Screening method for tuberculosis infection samples and biomarkers and probe sets for use therein

Abstract

Provided are: a biomarker whereby useful information about the presence / absence of infection can be provided in a short time using urine, which is easily collected, as a specimen sample; a method for screening a tuberculosis infection sample using said marker; and a probe kit that can be used in said screening. In the present invention, miRNA included in urine collected from an active tuberculosis patient and a healthy person is comprehensively detected and analyzed, and on the basis of the result of searching for miRNA that is specifically over- / under-expressed depending on the presence or absence of tuberculosis infection, miRNA expressed at a high frequency relative to that of a healthy person (SEQ ID NO: 1-25 and 45-55) and miRNA expressed at a low frequency relative to that of a healthy person (SEQ ID NO: 26-45 and 56-75) are specified as biomarkers. Nucleotides that can hybridize with these biomarkers are used as probes.

Description

[Technical Field]

[0001] The present invention relates to the use of miRNAs, whose expression frequency differs depending on whether or not a patient is infected with tuberculosis, as biomarkers. Specifically, the present invention relates to biomarkers that provide information to assist in the diagnosis of active tuberculosis, and which can be used with urine as a specimen, as well as testing and screening methods and test probe sets that use the same. [Background technology]

[0002] It is said that one-quarter of the world's population is infected with tuberculosis, and the infection is particularly widespread in developing countries. For this reason, there is a need to develop a rapid and simple method for testing for tuberculosis infection that can be implemented in developing countries.

[0003] In Japan, the most commonly used diagnostic method for tuberculosis infection is the QuantiFERON® TB or T-Spot® TB test, which measures interferon-γ (IFN-γ) produced by lymphocytes in response to antigen stimulation with ESAT-6, CFP-10, or other antigens. This test involves drawing blood from a subject, isolating lymphocytes, dispensing a fixed amount of the blood onto a culture plate coated with anti-human interferon-γ antibodies, adding the tuberculosis-specific antigens ESAT-6 and CFP-10, and culturing the plate for approximately 20 hours. The number of INF-γ-producing cells bound to the anti-human INF-γ antibodies is then counted. Because this method is not affected by BCG vaccination or nontuberculous mycobacteria, it has the advantage of being more specific than the tuberculin test. However, because it requires culture, it takes approximately two days for a result to be determined. Furthermore, because it requires equipment for cell isolation and cell culture, the cost per sample is high, making it difficult to deploy in developing countries. Furthermore, in patients who are infected with not only Mycobacterium tuberculosis but also HIV (human immunodeficiency virus) (those with both HIV and tuberculosis infections), the number of INF-γ-producing cells is low due to immunodeficiency, so even though the patient is infected with tuberculosis, the test result may be false negative.

[0004] Furthermore, the MPB64 antibody is known as an antibody specific to patients with active tuberculosis. A method for determining whether or not a patient has active tuberculosis is also known, using this antibody as a marker to detect the amount of MPB64 antibody in plasma, for example, by the dot blot assay. It has been reported that the MPB64 antibody is also present in urine and correlates with results obtained using serum as a measurement sample (Non-Patent Document 1: Yasuko Tamada et al., Microbiol Immunol 2012;56:740-747). Testing methods using the MPB64 antibody as a marker can use urine, which is easy to collect, as a measurement sample, making them suitable for quickly determining the presence or absence of tuberculosis infection in developing countries where tuberculosis infection is likely to spread. However, cross-reactive antibodies are known to exist, and false-positive test results may occur in BCG-vaccinated individuals.

[0005] Sputum culture tests conducted on samples taken from subjects are highly accurate in determining whether or not tuberculosis bacteria are present, but because they require the necessary equipment and a culture period of 1 to 4 weeks, they are difficult to use as a rapid testing or diagnostic method.

[0006] Under these circumstances, there is a need to develop a rapid testing and diagnostic method for testing for tuberculosis infection in developing countries that does not require cell culture, uses minimally invasive samples, and is less likely to produce erroneous test results such as false positives or false negatives.

[0007] In recent years, the use of miRNAs as biomarkers has been proposed, in addition to antibodies, cells, cytokines, etc. Abnormal miRNA expression has been shown to be involved in the onset and malignant progression of many diseases. Furthermore, because miRNAs can be detected in body fluids such as blood, their use as liquid biopsies has attracted attention. In recent years, various reports have been made on the use of miRNAs as diagnostic markers, primarily in the field of cancer (Patent Document 1: JP-A-2010-534480, Patent Document 2: WO2015 / 133477 (pancreatic cancer), Patent Document 3: JP-A-2018-505656 (prostate cancer), etc.).

[0008] Several unique miRNAs have also been reported in tuberculosis patients (Non-Patent Document 2: Naveed Sabir et al., Frontiers in Microbiology vol. 9, March 2018), and Table 1 in Non-Patent Document 2 introduces miRNAs that could be candidates for biomarkers using human peripheral blood, whole blood, serum, or macrophages as samples. However, even when focusing on human peripheral blood and serum, the miRNAs listed in Table 1 vary depending on the reference literature, and it has not been clarified what level of specificity each miRNA has as a diagnostic marker or how to determine it.

[0009] Furthermore, although blood is an excellent specimen for liquid biopsy, it must be drawn for testing, and as a testing method in developing countries, it is limited to patients who are strongly suspected of having tuberculosis infection, and its application as a testing method for widely screening tuberculosis-infected patients is limited. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Special Publication No. 2010-534480 [Patent Document 2] WO2015 / 133477 publication [Patent Document 3] Special Publication No. 2018-505656 [Non-patent literature]

[0011] [Non-Patent Document 1] Yasuko Tamada et.al., “Diagnosis of active tuberculosis using MBP64, a specific antigen of Mycobacterium bovis”, Microbiol immunol 2012;56:740-747 [Non-patent document 2] Naveed Sabir et.al., “miRNAs in Tuberculosis: New Avenues for Diagnosis and Host-Directed Therapy”, Frontiers in Microbiology vol.9, March 2018 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention provides a biomarker that can provide useful information about the presence or absence of infection in a short period of time using minimally invasive liquid biopsy, particularly urine, which is an easy sample to collect, as well as a method for determining and screening for the presence or absence of tuberculosis infection using the marker and a screening probe set. [Means for solving the problem]

[0013] The present inventors have comprehensively detected and analyzed miRNAs contained in urine collected from patients with active tuberculosis and healthy individuals, and have completed the present invention based on the results of searching for miRNAs that are specifically over- or under-expressed depending on whether or not the patient is infected with tuberculosis.

