WO2021261373A1 - Procédé de dépistage d'une infection tuberculeuse dans un échantillon, et biomarqueur et jeu de sondes utilisés dans ce dernier - Google Patents

Procédé de dépistage d'une infection tuberculeuse dans un échantillon, et biomarqueur et jeu de sondes utilisés dans ce dernier Download PDF

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WO2021261373A1
WO2021261373A1 PCT/JP2021/023027 JP2021023027W WO2021261373A1 WO 2021261373 A1 WO2021261373 A1 WO 2021261373A1 JP 2021023027 W JP2021023027 W JP 2021023027W WO 2021261373 A1 WO2021261373 A1 WO 2021261373A1
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mirna
sequence
sample
tuberculosis
seq
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靖士 神田
利正 西山
敬紀 下埜
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学校法人関西医科大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention provides information regarding the use of miRNA whose expression frequency differs depending on the presence or absence of tuberculosis infection as a biomarker, specifically, for supporting active tuberculosis diagnosis, in which urine can be used as a sample sample.
  • the biomarker to be used a test / screening method using the biomarker, and a probe set for test.
  • a test method for measuring interferon gamma (INF- ⁇ ) produced from lymphocytes by antigen stimulation with ESAT-6, CFP-10, etc. (Quantiferon (Quantiferon (Quantiferon) (Registered trademark) TB, T-spot (registered trademark) TB) are generally used.
  • blood is collected from the subject, lymphocytes are separated, a certain amount is dispensed into a culture plate coated with anti-human interferon gamma antibody, and then M. tuberculosis-specific antigens ESAT-6 and CFP-10 are added.
  • the number of INF- ⁇ -producing cells bound to the anti-human INF- ⁇ antibody is measured. Since it is not affected by BCG inoculation or nontuberculous mycobacteria, it is characterized by high specificity compared to the tuberculin reaction, but it takes about 2 days to determine because it requires culture. In addition, since a device for cell separation and cell culture is required, the cost per sample is high, and it is difficult to disseminate it in developing countries.
  • INF- ⁇ is a tuberculosis-infected patient due to immunodeficiency in patients who are infected with HIV (human immunodeficiency virus) as well as tuberculosis bacteria (coinfected persons with HIV infection and tuberculosis infection).
  • HIV human immunodeficiency virus
  • tuberculosis bacteria coinfected persons with HIV infection and tuberculosis infection.
  • MPB64 antibody is known as an antibody specific to active tuberculosis patients, and the state of active tuberculosis can be detected by detecting the amount of MPB64 antibody in plasma using such antibody as a marker, for example, by dot blotting. A method of determining whether or not the antibody is present is also known. It has been reported that the MPB64 antibody is also present in urine and correlates with the result of using serum as a measurement sample (Non-Patent Document 1: Yasuko Tamada et.al., Microbiol immunol 2012; 56: 740). -747).
  • urine that can be easily collected can be used as a measurement sample, which is suitable as a method that can quickly determine the presence or absence of infection in developing countries where tuberculosis infection is likely to spread. Is.
  • the existence of antibodies that show cross-reactivity is also known, and in the case of BCG vaccinated persons, test results such as false positives may appear.
  • test by sputum culture collected from the subject is highly accurate because it determines the presence or absence of tubercle bacillus, but it requires a rapid test / diagnosis method because it requires equipment for culture and a culture period of 1 to 4 weeks. It is difficult to use as.
  • miRNA As a biomarker separately from antibodies, cells, cytokines and the like. Abnormal expression of miRNA has been clarified to be involved in the onset and malignant transformation of many diseases, and since miRNA can be detected in body fluids such as blood, its use as a liquid biopsy is attracting attention. .. In recent years, various uses of miRNA as a diagnostic marker have been reported mainly for cancer (Patent Document 1: Japanese Patent Application Laid-Open No. 2010-534480, Patent Document 2: WO2015 / 133477A (pancreatic cancer), Patent Document 3). : Japanese Patent Publication No. 2018-505656 (prostate cancer), etc.).
  • Non-Patent Document 2 shows human peripheral blood. , Whole blood, serum, or macrophages as specimens, and miRNAs that can be candidates for biomarkers have been introduced. However, regarding the miRNAs shown in Table 1, even if attention is paid to human peripheral blood and serum, they are different according to the references, and which miRNA has a certain degree of specificity as a diagnostic marker. It has not been clarified whether it is or how to judge it.
  • blood as a sample sample is excellent as a liquid biopsy
  • blood sampling is required for testing, and the testing method in developing countries is limited to patients with a strong suspicion of tuberculosis infection. It has limited application as a test method for broadly screening patients with tuberculosis infection.
  • the present invention uses a less invasive liquid biopsy, particularly a biomarker that can provide useful information about the presence or absence of infection in a short time using urine, which is easy to collect a sample, and a tuberculosis infection using the marker. It is an object of the present invention to provide a method for determining the presence or absence, a screening method, and a probe set for screening.
  • the present inventors comprehensively detected and analyzed miRNA contained in urine collected from patients with active tuberculosis and healthy subjects, and searched for miRNA specifically over / decreased depending on the presence or absence of tuberculosis infection. Based on the results, the present invention was completed.
