, deproteinization using methanol. The method of deproteinization is known and can be performed according to a standard method. For example, the deproteinization is not particularly limited, but is performed by adding 0.2 to 20 times, preferably 1 to 5 times the amount of ethanol or methanol to a sample (preferably a body fluid sample) and reacting for a sufficient time (for example, 15 minutes) for protein denaturation, followed by centrifugation under conditions sufficient to precipitate the denatured protein (for example, 12,000×g for 15 minutes), and recovering the supernatant (organic solvent layer), thereby obtaining a deproteinized sample. The deproteinized sample can be used as it is or after drying using a centrifugal evaporator or the like and dissolving in an appropriate solvent such as distilled water for LC.

[0036] As the method for desalting, any known desalting method used in analysis can be appropriately used. In addition, by using the above-mentioned protein removal treatment method, it is possible to use the method for desalting as well.

[0037] In one embodiment of the method of the present invention, the t 6 A and / or ms 2 t 6 Detect the amount of A. t 6 A and / or ms 2 t 6 The amount of A may be detected with or without correction using an internal standard, but detection can be performed with higher accuracy by using an internal standard. The internal standard is not limited to, but may include adenosine in plasma or serum when the sample is a blood sample, for example, plasma or serum, and may include creatinine, urea nitrogen, uric acid, adenosine, and 3-amino-3-carboxypropyluridine (acp) in urine when the sample is urine. 3Using the measured values ​​of these internal standards, the measured t 6 A and / or ms 2 t 6 By correcting the amount of A, the target substance can be detected more accurately. 6 A and / or ms 2 t 6 The amount of A may also be detected using a previously prepared calibration curve.

[0038] When blood samples, such as plasma or serum, are used and corrected using serum adenosine as an internal standard, the t 6 If the amount of A is at least 3 times, preferably 5 times, more preferably 10 times, greater than serum adenosine, it can be determined that the subject from whom the blood sample is derived is infected with or may be infected with COVID-19. 2 t 6 If the amount of A is at least 2-fold, preferably 3-fold, and more preferably 5-fold greater than serum adenosine, it can be determined that the subject from which the blood sample was derived is infected with or at risk of being infected with COVID-19.

[0039] Urine samples were used, and 3-amino-3-carboxypropyluridine (acp 3 When corrected using U), the t 6 The amount of A is acp 3 When the ms U concentration is at least 50 times, preferably 80 times, and more preferably more than 100 times higher than that of the ms U concentration, it can be determined that the subject from which the urine sample is derived is infected with COVID-19 or is at risk of being infected with COVID-19. 2 t 6 The amount of A is acp 3 If the antibody titer is at least 5 times, preferably 8 times, and more preferably more than 10 times higher than U, it can be determined that the subject from which the blood sample is derived is infected with or at risk of being infected with COVID-19.

[0040] The method of the present invention comprises: 6 A and / or ms 2t 6 Correction (e.g., correction using an internal standard) may not be performed when detecting the amount of A. In such a case, the t 6 The amount of A and t 6 The average value of the amount of A is preset, and if the amount in a sample derived from a subject is higher than that value, it can be determined that the subject is infected with COVID-19 or is at risk of being infected. 2 t 6 The same applies when detecting the amount of A.

[0041] In one embodiment of the method of the present invention, the t 6 A and / or ms 2 t 6 The amount of A can also be detected by ELISA. ELISA is a method in which a specific antibody is bound to a target antigen contained in a sample and detection and quantification are performed using an enzyme reaction. 6 Antibodies against A and / or ms 2 t 6 Detection can be performed using an antibody against A. The antibody against the modified nucleoside can be an antibody produced according to a conventional method, or an antibody produced by a contract or a commercially available antibody. The antibody can be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody.

[0042] In the method of the present invention, t 6 A and / or ms 2 t 6 The amount of A can be detected by a conventional method, for example, a direct method, an indirect method, a sandwich method, or a competitive method, but the sandwich method is preferred.

