WO2021153789A1 - Method for detecting intrauterine infection - Google Patents
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- WO2021153789A1 WO2021153789A1 PCT/JP2021/003405 JP2021003405W WO2021153789A1 WO 2021153789 A1 WO2021153789 A1 WO 2021153789A1 JP 2021003405 W JP2021003405 W JP 2021003405W WO 2021153789 A1 WO2021153789 A1 WO 2021153789A1
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- 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
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- 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
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Definitions
- the present invention relates to a method for detecting an intrauterine infection.
- Intrauterine infection is an inflammatory disease that occurs when bacteria in the vagina move into the uterus and amniotic fluid is infected with the bacteria. It is known that intrauterine infection causes abnormalities in the course of pregnancy such as premature rupture of water, miscarriage, and premature birth, and has a long-term effect on the development and development of the fetal or postnatal infant. Diagnosis of intrauterine infection is made by a culture test of amniotic fluid and a histopathological test of the placenta. However, bacteria that cannot be cultured cannot be detected, and even if they can be cultured, their quantification may be lost. In addition, amniotic fluid collection is difficult to carry out easily due to the invasion of the subject. Moreover, since histopathological examination cannot be performed before delivery, it is extremely difficult to accurately diagnose the severity of intrauterine infection before delivery.
- An object of the present invention is to provide a method for detecting an intrauterine infection that can detect an intrauterine infection based on a sample derived from a subject.
- the first aspect is a method for detecting an intrauterine infection, which comprises measuring the expression level of at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p in a sample derived from a subject. be.
- a second aspect is a diagnostic agent for intrauterine infections comprising at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p.
- a third aspect is a kit for detecting intrauterine infections comprising a polynucleotide having a sequence complementary to at least a portion of at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p. Is.
- the present invention it is possible to provide a method for detecting an intrauterine infection that can detect an intrauterine infection based on a sample derived from a subject.
- (A) is a diagram showing the expression level of miR-4535 in amniotic fluid
- (B) is a diagram showing the expression level of miR-1915-5p in amniotic fluid
- (C) is a diagram showing the expression level of 16S-rDNA in amniotic fluid. It is a figure which shows the detected amount of.
- (A) is a diagram showing the expression level of miR-4535 in amniotic fluid
- (B) is a diagram showing the expression level of miR-1915-5p in amniotic fluid
- (C) is a diagram showing the expression level of 16S-rDNA in amniotic fluid. It is a figure which shows the detected amount of.
- (A) is a diagram showing the relationship between the expression level of miR-4535 in amniotic fluid and the detected amount of 16S-rDNA
- (B) is the expression level of miR-1915-5p in amniotic fluid and the detected amount of 16S-rDNA. It is a figure which shows the relationship of.
- (A) is a diagram showing the relationship between the expression level of miR-4535 in amniotic fluid and the white blood cell count in maternal blood
- (B) is the expression level of miR-1915-5p in amniotic fluid and maternal blood. It is a figure which shows the relationship of the white blood cell count.
- (A) is a diagram showing the relationship between the expression level of miR-4535 in amniotic fluid and CRP in maternal blood
- (B) is a diagram showing the expression level of miR-1915-5p in amniotic fluid and CRP in maternal blood. It is a figure which shows the relationship of.
- (A) is a diagram showing the relationship between the expression level of miR-4535 in amniotic fluid and the white blood cell count in fetal blood
- (B) is the expression level of miR-1915-5p in amniotic fluid and fetal blood. It is a figure which shows the relationship of the white blood cell count.
- (A) is a diagram showing the relationship between the expression level of miR-4535 in amniotic fluid and CRP in fetal blood
- (B) is a diagram showing the expression level of miR-1915-5p in amniotic fluid and CRP in fetal blood. It is a figure which shows the relationship of.
- (A) is a diagram showing the relationship between the expression level of miR-4535 in the maternal blood and the malignancy of chorioamnionitis
- (B) is the expression level of miR-1915-5p in the maternal blood. It is a figure which shows the relationship with the malignancy of chorioamnionitis.
- (A) is a diagram showing the relationship between the expression levels of miR-4535 in blood and amniotic fluid, and (B) shows the relationship between the expression levels of miR-1915-5p in blood and amniotic fluid. It is a figure.
- (A) is a diagram showing the relationship between the leukocyte count of a newborn baby and the expression level of miR-4535 in amniotic fluid, and (B) is the relationship between the leukocyte count of a newborn baby and the expression level of miR-1195-5p in amniotic fluid.
- (C) shows the relationship between the leukocyte count of the newborn and the detected amount of 16SrDNA in amniotic fluid
- (D) shows the relationship between the leukocyte count of the newborn and the detected amount of IL-6 in the amniotic fluid.
- Is. It is a figure which shows the relationship between the expression level of miR-4535 in maternal blood, and the malignancy of chorioamnionitis. It is a figure which shows the relationship of the expression level of miR-4535 in maternal blood and amniotic fluid. It is a figure which shows the ROC curve by the expression level of miR-4535 in the blood of a mother's womb. It is a figure which shows the ROC curve by the expression level of miR-4535 in the blood of a mother's womb.
- microRNA may be abbreviated as miRNA.
- embodiments of the present invention will be described in detail. However, the embodiments shown below exemplify a method for detecting an intrauterine infection for embodying the technical idea of the present invention, and the present invention is limited to the method for detecting an intrauterine infection shown below. Not done.
- a method for detecting intrauterine infection is at least one type of microRNA selected from the group consisting of miR-4535 and miR-1915-5p in a sample derived from a subject (hereinafter, these are collectively referred to as “). It includes a first step of measuring the expression level of (also referred to as "specific microRNA").
- the expression level of a specific microRNA contained in a sample derived from a subject is measured, and the presence of intrauterine infection in the subject is detected based on the expression level.
- the specific microRNA is at least one selected from the group consisting of miR-4535 and miR-1915-5p.
- the role of these specific microRNAs in vivo is unknown.
- the expression levels of these specific microRNAs in amniotic fluid were elevated in subjects with intrauterine infection, and that 16S-rDNA was detected in amniotic fluid and in the blood of the subjects or in the blood of the subjects.
- intrauterine infection can be detected by measuring the expression level of a specific microRNA in the blood of a subject.
- the expression level of a specific microRNA which is at least one selected from the group consisting of miR-4535 and miR-1915-5p, is measured in a sample derived from a subject.
- the target in the method for detecting an intrauterine infection may be a pregnant human woman, for example, a pregnant woman in the perinatal period, or a pregnant woman in the perinatal period in which imminent preterm birth is a concern.
- the sample derived from the subject may be a sample derived from the body fluid of the subject, and may be the body fluid itself collected from the subject or a sample obtained by subjecting the collected body fluid to a treatment normally performed.
- the body fluid include blood, lymph, tissue fluid, body cavity fluid such as ascites, digestive fluid such as saliva, runny nose, urine, vaginal fluid, amniotic fluid, and the like, and at least one selected from the group consisting of these. You can.
- the body fluid as a sample may be at least one selected from the group consisting of, for example, blood, urine and amniotic fluid.
- the sample derived from blood may be the blood itself collected from the subject, or the collected blood may be subjected to a treatment such as centrifugation which is usually performed.
- the sample derived from blood may be whole blood, serum or plasma. Further, from the viewpoint of convenience, serum or plasma prepared by a conventional method from blood collected from a subject may be used, and serum may be preferable.
- miR-4535 may be hsa-miR-4535 and may have the nucleotide sequence of SEQ ID NO: 1.
- miR-1955-5p may be hsa-miR-1955-5p and may have the base sequence of SEQ ID NO: 2.
- RNA can be extracted from the sample using a commercially available RNA extraction kit such as miRNAeasy Serum / Plasma Kit (manufactured by Qiagen) or miRCURY TM RNA Isolation Kit (manufactured by EXIQON) according to the procedure in the attached instruction manual. It can be carried out.
- miRNAeasy Serum / Plasma Kit manufactured by Qiagen
- miRCURY TM RNA Isolation Kit manufactured by EXIQON
- the expression level of the specific microRNA can be measured by, for example, quantitative PCR, next-generation sequencer, DNA microarray, enzyme-linked immunosorbent assay (EIA method), or the like.
- the expression level of the specific microRNA to be measured may be an absolute expression level or a relative expression level.
- RNA can be measured.
- the expression level of the specific microRNA measured by quantitative PCR may be, for example, an absolute expression level such as copy number, or a relative expression level in which the copy number is standardized.
- next-generation sequencer When measuring the expression level of a specific microRNA using a next-generation sequencer, for example, cDNA is prepared from the extracted RNA, an amplicon is obtained by PCR, and amplicon sequencing is performed using the next-generation sequencer. .. From the obtained sequencing data, low quality reads and the like are deleted by quality control, and the expression level is measured by the number of reads having a desired base sequence.
- the next-generation sequencer is a term that is contrasted with a capillary sequencer that uses the Sanger method. Next-generation sequencers use sequencing principles such as synthetic sequencing, pyrosequencing, and ligase reaction sequencing.
- next-generation sequencer examples include a MiSeq (registered trademark) system (illmina), a HiSeq (registered trademark) system (illmina), and an IonPGM (registered trademark) system (Life Technology). Amplicon sequencing with a next-generation sequencer can be performed according to the manufacturer's recommended protocol.
- the extracted RNA is fluorescently labeled using a commercially available kit, and a sequence complementary to the specific microRNA arranged on the DNA microarray is obtained.
- the expression level of the specific microRNA can be measured by measuring the fluorescence intensity after hybridizing with the probe to be possessed.