  • the method for screening a tuberculosis-infected sample of the present invention is a miRNA (miRNA group consisting of sequence No: 1 to sequence No: 25, or sequences No: 49 to 55 and sequence 3, sequence 3 contained in a sample derived from the urine of a subject.
  • a miRNA miRNA group consisting of sequence No: 1 to sequence No: 25, or sequences No: 49 to 55 and sequence 3, sequence 3 contained in a sample derived from the urine of a subject.
  • At least two types of miRNA selected above include at least one type of miRNA selected from the group consisting of sequence Nos. 3, 6, 9, 10, 13, and 14.
  • the detection step is preferably a step of separating into positive samples when the expression frequency in the sample is higher than a predetermined value. Further, the detection step is a step of separating into positive samples when the content ratio of the detected miRNA to the internal control miRNA (sequence No: 46 to sequence No: 48) is higher than a predetermined value. It is preferable to have.
  • At least one miRNA selected from miRNA (a miRNA group consisting of sequences No: 26 to No: 45, or a miRNA group consisting of sequences No: 56 to 75 and sequences No: 29, 40), or the miRNA thereof. It may include a second step of qualitatively and / or quantitatively detecting miRNAs having 70% or more sequence homology. In this case, it is preferable to contain the miRNA of either sequence No: 29 or sequence No: 40.
  • the screening method of the present invention is selected from the group consisting of sequence No: 1 to sequence No: 25 and sequence No: sequence No: 49 to sequence No: 55 contained in the sample derived from the urine of the subject.
  • the test probe set of the present invention is a test probe set for separating a positive sample of active tuberculosis from a sample derived from the urine of a subject. Hybridizes with any one of the miRNAs (SEQ ID NO: 1 to SEQ ID NO: 25 and Sequence No: 49 to SEQ ID NO: 55) shown in Table 1-1 or Table 1-2 under stringent conditions.
  • This is a test probe set containing two or more kinds of oligonucleotide probes A capable of soaking.
  • any one of the miRNAs (SEQ ID NO: 26 to Sequence No: 45 and Sequence No: 56 to SEQ ID NO: 75) shown in Table 2-1 or Table 2-2. It may be a probe set containing an oligonucleotide probe D capable of hybridizing.
  • the sample derived from the urine of the subject can be used not only as a test probe set for separating positive samples but also as a probe set for separating positive sample groups and negative sample groups of active tuberculosis. ..
  • the probe set of the present invention may be a combination of one belonging to the nucleotide probe A and one belonging to the nucleotide probe D from another viewpoint.
  • the test probe set of the present invention further serves as a probe (oligonucleotide probe for internal control) for detecting miRNA for internal control (sequence No: 46 to sequence No. 48), and the sequence No: 46 to sequence No .: It may contain an oligonucleotide probe capable of hybridizing with any one of the species shown in 48 under stringent conditions.
  • the test probe set of the present invention is a combination of the above-mentioned oligonucleotide probe A and the oligonucleotide probe for internal control; or a combination of oligonucleotide probe A, oligonucleotide probe D, and oligonucleotide probe for internal control.
  • tuberculosis refers to an infectious disease of Mycobacterium tuberculosis.
  • symptoms such as coughing and weight loss already appearing.
  • clinical findings such as symptoms, chest X-ray findings, and bacterial test findings, the determination of the presence or absence of latent tuberculosis infected with Mycobacterium tuberculosis is not included.
  • miRNA refers to a miRNA specified by a sequence ID (sequence No.). Unless otherwise specified, it corresponds to mature miRNA, which is a non-coding RNA of about 16 to 25 bases, as well as early transcripts in which the miRNA gene is transcribed by RNA polymerase II, and has a hairpin loop structure. This is a concept that includes pre-miRNA, which is a precursor of miRNA, in a state where this pre-mRNA is partially cleaved by RNase III-like Drosha.
  • oligonucleotide probe capable of hybridizing under stringent conditions refers to a nucleotide having a complementary sequence to each sequence of the target miRNA, or 70% or more, preferably 80% or more of the complementary sequence. % Or more, more preferably 90% or more of the nucleotides having a matching sequence, still more preferably 95% or more, particularly preferably 99% or more of the nucleotides having a matching sequence.
  • the screening method for tuberculosis-infected samples of the present invention uses miRNA as a biomarker, it is not necessary to perform cell culture or the like when preparing a sample for executing the method, and rapid determination is possible. Further, since urine that does not need to be collected by an expert is used as the sample sample for measurement, it is useful as a test method that requires a large amount of sample to be tested quickly and inexpensively.
  • the miRNAs that can be used as biomarkers in the method for screening tuberculosis-infected samples of the present invention include miRNAs having the sequences shown in Table 1-1, Table 1-2 and Table 2-1 and Table 2-2, and the miRNAs.
  • the miRNAs listed in Table 1-1 and Table 1-2 (sequence No: 1 to sequence No: 25, sequence No: 49 to sequence No: 55, and the group consisting of these is collectively referred to as "Group A".
