JP7796416B2 - Screening method for tuberculosis infection samples and biomarkers and probe sets for use therein - Google Patents

Description

The present invention relates to the use of miRNAs, whose expression frequency differs depending on whether or not a person 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 sample, as well as testing and screening methods and testing probe sets that use the same.

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 for the development of a rapid and simple method for testing for tuberculosis infection that can be implemented in developing countries.

In Japan, the most commonly used diagnostic method for tuberculosis infection is the QuantiFERON® TB or T-Spot® TB, which measures interferon-γ (IFN-γ) produced by lymphocytes in response to antigenic stimulation with ESAT-6, CFP-10, or other antigens. This method involves drawing blood from a subject, isolating lymphocytes, dispensing a fixed amount of the lymphocytes onto a culture plate coated with anti-human interferon-γ antibodies, adding the tuberculosis-specific antigens ESAT-6 and CFP-10, and culturing the cells for approximately 20 hours. The number of IFN-γ-producing cells bound to the anti-human INF-γ antibodies is then counted. This method is characterized by its high specificity compared to the tuberculin test, as it is not affected by BCG vaccination or nontuberculous mycobacteria. 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 popularize in developing countries.
Furthermore, in patients who are infected with not only tuberculosis bacteria 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, even though the patient is infected with tuberculosis, so there is a problem that the test result may be false negative.

Furthermore, the MPB64 antibody is known to be specific to patients with active tuberculosis. A method is also known in which the amount of MPB64 antibody in plasma is detected using this antibody as a marker, for example, by the dot blot assay, to determine whether or not a patient has active tuberculosis. The MPB64 antibody is also present in urine, and it has been reported to correlate 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 allow for the use of urine, which is easy to collect, as a measurement sample, making them ideal 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.

Tests using sputum culture collected from subjects are highly accurate in determining whether or not tuberculosis bacteria are present, but because the necessary equipment and a culture period of one to four weeks are required, it is difficult to use as a rapid testing or diagnostic method.

In 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.

In recent years, it has been proposed to use miRNA as a biomarker, separate from antibodies, cells, cytokines, etc. Abnormal expression of miRNA has been shown to be involved in the onset and malignant progression of many diseases, and because miRNA can be detected in body fluids such as blood, its use as a liquid biopsy has attracted attention.
In recent years, various reports have been made on the use of miRNA as a diagnostic marker, mainly 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.).

Several miRNAs specific to tuberculosis patients have also been reported (Naveed Sabir et al., Frontiers in Microbiology vol. 9, March 2018), and Table 1 of that publication 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 yet been clarified what level of specificity each miRNA has as a diagnostic marker, or how to determine its specificity.

In addition, 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.

Special Publication No. 2010-534480 WO2015/133477 publication Special table 2018-505656 publication

Yasuko Tamada et.al., “Diagnosis of active tuberculosis using MBP64, a specific antigen of Mycobacterium bovis”, Microbiol immunol 2012;56:740-747 Naveed Sabir et.al., “miRNAs in Tuberculosis: New Avenues for Diagnosis and Host-Directed Therapy”, Frontiers in Microbiology vol.9, March 2018

The present invention aims to provide 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.

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

In other words, 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 sequence numbers 1 to 25, or a miRNA group consisting of sequence numbers 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 homology to the miRNAs.

It is preferable that the at least two types of miRNA selected above include at least one type of miRNA selected from the group consisting of sequence numbers 3, 6, 9, 10, 13, and 14.

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. Furthermore, the detection step is preferably a step of classifying a sample as positive if the content ratio of the detected miRNA to the internal control miRNA (SEQ ID NO: 46 to SEQ ID NO: 48) is higher than a predetermined value.

The method may further include a second step of qualitatively and/or quantitatively detecting at least one miRNA selected from miRNAs (a miRNA group consisting of SEQ ID NO: 26 to SEQ ID NO: 45, or a miRNA group consisting of SEQ ID NO: 56 to SEQ ID NO: 75 and SEQ ID NO: 29 and SEQ ID NO: 40), or a miRNA having 70% or more sequence identity with the miRNA. In this case, it is preferable that the miRNA contains either SEQ ID NO: 29 or SEQ ID NO: 40.