[0014] That is, the screening method for tuberculosis-infected samples of the present invention includes a first step of qualitatively and / or quantitatively detecting at least two miRNAs selected from miRNAs (a miRNA group consisting of sequences No. 1 to No. 25, or a miRNA group consisting of sequences Nos. 49 to 55 and sequences 3, 6, 9, 10, 13, and 14) contained in a sample derived from the urine of a subject, or miRNAs having 70% or more sequence identity to the miRNAs.

[0015] The at least two miRNAs selected above preferably include at least one miRNA selected from the group consisting of SEQ ID NOs: 3, 6, 9, 10, 13, and 14.

[0016] The detection step is preferably a step of classifying a sample as positive if the expression frequency in the sample is higher than a predetermined value, and also preferably a step of classifying a sample as positive if the content ratio of the detected miRNA to the internal control miRNAs (SEQ ID NOs: 46 to 48) is higher than a predetermined value.

[0017] Furthermore, the method may include a second step of qualitatively and / or quantitatively detecting at least one miRNA selected from the miRNA group consisting of SEQ ID NOs: 26 to 45, or the miRNA group consisting of SEQ ID NOs: 56 to 75 and SEQ ID NOs: 29 and 40, or a miRNA having 70% or more sequence identity with the miRNA. In this case, the miRNA preferably contains either SEQ ID NO: 29 or SEQ ID NO: 40.

[0018] In another aspect, the screening method of the present invention comprises: a first step of qualitatively and / or quantitatively detecting, contained in a sample derived from the urine of a subject, one miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 25 and SEQ ID NO: 49 to SEQ ID NO: 55, or a miRNA having 70% or more sequence identity to the miRNA; and a second step of qualitatively and / or quantitatively detecting, contained in a sample derived from the urine of a subject, one miRNA selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 45 and SEQ ID NO: 56 to SEQ ID NO: 75, or a miRNA having 70% or more sequence identity to the miRNA.

[0019] The test probe set of the present invention is a test probe set for distinguishing samples derived from urine of a subject from samples positive for active tuberculosis, comprising: This is a test probe set containing two or more types of oligonucleotide probe A that can hybridize under stringent conditions to any one of the miRNAs (sequence No. 1 to sequence No. 25, and sequence No. 49 to sequence No. 55) shown in Table 1-1 or Table 1-2.

[0020] Furthermore, the probe set may include an oligonucleotide probe D that can hybridize under stringent conditions with any one of the miRNAs (SEQ ID NO: 26 to SEQ ID NO: 45 and SEQ ID NO: 56 to SEQ ID NO: 75) shown in Table 2-1 or Table 2-2. In this case, the probe set can be used not only as a test probe set for distinguishing positive samples from urine samples derived from subjects, but also as a probe set for distinguishing positive and negative samples for active tuberculosis.

[0021] From another viewpoint, the probe set of the present invention may be a combination of one type of nucleotide probe A and one type of nucleotide probe D.

[0022] The testing probe set of the present invention may further include an oligonucleotide probe capable of hybridizing under stringent conditions to any one of SEQ ID NOs: 46 to 48 as a probe for detecting the internal control miRNA (SEQ ID NOs: 46 to 48) (internal control oligonucleotide probe). In this case, the testing probe set of the present invention is a combination of the above-mentioned oligonucleotide probe A and the internal control oligonucleotide probe; or a combination of oligonucleotide probe A, oligonucleotide probe D, and the internal control oligonucleotide probe.

[0023] As used herein, "tuberculosis" refers to an infection caused by Mycobacterium tuberculosis. It refers to an active state of tuberculosis in which symptoms such as coughing and weight loss have already appeared and tuberculosis bacteria can be detected in sputum. However, it does not cover the determination of latent tuberculosis, in which there are no symptoms or abnormal clinical findings such as chest X-ray or bacterial test results, but the patient is infected with tuberculosis bacteria.

[0024] As used herein, "miRNA (microRNA)" refers to the miRNA identified by a sequence ID (sequence number), when the miRNA is identified by the sequence. Unless otherwise specified, the term includes mature miRNA, which is a non-coding RNA of approximately 16 to 25 bases, as well as pri-miRNA, which is an initial transcription product of miRNA genes transcribed by RNA polymerase II and has a hairpin loop structure, and pre-miRNA, which is a precursor of miRNA and is formed when this pri-mRNA is partially cleaved by the RNase III-like Drosha.

[0025] As used herein, "oligonucleotide probes capable of hybridizing under stringent conditions" refers to probes consisting of nucleotides having a complementary sequence to each sequence of the target miRNA, or nucleotides having a sequence that is 70% or more, preferably 80% or more, more preferably 90% or more identical to the complementary sequence, even more preferably 95% or more, and particularly preferably 99% or more identical. [Effects of the Invention]

[0026] The method for screening tuberculosis-infected samples of the present invention uses miRNA as a biomarker, eliminating the need for cell culture or other procedures for sample preparation, enabling rapid determination. Furthermore, because urine is used as the test sample for measurement, which does not require expert collection, the method is useful as a testing method that requires rapid and inexpensive testing of a large number of samples. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 2] FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 3]FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 4] FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 5] FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 6] FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 7] FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 8] FIG. 1 is a plot showing the results of the expression frequency of miRNAs in Group A. [Figure 9] FIG. 1 is a plot showing the results of miRNA expression frequency in Group A (excluding HIV patients). [Figure 10] FIG. 1 is a plot showing the results of miRNA expression frequency in Group A (excluding HIV patients). [Figure 11] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 12] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 13] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 14] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 15] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 16] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 17] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 18] FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group D. [Figure 19] FIG. 1 is a plot diagram showing statistically processed miRNA expression frequency results obtained in the examples in relation to the expression frequency folds in healthy subjects. DETAILED DESCRIPTION OF THE INVENTION

[0028] <Biomarkers for determining tuberculosis infection> The miRNAs that can be used as biomarkers in the screening method for tuberculosis-infected samples of the present invention are miRNAs having the sequences shown in Tables 1-1, 1-2, 2-1, and 2-2, and miRNAs that have sequence homology of 70% or more, preferably 80% or more, and more preferably 90% or more to the miRNAs.