- Measuring the expression level of a specific microRNA includes not only measuring the expression level of a mature microRNA, but also measuring the expression level of a transcript of a gene encoding the specific microRNA or a processing product thereof. May be good.
- the primary transcript of a gene encoding a microRNA is called an early transcript (pri-miRNA) and has a stem-loop hairpin structure.
- the pri-miRNA is converted into a precursor (pre-miRNA) of a mature miRNA having a stem-loop structure by an enzyme called Drosha like RNaseIII.
- the pre-miRNA is transported out of the cell nucleus and spliced with an enzyme called Dicer to become a 20 to 25 base long double-stranded mature miRNA.
- transcripts of genes encoding microRNAs or processing products thereof include tri-miRNAs, pre-miRNAs and mature miRNAs.
- To measure the expression level of double-stranded mature miRNA not only the expression level of single-stranded mature miRNA but also the expression level of single-stranded RNA forming a complementary pair with it should be measured. May be included.
- the primary transcript of the gene encoding hsa-miR-4535 may have, for example, the nucleotide sequence of SEQ ID NO: 3. Further, the RNA forming a complementary pair with hsa-miR-4535 may have, for example, the base sequence of SEQ ID NO: 4.
- the primary transcript of the gene encoding hsa-miR-915-5p may have, for example, the nucleotide sequence of SEQ ID NO: 5. Further, hsa-miR-1915-3p, which forms a complementary pair with hsa-miR-1915-5p, may have, for example, the base sequence of SEQ ID NO: 6.
- microRNA having a biological function equivalent to that of the specific microRNA for example, homologues, homologues such as orthologs, variants such as gene polymorphisms, or derivatives thereof. May include measuring the expression level of.
- homologues, homologues or derivatives of microRNAs can be specifically identified with reference to miRBase (http://www.mirbase.org/).
- the method for detecting an intrauterine infection may include a second step of associating the expression level of the specific microRNA in the sample with the presence of the intrauterine infection.
- the sample may be associated with the presence of an intrauterine infection. That is, the method for detecting intrauterine infection may be a method for diagnosing intrauterine infection, or may be a method for assisting in diagnosing intrauterine infection.
- a method appropriately selected from known methods can be applied to the association in the second step.
- the control healthy pregnant woman here means a pregnant woman whose intrauterine infection is known to be absent in advance. Specifically, for example, a pregnant woman who did not give birth prematurely and did not find any findings of chorioamnionitis, a pregnant woman who did not find any complications, and a severity classification (Blanc classification) in chorioamnionitis was 0 or I. It may be a pregnant woman, a pregnant woman less than 17 weeks pregnant, or the like.
- a high expression level means that the expression of specific microRNA in a sample derived from the subject is higher than the normal detection amount (cutoff value) set to distinguish between a healthy pregnant woman and a subject having an intrauterine infection. The amount is larger.
- the cutoff value for example, ROC analysis of the expression level of specific microRNA in a sample group derived from a subject having an intrauterine infection and the expression level of specific microRNA in a sample group derived from a control healthy pregnant woman, etc. It can be set by attaching to. Further, the cutoff value can be set as, for example, a value obtained by adding a value obtained by multiplying the standard deviation by 2 or 3 to the average value of the expression level of a healthy pregnant woman, and further, sensitivity (detection rate) and specificity (detection rate) and specificity ( A value that satisfies (low false positive rate) in a well-balanced manner can be appropriately set.
- the cutoff value may be set for the expression levels of miR-4535 and miR-1915-5p, respectively, and the presence of intrauterine infection calculated from the expression levels of miR-4535 and miR-1915-5p can be determined. It may be set for the judgment index to be judged.
- the method for detecting intrauterine infection may further include a third step of detecting the 16S ribosomal RNA gene (16S rDNA) contained in a sample derived from the target amniotic fluid.
- the third step may include a step of collecting amniotic fluid from the subject and a step of detecting the 16S ribosomal RNA gene from the sample derived from the collected amniotic fluid.
- the subject from which the amniotic fluid is collected in the third step may be the subject from which the sample associated with the presence of the intrauterine infection was collected in the second step. That is, the target for collecting amniotic fluid may be a pregnant woman who is expected to have an intrauterine infection.
- DNA is extracted from a sample derived from the collected sheep water by a conventional method, and the 16S ribosomal RNA gene is comprehensively amplified by using a universal primer for the 16S ribosomal RNA gene. Can be carried out at.
- the 16S ribosomal RNA gene in amniotic fluid which is considered to be sterile, the progress of amniotic fluid infection can be more accurately diagnosed.
- the third step may include a step of identifying a group of microorganisms detected in amniotic fluid based on the 16S ribosomal RNA gene detected in amniotic fluid. Identification of the microbial community can be performed, for example, by quantitative and comprehensive 16S ribosomal RNA gene analysis using a next-generation sequencer. Specifically, for example, a group of microorganisms contained in amniotic fluid can be identified by referring to the description in JP-A-2017-209063.
- the group of microorganisms identified may be, for example, the following microorganisms related to chorioamnionitis.
- Chorioamnionitis-related microorganisms include, for example, Ureaplasma parvum, Mycoplasma hominis, Gardnerella vaginalis, Lactobacillus jensenii, and Hemophilus. Includes influenzae, Sneathia sanguinegens, Capnocytophaga sprode, Bacteroides fragilis, and Streptococcus agalactiae.
- a subject in which an intrauterine infection is detected may be subjected to measures such as follow-up and intervention depending on the progress of the infection.
- the treatment method for intrauterine infection is at least one type of microRNA selected from the group consisting of miR-4535 and miR-1915-5p contained in a sample derived from a subject (hereinafter, specific microRNA).
- the first step of measuring the expression level of (also referred to as), the second step of associating the expression level of the specific microRNA with the presence of intrauterine infection in the subject, and the fourth step of treating the subject in which the intrauterine infection is present. May include.
- the method for treating an intrauterine infection may further include a third step of detecting the 16S ribosomal RNA gene contained in a sample derived from the amniotic fluid of interest before the fourth step.
- the method for treating intrauterine infection may be a method for preventing neonatal infection or a method for preventing neonatal disease (for example, FIRS).
- the subject-specific treatment may be any treatment performed for the intrauterine infection, and is caused by, for example, treatment, improvement, suppression of progression (prevention of deterioration), prevention, or intrauterine infection of the uterine infection. Relief of symptoms that occur.
- Specific treatments include, for example, administration of antibacterial agents, administration of uterine contraction-suppressing agents, administration of steroids, administration of labor-promoting agents, induction of delivery, cesarean section, hysterotomy, dilation and curettage, hysterectomy. Techniques can be mentioned.
- a diagnostic agent for intrauterine infection comprises at least one specific microRNA selected from the group consisting of miR-4535 and miR-1915-5p.
- the diagnostic agent for intrauterine infection may be applied to a sample derived from the subject to be diagnosed.
- the expression level of at least one specific microRNA contained in the diagnostic agent can be measured from a sample derived from the diagnostic target, and intrauterine infection can be detected based on the expression level. That is, the diagnostic agent may be a diagnostic biomarker for intrauterine infection.
- Intrauterine infection detection kit contains a sequence complementary to at least a part of a specific microRNA, which is at least one selected from the group consisting of miR-4535 and miR-1915-5p. Consists of a polynucleotide having.
- the kit for detecting an intrauterine infection can detect an intrauterine infection in a subject by applying it to a sample derived from the detection target.
- a primer set for PCR capable of amplifying the specific microRNA by the PCR method, and a carrier such as beads that can hybridize with the specific microRNA. Includes immobilized polynucleotides and the like.
- the present invention includes the use of specific microRNAs in the detection of intrauterine infections, the use of specific microRNAs in the treatment of intrauterine infections, and the specific microRNAs used in the detection of intrauterine infections.
- placental pathological examination was performed and divided into two groups: 37 cases in the chorioamnionitis group (Blanc classification stage II or higher) and 12 cases in the non-chorionic amniotic inflammation group (Blanc classification stage I or lower).
- RNA was extracted from the amniotic fluid collected from the subject using miRNAeasy Serum / Plasma Kit (manufactured by Qiagen) according to the attached protocol, and cDNA was prepared from the extracted RNA.
- cDNA was prepared from the extracted RNA.
- the rDNA was quantified. 2 ⁇ L of cDNA was diluted with 18 ⁇ L of Bio-Rad QX200 reagent and divided into approximately 20,000 droplets using the QX200 Droplet Generator.
- PCR was run on a 96-well plate at 95 ° C. for 5 minutes, followed by 40 cycles of 95 ° C. for 30 seconds and 52 ° C. for 1 minute, then at 4 ° C. for 5 minutes, at 90 ° C. for 5 minutes, and then at 2 ° C./sec. The temperature was lowered to 4 ° C. Fluorescence was detected by the QX200 Droplet Reader and analyzed using Bio-Rad QuantaSoft software to calculate the copy number of microRNA per ⁇ L of sample.
- miR-1915-5p (121120_mat) and miR-4535 (464284_mat) were used as primers for microRNA, and the following SEQ ID NOs: 7 and 8 were used as primers for 16S-rDNA.
- the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were significantly increased by intrauterine infection with miCAM.
- the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were strongly correlated with the amount of 16S-rDNA in amniotic fluid.
- the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in maternal blood were significantly increased due to exacerbation of intrauterine infection.
- the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in blood correlated with their expression levels in amniotic fluid.
- intrauterine infection can be detected by measuring the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in blood.