In another aspect, the screening method of the present invention includes a first step of qualitatively and/or quantitatively detecting one type of 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 with said miRNA, contained in a sample derived from the urine of a subject; and a second step of qualitatively and/or quantitatively detecting one type of 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 with said miRNA.

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 (SEQ ID NO: 1 to SEQ ID NO: 25, and SEQ ID NO: 49 to SEQ ID NO: 55) shown in Table 1-1 or Table 1-2.

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

In addition, from another perspective, 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.

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 NO: 46 to SEQ ID NO: 48 as a probe for detecting an internal control miRNA (SEQ ID NO: 46 to SEQ ID NO: 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.

In this specification, "tuberculosis" refers to an infection caused by the tuberculosis bacillus. 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, this does not include 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.

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 concept 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 transcribed from a miRNA gene 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.

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.

The screening method for tuberculosis infection samples of the present invention uses miRNA as a biomarker, eliminating the need for cell culture or other procedures to prepare samples for the method, allowing for rapid determination. Furthermore, because urine is used as the sample for measurement, which does not require expert collection, the method is useful as a testing method that requires rapid and inexpensive testing of large quantities of samples.

FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the results of the expression frequency of miRNAs in Group A. FIG. 10 is a plot showing the miRNA expression frequency results for Group A (excluding HIV patients). FIG. 10 is a plot showing the miRNA expression frequency results for Group A (excluding HIV patients). FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 10 is a plot showing the results of miRNA expression frequency in group D. FIG. 1 is a plot diagram showing statistically processed miRNA expression frequency results obtained in the examples in relation to the expression frequency ratio in healthy subjects.

<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 70% or more, preferably 80% or more, and more preferably 90% or more sequence homology with the miRNAs.

The miRNAs listed in Tables 1-1 and 1-2 (miRNAs SEQ ID NO: 1 to SEQ ID NO: 25, 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 compared to healthy individuals. Of these, miRNAs belonging to Group A1 (SEQ ID NO: 1 to SEQ ID NO: 11) showed 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) showed 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 NO: 49 to SEQ ID NO: 55, and SEQ ID NO: 3, 6, 9, 10, 13, and 14) have a p<0.05 and a Q value (FDR)<0.3 in Fisher's exact test, indicating that their expression frequency in samples from M. tuberculosis-infected individuals is higher than that in healthy individuals. Therefore, miRNAs selected from the A1 group or the Af group are more preferable as biomarkers for screening tuberculosis-positive samples.

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.

In addition, 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, given their high expression frequency regardless of whether or not HIV co-infection is present and the difference from healthy individuals is significant.

The miRNAs listed in Tables 2-1 and 2-2 (sequence No. 26 to 45, sequence No. 56 to 75, the group consisting of which is sometimes collectively referred to as "Group D") are miRNAs whose expression frequency tends to decrease with tuberculosis infection compared to that in healthy individuals.

In this specification, the term "healthy individual" refers to a sample derived from an individual who is tuberculosis-negative, meaning that 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.

Of 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 (a significant difference is indicated at a level less than 0.05), while the miRNAs belonging to Group D2 (SEQ ID NO: 32 to SEQ ID NO: 45) had a significance level of p<0.1 (a significant difference is indicated at a level 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.

Furthermore, among the miRNAs belonging to group D, miRNAs belonging to group Df (sequence numbers 56 to 75, and 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 a 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.

In particular, sequence numbers 29 (hsa-miR-451a) and 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 sequence number 29 (hsa-miR-451a) or sequence number 40 (hsa-miR-769-5p), and more preferably sequence number 29 (hsa-miR-451a).

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

It is also possible to use a combination of miRNAs selected from Group A and Group D. 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 results, such as false positives and false negatives, can be reduced.

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.