[0029] [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0030] The miRNAs listed in Tables 1-1 and 1-2 (miRNAs of SEQ ID NO: 1 to SEQ ID NO: 25 and SEQ ID NO: 49 to SEQ ID NO: 55, collectively referred to as "Group A") are expressed at higher frequencies in samples from tuberculosis-infected individuals than in healthy individuals. Of these, miRNAs in Group A1 (SEQ ID NO: 1 to SEQ ID NO: 11) had a significance level of p<0.05 in Welch's t-test (significant difference at a level of less than 0.05), and miRNAs in Group A2 (SEQ ID NO: 12 to SEQ ID NO: 25) had a significance level of p<0.1 (significant difference at a level of less than 0.1). Furthermore, the miRNAs belonging to the Af group (SEQ ID NOs: 49 to 55, and SEQ ID NOs: 3, 6, 9, 10, 13, and 14) have a higher expression frequency in samples from M. tuberculosis-infected individuals than in healthy individuals, as determined by Fisher's exact test with a p<0.05 and Q value (FDR)<0.3. Therefore, miRNAs selected from the A1 group or the Af group are more preferred as biomarkers for screening tuberculosis-positive samples.

[0031] In particular, sequences No. 3 (hsa-miR-424-5p), 6 (hsa-miR-92b-3p), 9 (hsa-miR-199a-5p), 10 (hsa-miR-1972), 13 (hsa-miR-373-3p), and 14 (hsa-miR-1268a) belong to the A1 or A2 group and also to the Af group, and therefore are highly likely to be positive for tuberculosis infection, making them useful biomarkers for screening tuberculosis-positive samples.

[0032] Furthermore, from the viewpoint that their expression frequency is high regardless of whether or not HIV co-infection is present and that there is a large difference between them and healthy individuals, hsa-miR-196a-5p, hsa-miR-500a-3p, hsa-miR-199a-5p, and hsa-miR-424-5p are preferably used, with hsa-miR-196a-5p and hsa-miR-500a-3p being more preferred.

[0033] The miRNAs listed in Tables 2-1 and 2-2 (sequences No. 26 to 45, and sequences No. 56 to 75, collectively referred to as "Group D") are miRNAs whose expression frequency tends to decrease with tuberculosis infection compared to that of healthy individuals.

[0034] As used herein, the term "healthy subject" refers to a sample derived from a subject who is tuberculosis-negative, i.e., tuberculosis bacteria are not detected in the sputum, and who can be determined to be AIDS-negative based on the proportion of CD4-positive T cells.

[0035] Among the miRNAs belonging to Group D, the miRNAs belonging to Group D1 (SEQ ID NO: 26 to SEQ ID NO: 31) had a significance level of p<0.05 in Welch's t-test (significant difference at less than 0.05), and the miRNAs belonging to Group D2 (SEQ ID NO: 32 to SEQ ID NO: 45) had a significance level of p<0.1 (significant difference at less than 0.1). Therefore, as biomarkers belonging to Group D, hsa-miR-107, hsa-miR-6887-5p, hsa-miR-196b-5p, hsa-miR-451a, and hsa-miR-132-3p belonging to Group D1 are preferably used, with hsa-miR-6887-5p being more preferred.

[0036] Furthermore, among the miRNAs belonging to Group D, miRNAs belonging to Group Df (SEQ ID NOs: 56 to 75, 29 and 40) are miRNAs whose expression frequency in samples from tuberculosis-infected individuals is half or less than that of healthy individuals, with p<0.01 and Q value (FDR)<0.08 in Fisher's exact test. Therefore, miRNAs selected from Group D1 or Group Df are more preferable as biomarkers for screening tuberculosis-positive samples.

[0037] In particular, SEQ ID NO: 29 (hsa-miR-451a) and SEQ ID NO: 40 (hsa-miR-769-5p) belong to both the D1 or D2 group and the Df group. Therefore, when using a biomarker from group D, it should contain at least one of SEQ ID NO: 29 (hsa-miR-451a) and SEQ ID NO: 40 (hsa-miR-769-5p), more preferably SEQ ID NO: 29 (hsa-miR-451a).

[0038] The above miRNAs can be used as markers for determining tuberculosis infection, but two or more types can be used in combination. By using two or more types of Group A miRNAs, which serve as biomarkers for determining whether a patient is tuberculosis positive, it is possible to reduce errors due to variations in test samples.

[0039] Alternatively, miRNAs selected from Group A and Group D may be used in combination. By combining the two groups, whose expression frequencies increase or decrease inversely with tuberculosis infection, more reliable results (tuberculosis positive or tuberculosis negative) can be obtained. Furthermore, even in the case of HIV infection, the risk of erroneous determinations, such as false positives and false negatives, can be reduced.

[0040] When selecting two or more types from Group A, two or more types may be selected from miRNAs identified from the results of analysis using a common testing method, or a combination of two or more types selected from different groups may be used. Regardless of whether the miRNA group in Table 1-1 or Table 1-2 is used, it is preferable that at least one type of miRNA of sequence Nos. 3, 6, 9, 10, 13, 14, or 16 is included.

[0041] <Inspection probe set> The test probe set of the present invention is a test probe set for distinguishing samples derived from urine of a subject from those positive for active tuberculosis, and is a set combining probes for detecting the above-mentioned biomarkers. Specifically, it includes two or more of the following test probes:

[0042] a) Oligonucleotide probe A capable of hybridizing under stringent conditions with any one of miRNAs included in Group A (SEQ ID NO: 1 to SEQ ID NO: 25, SEQ ID NO: 49 to SEQ ID NO: 55) shown in Table 1-1 or Table 1-2

[0043] b) Oligonucleotide probe D capable of hybridizing under stringent conditions with any one of the miRNAs of group D shown in Table 2-1 or Table 2-2 (SEQ ID NO: 26 to SEQ ID NO: 45, SEQ ID NO: 56 to SEQ ID NO: 75).

[0044] c) An internal control oligonucleotide probe capable of hybridizing under stringent conditions with at least one of the miRNAs (SEQ ID NO: 46 to SEQ ID NO: 48) shown in Table 3.

[0045] [Table 3]

[0046] Here, an oligonucleotide probe that can hybridize under stringent conditions is a nucleotide having a complementary sequence to each sequence of the target miRNA, or a nucleotide having a sequence that is 70% or more, preferably 80% or more, more preferably 90% or more identical to the complementary sequence, even more preferably 95% or more, and particularly preferably 99% or more identical.