[0043] In one embodiment of the present invention, the method of the present invention can be used to determine whether a subject is infected with or at risk of being infected with COVID-19. In one embodiment of the method of the present invention, the t 6 A and / or ms 2 t 6 By comparing the amount of A with a predetermined reference value, it is possible to determine whether the subject is at risk of or has COVID-19. The predetermined reference value can be, but is not limited to, for example, a t detected in a healthy subject (healthy person). 6 A and / or ms 2 t 6 The cutoff value (reference value) can be the value of A, or a value obtained by adding a value to that value to effectively eliminate false positives. 6 A and / or ms 2 t 6 If the amount of A is significantly increased, it can be determined that the subject is infected with or at risk of being infected with COVID-19. Whether or not it is significantly increased can be determined appropriately depending on the sensitivity required for the detection method, and can be set, for example, to 2, 3, 5, 10 times, etc., the average value for healthy people. The cutoff value can be appropriately set by a person skilled in the art from the viewpoints of sensitivity, specificity, morbidity (infection) positive predictive value, morbidity (infection) negative predictive value, etc. For example, the cutoff value can be set based on ROC curve analysis. In addition, the cutoff value of the method of the present invention can be determined from the results by determining whether the subject is positive or negative using other known COVID-19 detection methods (e.g., PCR method) and comparing the results with data obtained when the same subject is measured using the method of the present invention.

[0044] In another embodiment of the present invention, 6 A and / or ms 2 t 6Detection of A can be used to determine whether a subject is infected with COVID-19, as well as to determine the severity of the subject. The severity of the subject can be determined, for example, as mild, moderate, or severe, but is not limited thereto. In one embodiment of the method of the present invention, the t measured in a sample from the subject is 6 A and / or ms 2 t 6 The severity of the subject can be determined by comparing the amount of A with predetermined reference values ​​for each severity.

[0045] In another embodiment of the present invention, 6 A and / or ms 2 t 6 Detection of A can be used to assess the efficacy of treatment in a subject suffering from COVID-19. Assessment of the efficacy of treatment in a subject can be, for example, based on the t measured in a sample from the subject. 6 A and / or ms 2 t 6 This can be done by comparing the amount of A before and after treatment, and based on the results, the treatment policy or dosing regimen in the subject can be determined or changed.

[0046] In another embodiment of the present invention, 6 A and / or ms 2 t 6 The detection result of A can be used to determine the pathological change or prognosis prediction of a subject suffering from COVID-19. More specifically, when a subject suffering from COVID-19 has mild or moderate symptoms, preferably mild symptoms, the t measured in the subject's sample can be used to determine the pathological change or prognosis prediction of a subject suffering from COVID-19. 6 A and / or ms 2 t 6 Based on the value of A, it is possible to predict whether the subject will develop severe symptoms. For example, it can be determined that the higher the measured value, the higher the risk of the subject developing severe symptoms. Alternatively, it is possible to predict whether the subject's t 6 A and / or ms 2 t 6If the amount of A is significantly increased, it can be determined that the subject is at high risk of developing severe symptoms. Whether or not the amount of A is significantly increased can be appropriately determined depending on the sensitivity required for the detection method. The cutoff value can be appropriately set by a person skilled in the art from the viewpoints of sensitivity, specificity, and prediction accuracy of severe symptoms.

[0047] For example, it can be set based on ROC curve analysis. In addition, the pathological changes or prognosis of the detection subject can be monitored, and the results can be compared with data obtained when the same subject is measured using the method of the present invention, and the cutoff value of the present method can be determined from the results. Based on the results, the treatment policy and drug regimen for the subject can be determined or changed. The drug regimen can be changed by referring to, for example, the "Guide to the Treatment of Novel Coronavirus Infection" by the Ministry of Health, Labor and Welfare, the WHO treatment guidelines, the treatment guidelines published by the National Institutes of Health and other countries, and these guidelines, including their revised versions, are incorporated into this specification.

[0048] The present invention will be described in more detail with reference to the following examples. However, the examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention in any way. EXAMPLES

[0049] (Example 1) Comprehensive analysis of modified nucleosides in SARS-CoV-2-infected cells We analyzed modified nucleosides in mammalian cells infected with SARS-CoV-2 as follows. Cell culture medium containing ACE2-overexpressing HEK293 cells (3x10 5 cells) in SARS-CoV2 solution (6x10 6The cells were infected with 50 μL of SARS-CoV2 (VP / mL), and 18 and 24 hours after infection, the RNA was extracted from the cells with Trizol. The RNA concentration was adjusted to 1,000 ng / μL using a spectrophotometer (NanoDrop ND-1000). The RNA was then degraded to nucleosides using RNase, and 2 μL of the sample was comprehensively analyzed for modified nucleosides using an ultrafast triple quadrupole mass spectrometer (Shimadzu LCMS-8050). The results showed that SARS-CoV2 infection was accompanied by t 6 A and ms 2 t 6 The structure of each is shown in Figure 1. 6 A increased 6-10 times with infection, and ms 2 t 6 A was increased 3-5 times. The results are shown in Figure 2.