<Test 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:

a) Oligonucleotide probe A capable of hybridizing under stringent conditions with any one of the 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

b) Oligonucleotide probe D capable of hybridizing under stringent conditions to any one of the miRNAs of group D (sequence No.: 26 to sequence No.: 45, sequence No.: 56 to sequence No.: 75) shown in Table 2-1 or Table 2-2

c) An internal control oligonucleotide probe capable of hybridizing under stringent conditions with at least one of the miRNAs (sequences No. 46 to 48) shown in Table 3

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.

Any combination that includes at least oligonucleotide probe A may be used. 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 internal control oligonucleotide probe; a combination of one type of oligonucleotide probe A, one type of oligonucleotide probe D, and an internal control oligonucleotide probe; and a combination of two or more types of oligonucleotide probe A, one or more types of oligonucleotide probe D, and an internal control oligonucleotide probe.

When multiple types of oligonucleotide probe A are combined, it is preferable that at least one type contains an miRNA selected from the group consisting of sequence numbers 3, 6, 9, 10, 13, 14, and 16.

The accuracy of the determination can be improved by combining multiple types of oligonucleotide probes. In particular, by detecting at least one type of miRNA using 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.

Furthermore, the internal control can reduce the influence of HIV infection, which has the effect of reducing the risk of false positives and false negatives.
That is, the amount of miRNA contained in the 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, which may result 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.

Furthermore, depending on the type of biomarker, the expression frequency may be low, making it difficult to distinguish between positive and negative samples. Furthermore, in the case of HIV infection, among the Group A biomarkers, the expression frequency is higher in HIV infection, which may result in a false positive, where a person is classified as tuberculosis positive even when they are not. Conversely, in the case of HIV infection, among the Group A biomarkers, there are biomarkers that tend to be expressed less frequently in HIV infection, which may result in a false negative, where a person is classified as tuberculosis negative even when they are tuberculosis positive. In this regard, if the ratio to the internal control is used as an indicator, it is possible to eliminate the influence of HIV infection.

The internal control oligonucleotide probe is selected appropriately depending on the type of oligonucleotide probe A or D to be combined, so that the combination will clearly show the influence of HIV and the difference from healthy individuals. For example, when hsa-miR-196a is used as the biomarker, combining it with hsa-miR-423 as the internal control will clearly show the difference between tuberculosis positive and negative results, thereby 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 influence of HIV in HIV-infected samples, thereby reducing the risk of false positives and false negatives due to HIV infection.

These nucleotide probes preferably have a label that can be detected when hybridized with the target miRNA. The label may be a known label that is preferably used for 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.

<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 is a sample that 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.

Urine tends to contain fewer exosomes containing miRNA than blood, but compared to other bodily fluids, it is possible to collect larger amounts. 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, etc. Therefore, using urine as a sample is suitable for testing in developing countries.

Like other bodily fluids such as blood, urine contains exosomes containing mature miRNAs that serve as biomarkers. Exosomes are a type of membrane vesicle secreted by cells, and generally measure 30-150 nm in diameter when observed under an electron microscope. In addition to mature miRNAs, they also contain pri-miRNAs, pre-miRNAs, coding-mRNAs, DNA, enzymes, cytoskeletal proteins, signaling molecules, and more.

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.

In the determination methods described below, when expression frequency is used as an index, it is desirable that a certain number or more types of miRNA be 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 or more types of miRNA are detected to a quantifiable level, and even urine can meet this requirement.

Examples of urine-derived samples include exosome-enriched or roughly purified fractions (hereinafter referred to as "exosome-rich fractions"), as well as samples from which RNA has been extracted. When detecting miRNA markers, it is preferable to use samples from which RNA has been directly extracted as measurement samples.

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 the like, 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.

Commercially available separation kits include the ExoQuick ^™ Exosome precipitation solution series (e.g., ExoQuick-TC, ExoQuick-CG, etc., all manufactured by System Biosciences) as exosome separation kits, and 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, as separation kits for obtaining nucleic acid extracted samples from body fluids.
Alternatively, a method (nano-biodevice method) may be used in which exosomes are collected simply by sending an exosome-containing sample (urine) to a nanowire structure.