  • the frequency of expression in specimens from tuberculosis-infected individuals is higher than that in healthy individuals.
  • miRNA belonging to the A1 group (sequence No: 1 to sequence No: 11) has a significance level of p ⁇ 0.05 in Welch's t-test (less than 0.05, there is a significant difference).
  • the significance level is p ⁇ 0.1 (less than 0.1, there is a significant difference).
  • miRNAs belonging to the Af group have p ⁇ 0.05 and p ⁇ 0.05 in Fisher's accurate establishment test.
  • Q value (FDR) ⁇ 0.3 it can be said that the frequency of expression in specimens from M. tuberculosis-infected individuals is higher than that in healthy subjects. Therefore, as a biomarker for screening a tuberculosis-positive sample, miRNA selected from the A1 group or the Af group is more preferable.
  • Hsa-miR-196a-5p, hsa-miR-500a-3p, hsa-miR-199a-5p from the viewpoint that the frequency of occurrence is high regardless of the presence or absence of HIV coinfection and the difference from healthy subjects is large.
  • Hsa-miR-424-5p are preferably used, and more preferably hsa-miR-196a-5p and hsa-miR-500a-3p.
  • the miRNAs listed in Table 2-1 and Table 2-2 (SEQ ID NO: 26 to SEQ ID NO: 45, SEQ ID NO: 56 to SEQ ID NO: 75, and the group consisting of these may be collectively referred to as "D group". ) Is a miRNA whose expression frequency tends to decrease due to tuberculosis infection as compared with the expression frequency of healthy subjects.
  • the term "healthy person” as used herein refers to a sample derived from a person who is negative for tuberculosis infection in which tubercle bacillus is not detected in sputum and who can be determined to be AIDS-negative based on the proportion of CD4-positive T cells.
  • the miRNA belonging to the D1 group (sequence No: 26 to sequence No: 31) has a significance level of p ⁇ 0.05 in Welch's t-test (significant below 0.05). (There is a difference), and in the miRNA of the D2 group (sequence No: 32 to sequence No: 45), the significance level is p ⁇ 0.1 (less than 0.1, there is a significant difference).
  • biomarkers belonging to the D group hsa-miR-107, hsa-miR-6887-5p, hsa-miR-196b-5p, hsa-miR-451a, hsa-miR-132-3p belonging to D1 are used. It is preferably used, and more preferably hsa-miR-6887-5p.
  • the miRNAs belonging to the D group have p ⁇ 0.01 and Q in Fisher's accurate establishment test.
  • the sequence No: 29 (hsa-miR-451a) and the sequence No: 40 (hsa-miR-769-5p) belong to both the D1 group or the D2 group and the Df group. Therefore, when using the D group biomarker, at least one of SEQ ID NO: 29 (hsa-miR-451a) or SEQ ID NO: 40 (hsa-miR-769-5p), more preferably SEQ ID NO:: Includes 29 (hsa-miR-451a).
  • the above miRNA can be used as a marker for determining tuberculosis infection, but two or more types are used in combination.
  • two or more types of miRNA of group A which is a biomarker for determining positive for tuberculosis, in combination, it is possible to suppress an error due to variation in sample samples.
  • miRNAs selected from each of the A group and the D group may be used in combination.
  • two groups in which the increase / decrease in the frequency of occurrence is reversed due to tuberculosis infection more reliable determination results (tuberculosis positive or tuberculosis negative) can be obtained. Further, even in the case of HIV infection, the risk of false positives and false negatives can be reduced.
  • two or more species may be selected from the miRNAs identified from the results analyzed by a common assay method, or a combination of two or more species selected from different groups may be selected. good. Regardless of whether the miRNA group shown in Table 1-1 or Table 1-2 is adopted, it is preferable that at least one type of miRNA of sequence No. 3, 6, 9, 10, 13, 14, 16 is contained.
  • the test probe set of the present invention is a test probe set for separating a positive sample of active tuberculosis from a sample derived from the urine of a subject, and is a set in which a probe for detecting the above biomarker is combined. be. Specifically, it includes two or more of the following inspection probes.
  • Oligonucleotide probe for internal control that can hybridize with at least one of the miRNAs (SEQ ID NO: 46 to Sequence No: 48) shown in Table 3 under stringent conditions.
  • the oligonucleotide probe that can hybridize under stringent conditions is a nucleotide having a complementary sequence to each sequence of the target miRNA, or 70% or more, preferably 80% or more, more than the complementary sequence. It is preferably a nucleotide having 90% or more matching sequences, more preferably 95% or more, and particularly preferably 99% or more matching sequences.
  • the mode of combination may be any mode containing at least oligonucleotide probe A.
  • a combination of two or more kinds of oligonucleotide probe A; a combination of one kind of oligonucleotide probe A and one kind of oligonucleotide probe D; two or more kinds of oligonucleotide probe A and one kind of oligonucleotide probe D The above combination; a combination of two or more kinds of oligonucleotide probe A and an oligonucleotide probe for internal control; one kind of oligonucleotide probe A, one kind of oligonucleotide probe D, and a combination of an oligonucleotide probe for internal control; an oligonucleotide.