[0050] Example 2: Detection of modified nucleic acids in patients with COVID-19 infection Modified nucleic acids were analyzed in patients with COVID-19 infection as follows: Serum and urine samples were collected from 30 patients diagnosed with COVID-19 when they were admitted to a COVID-19 designated medical institution and stored in a freezer at -30°C. After thawing at room temperature, 100 μL was deproteinized and desalted using a column (Nanosep with 3K Omega). 2 μL of the sample was used to comprehensively analyze modified nucleosides using an ultrafast triple quadrupole mass spectrometer (Shimadzu LCMS-8050). In addition, serum and urine from healthy subjects and urine from patients with bacterial infections, influenza virus infections, and post-surgery fever were also analyzed using the same method. In addition, serum measurement results were corrected with serum adenosine levels, and urine measurement results were corrected with urinary 3-amino-3-carboxypropyluridine (acp3U). The results using serum are shown in Figure 3. t in the serum of COVID-19 patients 6 A amount and ms 2 t 6 The amount of A was significantly higher than in healthy subjects.

[0051] The urinary t 6When comparing the amount of A, it was found that the levels were similar to those of healthy subjects (controls) in patients with bacterial infections, patients with other viral infections such as influenza, and patients with fevers other than infectious diseases such as postoperative fever, but were significantly elevated in COVID-19 patients. The results are shown in Figure 4.

[0052] Regarding COVID-19 patients, urinary t 6 The ROC curve for the amount of A was drawn, and the sensitivity, specificity, etc. were examined. When the cutoff value was set at 52.57, the sensitivity was 100%, the specificity was 96.43%, and the likelihood ratio was 28. The results are shown in Figure 5.

[0053] Next, the urinary ms of COVID-19 infected patients and patients with fever were 2 t 6 When comparing the amount of A, t 6 As in A, the levels were similar to those of healthy subjects in patients with bacterial infections, patients with viral infections other than COVID-19 such as influenza, and patients with fevers other than infectious diseases such as postoperative fever, but were significantly elevated in COVID-19 patients. The results are shown in Figure 6.

[0054] Urinary MS in COVID-19 patients 2 t 6 The ROC curve for the amount of A was drawn, and the sensitivity, specificity, etc. were examined. When the cutoff value was set at 9.269, the sensitivity was 99.3%, the specificity was 93.33%, and the likelihood ratio was 14.9. The results are shown in Figure 7.

[0055] (Example 3) Detection of modified nucleic acids in COVID-19 infected patients and healthy individuals (1) In the same manner as in Example 2, serum and urinary t were measured using samples from COVID-19 infected patients (17 people) and healthy individuals (14 people). 6 A amount and ms 2 t 6The amount of A was measured. The results are shown in Figure 8. However, no correction using an internal standard was performed. Even without correction, COVID-19 infection in the subjects could be significantly detected.

[0056] (Example 4) Detection of modified nucleic acids in COVID-19 infected patients and healthy individuals (2) In the same manner as in Example 2, serum and urinary t were measured using samples from COVID-19 infected patients (28 people) and healthy individuals (21 people). 6 A amount and ms 2 t 6 The amount of adenosine in serum was measured, and the sensitivity was compared between urine and serum. The serum measurement results were corrected for serum adenosine levels, and the urine measurement results were corrected for urinary 3-amino-3-carboxypropyluridine (ACP 3 The results are shown in Figure 9. In both urine and serum samples, t 6 A amount and ms 2 t 6 Both amounts of A were able to significantly detect COVID-19 infection in subjects, but detection sensitivity was superior when urine samples were used.

[0057] Example 5: Detection of modified nucleic acids in severely infected COVID-19 patients In the same manner as in Example 2, serum t 6 A amount and ms 2 t 6 The amount of A was measured. However, no correction using an internal standard was performed. The results are shown in Figure 10. The amount of each modified nucleoside was significantly increased in severe patients compared to asymptomatic patients and mildly symptomatic patients. It was found that the determination method of the present invention can also be used to confirm severe patients.