<Detection and quantification of miRNA>
Methods for detecting the target miRNA from the RNA-containing sample prepared as described above include, for example, RT-PCR, microarray, Northern blotting, next-generation sequencing, and liquid-phase nucleic acid hybridization.
When it is necessary to detect most of the miRNAs contained in a sample (for example, when expression frequency is used as a determination index), RT-PCR, microarray, next-generation sequencer, etc. are used.

Methods for detection and quantification using hybridization include microarray methods, Northern blotting, liquid-phase nucleic acid hybridization, etc. A miRNA that serves as a biomarker can be detected or quantified by utilizing hybridization with a specific miRNA.
The hybridization method is preferable as a simple test method because it can directly detect miRNAs that can hybridize with probes. By using the probe set of the present invention, specific miRNAs can be detected/quantified quickly and at a relatively low cost.

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

Furthermore, using a next-generation sequencing system, it is possible to comprehensively detect and quantify RNA contained in the prepared RNA measurement sample.
When the abundance ratio of a marker miRNA is used as a judgment index, 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.

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 while short oligonucleotide probes that ligate to each other are ligated to a primer sequence by ligation), or ion semiconductor sequencing (detection of cluster sequences using hydrogen ion release during the sequencing reaction).

cDNA synthesis can be performed using conventional methods or using commercially available kits, such as the Ion Total RNA-seq kit v2 for small RNA libraries (Thermo Fisher).

Alternatively, a system for multiplex miRNA analysis from body fluids without an RNA purification step may be used. For example, Abcam's Firefly ^™ technology is a system that simultaneously profiles miRNAs using a flow cytometer, and is said to be capable of analyzing body fluids such as plasma and urine using a Multiplex Circulating miRNA Assay.

Sequenced miRNAs are identified by comparing them with reference sequences, and their relative abundance can be quantified according to the number of reads of the identified miRNA. The total amount of identified miRNA 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.

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 16-25 bp, a match of 10 or more bases represents a sequence homology of 70% or more, and the homology is even higher when 10 or more consecutive bases match.

Even if miRNAs originate from different gene loci or different precursors (pri-miRNA or pre-miRNA), the mature miRNAs obtained ultimately may differ in sequence by only one or a few bases and therefore may be identified as a common mature miRNA. Furthermore, if pri-miRNA and pre-miRNA are contained in the measurement sample, mismatches of several bases will occur depending on 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.

A method may also be used in which the PCR method is combined with immunochromatography to detect the marker by color development.

In addition, using the microarray method, for example, 3D-Gene (registered trademark) can be used to specifically extract miRNA from body fluids, and more than 800 types of miRNA can be detected at once using a highly sensitive DNA chip (microarray).

In methods that utilize hybridization, the method for detecting the presence or absence of hybridization is selected appropriately depending on the type of label on 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.

<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.

The screening method of the present invention includes 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 that share 70% or more sequence identity with the miRNAs. The miRNAs to be detected are preferably a combination including one selected from SEQ ID NO: 3, 6, 9, 10, 13, 14.

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 that share 70% or more sequence identity with said miRNAs. 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, it is preferable that the miRNA includes miRNA of SEQ ID NO: 29 or SEQ ID NO: 40.

While only one of Step 1 and Step 2 may be performed, it is preferable to perform both Step 1 and Step 2. 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 positive tuberculosis test 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 negative tuberculosis test is high.

Here, when the first and second steps are included, the order of the first and second steps 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 and second steps may be performed simultaneously.

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 sequence No. 1 to sequence No. 25, or sequence No. 49 to sequence No. 55.

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 a 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.