[0047] The combination may be any combination that includes at least oligonucleotide probe A. Specific examples include a combination of two or more types of oligonucleotide probe A; a combination of one type of oligonucleotide probe A and one type of oligonucleotide probe D; a combination of two or more types of oligonucleotide probe A and one or more types of oligonucleotide probe D; a combination of two or more types of oligonucleotide probe A and an oligonucleotide probe for internal control; a combination of one type of oligonucleotide probe A, one type of oligonucleotide probe D, and an oligonucleotide probe for internal control; and a combination of two or more types of oligonucleotide probe A, one or more types of oligonucleotide probe D, and an oligonucleotide probe for internal control.

[0048] In the case of a combination of multiple types of oligonucleotide probes A, it is preferable that at least one type contains a miRNA selected from the group consisting of SEQ ID NOs: 3, 6, 9, 10, 13, 14, and 16.

[0049] Combining multiple types of oligonucleotide probes can improve the accuracy of the determination. In particular, by detecting at least one type of miRNA with probe D selected from the group consisting of b), it is possible not only to determine whether a sample is positive or not, but also to distinguish the sample into samples with a high probability of being positive and samples with a high probability of being negative.

[0050] Furthermore, the internal control can reduce the influence of HIV infection, which also has the effect of reducing the risk of false positives and false negatives. In other words, the amount of miRNA contained in a specimen sample varies depending on the individual sample. In particular, when a miRNA with a low expression frequency is used as a biomarker, the detected miRNA content may be low or the total amount of miRNA may not be accurately measured, resulting in a false negative or positive result. In this case, the ratio with the internal control can be used to suppress individual differences between samples.

[0051] In addition, some biomarkers have low expression frequencies, making it difficult to distinguish between positive and negative samples. Furthermore, in the case of HIV infection, the expression frequencies of group A biomarkers are higher in HIV infection, which may result in false positives, where a patient is classified as tuberculosis positive even when they are not. Conversely, in the case of HIV infection, some group A biomarkers tend to be expressed less frequently in HIV infection, which may result in false negatives, where a patient is classified as tuberculosis negative even when they are tuberculosis positive. In this regard, using the ratio to the internal control as an index makes it possible to eliminate the influence of HIV infection.

[0052] The internal control oligonucleotide probe is appropriately selected depending on the type of oligonucleotide probe A or D to be combined, so that the combination will significantly differentiate between HIV and healthy individuals. For example, when hsa-miR-196a is used as the biomarker, combining it with hsa-miR-423 as the internal control will significantly differentiate between tuberculosis positive and negative results, improving the accuracy of the determination. Furthermore, when hsa-miR-107 is used as the D group biomarker, combining it with hsa-miR-21 as the internal control will reduce the HIV influence of HIV-infected samples, thereby reducing the risk of false positives and false negatives due to HIV infection.

[0053] These nucleotide probes preferably have a label that can be detected when hybridized with the target miRNA. The label can be any known label that is suitable for use in nucleotide probes, such as a fluorescent dye or gold colloid. The use of such labels allows not only qualitative detection of the target marker, but also quantification by known methods.

[0054] <Preparation of specimens and test samples> The specimen to be tested is urine collected from a subject. By using urine as a test sample from among human body fluids, collection of specimen samples, such as blood samples, does not depend on a specific skilled worker, and the screening method of the present invention can be applied as a simple screening method for comprehensively testing a large number of samples. Although saliva can be collected without relying on skilled workers, it can be difficult to collect from subjects with dry mouths, such as elderly people. In this regard, urine is an excellent sample that can be easily collected from all subjects.

[0055] Although urine tends to contain fewer miRNA-containing exosomes than blood, it is possible to collect larger amounts of urine than other bodily fluids. Furthermore, since no blood collection equipment such as syringes is required, there is no risk of infection with blood-borne viral infections due to needlestick injuries, making urine a suitable specimen for testing in developing countries.

[0056] Like other bodily fluids such as blood, urine contains exosomes containing mature miRNAs, which serve as biomarkers. Exosomes are membrane vesicles secreted by cells and generally measure 30–150 nm in diameter when observed under an electron microscope. In addition to mature miRNAs, exosomes contain pri-miRNAs, pre-miRNAs, coding-mRNAs, DNA, enzymes, cytoskeletal proteins, and signaling molecules.

[0057] The miRNAs used as biomarkers in the testing and screening methods of the present invention were discovered as a result of searching for miRNAs contained in urine that are specifically over- or under-expressed in tuberculosis-infected / non-infected individuals, and therefore their usefulness is not diminished even when urine is used as a sample.

[0058] In the determination method described below, when expression frequency is used as an index, it is desirable that a predetermined number or more types of miRNAs are detected to a quantifiable (or readable) level, although this depends on the method for preparing the measurement sample. Therefore, depending on the screening method, it is necessary to use a sample in which at least 100 types of miRNAs are detected to a quantifiable level, and even urine can meet this requirement.

[0059] Examples of urine-derived samples include exosome-enriched or roughly purified fractions (hereinafter referred to as "exosome-rich fractions"), samples from which RNA has been extracted, etc. Among these, when detecting miRNA markers, it is preferable to use a sample from which RNA has been directly extracted as the measurement sample.

[0060] A measurement sample (isolated RNA) can be prepared from the urine of a subject by a conventionally known method. For example, exosomes can be isolated from body fluids by size exclusion chromatography, centrifugation, or other methods, and RNA can be extracted from the resulting exosome-rich fraction. Alternatively, exosomes can be isolated using commercially available kits, or RNA can be isolated directly.

[0061] Commercially available exosome isolation kits include ExoQuick TM Examples of kits that can be used to obtain nucleic acid extracted samples from body fluids include the Exosome precipitation solution series (e.g., ExoQuick-TC, ExoQuick-CG, etc., both manufactured by System Biosciences), and isolation kits such as the miRNeasy mini kit (manufactured by Qiagen), MagMax mirVana Total RNA kit (Thermo Fisher), and Magtration (registered trademark) (Precision System Science), which uses magnetic particles. Alternatively, a method of recovering exosomes (nanobiodevice method) by simply feeding an exosome-containing sample (urine) to a nanowire structure may be used.

[0062] <Detection and quantification of miRNA> Examples of methods for detecting a target miRNA from the RNA-containing sample prepared as described above include, for example, RT-PCR method, microarray method, Northern blot method, next-generation sequencer method, liquid-phase nucleic acid hybridization, and the like. When it is necessary to detect most of the miRNAs contained in the sample (for example, when the expression frequency is adopted as a determination index), the RT-PCR method, microarray method, next-generation sequencer method, or the like is adopted.