- Fetal inflammatory reaction syndrome was defined as a postpartum neonatal leukocyte count of less than 5000 cells / ⁇ L or 20000 cells / ⁇ L or higher, and 57 pregnant women were classified as normal (non-FIRS) in other cases.
- the expression levels of hsa-miR-4535 and hsa-miR-1915-5p, the detected amount of 16S-rDNA, and the IL-6 concentration were measured in the amniotic fluid of the classified pregnant women, respectively.
- the results are shown in Table 4 and FIG. Table 4 shows the average value of the measured values in each group, the 95% confidence interval, and the P value, and FIG. 10 shows the distribution of the measured values.
- the IL-6 concentration is one of the indexes of FIRS, and was measured by absorbance at 450 nm using ELISA-Kit (Quantikine (R) HS, R & D Systems Minneapolis, USA).
- FIRS and non-FIRS could be distinguished with a significant difference by measuring the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid.
- the cutoff value was set by ROC analysis to evaluate the diagnostic accuracy of FIRS. The results are shown in Table 5.
- the AUC is an area value under the ROC curve, and the larger the value, the better the diagnostic accuracy.
- Intrauterine infection was detected using the expression level of hsa-miR-4535 in blood.
- the ROC curve between stage III and stage 0-I of chorioamnionitis is shown in FIG. 13A, and the ROC curve between stage III and stage 0-II is shown in FIG. 13B.
- Table 6 shows Youden Index, sensitivity, and specificity.
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Abstract
Provided is a method for detecting an intrauterine infection, whereby it becomes possible to detect an intrauterine infection on the basis of a sample from a subject. The method for detecting an intrauterine infection comprises measuring the expression amount of at least one type of microRNA selected from the group consisting of miR-4535 and miR-1915-5p in a sample from a subject. The method for detecting an intrauterine infection may include associating the measured expression amount of the mRNA with the occurrence of the intrauterine infection.
Description
本発明は、子宮内感染の検出方法に関する。
The present invention relates to a method for detecting an intrauterine infection.
子宮内感染とは、膣の中にいる細菌が子宮内へ移動して羊水が細菌に感染して生じる炎症性疾患である。子宮内感染は、前期破水、流産、早産などの妊娠経過の異常を引き起こすとともに、胎児、あるいは出生後の児の発育、発達に長期にわたって影響を及ぼすことが知られている。子宮内感染の診断は、羊水の培養検査、胎盤の病理組織学的検査により診断される。しかしながら、培養できない細菌は検出できず、培養できても定量性が失われる場合がある。また、羊水採取は対象に対する侵襲を伴い、容易に実施することが困難である。更に、病理組織学的検査は分娩前に施行不可能であるため、分娩前に正確に子宮内感染の重症度を診断することは極めて困難である。
Intrauterine infection is an inflammatory disease that occurs when bacteria in the vagina move into the uterus and amniotic fluid is infected with the bacteria. It is known that intrauterine infection causes abnormalities in the course of pregnancy such as premature rupture of water, miscarriage, and premature birth, and has a long-term effect on the development and development of the fetal or postnatal infant. Diagnosis of intrauterine infection is made by a culture test of amniotic fluid and a histopathological test of the placenta. However, bacteria that cannot be cultured cannot be detected, and even if they can be cultured, their quantification may be lost. In addition, amniotic fluid collection is difficult to carry out easily due to the invasion of the subject. Moreover, since histopathological examination cannot be performed before delivery, it is extremely difficult to accurately diagnose the severity of intrauterine infection before delivery.
上記に関連して、羊水から特定の微生物を検出することで子宮内感染の一病態である絨毛膜羊膜炎の存在を検出する方法が提案されている（例えば、特開２０１７－２０９０６３号公報参照）。
In relation to the above, a method for detecting the presence of chorioamnionitis, which is a pathological condition of intrauterine infection, by detecting a specific microorganism in amniotic fluid has been proposed (see, for example, JP-A-2017-209063). ).
本発明は、対象に由来する試料に基づいて子宮内感染を検出することができる子宮内感染の検出方法を提供することを目的とする。
An object of the present invention is to provide a method for detecting an intrauterine infection that can detect an intrauterine infection based on a sample derived from a subject.
第１の態様は、対象に由来する試料におけるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡの発現量を測定することを含む子宮内感染の検出方法である。第２の態様は、ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡを含む子宮内感染の診断剤である。第３の態様は、ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡの少なくとも一部と相補的な配列を有するポリヌクレオチドを含む子宮内感染の検出用キットである。
The first aspect is a method for detecting an intrauterine infection, which comprises measuring the expression level of at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p in a sample derived from a subject. be. A second aspect is a diagnostic agent for intrauterine infections comprising at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p. A third aspect is a kit for detecting intrauterine infections comprising a polynucleotide having a sequence complementary to at least a portion of at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p. Is.
本発明によれば、対象に由来する試料に基づいて子宮内感染を検出することができる子宮内感染の検出方法を提供することができる。
According to the present invention, it is possible to provide a method for detecting an intrauterine infection that can detect an intrauterine infection based on a sample derived from a subject.
本明細書において「工程」との語は、独立した工程だけではなく、他の工程と明確に区別できない場合であってもその工程の所期の目的が達成されれば、本用語に含まれる。またマイクロＲＮＡをｍｉＲＮＡと略記することがある。以下、本発明の実施形態を詳細に説明する。ただし、以下に示す実施形態は、本発明の技術思想を具体化するための、子宮内感染の検出方法を例示するものであって、本発明は、以下に示す子宮内感染の検出方法に限定されない。
In the present specification, the term "process" is included in this term not only as an independent process but also as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes. .. In addition, microRNA may be abbreviated as miRNA. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments shown below exemplify a method for detecting an intrauterine infection for embodying the technical idea of the present invention, and the present invention is limited to the method for detecting an intrauterine infection shown below. Not done.
子宮内感染の検出方法
子宮内感染の検出方法は、対象に由来する試料におけるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡ（以下、これらを併せて「特定マイクロＲＮＡ」ともいう）の発現量を測定する第１工程を含む。 Method for detecting intrauterine infection A method for detecting intrauterine infection is at least one type of microRNA selected from the group consisting of miR-4535 and miR-1915-5p in a sample derived from a subject (hereinafter, these are collectively referred to as “). It includes a first step of measuring the expression level of (also referred to as "specific microRNA").
子宮内感染の検出方法は、対象に由来する試料におけるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡ（以下、これらを併せて「特定マイクロＲＮＡ」ともいう）の発現量を測定する第１工程を含む。 Method for detecting intrauterine infection A method for detecting intrauterine infection is at least one type of microRNA selected from the group consisting of miR-4535 and miR-1915-5p in a sample derived from a subject (hereinafter, these are collectively referred to as “). It includes a first step of measuring the expression level of (also referred to as "specific microRNA").
子宮内感染の検出方法においては、対象に由来する試料に含まれる特定マイクロＲＮＡについてその発現量を測定し、その発現量に基づいて対象における子宮内感染の存在を検出する。これにより、胎盤の病理学的所見を得ることなく子宮内感染を検出することができる。特定マイクロＲＮＡは、ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種である。これらの特定マイクロＲＮＡの生体内での役割は不詳である。しかしながら、本発明者らは、これらの特定マイクロＲＮＡの羊水中における発現量が、子宮内感染が認められる対象において上昇していること、更に羊水中における１６Ｓ－ｒＤＮＡ検出量及び対象の血液中または羊水中における特定マイクロＲＮＡの発現量と相関することを見いだした。この知見に基づいて、例えば、対象の血液中における特定マイクロＲＮＡの発現量を測定することで子宮内感染を検出することができる。
In the method for detecting intrauterine infection, the expression level of a specific microRNA contained in a sample derived from a subject is measured, and the presence of intrauterine infection in the subject is detected based on the expression level. This makes it possible to detect intrauterine infection without obtaining pathological findings of the placenta. The specific microRNA is at least one selected from the group consisting of miR-4535 and miR-1915-5p. The role of these specific microRNAs in vivo is unknown. However, we found that the expression levels of these specific microRNAs in amniotic fluid were elevated in subjects with intrauterine infection, and that 16S-rDNA was detected in amniotic fluid and in the blood of the subjects or in the blood of the subjects. We found that it correlates with the expression level of specific microRNAs in amniotic fluid. Based on this finding, for example, intrauterine infection can be detected by measuring the expression level of a specific microRNA in the blood of a subject.
第１工程
第１工程では、対象に由来する試料におけるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種である特定マイクロＲＮＡの発現量を測定する。子宮内感染の検出方法における対象としては、ヒトの妊婦であればよく、例えば、周産期の妊婦であってよく、切迫早産が懸念される周産期の妊婦であってよい。 First Step In the first step, the expression level of a specific microRNA, which is at least one selected from the group consisting of miR-4535 and miR-1915-5p, is measured in a sample derived from a subject. The target in the method for detecting an intrauterine infection may be a pregnant human woman, for example, a pregnant woman in the perinatal period, or a pregnant woman in the perinatal period in which imminent preterm birth is a concern.
第１工程では、対象に由来する試料におけるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種である特定マイクロＲＮＡの発現量を測定する。子宮内感染の検出方法における対象としては、ヒトの妊婦であればよく、例えば、周産期の妊婦であってよく、切迫早産が懸念される周産期の妊婦であってよい。 First Step In the first step, the expression level of a specific microRNA, which is at least one selected from the group consisting of miR-4535 and miR-1915-5p, is measured in a sample derived from a subject. The target in the method for detecting an intrauterine infection may be a pregnant human woman, for example, a pregnant woman in the perinatal period, or a pregnant woman in the perinatal period in which imminent preterm birth is a concern.