As will be described later, by using the ratio to the internal control as the judgment indicator, it is also possible to reduce the risk of false positives and false negatives in HIV-infected individuals.
In the case of HIV infection, depending on the type of biomarker, some biomarkers have a high expression frequency (e.g., hsa-miR-196a-5p, hsa-miR-500a-3p) and others have a low expression frequency (e.g., hsa-miR-16-5p, hsa-miR-6887-5p). By using the ratio of such biomarkers to the internal control as a judgment index, the effect of HIV infection on the expression frequency can be reduced, and the risk of false positives and false negatives in HIV-infected individuals can be reduced.

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

The first step in the screening method of the invention involves distinguishing between positive and negative samples, in which a sample is classified as positive if the expression frequency of the miRNA to be detected in the sample is higher than a predetermined value.Specific examples of such determination methods include the following:

(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 if the content ratio (or amount) is higher than a predetermined threshold, the sample is classified into a positive sample group.

The ratio of the number of reads of the miRNA to be tested (biomarker) to the total number of reads when read using 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.

When carrying out the first determination method, all miRNAs or miRNAs similar thereto contained in the measurement sample are measured, and the types and amounts of each are measured to calculate the total amount (total number of reads). The expression frequency (abundance ratio) of the miRNA used as a marker is then calculated and compared with a threshold. The threshold is set to be the average expression frequency of the target marker in healthy individuals, and is set appropriately depending on the type of biomarker used. A positive determination is made when a significance level of p<0.1, preferably p<0.05, more preferably p<0.01 can be achieved by comparing with the threshold.

If the expression frequency of healthy individuals (control group) can be used as a preset threshold, the content of the target marker miRNA can be compared instead of the expression frequency. 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 can be classified as positive.

(2) Second Determination 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 the ratio is higher than a threshold value. The ratio can be calculated by quantifying the internal control and the miRNA used as a marker, making it possible to perform screening 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.

Furthermore, in the second determination method using an internal control, the selection of an 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 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 as the determination indicators.
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 same miRNA to the internal control miRNA in healthy individuals).

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, false negatives, in which samples that should be classified as positive are classified into the negative sample group, can be avoided, and the reliability of the negativity of samples classified as negative can be increased.

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

To 3 ml of urine, 2.4 ml of lysis buffer was added, followed by shaking (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, followed by shaking (500 rpm) for 5 minutes.
To the resulting solution, 5.76 ml of isopropanol was added, pipetted, and then incubated for 20 minutes (200 rpm). After separation on a magnetic stand, the supernatant was discarded, and the RNA was rebound to the RNA binding beads by adding a washing solution and washing by shaking. Turbo DNase ^™ solution was then added, and the rebound RNA was again separated 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 supernatant was then left on a magnetic stand and used as the measurement sample.

[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).

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.
In comparison and identification with the reference sequence, if 10 or more consecutive bases matched, the miRNA was identified as corresponding to the reference sequence.

When the detected miRNAs were arranged in order of expression frequency, it was possible to quantify more than 200 types of miRNAs from each prepared sample.

[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 Mycobacterium tuberculosis contained in each subject's sputum and analysis using the GeneXpert® system. The presence or absence of Mycobacterium tuberculosis 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 Mycobacterium 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.

[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 an expression level that was 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 No. 1 to sequence No. 11 were selected as miRNAs belonging to the A1 group, and sequence No. 12 to sequence No. 25 were selected as miRNAs belonging to the A2 group.

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 statistically significant 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, sequence numbers 26 to 31 were selected as miRNAs belonging to the D1 group, and sequence numbers 32 to 45 were selected as miRNAs belonging to the D2 group.

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 level 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.

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 and expression folds relative to healthy subjects for miRNAs belonging to group D1 are shown in Table 6.

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.

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 compared to 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 even in patients without HIV coinfection (patients infected with tuberculosis alone), the expression frequency was higher than in healthy subjects.

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 impact of HIV infection could be reduced (for example, in the case of miR196a-5p, 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).