[0063] Examples of methods for detection and quantification using hybridization include the microarray method, Northern blot method, liquid-phase nucleic acid hybridization, and the like. By utilizing hybridization with a specific miRNA, the miRNA serving as a biomarker may be detected or quantified. In the method using hybridization, since miRNAs that can directly hybridize with a probe can be detected, it is preferable as a simple inspection method. By using the probe set of the present invention, detection / quantification of a specific miRNA can be performed quickly at a relatively low cost.

[0064] In the RT-PCR method, using the extracted RNA as a template, for example, TaqMan TM miRNA RT Kit (manufactured by Applied Biosystems) is used to synthesize cDNA, and the target miRNA can be detected and quantified by real-time quantitative PCR for such cDNA.

[0065] In addition, by using a next-generation sequencing system, RNAs contained in a measurement sample prepared for RNA can be comprehensively detected and quantified. When the abundance ratio of a marker miRNA is used as a judgment indicator, it is necessary to measure the total amount of miRNA contained in the sample. In such cases, it is preferable to use a next-generation sequencer that can comprehensively detect and analyze miRNA.

[0066] When using a next-generation sequencing system, a cDNA library (a group of DNA clusters) is prepared by performing a reverse transcription reaction on total RNA, and then individual clusters in the prepared DNA library are sequenced. Sequencing methods may include sequencing-by-synthesis (nucleotides are reversibly terminated and one nucleotide is incorporated and analyzed per cycle), pyrosequencing (the sequencing reaction is monitored via the release of pyrophosphate upon nucleic acid incorporation), ligation (sequencing is performed using short oligonucleotide probes that ligate to primer sequences using ligase), or ion semiconductor sequencing (detection of cluster sequences using hydrogen ion release during the sequencing reaction).

[0067] cDNA synthesis can be performed by conventionally known methods, or may be performed using a commercially available kit, for example, Ion Total RNA-seq kit v2 for small RNA libraries (Thermo Fisher).

[0068] Alternatively, a system for multiplexing miRNA analysis from body fluids without an RNA purification step may be used. For example, Abcam's Firefly TM The technology is a system that uses a flow cytometer to simultaneously profile miRNAs, and is said to be capable of analyzing them using a Multiplex Circulating miRNA Assay with body fluids such as plasma and urine as samples.

[0069] The sequenced miRNAs are identified by matching them with reference sequences, and their relative abundance can be quantified according to the number of reads of the identified miRNAs. The total amount of identified miRNAs is calculated from the total number of reads, and the abundance ratio (expression frequency) of the marker can be calculated from the number of reads of the miRNA used as a marker relative to the total amount of the miRNA.

[0070] The reference sequence can be sequence data registered in a miRNA database, such as miRBase (http: / / www.mirbase.org). Comparison and identification with the reference sequence is not limited to a perfect match; identification can also be based on a match of 10 or more consecutive bases. Since the miRNA used as a marker is 16 to 25 bp, a match of 10 or more bases indicates a sequence homology of 70% or more, and a match of 10 or more consecutive bases indicates even higher homology.

[0071] Even if miRNAs originate from different gene loci or different precursors (pri-miRNA or pre-miRNA), the mature miRNAs obtained may differ only by one or several bases in their sequences and thus may be identified as a common mature miRNA. Furthermore, when pri-miRNA and pre-miRNA are contained in a measurement sample, mismatches of several bases occur at the read site, but the final mature miRNAs will have a common sequence and can therefore be identified and quantified as the same type of miRNA.

[0072] A method in which the marker is detected by color development by combining PCR and immunochromatography may also be used.

[0073] In addition, the microarray method, for example, uses 3D-Gene (registered trademark) to specifically extract miRNA from body fluids, and then uses a highly sensitive DNA chip (microarray) to detect more than 800 types of miRNA at once.

[0074] In methods that utilize hybridization, the method for detecting the presence or absence of hybridization is appropriately selected depending on the type of label of the probe used. For example, when a fluorescent label is used, the presence or absence of fluorescence emitted by hybrid formation can be detected by visual observation. In addition, the content and content ratio can be calculated by scaling the fluorescence intensity, etc.

[0075] <Screening method for active tuberculosis samples> The screening method of the present invention is a method for classifying specimens into a group that may be positive for active tuberculosis and a negative group, and can be used as a screening method for separating a large number of specimens into a group of positive samples and a group of negative samples, as in a health checkup, or as a simple testing method for the initial examination of patients suspected of having tuberculosis.

[0076] The screening method of the present invention comprises a first step of qualitatively and / or quantitatively detecting biomarkers contained in a sample derived from the urine of a subject, i.e., at least two miRNAs selected from the miRNAs shown in Table 1-1 (SEQ ID NO: 1 to SEQ ID NO: 25) or the miRNAs shown in Table 1-2 (SEQ ID NO: 3, 6, 9, 10, 13, 14 and SEQ ID NO: 49 to SEQ ID NO: 55), or miRNAs having 70% or more sequence identity to the miRNAs. The miRNAs to be detected are preferably a combination including one selected from SEQ ID NO: 3, 6, 9, 10, 13, and 14.

[0077] The screening method of the present invention preferably further comprises a second step of qualitatively and / or quantitatively detecting at least one miRNA selected from the miRNAs of Group D shown in Table 2-1 or Table 2-2 (SEQ ID NO: 26 to SEQ ID NO: 45, SEQ ID NO: 56 to SEQ ID NO: 75), or miRNAs sharing 70% or more sequence identity with the miRNA. The miRNAs detected in the second step are preferably one or a combination of two or more miRNAs selected from SEQ ID NO: 26 to SEQ ID NO: 31 belonging to Group D1, or one or a combination of two or more miRNAs selected from Group Df. In this case, the miRNA preferably includes the miRNA of SEQ ID NO: 29 or SEQ ID NO: 40.

[0078] Either step 1 or step 2 may be performed, but both steps 1 and 2 are preferably performed. For example, if the expression frequency of the biomarker detected in step 1 is high and the expression frequency of the biomarker detected in step 2 is low, the accuracy of determining that the probability of a tuberculosis positive is high increases. Furthermore, if the expression frequency of miRNA in step 1 is low and the expression frequency of miRNA in group D in step 2 is high, it can be determined that the probability of a tuberculosis negative is high.