  • Examples thereof include two or more types of probe A, one or more types of oligonucleotide probe D, and a combination of oligonucleotide probes for internal control.
  • oligonucleotide probe A it is preferable that at least one type contains a miRNA selected from the group consisting of sequence Nos. 3, 6, 9, 10, 13, 14, and 16.
  • Judgment accuracy can be improved by combining multiple types of oligonucleotide probes.
  • probe D selected from the group consisting of b
  • the influence of HIV infection can be reduced by the internal control, there is also an effect that the risk of false positives and false negatives can be reduced. That is, the amount of miRNA contained in the sample sample differs depending on the individual sample. In particular, when miRNA having a low expression frequency is used as a biomarker, it may be erroneously determined as negative or positive because the detected miRNA content is low or the total amount of miRNA may not be measured correctly. In this case, individual differences in the sample can be suppressed by adopting the ratio with the internal control.
  • the oligonucleotide probe for internal control is appropriately selected so as to be a combination in which the influence of HIV and the difference from a healthy person are remarkable, depending on the type of oligonucleotide probe A or D to be combined.
  • hsa-miR-196a is used as a biomarker
  • hsa-miR-423 is used as an internal control
  • the difference between tuberculosis positive and negative becomes remarkable, and the determination accuracy can be improved.
  • hsa-miR-107 is used as the D group biomarker
  • the effect of HIV on the HIV-infected sample can be reduced by combining hsa-miR-21 as the internal control. It is possible to reduce the risk of false positives and false negatives due to infection.
  • nucleotide probes preferably have a label that can be detected when hybridized with the target miRNA.
  • a known label preferably used for the nucleotide probe, for example, a fluorescent dye, a gold colloid, or the like can be used.
  • the label not only the qualitative detection of the target marker but also the quantification can be applied by a known method.
  • the sample to be tested is urine collected from the subject.
  • urine By using urine as a test sample among human body fluids, it is not necessary to rely on a specific technician for collecting a sample sample such as blood sampling, and the screening method of the present invention is for comprehensively inspecting a large number of samples. It can be applied to a simple screening method.
  • Saliva is also a sample that can be collected without depending on a specific technician, but it may not be easy to collect in a dry mouth subject such as an elderly person. In this respect, urine is excellent as a sample sample that can be easily collected from all subjects.
  • Urine tends to contain less miRNA-containing exosomes than blood, but it can be collected in large quantities compared to other body fluids, and it does not require a blood collection device such as a syringe. Since there is no risk of blood-borne viral infections due to needlestick accidents, it is preferable to use urine as a sample as a test method to be carried out in developing countries.
  • Urine contains exosomes containing mature miRNAs that serve as biomarkers, similar to other body fluids such as blood. Exosomes are one of the membrane vesicles secreted from cells, generally 30-150 nm in diameter as observed by electron microscopy, and in addition to mature miRNAs, pri-miRNAs, pre-miRNAs, coding. -Contains mRNA, DNA, enzymes, cytoskeletal proteins, signal molecules, etc.
  • the miRNA as a biomarker used in the test / screening method of the present invention was discovered as a result of searching for miRNA specifically over- or decreased in urinary infected / non-infected persons. Therefore, even if urine is used as a sample, its usefulness is not impaired.
  • the number of types of miRNA detected to a quantifiable degree is a predetermined number or more, although it depends on the method for preparing a sample for measurement. Is desired. 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 in a quantifiable manner, and this requirement can be satisfied even with urine.
  • urine-derived samples include fractions in which exosomes are concentrated or crudely purified (hereinafter referred to as "exosome-rich fractions"), and samples from which RNA has been extracted. Of these, when detecting miRNA as a marker, it is preferable to use a sample from which RNA is directly extracted as a measurement sample.
  • RNA isolation As a method for preparing a measurement sample (RNA isolate) from the urine of a subject, a conventionally known method can be used. For example, exosomes may be separated from body fluids by size exclusion chromatography, centrifugation, etc., and RNA may be extracted from the resulting exosome-rich fraction, or exosomes may be separated using a commercially available kit. Alternatively, RNA isolation may be performed directly.
  • ExoQuick TM Exosome precipitation solution series for example, ExoQuick-TC, ExoQuick-CG, etc., all manufactured by System Biosciences
  • nucleic acid extraction samples are obtained from body fluids.
  • miRNeasy mini kit manufactured by Qiagen
  • MagMax mirVana Total RNAkit manufactured by Thermo Fisher
  • Magtration registered trademark
  • magnetic particles manufactured by Prescription System Science
  • Examples of the method for detecting the target miRNA from the RNA-containing sample prepared as described above include RT-PCR method, microarray method, Northern blotting method, next-generation sequencer method, liquid phase nucleic acid hybridization and the like.
  • the RT-PCR method, the microarray method, the next-generation sequencer method, or the like is adopted.
  • Examples of the method for detection and quantification using hybridization include a microarray method, a Northern blotting method, and a liquid phase nucleic acid hybridization.