[0058] (Example 6) Prediction of pathological changes in patients infected with COVID-19 In the same manner as in Example 2, samples from COVID-19 infected patients (21 patients) diagnosed with mild symptoms were used to measure ms in the serum at the time of admission. 2 t 6The amount of ms A was measured. However, no correction was made using an internal standard. After that, the pathological changes of each patient who received treatment were followed up, and the ms A amount was measured. 2 t 6 The 15 patients with low A levels were discharged without progression, whereas the 15 patients with low A levels were discharged without progression. 2 t 6 Six patients with high A levels became seriously ill or died. The serum MS A levels at the time of admission of patients who were discharged without progression (Recovered) and patients who became seriously ill or died (Severed / Death) were 2 t 6 The results of measuring the amount of A are shown in Figure 11. These results demonstrate that the marker and assessment method of the present invention can predict the prognosis of patients infected with COVID-19. [Industrial Applicability]

[0059] According to the present invention, it is possible to determine whether a subject from which a sample is derived is at risk of or has COVID-19 by detecting modified nucleosides in an easily obtainable sample such as serum or urine. This makes the diagnosis of COVID-19 more convenient and easy. The method of the present invention can also be used to determine the severity and prognosis of COVID-19.

Claims

1. A method for determining whether a mammalian subject is at risk for or has COVID-19, comprising detecting 6-threonylcarbamoyl adenosine (t ６ A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms ２ t ６ A) detecting an amount of a modified nucleoside that is A) and providing the detected amount of the modified nucleoside for said determination.

2. The method according to claim 1, further comprising a step of comparing the amount of the detected modified nucleoside with a first reference value for determining that a person with a high amount of the modified nucleoside is at risk of having or has COVID-19, and that a person with a low amount of the modified nucleoside is not having COVID-19.

3. The method according to claim 1, further comprising a step of comparing the amount of the detected modified nucleoside with a second reference value for assessing that a high amount of the modified nucleoside indicates a severe degree of COVID-19 disease, and a low amount of the modified nucleoside indicates a mild degree of COVID-19 disease.

4. When the amount of the modified nucleoside detected in the subject is greater than the first reference value and the subject has a mild symptom, The method of claim 2, further comprising a step of comparing the amount of the modified nucleoside detected in the subject with a third reference value for predicting a subsequent change in the subject's pathological condition, in which a high amount of the modified nucleoside will result in a worsening of the condition or death, and a low amount of the modified nucleoside will result in recovery without progression.

5. A method for evaluating the efficacy of treatment in a mammal suffering from COVID-19, comprising: detecting the presence of 6-threonylcarbamoyl adenosine (t ６ A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms ２ t ６ A) detecting an amount of a modified nucleoside; and providing the detected amount of the modified nucleoside for said evaluation.

6. The method of claim 1 or claim 5, wherein the sample is plasma, serum, or urine.

7. The method according to claim 1 or claim 5, wherein the amount of the modified nucleoside is detected by tandem mass spectrometry (MS / MS).

8. The method according to claim 7 , wherein the sample has been subjected to deproteinization and desalting treatment.

9. The method according to claim 7, wherein the sample is plasma or serum, and the step of detecting the amount of the modified nucleoside includes a step of correcting the measurement result by the amount of adenosine in the plasma or serum.

10. The method according to claim 7, wherein the sample is urine, and the step of detecting the amount of the modified nucleoside includes a step of correcting the measurement result with the amount of at least one substance selected from the group consisting of creatinine, urea nitrogen, uric acid, adenosine, and 3-amino-3-carboxypropyluridine in the urine.

11. The method according to claim 1 or claim 5, wherein the amount of the modified nucleoside is detected by an ELISA method.

12. The method of claim 1 or claim 5, wherein the subject is a human.

13. The method of claim 12, wherein the subject is a patient with fever.

14. A method for determining whether a mammalian subject is a candidate for treatment for COVID-19, comprising detecting 6-threonylcarbamoyl adenosine (t ６ A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms ２ t ６ A) detecting an amount of a modified nucleoside that is A) and providing the detected amount of the modified nucleoside for said determination.

15. 6-Threonylcarbamoyl adenosine (t ６ A) and / or 2-thiomethyl, 6-threonylcarbamoyl adenosine (ms ２ t ６ A) A marker for severe symptoms or a predictive marker for severe symptoms of COVID-19.

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