[0079] Here, when the first step and the second step are included, the order of the first step and the second step is not particularly limited, and the first step may be performed after the second step, or the second step may be performed after the first step, or the first step and the second step may be performed simultaneously.

[0080] When both the first and second steps are performed, the miRNA detected in the first step may be only one of the miRNAs represented by SEQ ID NO: 1 to SEQ ID NO: 25 and SEQ ID NO: 49 to SEQ ID NO: 55.

[0081] In the screening method of the present invention, it is preferable to detect the internal control miRNAs (SEQ ID NO: 46 to SEQ ID NO: 48) shown in Table 3 in addition to detecting the miRNAs that serve as biomarkers. When the expression frequency ratio relative to the internal standard is used as the judgment index for distinguishing between positive sample groups or positive and negative sample groups, the internal control miRNA shown in Table 3 is also detected.

[0082] As will be described later, using the ratio to the internal control as the judgment indicator also makes it possible to reduce the risk of false positives and false negatives in HIV-infected individuals. In the case of HIV infection, some biomarkers show increased expression (e.g., hsa-miR-196a-5p, hsa-miR-500a-3p) and others show decreased expression (e.g., hsa-miR-16-5p, hsa-miR-6887-5p). For such biomarkers, using the ratio to the internal control as the assessment index can reduce the impact of HIV infection on expression frequency and reduce the risk of false positives and false negatives in HIV-infected individuals.

[0083] In particular, the internal control ratio of miRNAs in Group D is a miRNA whose expression frequency is significantly lower than that of the internal control in HIV-positive samples, so those with a low internal control ratio can be classified as having a high possibility of HIV infection. Together with cases where the expression frequency is high in Step 1, it can be considered that there is a high possibility of tuberculosis / HIV coinfection, or that the result in Step 1 is a false positive due to HIV infection.

[0084] In the first step of the screening method of the invention, the separation of positive and negative samples is a step of separating a sample into positive samples if the expression frequency of the detected miRNA in the sample is higher than a predetermined value.Specific examples of the determination method include the following.

[0085] (1) First determination method The first determination method is a method in which the content ratio (or amount) of miRNA used as a biomarker is used as a determination index, and samples are classified into a positive sample group when the content ratio (or amount) is higher than a preset threshold.

[0086] The ratio of the number of reads of the miRNA to be tested (biomarker) to the total number of reads when read by a next-generation sequencer corresponds to the content of the miRNA to be tested relative to the total content of miRNA (usually 200 to 500 types) contained in detectable amounts in the sample.

[0087] In carrying out the first determination method, all miRNAs or the types of miRNAs similar thereto contained in the measurement sample and their respective contents are measured, and the total content (total number of reads) is calculated. The expression frequency (abundance ratio) of the miRNA used as a marker is then calculated and compared with a threshold. The threshold is set as the average expression frequency of the target marker in healthy individuals and is appropriately set depending on the type of biomarker used. A positive result is determined when the significance level compared with the threshold is p<0.1, preferably p<0.05, and more preferably p<0.01.

[0088] When the expression frequency of healthy individuals (control group) can be used as a preset threshold, the content may be compared instead of the expression frequency of the target marker miRNA. In this case, there is no need to comprehensively read miRNAs; the content of the target miRNA can be compared with the content of the control using a test probe set. The threshold is set to a content that is approximately 1.5 times, preferably 2 times, the average expression level of the corresponding miRNA in healthy individuals, and samples that are calculated to be equal to or greater than this threshold are classified as positive samples.

[0089] (2)Second judgment method The second determination method uses the ratio of the expression level of the biomarker miRNA to be detected relative to the expression level of the miRNA used as an internal control as a determination index, and determines a positive result when this ratio is higher than a threshold. The ratio can be calculated by quantifying the internal control and the miRNA used as a marker, making screening possible using the probe set of the present invention. Comprehensive detection and quantification of miRNAs contained in a measurement sample requires special equipment such as a next-generation sequencer, and the testing time is long, resulting in high costs per sample. In this regard, the screening method using the probe set of the present invention is suitable for testing a large number of samples in a short period of time. Therefore, it can be used as a testing method that can be widely implemented in developing countries and can be applied to health checkups that require rapid diagnosis of a large number of subjects.

[0090] Furthermore, in the second determination method using an internal control, the influence of HIV infection can be reduced by selecting an internal control, which has the effect of reducing the risk of false positives and false negatives.

[0091] In the second step, the separation of positive and negative samples is carried out by separating samples into positive samples when the expression frequency of the detected miRNA in the sample is lower than a predetermined value. The determination indicators used in the first and second determination methods in the first step can be used. That is, in the first determination method, a positive result is determined when the expression frequency of the detected target miRNA is lower than a threshold value (the average expression frequency of the same miRNA in healthy individuals), whereas in the second determination method, a positive result is determined when the ratio of the expression frequency of the detected target miRNA to the internal control miRNA is lower than a threshold value (the ratio of the expression frequency of the same miRNA in healthy individuals to the internal control miRNA).

[0092] Furthermore, based on the judgment results (positive (+) or negative (-)) from the first and second steps, it is possible to classify the samples into three types: positive, negative, and possible positive, according to Table 4 below. By classifying samples judged as positive and possible positive in Table 4 into the positive sample group, it is possible to avoid false negatives, in which samples that should be classified as positive are classified into the negative sample group, and the reliability of the negativity of samples classified as negative is increased.

[0093] [Table 4] [Example]

[0094] [RNA sample preparation] Urine samples (3 ml) were collected from three healthy individuals and ten patients with active tuberculosis infection (four of whom were co-infected with HIV). TM Total RNA samples were extracted according to the protocol in the mirVana Total RNA-Seq kit (Thermo Fisher Scientific) operating manual (urine samples). The specific procedure was as follows.

[0095] To 3 ml of urine, 2.4 ml of lysis buffer was added and the mixture was shaken (500 rpm) for 7 minutes. To the resulting mixture, 350 μl of a separately prepared binding bead mix (a mixture of 234 μl of RNA binding beads and 116 μl of lysis / binding enhancer) was added and the mixture was shaken (500 rpm) for 5 minutes. To the resulting solution, 5.76 ml of isopropanol was added, and the mixture was pipetted and then incubated for 20 minutes (200 rpm). The mixture was then placed on a magnetic stand to separate the particles. The supernatant was discarded, and the mixture was washed by adding a washing solution and shaking. Then, the mixture was washed with Turbo DNase. TM The RNA was re-bound to the RNA Binding Beads by adding the solution, and the re-bound RNA was separated again using a magnet. After repeated separation and washing, elution buffer was added to the dried RNA Binding Beads and incubated at 65°C for 5 minutes. The mixture was placed on a magnetic stand, and the resulting supernatant was used as the measurement sample.