対象に由来する試料は、対象の体液に由来する試料であればよく、対象から採取した体液自体であっても、採取した体液に通常行われる処理を行ったものであってもよい。体液としては、例えば、血液、リンパ液、組織液、腹水等の体腔液、唾液等の消化液、鼻水、尿、膣液、羊水等が挙げられ、これらからなる群から選択される少なくとも１種であってよい。これらの中でも、試料となる体液は、例えば、血液、尿及び羊水からなる群から選択される少なくとも１種であってよい。
The sample derived from the subject may be a sample derived from the body fluid of the subject, and may be the body fluid itself collected from the subject or a sample obtained by subjecting the collected body fluid to a treatment normally performed. Examples of the body fluid include blood, lymph, tissue fluid, body cavity fluid such as ascites, digestive fluid such as saliva, runny nose, urine, vaginal fluid, amniotic fluid, and the like, and at least one selected from the group consisting of these. You can. Among these, the body fluid as a sample may be at least one selected from the group consisting of, for example, blood, urine and amniotic fluid.
血液に由来する試料は、対象から採取した血液自体であっても、採取した血液に通常行われる遠心分離等の処理を行ったものであってもよい。血液に由来する試料は、全血、血清又は血漿のいずれであってよい。また、簡便性の点から、対象から採取した血液から常法により調製した血清又は血漿であってよく、好ましくは血清であってよい。
The sample derived from blood may be the blood itself collected from the subject, or the collected blood may be subjected to a treatment such as centrifugation which is usually performed. The sample derived from blood may be whole blood, serum or plasma. Further, from the viewpoint of convenience, serum or plasma prepared by a conventional method from blood collected from a subject may be used, and serum may be preferable.
発現量の測定対象である特定マイクロＲＮＡのうち、ｍｉＲ－４５３５は、ｈｓａ－ｍｉＲ－４５３５であってよく、配列番号１の塩基配列を有していてよい。また、ｍｉＲ－１９１５－５ｐは、ｈｓａ－ｍｉＲ－１９１５－５ｐであってよく、配列番号２の塩基配列を有していてよい。
Among the specific microRNAs whose expression levels are to be measured, miR-4535 may be hsa-miR-4535 and may have the nucleotide sequence of SEQ ID NO: 1. Further, miR-1955-5p may be hsa-miR-1955-5p and may have the base sequence of SEQ ID NO: 2.
試料に含まれる特定マイクロＲＮＡの発現量は、例えば、試料からＲＮＡを抽出し、抽出されたＲＮＡに含まれる特定マイクロＲＮＡを定量または半定量することで測定される。試料からのＲＮＡの抽出は、ｍｉＲＮＡｅａｓｙ Ｓｅｒｕｍ／Ｐｌａｓｍａ Ｋｉｔ（Ｑｉａｇｅｎ社製）、ｍｉＲＣＵＲＹＴＭ ＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（ＥＸＩＱＯＮ社製）等の市販のＲＮＡ抽出キットを用いて、付属の取り扱い説明書の手順に準じて行うことができる。
The expression level of the specific microRNA contained in the sample is measured, for example, by extracting RNA from the sample and quantifying or semi-quantifying the specific microRNA contained in the extracted RNA. RNA can be extracted from the sample using a commercially available RNA extraction kit such as miRNAeasy Serum / Plasma Kit (manufactured by Qiagen) or miRCURY TM RNA Isolation Kit (manufactured by EXIQON) according to the procedure in the attached instruction manual. It can be carried out.
特定マイクロＲＮＡの発現量は、例えば、定量ＰＣＲ、次世代シーケンサー、ＤＮＡマイクロアレイ、酵素免疫測定法（ＥＩＡ法）等で測定することができる。測定される特定マイクロＲＮＡの発現量は、絶対的発現量であっても相対的発現量であってもよい。
The expression level of the specific microRNA can be measured by, for example, quantitative PCR, next-generation sequencer, DNA microarray, enzyme-linked immunosorbent assay (EIA method), or the like. The expression level of the specific microRNA to be measured may be an absolute expression level or a relative expression level.
定量ＰＣＲには、リアルタイムＰＣＲ（ＲＴ－ＰＣＲ）、デジタルＰＣＲ等の公知の手法を適用することができる。定量ＰＣＲで特定マイクロＲＮＡの発現量を測定する場合、例えば、抽出されたＲＮＡからｃＤＮＡを調製し、ｃＤＮＡを特定マイクロＲＮＡに特異的なプライマーセットを用いて定量ＰＣＲにより増幅することで、特定マイクロＲＮＡの発現量を測定することができる。定量ＰＣＲで測定される特定マイクロＲＮＡの発現量は、例えば、コピー数のような絶対発現量であってよく、コピー数を規格化した相対発現量であってもよい。
Known methods such as real-time PCR (RT-PCR) and digital PCR can be applied to quantitative PCR. When measuring the expression level of a specific microRNA by quantitative PCR, for example, by preparing cDNA from the extracted RNA and amplifying the cDNA by quantitative PCR using a primer set specific to the specific microRNA, the specific micro The expression level of RNA can be measured. The expression level of the specific microRNA measured by quantitative PCR may be, for example, an absolute expression level such as copy number, or a relative expression level in which the copy number is standardized.
次世代シーケンサーを用いて特定マイクロＲＮＡの発現量を測定する場合、例えば、抽出されたＲＮＡからｃＤＮＡを調製し、ＰＣＲによってアンプリコンを得て、次世代シーケンサーを用いてアンプリコンシーケンシングを実施する。得られたシーケンシングデータから、クオリティコントロールにより、低クオリティリードなどを削除した上で、所望の塩基配列を有するリード数によって発現量が測定される。ここで次世代シーケンサーとは、サンガー法を用いるキャピラリーシーケンサーと対比される用語である。次世代シーケンサーでは、合成シーケンシング法、パイロシーケンシング法、リガーゼ反応シーケンシング法等のシーケンシング原理が用いられる。次世代シーケンサーとして具体的には、ＭｉＳｅｑ（登録商標）システム（ｉｌｌｍｉｎａ社）、ＨｉＳｅｑ（登録商標）システム（ｉｌｌｍｉｎａ社）、ＩｏｎＰＧＭ（登録商標）システム（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｙ社）等が挙げられる。次世代シーケンサーによるアンプリコンシーケンシングは、メーカー推奨プロトコールに従って実施することができる。
When measuring the expression level of a specific microRNA using a next-generation sequencer, for example, cDNA is prepared from the extracted RNA, an amplicon is obtained by PCR, and amplicon sequencing is performed using the next-generation sequencer. .. From the obtained sequencing data, low quality reads and the like are deleted by quality control, and the expression level is measured by the number of reads having a desired base sequence. Here, the next-generation sequencer is a term that is contrasted with a capillary sequencer that uses the Sanger method. Next-generation sequencers use sequencing principles such as synthetic sequencing, pyrosequencing, and ligase reaction sequencing. Specific examples of the next-generation sequencer include a MiSeq (registered trademark) system (illmina), a HiSeq (registered trademark) system (illmina), and an IonPGM (registered trademark) system (Life Technology). Amplicon sequencing with a next-generation sequencer can be performed according to the manufacturer's recommended protocol.
ＤＮＡマイクロアレイを用いて特定マイクロＲＮＡの発現量を測定する場合、例えば、抽出されたＲＮＡを、市販のキットを用いて蛍光標識し、ＤＮＡマイクロアレイ上に配置された特定マイクロＲＮＡと相補的な配列を有するプローブとハイブリダイズさせた後、蛍光強度を測定することで、特定マイクロＲＮＡの発現量を測定することができる。
When measuring the expression level of a specific microRNA using a DNA microarray, for example, the extracted RNA is fluorescently labeled using a commercially available kit, and a sequence complementary to the specific microRNA arranged on the DNA microarray is obtained. The expression level of the specific microRNA can be measured by measuring the fluorescence intensity after hybridizing with the probe to be possessed.
酵素免疫測定法により、特定マイクロＲＮＡの発現量を測定する場合、例えば、特定マイクロＲＮＡに相補的な配列を有する標識されたＤＮＡプローブと特定マイクロＲＮＡとのハイブリッド鎖を、ハイブリッド鎖に特異的な抗体に結合させた後、ＤＮＡプローブの標識を検出することでマイクロＲＮＡの発現量の発現量を測定することができる。
When the expression level of a specific microRNA is measured by an enzyme immunoassay, for example, a hybrid strand of a labeled DNA probe having a sequence complementary to the specific microRNA and the specific microRNA is specific to the hybrid strand. After binding to the antibody, the expression level of the microRNA expression level can be measured by detecting the label of the DNA probe.