  • a microarray method By utilizing hybridization with a specific miRNA, miRNA as a biomarker may be detected or quantified.
  • the method using hybridization is preferable as a simple test method because it can directly detect miRNA that can hybridize with the probe. By using the probe set of the present invention, it is possible to quickly detect / quantify a specific miRNA at a relatively low cost.
  • cDNA is synthesized using the extracted RNA as a template, for example, using the TaqMan TM miRNA RT Kit (manufactured by Applied Biosystems), and the target miRNA is detected by real-time quantitative PCR for such cDNA. Can be quantified.
  • RNA contained in the prepared measurement sample for RNA can be comprehensively detected and quantified using the next-generation sequential system.
  • the abundance ratio of miRNA used as a marker is adopted as a judgment index, it is necessary to measure the total amount of miRNA contained in the sample.
  • a next-generation sequencer capable of comprehensively detecting and analyzing miRNA is used. It is preferable to use it.
  • a cDNA library (clusters of DNA) is prepared by performing a reverse transcription reaction on all RNA, and individual clusters of the prepared DNA library are sequenced.
  • the sequencing method may be sequencing-by-synthesis (nucleotides are reversibly terminated and one nucleotide is taken up and analyzed per cycle) or pyrosequencing (pyrophosphate release during nucleic acid uptake). (Sequencing reaction is monitored via), ligation (sequencing while ligating to the primer sequence using a short-chain oligonucleotide probe that is linked to each other), ion semiconductor sequencing (hydrogen in the sequencing reaction). Cluster sequence detection using ion emission) may be performed.
  • the synthesis of cDNA can be performed by a conventionally known method, and may be performed using a commercially available kit. For example, it should be performed using Ion Total RNA-seq kit v2 for small RNA libraries (Thermo Fisher). Can be done.
  • a system for multiplex miRNA analysis from body fluids without the RNA purification step may be utilized.
  • Abcam's Firefly TM technology is a system for simultaneously profiling miRNAs using a flow cytometer, which can be analyzed using a Multiplex Circulating miRNA assay using body fluids such as plasma and urine as samples. ..
  • the sequenced miRNA can be identified by collation with the reference sequence, and the relative abundance can be quantified according to the number of reads of the identified miRNA.
  • the total amount of identified miRNA can be 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 adopted as the marker with respect to the total amount of the miRNA.
  • sequence data registered in the miRNA registered data bank for example, miRBase (http://www.mirbase.org) can be used.
  • miRBase http://www.mirbase.org
  • collation and identification with the reference sequence not only perfect matching but also matching of 10 or more consecutive bases may be used for identification. Since the miRNA used as a marker is 16 to 25 bp, the match of 10 or more bases has a sequence homology of 70% or more, and the homology when 10 or more consecutive bases match is further higher.
  • the final mature miRNA obtained has only one to several sequences different. It may be identified as a common mature miRNA.
  • the measurement sample contains pri-miRNA and pre-miRNA, several base mismatches occur depending on the read site, but the final mature miRNA has a common sequence. Therefore, it can be identified and quantified as the same type of miRNA.
  • a method may be used in which a marker is detected by coloration by combining a PCR method and an immunochromatography method.
  • microarray method for example, 3D-Gene (registered trademark) is used to specifically extract miRNA from body fluids, and a high-sensitivity DNA chip (microarray) is used to detect more than 800 types of miRNA at one time. Is possible.
  • the method for detecting the presence or absence of hybridization is appropriately selected according to the type of label of the probe used. For example, when a fluorescent label is used, it can be detected by visually observing the presence or absence of fluorescence emitted by hybrid formation. Further, it is possible to calculate the content and the content ratio by scaling the fluorescence intensity and the like.
  • the screening method of the present invention is a method of classifying a sample as a sample into a positive possibility group and a negative group of active tuberculosis, and a large number of samples are classified into a positive sample group and a negative sample group as in a medical examination. It can be adopted as a screening method for sorting and a simple test method as the initial diagnosis of a patient suspected of having tuberculosis.
  • the screening method of the present invention is shown in a biomarker contained in a sample derived from the urine of a subject, that is, miRNA (SEQ ID NO: 1 to Sequence No: 25) shown in Table 1-1 or Table 1-2. At least two miRNAs selected from the miRNAs shown in the miRNAs (SEQ ID NO: 3, 6, 9, 10, 13, 14 and Sequence No: 49 to Sequence No: 55), or 70% with the miRNA.
  • the first step of qualitatively and / or quantitatively detecting miRNA having the above sequence homology is included.
  • the miRNA to be detected is preferably a combination containing one selected from sequence Nos .: 3,6,9,10,13,14.
  • the screening method of the present invention is further selected from at least the D group miRNAs (SEQ ID NO: 26 to SEQ ID NO: 45, SEQ ID NO: 56 to SEQ ID NO: 75) shown in Table 2-1 or Table 2-2. It is preferable to include a second step of qualitatively and / or quantitatively detecting one type of miRNA, or a miRNA having 70% or more sequence homology with the miRNA.