[0096] [Sequence analysis method] Using the RNA extraction sample obtained above, adapters were added and reverse transcription was performed according to the operating manual for the Ion Total RNA-Seq kit v2 for small RNA Libraries (Thermo Fisher), to prepare a cDNA library for next-generation sequencers. The sequence of the prepared cDNA library was analyzed using a next-generation sequencer, Ion Torrent PGM (Thermo Fisher).

[0097] The obtained sequences were comprehensively compared with miRNAs registered in the miRBase database (a data bank site) as reference sequences, and the number of reads for each miRNA was calculated to quantify the miRNAs in the sample, and the ratio of the number of reads to the total miRNA was calculated. When comparing and identifying with the reference sequence, if 10 or more consecutive bases matched, the miRNA was identified as corresponding to the reference sequence.

[0098] The detected miRNAs were arranged in order of expression frequency, and it was possible to quantify more than 200 types of miRNAs from each prepared sample.

[0099] [Confirmation of tuberculosis and HIV infection in specimens] The presence or absence of tuberculosis infection in the 13 subjects subjected to the sequence analysis was confirmed by culture testing of the tuberculosis bacteria contained in each subject's sputum and analysis using the GeneXpert® system. The presence or absence of tuberculosis bacteria was confirmed using the GeneXpert® system by amplifying RNA contained in the sputum by PCR and then examining it using a probe targeting the drug resistance region of the rpoB gene of M. tuberculosis. The presence or absence of HIV infection was examined by measuring the proportion of CD4-positive T cells using FACS Presto (registered trademark) from BD Biosciences.

[0100] [Analysis of sequence analysis results: Welch's T analysis] Regarding the expression frequency of the identified miRNAs, Welch's T analysis was used to classify miRNAs that were found to have a high expression frequency in tuberculosis-positive samples and a low expression frequency in healthy samples into Group A. Furthermore, miRNAs with a statistical significance level of P less than 0.05 and a statistical significance level of P less than 0.06 and showing expression levels more than twice that of healthy samples into Group A1, and miRNAs with a P less than 0.1 into Group A2. As a result, sequence Nos. 1 to 11 were selected as miRNAs belonging to the A1 group, and sequence Nos. 12 to 25 were selected as miRNAs belonging to the A2 group.

[0101] Furthermore, miRNAs that are highly expressed in healthy individuals but whose expression frequency decreases due to tuberculosis infection were classified into group D, with those with a statistical significance level (P value less than 0.05) classified as group D1, and those with a P value less than 0.1 classified as group D2. As a result, SEQ ID NO: 26 to SEQ ID NO: 31 were selected as miRNAs belonging to the D1 group, and SEQ ID NO: 32 to SEQ ID NO: 45 were selected as miRNAs belonging to the D2 group.

[0102] The results (plots) for the number of reads, expression frequency, and internal control ratio for miRNAs belonging to Group A are shown in Figures 1 to 8. Furthermore, the P values ​​and expression folds relative to healthy individuals for miRNAs belonging to Group A1 are shown in Table 5. Furthermore, plots of the results for miRNA-196a-5p, miRNA-424-5p, miRNA-500a-3p, and miRNA-199a-5p, excluding HIV-infected patients, are shown in Figures 9 and 10.

[0103] Furthermore, the results (plots) of the number of reads, expression frequency, and internal control ratio of miRNAs (sequence numbers 26-45) classified into group D are shown in Figures 11 to 18. Furthermore, the P values ​​of miRNAs belonging to group D1 and the expression folds relative to healthy individuals are shown in Table 6.

[0104] In the plot, (TB-) indicates the results of samples from healthy individuals, and (TB+) indicates the results of samples from tuberculosis-positive patients. Among the TB+ sample results, the results of HIV-infected patients are plotted as white squares (□). In each figure, the vertical axis of the leftmost plot shows the number of reads, and the vertical axis of the second plot shows the expression frequency (%). The third, fourth, and fifth plots from the left show the expression frequency ratio (fold) relative to the internal controls hsa-miR-30c (SEQ ID NO: 46), hsa-miR-423-5p (SEQ ID NO: 47), and hsa-miR-21 (SEQ ID NO: 48), respectively.

[0105] [Table 5]

[0106] [Table 6]

[0107] As can be seen from Figures 1 to 8 and Table 5, the expression levels of miRNAs belonging to Group A1 were higher in tuberculosis patients than in healthy subjects, and a similar trend was observed in their expression frequencies. In the case of HIV coinfection, the expression levels and frequencies of miRNAs belonging to Group A1 tended to be even higher, but by using the ratio with the internal control, it was found that the expression frequencies were higher than those of healthy subjects even in patients not coinfected with HIV (patients infected with tuberculosis alone).

[0108] In HIV-infected individuals, the expression levels of these miRNAs tended to be higher, but by using the ratio to the internal control, it was found that the effect of HIV infection could be reduced (for example, in the case of miR196a-5p, by using the ratio to the internal control as an indicator, it was found that the expression frequency was significantly higher in patients with tuberculosis alone).

[0109] Furthermore, as can be seen from Figures 9 and 10, among the A1 group, miR196a-5p, miR424-5p, miR500a-3p, and miR199a-5p are expressed at higher levels in tuberculosis infection, regardless of HIV infection. Therefore, by using these as biomarkers, and by using the ratios to their respective appropriate internal controls as indicators, it is possible to distinguish tuberculosis-only positive patient samples.

[0110] As can be seen from Figures 11 to 18, the expression frequency of miRNAs belonging to Group D is particularly low in cases of HIV co-infection. Furthermore, the ratio to the internal control is significantly lower than that of healthy individuals. Regarding miR1290 (sequence No. 31), the following results were obtained by statistical processing using Mann-Whitney, and it was confirmed that it was a biomarker with a significant difference in the D1 group. P value: 0.007 Expression ratio relative to healthy subjects: 0.56 Mann-Whitney statistical analysis excluding HIV-coinfected patients P value: 0.034 Expression ratio relative to healthy subjects: 0.46

[0111] Therefore, it is clear that the detection of both groups A and D can improve the reliability of screening for tuberculosis-positive samples. Similarly, the detection of both groups A and D can enable differential screening of tuberculosis-negative samples.