特定マイクロＲＮＡの発現量の測定には、成熟マイクロＲＮＡの発現量を測定することのみならず、特定マイクロＲＮＡをコードする遺伝子の転写産物又はそのプロセシング産物の発現量を測定することが含まれてもよい。一般にマイクロＲＮＡをコードする遺伝子の一次転写産物は初期転写産物（ｐｒｉ－ｍｉＲＮＡ）と呼ばれ、ステムループのヘアピン構造を有する。ｐｒｉ－ｍｉＲＮＡはＲＮａｓｅＩＩＩ様のＤｒｏｓｈａと呼ばれる酵素によってステムループ構造をもつ成熟ｍｉＲＮＡの前駆体（ｐｒｅ－ｍｉＲＮＡ）に変換される。ｐｒｅ－ｍｉＲＮＡは細胞核の外に輸送され、Ｄｉｃｅｒと呼ばれる酵素のスプライシングによって、２０から２５塩基長の２本鎖の成熟ｍｉＲＮＡとなる。したがって、マイクロＲＮＡをコードする遺伝子の転写産物又はそのプロセッシング産物は、ｐｒｉ－ｍｉＲＮＡ、ｐｒｅ－ｍｉＲＮＡ及び成熟ｍｉＲＮＡを包含する。なお、２本鎖の成熟ｍｉＲＮＡの発現量の測定には、１本鎖の成熟ｍｉＲＮＡの発現量を測定することのみならず、それと相補対を形成する１本鎖ＲＮＡの発現量を測定することが含まれてよい。
Measuring the expression level of a specific microRNA includes not only measuring the expression level of a mature microRNA, but also measuring the expression level of a transcript of a gene encoding the specific microRNA or a processing product thereof. May be good. Generally, the primary transcript of a gene encoding a microRNA is called an early transcript (pri-miRNA) and has a stem-loop hairpin structure. The pri-miRNA is converted into a precursor (pre-miRNA) of a mature miRNA having a stem-loop structure by an enzyme called Drosha like RNaseIII. The pre-miRNA is transported out of the cell nucleus and spliced with an enzyme called Dicer to become a 20 to 25 base long double-stranded mature miRNA. Thus, transcripts of genes encoding microRNAs or processing products thereof include tri-miRNAs, pre-miRNAs and mature miRNAs. To measure the expression level of double-stranded mature miRNA, not only the expression level of single-stranded mature miRNA but also the expression level of single-stranded RNA forming a complementary pair with it should be measured. May be included.
ｈｓａ－ｍｉＲ－４５３５をコードする遺伝子の一次転写産物は、例えば、配列番号３の塩基配列を有していてよい。また、ｈｓａ－ｍｉＲ－４５３５と相補対を形成するＲＮＡは、例えば、配列番号４の塩基配列を有していてよい。ｈｓａ－ｍｉＲ－１９１５－５ｐをコードする遺伝子の一次転写産物は、例えば、配列番号５の塩基配列を有していてよい。また、ｈｓａ－ｍｉＲ－１９１５－５ｐと相補対を形成するｈｓａ－ｍｉＲ－１９１５－３ｐは、例えば、配列番号６の塩基配列を有していてよい。
The primary transcript of the gene encoding hsa-miR-4535 may have, for example, the nucleotide sequence of SEQ ID NO: 3. Further, the RNA forming a complementary pair with hsa-miR-4535 may have, for example, the base sequence of SEQ ID NO: 4. The primary transcript of the gene encoding hsa-miR-915-5p may have, for example, the nucleotide sequence of SEQ ID NO: 5. Further, hsa-miR-1915-3p, which forms a complementary pair with hsa-miR-1915-5p, may have, for example, the base sequence of SEQ ID NO: 6.
また、特定マイクロＲＮＡの発現量の測定には、特定マイクロＲＮＡと生物学的な機能が同等であるマイクロＲＮＡ、例えばホモログ、オルソログ等の同族体、遺伝子多型などの変異体、又はこれらの誘導体の発現量を測定することが含まれてもよい。マイクロＲＮＡの同族体、変異体又は誘導体は、具体的には、miRBase（http://www.mirbase.org/）を参照して同定することができる。
Further, for measuring the expression level of a specific microRNA, microRNA having a biological function equivalent to that of the specific microRNA, for example, homologues, homologues such as orthologs, variants such as gene polymorphisms, or derivatives thereof. May include measuring the expression level of. Homologues, variants or derivatives of microRNAs can be specifically identified with reference to miRBase (http://www.mirbase.org/).
第２工程
子宮内感染の検出方法は、試料における特定マイクロＲＮＡの発現量と子宮内感染の存在とを関連付ける第２工程を含んでいてもよい。第２工程においては、試料と子宮内感染の存在とを関連付けてよい。すなわち、子宮内感染の検出方法は、子宮内感染の診断方法であってよく、子宮内感染の診断を補助する方法であってもよい。 Second Step The method for detecting an intrauterine infection may include a second step of associating the expression level of the specific microRNA in the sample with the presence of the intrauterine infection. In the second step, the sample may be associated with the presence of an intrauterine infection. That is, the method for detecting intrauterine infection may be a method for diagnosing intrauterine infection, or may be a method for assisting in diagnosing intrauterine infection.
子宮内感染の検出方法は、試料における特定マイクロＲＮＡの発現量と子宮内感染の存在とを関連付ける第２工程を含んでいてもよい。第２工程においては、試料と子宮内感染の存在とを関連付けてよい。すなわち、子宮内感染の検出方法は、子宮内感染の診断方法であってよく、子宮内感染の診断を補助する方法であってもよい。 Second Step The method for detecting an intrauterine infection may include a second step of associating the expression level of the specific microRNA in the sample with the presence of the intrauterine infection. In the second step, the sample may be associated with the presence of an intrauterine infection. That is, the method for detecting intrauterine infection may be a method for diagnosing intrauterine infection, or may be a method for assisting in diagnosing intrauterine infection.
第２工程における関連付けには、公知の手法から適宜選択される手法が適用できる。例えば、対象に由来する試料における特定マイクロＲＮＡの発現量と、対照となる健常妊婦に由来する試料における特定マイクロＲＮＡの発現量とを比較する工程と、対象に由来する試料における特定マイクロＲＮＡの発現量が、対照となる健常妊婦に由来する試料における特定マイクロＲＮＡの発現量よりも多い場合と子宮内感染の存在とを関連付ける工程とを含んでいてよい。
A method appropriately selected from known methods can be applied to the association in the second step. For example, a step of comparing the expression level of a specific microRNA in a sample derived from a subject with the expression level of a specific microRNA in a sample derived from a control healthy pregnant woman and the expression of the specific microRNA in a sample derived from the subject. It may include a step of associating the presence of an intrauterine infection with a case where the amount is greater than the expression level of a particular microRNA in a sample derived from a control healthy pregnant woman.
ここで対照となる健常妊婦とは、子宮内感染が存在していないことが予め判明している妊婦を意味する。具体的には、例えば、早産を来さず絨毛膜羊膜炎の所見を認めなかった妊婦、合併症を認めなかった妊婦、絨毛膜羊膜炎における重症度分類（Ｂｌａｎｃ分類）が０又はＩである妊婦、妊娠１７週未満の妊婦等であってよい。また、発現量が多いとは、健常妊婦と子宮内感染が存在する対象とを区別するために設定された正常検出量（カットオフ値）よりも、対象に由来する試料における特定マイクロＲＮＡの発現量のほうが多いことである。
The control healthy pregnant woman here means a pregnant woman whose intrauterine infection is known to be absent in advance. Specifically, for example, a pregnant woman who did not give birth prematurely and did not find any findings of chorioamnionitis, a pregnant woman who did not find any complications, and a severity classification (Blanc classification) in chorioamnionitis was 0 or I. It may be a pregnant woman, a pregnant woman less than 17 weeks pregnant, or the like. In addition, a high expression level means that the expression of specific microRNA in a sample derived from the subject is higher than the normal detection amount (cutoff value) set to distinguish between a healthy pregnant woman and a subject having an intrauterine infection. The amount is larger.
カットオフ値は、例えば、子宮内感染が存在する対象に由来する試料群における特定マイクロＲＮＡの発現量と、対照となる健常妊婦に由来する試料群における特定マイクロＲＮＡの発現量とをＲＯＣ解析等に付することよって設定することができる。また、カットオフ値は、例えば、健常妊婦の発現レベルの平均値に、標準偏差を２倍あるいは３倍した値を加算した値として定めることもでき、更に、感度（検出率）・特異性（偽陽性率の低さ）をバランスよく満たす値に適宜定めることができる。
For the cutoff value, for example, ROC analysis of the expression level of specific microRNA in a sample group derived from a subject having an intrauterine infection and the expression level of specific microRNA in a sample group derived from a control healthy pregnant woman, etc. It can be set by attaching to. Further, the cutoff value can be set as, for example, a value obtained by adding a value obtained by multiplying the standard deviation by 2 or 3 to the average value of the expression level of a healthy pregnant woman, and further, sensitivity (detection rate) and specificity (detection rate) and specificity ( A value that satisfies (low false positive rate) in a well-balanced manner can be appropriately set.
カットオフ値はｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐのそれぞれ発現量に対して設定してよく、ｍｉＲ－４５３５の発現量及びｍｉＲ－１９１５－５ｐの発現量から算出される子宮内感染の存在を判定する判定指数に対して設定してもよい。
The cutoff value may be set for the expression levels of miR-4535 and miR-1915-5p, respectively, and the presence of intrauterine infection calculated from the expression levels of miR-4535 and miR-1915-5p can be determined. It may be set for the judgment index to be judged.
第３工程
子宮内感染の検出方法は、対象の羊水に由来する試料に含まれる１６ＳリボソームＲＮＡ遺伝子（１６Ｓ ｒＤＮＡ）を検出する第３工程を更に含んでいてもよい。第３工程は、対象から羊水を採取する工程と、採取した羊水に由来する試料から１６ＳリボソームＲＮＡ遺伝子を検出する工程とを含んでよい。第３工程で羊水を採取する対象は、第２工程において子宮内感染の存在と関連付けられた試料が採取された対象であってよい。すなわち、羊水を採取する対象は、子宮内感染の存在が想定される妊婦であってよい。子宮内感染の存在が想定される対象から採取される羊水から１６ＳリボソームＲＮＡ遺伝子を検出することで、対象における子宮内感染の進行状況をより的確に診断することができる。 Third Step The method for detecting intrauterine infection may further include a third step of detecting the 16S ribosomal RNA gene (16S rDNA) contained in a sample derived from the target amniotic fluid. The third step may include a step of collecting amniotic fluid from the subject and a step of detecting the 16S ribosomal RNA gene from the sample derived from the collected amniotic fluid. The subject from which the amniotic fluid is collected in the third step may be the subject from which the sample associated with the presence of the intrauterine infection was collected in the second step. That is, the target for collecting amniotic fluid may be a pregnant woman who is expected to have an intrauterine infection. By detecting the 16S ribosomal RNA gene in amniotic fluid collected from a subject in which the presence of intrauterine infection is assumed, the progress of intrauterine infection in the subject can be more accurately diagnosed.