  • the miRNA detected in the second step is preferably sequences No: 26 to No: 31 belonging to the D1 group, one selected or a combination of two or more, or a combination of one or two or more selected from the Df group. Is. In this case, it is preferable to include miRNA of sequence No: 29 or sequence No: 40.
  • the first step and the second step may be only one of the steps, but preferably both the first step and the second step are performed.
  • the determination accuracy for determining that the probability of tuberculosis positivity is high is enhanced.
  • the expression frequency of miRNA in the first step is low and the expression frequency of the D group miRNA in the second step is high, it can be determined that there is a high possibility that the patient is tuberculosis negative.
  • the order of the first step and the second step is not particularly limited, and the second step may be performed after the first step or the second step. After performing the steps, the first step may be performed, or the first step and the second step may be performed at the same time.
  • the miRNA detected in the first step is one of the miRNAs represented by the sequences No: 1 to No. 25 and the sequences No: 49 to No. 55. You may just detect the species.
  • the miRNA for internal control SEQ ID NO: 46 to Sequence No: 48
  • the miRNA for internal control shown in Table 3 is also detected.
  • the ratio to the internal control as a determination index, it is possible to reduce the risk of false positives and false negatives of HIV-infected persons.
  • the frequency of expression is high (eg, hsa-miR-196a-5p, hsa-miR-500a-3p) or low (eg, hsa-miR-16-5p) depending on the type of biomarker. , Hsa-miR-6887-5p).
  • the internal control ratio of miRNA in Group D is that in HIV-positive samples, the expression frequency is significantly lower than the expression frequency of internal control. Therefore, those with a low internal control ratio can be infected with HIV. It should be sorted as having high sex. In addition to the high frequency of occurrence in the first step, it can be considered that there is a high possibility that the infection is tuberculosis / HIV co-infection or the result in the first step is a false positive due to the HIV infection.
  • the separation of a positive sample and a negative sample in the first step of the screening method of the present invention is a step of separating a positive sample into a positive sample when the expression frequency of the detected miRNA in the sample is higher than a predetermined value. Has the following determination methods.
  • the first determination method is a case where the content ratio (or content) of miRNA used as a biomarker is used as a determination index, and when it is higher than a preset threshold value, it is classified into a positive sample group. How to do it.
  • the ratio of the number of read miRNAs to be tested (biomarkers) to the total number of reads when read by the next-generation sequencer is miRNA containing a detectable amount in the sample (usually 200 to 500 types). ) Corresponds to the content of the miRNA to be tested with respect to the total content.
  • the type of total miRNA contained in the measurement sample or a miRNA close to it and the content of each are measured, and the total content (total number of reads) is calculated.
  • the expression frequency (presence ratio) of the miRNA used as a marker may be calculated and compared with the threshold value.
  • the threshold value to be set is the average frequency of expression of the target marker in a healthy person, and is appropriately set depending on the type of biomarker used. When it can be distinguished by p ⁇ 0.1, preferably p ⁇ 0.05, and more preferably p ⁇ 0.01 as the significance level as compared with the threshold value, it is determined to be positive.
  • the expression frequency of healthy subjects can be used as a preset threshold value
  • the content may be compared instead of the expression frequency of the target marker miRNA.
  • a threshold value a content of about 1.5 times, preferably 2 times the average value of the corresponding miRNA expression levels of healthy subjects is set, and when the calculated content is equal to or higher than the threshold value, a positive sample is used. You just have to sort it.
  • the second determination method uses the ratio of the biomarker miRNA to be detected to the miRNA expression level used as the internal control as a determination index, and determines that the ratio is positive when the ratio is higher than the threshold value. do. Since the ratio can be calculated by quantifying the miRNA as an internal control and a marker, screening using the probe set of the present invention becomes possible. In order to comprehensively detect and quantify miRNA contained in a measurement sample, a special device such as a next-generation sequencer is required, and the inspection time is long, so that the cost per sample is high. In this respect, the screening method using the probe set of the present invention is suitable as a method for inspecting a large number of samples in a short time. Therefore, it can be applied to a test method that can be widely performed in developing countries and a medical examination that requires a rapid diagnosis of a large number of subjects.
  • the influence of HIV infection can be reduced by selecting the internal control, so that there is also an effect of reducing the risk of false positives and false negatives.
  • Separation of a positive sample and a negative sample in the second step is a step of separating into a positive sample when the expression frequency of the detected miRNA in the sample is lower than a predetermined value, and the determination index is the first step.
  • the determination index used in the first and second determination methods can be used. That is, in the first determination method, it is determined to be positive when the expression frequency of the miRNA detected as a target is lower than the threshold value (the average expression frequency of the same miRNA in a healthy person). Further, in the second determination method, the miRNA detected as a target is positive when the ratio of the miRNA for internal control to the expression frequency is lower than the threshold value (magnification of the same miRNA for healthy subjects to the miRNA for internal control). judge.
  • RNA sample Urine (3 ml) was collected from 3 healthy subjects and 10 active tuberculosis-infected patients (4 of whom were co-infected with HIV infection). From the urine sample after cryopreservation, a total RNA extraction sample was obtained according to the protocol of the operation manual (urine sample) of MagMax TM mirVana Total RNA-Seq kit (Thermo Fisher). The specific procedure was as follows.