[0112] [Analysis of sequence analysis results Part 2: Statistical processing using Fisher's exact test] (1) RNA sample 2 Urine samples collected from 10 newly acquired Laotian patients with active tuberculosis (one of whom also had AIDS) and 6 healthy individuals were analyzed using the MagMax TMTotal RNA samples were extracted according to the protocol in the operating manual (urine samples) of the mirVana Total RNA-Seq kit (Thermo Fisher). The specific procedure has already been described.

[0113] Using the RNA extraction sample obtained above, adapters were added and reverse transcription was performed according to the operating manual for the Ion Total RNA-Seq kit v2 for small RNA Libraries (Thermo Fisher) as described above to prepare a cDNA library for next-generation sequencers. The sequence of the prepared cDNA library was analyzed using the next-generation sequencer Ion Torrent PGM (Thermo Fisher).

[0114] The obtained sequences were comprehensively compared with miRNAs registered in the miRBase database (a data bank site) as reference sequences, and the number of reads for each miRNA was calculated to quantify the miRNAs in the sample, and the ratio of the number of reads to the total miRNA was calculated. When comparing and identifying with the reference sequence, if 10 or more consecutive bases matched, the miRNA was identified as corresponding to the reference sequence.

[0115] The miRNA expression frequency data from the samples (10 active TB patients and 3 healthy controls) previously subjected to Mann-Whitney statistical processing was combined with the miRNA expression frequency data from additional samples (4 active TB patients and 6 healthy controls) obtained this time and analyzed using Fisher's exact test. For the miRNAs identified using Fisher's exact test (TCC-GUI software), the expression frequency fold increase in active TB patients compared to the expression frequency in healthy controls was examined. Figure 19 shows a scatter plot showing the relationship between the expression frequency fold increase of each miRNA and statistical significance.

[0116] In FIG. 19, the horizontal axis indicates M value=log2 (multiplication factor relative to the expression frequency in healthy subjects). In other words, an M value of 1 indicates that the expression frequency is twice that of healthy subjects. 10 logarithmic scale) is the logarithm of the P value (-log 10 (P value) Therefore, when the vertical axis value is 1, the P value is 0.1.

[0117] An open triangle (△) indicates an expression fold of 2 or more (M value > 1) with a significance level of P value < 0.05. An open circle (○) indicates an M value of less than -2 with a P value < 0.01. miRNAs plotted with an open triangle are effective miRNAs classified into the Af group, and miRNAs plotted with an open circle are effective miRNAs classified into the Df group.

[0118] The M value of each miRNA, the P value obtained from the analysis, and the Q value obtained from the analysis of the miRNAs classified into the Af group and the Df group as shown in Figure 19 are shown in Tables 7 and 8. In Table 8, a P value of less than 0.001 was essentially considered to be a P value of 0. The Q value is the minimum FDR threshold at which the test results are deemed significant, and corresponds to the proportion of differentially expressed miRNAs obtained in the test that are not actually differentially expressed miRNAs.

[0119] [Table 7]

[0120] [Table 8]

[0121] As can be seen from Table 7, the 13 miRNAs (Af group miRNAs) selected based on the results of analysis using Fisher's exact test and increased numbers of tuberculosis-positive and healthy subjects were useful as biomarkers for tuberculosis positivity, as their expression levels were more than twice as high as those of healthy subjects, with a significance level of p<0.05 and a Q value (FDR)<0.08. In particular, identifying two or more miRNAs belonging to the Af group enables highly accurate determination.

[0122] Furthermore, by combining and identifying with the Df group, it is possible to improve the accuracy of distinguishing between positive and negative. [Industrial Applicability]

[0123] The screening method for tuberculosis infection samples of the present invention can use urine samples as the specimen sample, which are minimally invasive and easy to collect for the subject, and can quickly obtain determination results without the need for culture or other procedures to determine tuberculosis infection, making it useful as a testing method for checking for the presence or absence of active tuberculosis infection in areas that lack adequate facilities, such as developing countries.

Claims

1. The miRNAs (sequence no. 1) shown in Table 1-1 contained in the urine samples of the subjects to 25) or at least two miRNAs selected from the miRNAs shown in Table 1-2 (Sequence Nos. 3, 6, 9, 10, 13, 14 and Sequence Nos. 49 to 55). A method for screening tuberculosis infected samples, comprising a first step of qualitatively and / or quantitatively detecting iRNA. Training method. Table 1-1 Table 1-2

2. The method for screening tuberculosis-infected samples according to claim 1, wherein the at least two miRNAs selected from Table 1-1 or Table 1-2 include at least one miRNA selected from the group consisting of sequence numbers 3, 6, 9, 10, 13, and 14.

3. 3. The method for screening tuberculosis-infected samples according to claim 1, wherein the detection step is a step of classifying a sample as positive if the expression frequency in the sample is higher than a predetermined value.

4. The detection step was carried out using the internal control miRNA (SEQ ID NO: 46) shown in Table 3. to sequence No. 48), the content ratio of the detected miRNA is higher than a predetermined value. The method for screening tuberculosis-infected samples according to any one of claims 1 to 3, further comprising the step of separating the samples into positive samples if the sample is positive. Table 3

5. Furthermore, miRNAs shown in Table 2-1 (Sequence No: 26 to Sequence No: 45) or Table 2-2 Selected from miRNAs shown in (Sequence Nos. 29, 40, 56 to 75) and a second step of qualitatively and / or quantitatively detecting at least one miRNA detected by the method. The method for screening tuberculosis-infected samples according to any one of claims 1 to 4, comprising: Table 2-1 Table 2-2

6. The at least one miRNA selected from the miRNAs shown in Table 2-1 or the miRNAs shown in Table 2-2 includes the miRNA of sequence No. 29 or sequence No.

40. A method for screening tuberculosis-infected samples according to claim 1.

7. Sequence No. 1 to Sequence No. 25 and Sequence No. Sequence N contained in a sample derived from the subject's urine A first step of qualitatively and / or quantitatively detecting one miRNA selected from the group consisting of SEQ ID NO: 49 to SEQ ID NO: 55; and From the group consisting of Sequence No. 26 to Sequence No. 45 and Sequence No. 56 to Sequence No. 75 A second step of qualitatively and / or quantitatively detecting one selected miRNA. A method for screening tuberculosis infection samples, comprising:

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