子宮内感染の検出方法は、対象の羊水に由来する試料に含まれる１６ＳリボソームＲＮＡ遺伝子（１６Ｓ ｒＤＮＡ）を検出する第３工程を更に含んでいてもよい。第３工程は、対象から羊水を採取する工程と、採取した羊水に由来する試料から１６ＳリボソームＲＮＡ遺伝子を検出する工程とを含んでよい。第３工程で羊水を採取する対象は、第２工程において子宮内感染の存在と関連付けられた試料が採取された対象であってよい。すなわち、羊水を採取する対象は、子宮内感染の存在が想定される妊婦であってよい。子宮内感染の存在が想定される対象から採取される羊水から１６ＳリボソームＲＮＡ遺伝子を検出することで、対象における子宮内感染の進行状況をより的確に診断することができる。 Third Step The method for detecting intrauterine infection may further include a third step of detecting the 16S ribosomal RNA gene (16S rDNA) contained in a sample derived from the target amniotic fluid. The third step may include a step of collecting amniotic fluid from the subject and a step of detecting the 16S ribosomal RNA gene from the sample derived from the collected amniotic fluid. The subject from which the amniotic fluid is collected in the third step may be the subject from which the sample associated with the presence of the intrauterine infection was collected in the second step. That is, the target for collecting amniotic fluid may be a pregnant woman who is expected to have an intrauterine infection. By detecting the 16S ribosomal RNA gene in amniotic fluid collected from a subject in which the presence of intrauterine infection is assumed, the progress of intrauterine infection in the subject can be more accurately diagnosed.
羊水における１６ＳリボソームＲＮＡ遺伝子の検出は、例えば、採取した羊水に由来する試料から常法によりＤＮＡを抽出し、１６ＳリボソームＲＮＡ遺伝子に対するユニバーサルプライマーを用いて、１６ＳリボソームＲＮＡ遺伝子を網羅的に増幅することで実施することができる。本来は無菌であると考えられる羊水から１６ＳリボソームＲＮＡ遺伝子が検出されることで、羊水感染の進行状態がより的確に診断される。
To detect the 16S ribosomal RNA gene in sheep water, for example, DNA is extracted from a sample derived from the collected sheep water by a conventional method, and the 16S ribosomal RNA gene is comprehensively amplified by using a universal primer for the 16S ribosomal RNA gene. Can be carried out at. By detecting the 16S ribosomal RNA gene in amniotic fluid, which is considered to be sterile, the progress of amniotic fluid infection can be more accurately diagnosed.
第３工程は、羊水から検出される１６ＳリボソームＲＮＡ遺伝子に基づいて、羊水から検出される微生物群を同定する工程を含んでいてもよい。微生物群の同定は、例えば、次世代シーケンサーを用いる定量的かつ網羅的な１６ＳリボソームＲＮＡ遺伝子解析により行うことができる。具体的には、例えば、特開２０１７－２０９０６３号公報の記載を参照して、羊水に含まれる微生物群を同定することができる。
The third step may include a step of identifying a group of microorganisms detected in amniotic fluid based on the 16S ribosomal RNA gene detected in amniotic fluid. Identification of the microbial community can be performed, for example, by quantitative and comprehensive 16S ribosomal RNA gene analysis using a next-generation sequencer. Specifically, for example, a group of microorganisms contained in amniotic fluid can be identified by referring to the description in JP-A-2017-209063.
同定される微生物群は、例えば、以下に示す絨毛膜羊膜炎関連微生物であってよい。絨毛膜羊膜炎関連微生物が羊水から検出されることで、対象における子宮内感染が絨毛膜羊膜炎まで進行していることが診断できる。
The group of microorganisms identified may be, for example, the following microorganisms related to chorioamnionitis. By detecting chorioamnionitis-related microorganisms in amniotic fluid, it can be diagnosed that intrauterine infection in the subject has progressed to chorioamnionitis.
絨毛膜羊膜炎関連微生物には、例えば、ウレアプラズマ・パルブム（Ureaplasma parvum）、マイコプラズマ・ホミニス（Mycoplasma hominis）、ガルドネレラ・バギナリス（Gardnerella vaginalis）、ラクトバチルス・ジェンセニイ（Lactobacillus jensenii）、ヘモフィルス・インフルエンザ（Haemophilus influenzae）、スネシア・サンギネジェンス（Sneathia sanguinegens）、カプノサイトファガ・スプチゲン（Capnocytophaga sputigene）、バクテロイデス・フラジリス（Bacteroides fragilis）、及びストレプトコッカス・アガラクチエ（Streptococcus agalactiae）が含まれる。
Chorioamnionitis-related microorganisms include, for example, Ureaplasma parvum, Mycoplasma hominis, Gardnerella vaginalis, Lactobacillus jensenii, and Hemophilus. Includes influenzae, Sneathia sanguinegens, Capnocytophaga sputigene, Bacteroides fragilis, and Streptococcus agalactiae.
子宮内感染の検出方法において、子宮内感染が検出された対象には、その進行状況等に応じて経過観察、治療介入等の処置を実施してもよい。
In the method for detecting an intrauterine infection, a subject in which an intrauterine infection is detected may be subjected to measures such as follow-up and intervention depending on the progress of the infection.
子宮内感染の処置方法
子宮内感染の処置方法は、対象に由来する試料に含まれるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡ（以下、特定マイクロＲＮＡともいう）の発現量を測定する第１工程と、特定マイクロＲＮＡの発現量と対象における子宮内感染の存在とを関連付ける第２工程と、子宮内感染が存在する対象を処置する第４工程とを含んでいてよい。子宮内感染の処置方法は、第４工程の前に、対象の羊水に由来する試料に含まれる１６ＳリボソームＲＮＡ遺伝子を検出する第３工程をさらに含んでいてもよい。 Treatment method for intrauterine infection The treatment method for intrauterine infection is at least one type of microRNA selected from the group consisting of miR-4535 and miR-1915-5p contained in a sample derived from a subject (hereinafter, specific microRNA). The first step of measuring the expression level of (also referred to as), the second step of associating the expression level of the specific microRNA with the presence of intrauterine infection in the subject, and the fourth step of treating the subject in which the intrauterine infection is present. May include. The method for treating an intrauterine infection may further include a third step of detecting the 16S ribosomal RNA gene contained in a sample derived from the amniotic fluid of interest before the fourth step.
子宮内感染の処置方法は、対象に由来する試料に含まれるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡ（以下、特定マイクロＲＮＡともいう）の発現量を測定する第１工程と、特定マイクロＲＮＡの発現量と対象における子宮内感染の存在とを関連付ける第２工程と、子宮内感染が存在する対象を処置する第４工程とを含んでいてよい。子宮内感染の処置方法は、第４工程の前に、対象の羊水に由来する試料に含まれる１６ＳリボソームＲＮＡ遺伝子を検出する第３工程をさらに含んでいてもよい。 Treatment method for intrauterine infection The treatment method for intrauterine infection is at least one type of microRNA selected from the group consisting of miR-4535 and miR-1915-5p contained in a sample derived from a subject (hereinafter, specific microRNA). The first step of measuring the expression level of (also referred to as), the second step of associating the expression level of the specific microRNA with the presence of intrauterine infection in the subject, and the fourth step of treating the subject in which the intrauterine infection is present. May include. The method for treating an intrauterine infection may further include a third step of detecting the 16S ribosomal RNA gene contained in a sample derived from the amniotic fluid of interest before the fourth step.
子宮内感染が検出された対象に、必要に応じた処置を施すことで、早産の抑制、妊娠期間の延長、新生児感染の抑制、胎児炎症反応症候群（ＦＩＲＳ）・新生児髄膜炎・新生児慢性肺疾患・脳室周囲白質軟化症・脳性麻痺・発達遅滞などの新生児疾患の抑制、障がい者の発生抑制等の効果を得ることができる。したがって、子宮内感染の処置方法は、新生児感染の予防方法であってもよく、新生児疾患（例えば、ＦＩＲＳ）の予防方法であってもよい。
Preterm birth can be suppressed, pregnancy can be extended, neonatal infection can be suppressed, fetal inflammatory reaction syndrome (FIRS), neonatal meningitis, and neonatal chronic lung can be treated as necessary for subjects in which intrauterine infection has been detected. It is possible to obtain effects such as suppression of neonatal diseases such as diseases, periventricular leukomalacia, cerebral palsy, and developmental delay, and suppression of the occurrence of persons with disabilities. Therefore, the method for treating intrauterine infection may be a method for preventing neonatal infection or a method for preventing neonatal disease (for example, FIRS).