  • Binding Beads Mix a mixed solution of 234 ⁇ l of RNA Binding Beads and 116 ⁇ l of Lysis / Binding Enhancer
  • the obtained solution 5.76 ml of isopropanol was added, pipetting was performed, and then the mixture was incubated for 20 minutes (200 rpm).
  • RNA Binding Beads After standing on a magnet stand and separating, the supernatant is rinsed, and the RNA is rebound to RNA Binding Beads by adding a washing solution, washing by shaking, and then adding a Turbo DNase TM solution, and the rebound RNA is re-magnetized. The separation operation was performed by. After repeated separation and washing, elution buffer was added to the dried RNA Binding Beads and incubated at 65 ° C. for 5 minutes. It was allowed to stand on a magnet stand, and the obtained supernatant was used as a measurement sample.
  • RNA extraction sample obtained above, attach an adapter according to the operation manual of Ion Total RNA-Seq kit v2 for small RNA Libraries (Thermo Fisher), perform reverse transcription reaction, and use it for the next-generation sequencer.
  • a cDNA library was created.
  • the prepared cDNA library was analyzed by the next-generation sequencer Ion Torrent PGM (Thermo Fisher).
  • the obtained sequence is comprehensively collated with the miRNA registered in a known miRNA (data bank site: miRBase) as a reference sequence, and the number of reads of each miRNA is calculated to quantify the miRNA in the sample. The ratio of total miRNA to the number of reads was calculated. Collation / identification with the reference sequence identified the miRNA corresponding to the reference sequence when 10 or more consecutive bases matched.
  • tuberculosis infection and HIV infection of specimens The presence or absence of tuberculosis infection in the 13 subjects subjected to the above sequence analysis was confirmed by a culture test of Mycobacterium tuberculosis contained in the sputum of each subject and analysis by the GeneXpert (registered trademark) system. The presence or absence of M. tuberculosis by the GeneXpert® system was confirmed by using a probe targeting the drug-resistant region of the rpoB gene of M. tuberculosis after amplifying the RNA contained in sputum by PCR. Regarding the presence or absence of HIV infection, the presence or absence of HIV infection was examined by measuring the proportion of CD4 positive T cells using FACS Presto (registered trademark) manufactured by BD bioscience.
  • miRNAs that are highly expressed in healthy subjects but whose expression frequency is low due to tuberculosis infection are classified into the D group, and those with a statistically significant level (P value less than 0.05) are classified into the D1 group and P value 0. Those less than 1 were designated as D2 group.
  • sequence No: 26 to sequence No: 31 were selected as the miRNA belonging to the D1 group
  • sequence No: 32 to sequence No: 45 were selected as the miRNA belonging to the D2 group.
  • FIGS. 1 to 8 The results (plot diagram) of the number of reads, expression frequency, and internal control ratio of miRNA belonging to group A are shown in FIGS. 1 to 8. Furthermore, Table 5 shows the P value of miRNA belonging to the A1 group and the expression level ratio for healthy subjects. In addition, plots of the results of miRNA-196a-5p, miRNA-424-5p, miRNA-500a-3p, and miRNA-199a-5p excluding HIV-infected patients are shown in FIGS. 9 and 10.
  • FIGS. 11 to 18 show the results (plot diagram) of the number of reads, the frequency of expression, and the internal control ratio of miRNAs (sequence No .: 26-45) classified into the D group. Furthermore, Table 6 shows the P value of miRNA belonging to the D1 group and the expression ratio for healthy subjects.
  • (TB-) is the result of a sample of a healthy subject
  • (TB +) is a result of a sample of a tuberculosis positive patient.
  • the results of HIV-infected patients are plotted as white squares ( ⁇ ).
  • the vertical axis of the leftmost plot shows the number of reads
  • the vertical axis of the second plot from the left shows the frequency of occurrence (%).
  • the third, fourth, and fifth plots from the left end are, in each order, hsa-miR-30c (SEQ ID NO: 46), hsa-miR-423-5p (SEQ ID NO: 47), hsa- as internal controls.
  • the magnification (times) of the expression frequency with respect to miR-21 is shown.
  • the expression level of miRNA belonging to the A1 group was higher in that of tuberculosis patients than in healthy subjects, and the same tendency was observed in the expression frequency.
  • miRNAs belonging to the A1 group tended to have higher expression levels and frequency of expression, but by adopting the ratio with internal control, patients without HIV coinfection (infection of tuberculosis alone). Even in the case of patients), it can be seen that the frequency of occurrence is higher than that of healthy subjects.
  • the expression level of these miRNAs tended to be high, but it can be seen that the influence of HIV infection can be reduced by adopting the ratio with the internal control (for example, in the case of miR196a-5p, it is internal. By using the control and ratio as indicators, it can be seen that the frequency of occurrence is significantly higher in patients with tuberculosis alone).