Furthermore, as can be seen from Figures 9 and 10, in the A1 group, miR196a-5p, miR424-5p, miR500a-3p, and miR199a-5p show increased expression levels in tuberculosis infection, regardless of whether HIV infection is present or not. Therefore, by using these as biomarkers, and by using the ratios to appropriate internal controls for each as indicators, it is possible to distinguish patient samples positive for tuberculosis alone.

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 used as an index, and it is found to be significantly lower than in healthy individuals.
Regarding miR1290 (SEQ ID NO: 31), the following results were obtained by statistical processing using Mann-Whitney, and it was confirmed that miR1290 is 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 co-infected patients
P value: 0.034 Expression ratio relative to healthy subjects: 0.46

This shows that detecting both group A and group D can improve the reliability of screening for tuberculosis-positive samples. Similarly, detecting both group A and group D can enable differential screening of tuberculosis-negative samples.

[Analysis of sequence analysis results Part 2: Statistical processing using Fisher's exact test]
(1) RNA sample 2
Total RNA samples were extracted from urine samples collected from 10 newly acquired Laotian patients with active tuberculosis (one of whom also had AIDS) and six healthy individuals, according to the protocol in the operating manual (urine samples) for the MagMax ^™ mirVana Total RNA-Seq kit (Thermo Fisher). The specific procedures were as previously described.

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 create a cDNA library for next-generation sequencing. The resulting cDNA library was then sequenced using the Ion Torrent PGM next-generation sequencer (Thermo Fisher).

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.
In comparison and identification with the reference sequence, if 10 or more consecutive bases matched, the miRNA was identified as corresponding to the reference sequence.

The miRNA expression frequency data from the samples (10 active tuberculosis patients and 3 healthy individuals) previously subjected to Mann-Whitney statistical processing was combined with the miRNA expression frequency data from additional samples (4 active tuberculosis patients and 6 healthy individuals) 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 tuberculosis patients compared to the expression frequency in healthy individuals was examined. Figure 19 shows a scatter plot illustrating the relationship between the expression frequency fold increase of each miRNA and statistical significance.

In Figure 19, the horizontal axis represents the M value = log ₂ (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. The vertical axis (logarithmic scale of log ₁₀ ) represents the logarithm of the P value (-log ₁₀ (P value)), which is an index of statistical significance. Therefore, a value of 1 on the vertical axis corresponds to a P value of 0.1.

An open triangle (△) indicates an expression fold of 2 or more (M value > 1) and a significance level P value < 0.05. An open circle (○) indicates an M value of less than -2 and 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.

The M value of each miRNA, the P value and Q value obtained from the analysis results for 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 judged to be significant, and corresponds to the proportion of differentially expressed miRNAs obtained in the test that are not actually differentially expressed miRNAs.

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

In addition, by combining and identifying with the Df group, it is possible to improve the accuracy of distinguishing between positive and negative results.

The screening method for tuberculosis infection samples of the present invention can use urine samples as the specimen, which are minimally invasive and easy to collect for the subject, and can quickly obtain results without the need for culture or other procedures to determine tuberculosis infection.Therefore, it is useful as a testing method for checking for the presence or absence of active tuberculosis infection in areas with poor facilities, such as developing countries.

Claims (7)

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 .
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. 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. The detection step was carried out using the internal control miRNA (SEQ ID NO: 46) shown in Table 3.
4. The method for screening tuberculosis-infected samples according to claim 1, wherein the sample is classified as positive if the content ratio of the detected miRNA to the miRNA of sequence No. 48 is higher than a predetermined value.
Furthermore, miRNAs shown in Table 2-1 (Sequence No: 26 to Sequence No: 45) or Table 2-2
The method for screening tuberculosis-infected samples according to any one of claims 1 to 4, further comprising a second step of qualitatively and/or quantitatively detecting at least one miRNA selected from the miRNAs shown in (SEQ ID NOs: 29, 40, 56 to 75).
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. Sequence No. 1 to Sequence No. 25 and Sequence No. Sequence N contained in a sample derived from the subject's urine
A method for screening a tuberculosis-infected sample, comprising: 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 a second step of qualitatively and/or quantitatively detecting 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.