ここで対象に応じた処置とは、子宮内感染について施される何らかの処置であればよく、例えば、子宮内感染の治療、改善、進行の抑制（悪化の防止）、予防、子宮内感染に起因する症状の緩和等が挙げられる。処置として具体的には、例えば、抗菌薬剤の投与、子宮収縮抑制薬剤の投与、ステロイド剤の投与、陣痛促進剤の投与、分娩誘発、帝王切開術、子宮切開術、子宮内容除去術、子宮摘出術などが挙げられる。
Here, the subject-specific treatment may be any treatment performed for the intrauterine infection, and is caused by, for example, treatment, improvement, suppression of progression (prevention of deterioration), prevention, or intrauterine infection of the uterine infection. Relief of symptoms that occur. Specific treatments include, for example, administration of antibacterial agents, administration of uterine contraction-suppressing agents, administration of steroids, administration of labor-promoting agents, induction of delivery, cesarean section, hysterotomy, dilation and curettage, hysterectomy. Techniques can be mentioned.
子宮内感染の診断剤
子宮内感染の診断剤は、ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種である特定マイクロＲＮＡを含んで構成される。子宮内感染の診断剤は、診断対象に由来する試料に適用されてよい。診断対象に由来する試料から、診断剤に含まれる特定マイクロＲＮＡの少なくとも１種の発現量を測定し、その発現量に基づいて子宮内感染を検出することができる。すなわち、診断剤は、子宮内感染の診断用バイオマーカーであってよい。 Diagnostic Agent for Intrauterine Infection A diagnostic agent for intrauterine infection comprises at least one specific microRNA selected from the group consisting of miR-4535 and miR-1915-5p. The diagnostic agent for intrauterine infection may be applied to a sample derived from the subject to be diagnosed. The expression level of at least one specific microRNA contained in the diagnostic agent can be measured from a sample derived from the diagnostic target, and intrauterine infection can be detected based on the expression level. That is, the diagnostic agent may be a diagnostic biomarker for intrauterine infection.
子宮内感染の診断剤は、ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種である特定マイクロＲＮＡを含んで構成される。子宮内感染の診断剤は、診断対象に由来する試料に適用されてよい。診断対象に由来する試料から、診断剤に含まれる特定マイクロＲＮＡの少なくとも１種の発現量を測定し、その発現量に基づいて子宮内感染を検出することができる。すなわち、診断剤は、子宮内感染の診断用バイオマーカーであってよい。 Diagnostic Agent for Intrauterine Infection A diagnostic agent for intrauterine infection comprises at least one specific microRNA selected from the group consisting of miR-4535 and miR-1915-5p. The diagnostic agent for intrauterine infection may be applied to a sample derived from the subject to be diagnosed. The expression level of at least one specific microRNA contained in the diagnostic agent can be measured from a sample derived from the diagnostic target, and intrauterine infection can be detected based on the expression level. That is, the diagnostic agent may be a diagnostic biomarker for intrauterine infection.
子宮内感染の検出用キット
子宮内感染の検出用キットは、ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種である特定マイクロＲＮＡの少なくとも一部と相補的な配列を有するポリヌクレオチドを含んで構成される。子宮内感染の検出用キットは、検出対象に由来する試料に適用することで、対象における子宮内感染を検出することができる。 Intrauterine infection detection kit The intrauterine infection detection kit contains a sequence complementary to at least a part of a specific microRNA, which is at least one selected from the group consisting of miR-4535 and miR-1915-5p. Consists of a polynucleotide having. The kit for detecting an intrauterine infection can detect an intrauterine infection in a subject by applying it to a sample derived from the detection target.
子宮内感染の検出用キットは、ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種である特定マイクロＲＮＡの少なくとも一部と相補的な配列を有するポリヌクレオチドを含んで構成される。子宮内感染の検出用キットは、検出対象に由来する試料に適用することで、対象における子宮内感染を検出することができる。 Intrauterine infection detection kit The intrauterine infection detection kit contains a sequence complementary to at least a part of a specific microRNA, which is at least one selected from the group consisting of miR-4535 and miR-1915-5p. Consists of a polynucleotide having. The kit for detecting an intrauterine infection can detect an intrauterine infection in a subject by applying it to a sample derived from the detection target.
特定マイクロＲＮＡの少なくとも一部と相補的な配列を有するポリヌクレオチドには、特定マイクロＲＮＡをＰＣＲ法で増幅することができるＰＣＲ用プライマーセット、特定マイクロＲＮＡとハイブリダイズ可能で、ビーズ等の担体に固定化されたポリヌクレオチド等が含まれる。
For polynucleotides having a sequence complementary to at least a part of the specific microRNA, a primer set for PCR capable of amplifying the specific microRNA by the PCR method, and a carrier such as beads that can hybridize with the specific microRNA. Includes immobilized polynucleotides and the like.
本発明は、別の態様として、子宮内感染の検出における特定マイクロＲＮＡの使用、子宮内感染の処置における特定マイクロＲＮＡの使用、子宮内感染の検出に使用される特定マイクロＲＮＡを包含する。
As another aspect, the present invention includes the use of specific microRNAs in the detection of intrauterine infections, the use of specific microRNAs in the treatment of intrauterine infections, and the specific microRNAs used in the detection of intrauterine infections.
以下、本発明を実施例により具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.
（試験例１）
福岡大学病院に切迫早産の診断で入院し、解析に十分な量の検体を採取できた未破水妊婦４９例を対象とした。また、妊娠１７週未満で羊水検査を希望し、解析に十分な量の残余検体があり、早産や絨毛膜羊膜炎を来さなかった健常妊婦１５例をコントロールとした。対象からは書面でのインフォームドコンセントに同意を受けた。対象からは検体として羊水及び血液を採取した。分娩後に胎盤の病理検査を実施して、絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例と非絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩ以下）１２例の２群に分けた。 (Test Example 1)
Forty-nine unbroken pregnant women who were admitted to Fukuoka University Hospital with a diagnosis of imminent preterm birth and were able to collect a sufficient amount of samples for analysis were included. In addition, 15 healthy pregnant women who requested amniocentesis at less than 17 weeks gestation, had a sufficient amount of residual sample for analysis, and did not develop preterm birth or chorioamnionitis were controlled. Subject consented to written informed consent. Amniotic fluid and blood were collected from the subjects as samples. After delivery, placental pathological examination was performed and divided into two groups: 37 cases in the chorioamnionitis group (Blanc classification stage II or higher) and 12 cases in the non-chorionic amniotic inflammation group (Blanc classification stage I or lower).
福岡大学病院に切迫早産の診断で入院し、解析に十分な量の検体を採取できた未破水妊婦４９例を対象とした。また、妊娠１７週未満で羊水検査を希望し、解析に十分な量の残余検体があり、早産や絨毛膜羊膜炎を来さなかった健常妊婦１５例をコントロールとした。対象からは書面でのインフォームドコンセントに同意を受けた。対象からは検体として羊水及び血液を採取した。分娩後に胎盤の病理検査を実施して、絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例と非絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩ以下）１２例の２群に分けた。 (Test Example 1)
Forty-nine unbroken pregnant women who were admitted to Fukuoka University Hospital with a diagnosis of imminent preterm birth and were able to collect a sufficient amount of samples for analysis were included. In addition, 15 healthy pregnant women who requested amniocentesis at less than 17 weeks gestation, had a sufficient amount of residual sample for analysis, and did not develop preterm birth or chorioamnionitis were controlled. Subject consented to written informed consent. Amniotic fluid and blood were collected from the subjects as samples. After delivery, placental pathological examination was performed and divided into two groups: 37 cases in the chorioamnionitis group (Blanc classification stage II or higher) and 12 cases in the non-chorionic amniotic inflammation group (Blanc classification stage I or lower).
対象から採取した羊水から、ｍｉＲＮＡｅａｓｙ Ｓｅｒｕｍ／Ｐｌａｓｍａ Ｋｉｔ（Ｑｉａｇｅｎ社製）を用い、添付のプロトコールに準じてＲＮＡを抽出し、抽出したＲＮＡからｃＤＮＡを調製した。調製したｃＤＮＡについて、デジタルドロップレットＰＣＲ（ＱＸ２００ Ｄｒｏｐｌｅｔ Ｄｉｇｉｔａｌ ＰＣＲシステム；バイオ・ラッド ラボラトリーズ株式会社）と、ＴａｑＭａｎ（Ｒ） ｍｉＲＮＡ Ａｓｓａｙを用いてｈｓａ－ｍｉＲ－４５３５、ｈｓａ－ｍｉＲ－１９１５－５ｐ及び１６Ｓ－ｒＤＮＡを定量した。ｃＤＮＡ２μＬをＢｉｏ－Ｒａｄ ＱＸ２００試薬１８μＬで希釈し、ＱＸ２００ＤｒｏｐｌｅｔＧｅｎｅｒａｔｏｒを用いて約２０，０００のドロップレットに分割した。ＰＣＲを９６ウェルプレート上、９５℃で５分の後、９５℃で３０秒と５２℃で１分の４０サイクルした後、４℃で５分、９０℃で５分、その後２℃／秒で４℃まで降温した。蛍光発光をＱＸ２００ ＤｒｏｐｌｅｔＲｅａｄｅｒで検出し、Ｂｉｏ－Ｒａｄ ＱｕａｎｔａＳｏｆｔソフトウェアを用いて解析して、試料１μＬあたりのマイクロＲＮＡのコピー数を算出した。なお、マイクロＲＮＡのプライマーとしてｍｉＲ－１９１５－５ｐ（１２１１２０＿ｍａｔ）及びｍｉＲ－４５３５（４６４２８４＿ｍａｔ）を用い、１６Ｓ－ｒＤＮＡのプライマーとして下記配列番号７及び８を用いた。結果を図１（Ａ）から（ｃ）に示す。ここで、有意差は、Ｍａｎｎ－ＷｉｔｎｅｙのＵ検定、又はＦｉｓｈｅｒの正確検定により検定した。
RNA was extracted from the amniotic fluid collected from the subject using miRNAeasy Serum / Plasma Kit (manufactured by Qiagen) according to the attached protocol, and cDNA was prepared from the extracted RNA. For the prepared cDNA, hsa-miR-4535, hsa-miR-1915-5p and 16S- using digital droplet PCR (QX200 Droplet Digital PCR system; Bio-Rad Laboratories Co., Ltd.) and TaqMan (R) miRNA Assay. The rDNA was quantified. 2 μL of cDNA was diluted with 18 μL of Bio-Rad QX200 reagent and divided into approximately 20,000 droplets using the QX200 Droplet Generator. PCR was run on a 96-well plate at 95 ° C. for 5 minutes, followed by 40 cycles of 95 ° C. for 30 seconds and 52 ° C. for 1 minute, then at 4 ° C. for 5 minutes, at 90 ° C. for 5 minutes, and then at 2 ° C./sec. The temperature was lowered to 4 ° C. Fluorescence was detected by the QX200 Droplet Reader and analyzed using Bio-Rad QuantaSoft software to calculate the copy number of microRNA per μL of sample. In addition, miR-1915-5p (121120_mat) and miR-4535 (464284_mat) were used as primers for microRNA, and the following SEQ ID NOs: 7 and 8 were used as primers for 16S-rDNA. The results are shown in FIGS. 1 (A) to 1 (c). Here, the significant difference was tested by Mann-Whitney U test or Fisher's exact test.