  • miR196a-5p, miR424-5p, miR500a-3p, and miR199a-5p have high expression levels in the case of tuberculosis infection regardless of the presence or absence of HIV infection. Become. Therefore, it can be seen that by using these as biomarkers and further by using the ratio with the internal control suitable for each as an index, a positive patient sample of tuberculosis alone can be separated.
  • the expression frequency of miRNAs belonging to the D group is low, especially in the case of HIV coinfection. Furthermore, using the ratio with internal control as an index, it can be seen that it is significantly lower than that of healthy subjects.
  • miR1290 SEQ ID NO: 31
  • the following results were obtained by statistical processing by Mann-Whitney, and it was confirmed that the biomarker was significantly different as the D1 group. P value: 0.007 Expression ratio for healthy subjects: 0.56
  • P-value 0.034 Expression rate for healthy subjects: 0.46
  • tuberculosis-negative samples can be sorted and screened by detecting both groups A and D.
  • RNA sample 2 The above-mentioned MagMax TM mirVana Total RNA-Seq kit (MagMax TM mirVana Total RNA-Seq kit) was collected from a total of 10 newly obtained Laotian active tuberculosis patients (one of whom was an AIDS duplication patient) and 6 healthy subjects. Total RNA extraction samples were obtained according to the protocol of the operation manual (urine sample) of Thermo Fisher). The specific procedure is as described above.
  • RNA extraction sample obtained above add an adapter and perform a reverse transcription reaction according to the operation manual of IonTotalRNA-Seqkitv2 for smallRNA Libraries (Thermo Fisher) in the same manner as above.
  • a cDNA library for the next-generation sequencer was prepared, and the sequence of the prepared cDNA library was analyzed by the next-generation sequencer Ion Torrent PGM (Thermo Fisher).
  • the obtained sequence is comprehensively collated with the miRNA registered in a known miRNA (data bank site: miRBase) as a reference sequence, and the number of reads of each miRNA is calculated to quantify the miRNA in the sample. The ratio of total miRNA to the number of reads was calculated. Collation / identification with the reference sequence identified the miRNA corresponding to the reference sequence when 10 or more consecutive bases matched.
  • the frequency of expression in active tuberculosis patients was examined when compared with the frequency of expression in healthy subjects.
  • FIG. 19 shows a scatter plot showing the relationship between the expression frequency ratio of each miRNA and the statistically significant difference.
  • the case where the expression ratio is 2 times or more (M value> 1) and the significance level P value ⁇ 0.05 is indicated by a white triangle ( ⁇ ). Further, the case where the M value is less than -2 and the P value is ⁇ 0.01 is indicated by a white circle ( ⁇ ).
  • the miRNAs plotted with white triangles are miRNAs that are effective as classified into the Af group, and the miRNAs plotted with white circles are miRNAs that are effective as classified into the Df group.
  • Tables 7 and 8 show the M value of each miRNA, the P value obtained from the analysis results, and the Q value of the miRNAs classified into the Af group and the miRNAs classified into the Df group according to FIG.
  • the Q value is the minimum FDR threshold value at which the test result is judged to be significant, and corresponds to the ratio of the expression-variable miRNA obtained by the test that is not actually considered to be the expression-variable miRNA. ..
  • the 13 types of miRNAs selected based on the analysis results of Fisher's exact test, which increased the number of tuberculosis-positive samples and healthy subjects, were compared with those of healthy subjects. Since the expression level is more than doubled and the significance level is p ⁇ 0.05 and Q value (FDR) ⁇ 0.08, it is useful as a miRNA used as a biomarker for tuberculosis positivity. In particular, by identifying two or more types of miRNAs belonging to the Af group, highly accurate determination becomes possible.
  • a urine sample that is minimally invasive to the subject and easy to collect can be used as the sample sample, and there is no need to culture or the like to determine tuberculosis infection, and the test can be performed quickly. Since the judgment result can be obtained, it is useful as a test method for checking the presence or absence of active tuberculosis infection in an unequipped area such as a developing country.

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Abstract

L'invention concerne : un biomarqueur grâce auquel des informations utiles concernant la présence/l'absence d'une infection peuvent être fournies en un court laps de temps à l'aide d'urine, qui est facilement collectée, en tant qu'échantillon ; un procédé de dépistage d'une infection tuberculeuse dans un échantillon à l'aide dudit marqueur ; et un kit de sonde qui peut être utilisé dans ledit dépistage. Dans la présente invention, du micro-ARN contenu dans de l'urine recueillie auprès d'un patient atteint d'une tuberculose active et d'une personne saine est détecté et analysé de manière complète, et sur la base du résultat de la recherche de micro-ARN qui est spécifiquement surexprimé/sous-exprimé en fonction de la présence ou de l'absence d'une infection tuberculeuse, le micro-ARN exprimé à une fréquence élevée par rapport à celui d'une personne saine (SEQ ID NO : 1-25 et 45-55) et le micro-ARN exprimé à une faible fréquence par rapport à celui d'une personne saine (SEQ ID NO : 26-45 et 56-75) sont spécifiés comme biomarqueurs. Des nucléotides qui peuvent s'hybrider avec ces biomarqueurs sont utilisés comme sondes.
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