羊水中のｍｉＲ４５３５及びｍｉＲ１９１５－５ｐの発現量は、子宮内感染の増悪に伴って有意に上昇した。
The expression levels of miR4535 and miR1915-5p in amniotic fluid increased significantly with the exacerbation of intrauterine infection.
（試験例２）
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例を、羊水から絨毛膜羊膜炎関連微生物が検出された群（ｍｉＣＡＭ）と検出されなかった群（ｎｏｎ－ｍｉＣＡＭ）に分割して、それぞれについてｈｓａ－ｍｉＲ－４５３５、ｈｓａ－ｍｉＲ－１９１５－５ｐ及び１６ＳｒＤＮＡを定量した。結果を図２（Ａ）から（Ｃ）に示す。また、図３（Ａ）及び（Ｂ）に特定マイクロＲＮＡの発現量と１６Ｓ－ｒＤＮＡの発現量の相関を示す。 (Test Example 2)
Thirty-seven patients in the chorioamnionitis group (Blanc classification stage II or higher) were divided into a group in which chorioamnionitis-related microorganisms were detected in amniotic fluid (miCAM) and a group in which no chorioamnionitis-related microorganisms were detected (non-miCAM). hsa-miR-4535, hsa-miR-1915-5p and 16SrDNA were quantified. The results are shown in FIGS. 2 (A) to 2 (C). In addition, FIGS. 3 (A) and 3 (B) show the correlation between the expression level of the specific microRNA and the expression level of 16S-rDNA.
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例を、羊水から絨毛膜羊膜炎関連微生物が検出された群（ｍｉＣＡＭ）と検出されなかった群（ｎｏｎ－ｍｉＣＡＭ）に分割して、それぞれについてｈｓａ－ｍｉＲ－４５３５、ｈｓａ－ｍｉＲ－１９１５－５ｐ及び１６ＳｒＤＮＡを定量した。結果を図２（Ａ）から（Ｃ）に示す。また、図３（Ａ）及び（Ｂ）に特定マイクロＲＮＡの発現量と１６Ｓ－ｒＤＮＡの発現量の相関を示す。 (Test Example 2)
Thirty-seven patients in the chorioamnionitis group (Blanc classification stage II or higher) were divided into a group in which chorioamnionitis-related microorganisms were detected in amniotic fluid (miCAM) and a group in which no chorioamnionitis-related microorganisms were detected (non-miCAM). hsa-miR-4535, hsa-miR-1915-5p and 16SrDNA were quantified. The results are shown in FIGS. 2 (A) to 2 (C). In addition, FIGS. 3 (A) and 3 (B) show the correlation between the expression level of the specific microRNA and the expression level of 16S-rDNA.
図２（Ａ）から（Ｃ）に示すように、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量は、ｍｉＣＡＭによる子宮内感染により有意に上昇した。図３（Ａ）及び（Ｂ）に示すように、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量は、羊水中の１６Ｓ－ｒＤＮＡ量と強く相関した。
As shown in FIGS. 2 (A) to 2 (C), the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were significantly increased by intrauterine infection with miCAM. As shown in FIGS. 3A and 3B, the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were strongly correlated with the amount of 16S-rDNA in amniotic fluid.
（試験例３）
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例について、対象から採取した血液中の白血球（ＷＢＣ）数及びＣＲＰを測定し、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量との関係を検討した。結果を図４（Ａ）から（Ｃ）、及び図５（Ａ）から（Ｃ）に示す。 (Test Example 3)
For 37 patients in the chorioamnionitis group (Blanc classification stage II or higher), the number of leukocytes (WBC) and CRP in the blood collected from the subjects were measured, and hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were measured. The relationship with the expression level of was examined. The results are shown in FIGS. 4 (A) to (C) and FIGS. 5 (A) to (C).
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例について、対象から採取した血液中の白血球（ＷＢＣ）数及びＣＲＰを測定し、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量との関係を検討した。結果を図４（Ａ）から（Ｃ）、及び図５（Ａ）から（Ｃ）に示す。 (Test Example 3)
For 37 patients in the chorioamnionitis group (Blanc classification stage II or higher), the number of leukocytes (WBC) and CRP in the blood collected from the subjects were measured, and hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were measured. The relationship with the expression level of was examined. The results are shown in FIGS. 4 (A) to (C) and FIGS. 5 (A) to (C).
羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量は、母体の炎症所見の増悪により有意に上昇した。
The expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were significantly increased due to exacerbation of maternal inflammatory findings.
（試験例４）
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例について、分娩後の臍帯血中の白血球（ＷＢＣ）数及びＣＲＰを測定し、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量との関係を検討した。結果を図６（Ａ）から（Ｃ）、及び図７（Ａ）から（Ｃ）に示す。 (Test Example 4)
For 37 patients in the chorioamnionitis group (Blanc classification stage II or higher), the number of leukocytes (WBC) and CRP in the cord blood after delivery were measured, and hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were measured. The relationship with the expression level of was examined. The results are shown in FIGS. 6 (A) to (C) and 7 (A) to (C).
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）３７例について、分娩後の臍帯血中の白血球（ＷＢＣ）数及びＣＲＰを測定し、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量との関係を検討した。結果を図６（Ａ）から（Ｃ）、及び図７（Ａ）から（Ｃ）に示す。 (Test Example 4)
For 37 patients in the chorioamnionitis group (Blanc classification stage II or higher), the number of leukocytes (WBC) and CRP in the cord blood after delivery were measured, and hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were measured. The relationship with the expression level of was examined. The results are shown in FIGS. 6 (A) to (C) and 7 (A) to (C).
羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量は、胎児の炎症所見の増悪により有意に上昇した。
The expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid were significantly increased by exacerbation of fetal inflammatory findings.
（試験例５）
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）７例及び非絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩ以下）５例について、血液中及び羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量を、上記と同様にして測定した。血液中の発現量の測定結果を図８（Ａ）及び（Ｂ）に示す。また、血液（Ｂｌｏｏｄ）中及び羊水（ａｍｎｉｏｔｉｃ ｆｌｕｉｄ）中の発現量の関係を図９（Ａ）及び（Ｂ）に示す。 (Test Example 5)
For 7 cases of chorioamnionitis group (Blanc classification stage II or higher) and 5 cases of non-chorionic amnionitis group (Blanc classification stage I or lower), hsa-miR-4535 and hsa-miR-1915- in blood and amniotic fluid. The expression level of 5p was measured in the same manner as above. The measurement results of the expression level in blood are shown in FIGS. 8 (A) and 8 (B). In addition, the relationship between the expression levels in blood and amniotic fluid is shown in FIGS. 9 (A) and 9 (B).
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）７例及び非絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩ以下）５例について、血液中及び羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量を、上記と同様にして測定した。血液中の発現量の測定結果を図８（Ａ）及び（Ｂ）に示す。また、血液（Ｂｌｏｏｄ）中及び羊水（ａｍｎｉｏｔｉｃ ｆｌｕｉｄ）中の発現量の関係を図９（Ａ）及び（Ｂ）に示す。 (Test Example 5)
For 7 cases of chorioamnionitis group (Blanc classification stage II or higher) and 5 cases of non-chorionic amnionitis group (Blanc classification stage I or lower), hsa-miR-4535 and hsa-miR-1915- in blood and amniotic fluid. The expression level of 5p was measured in the same manner as above. The measurement results of the expression level in blood are shown in FIGS. 8 (A) and 8 (B). In addition, the relationship between the expression levels in blood and amniotic fluid is shown in FIGS. 9 (A) and 9 (B).
母体血液中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量は、子宮内感染の増悪により有意に上昇した。また血液中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量は、羊水中のそれらの発現量と相関した。
The expression levels of hsa-miR-4535 and hsa-miR-1915-5p in maternal blood were significantly increased due to exacerbation of intrauterine infection. The expression levels of hsa-miR-4535 and hsa-miR-1915-5p in blood correlated with their expression levels in amniotic fluid.
以上から、血液中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量を測定することで、子宮内感染を検出できることが分かる。
From the above, it can be seen that intrauterine infection can be detected by measuring the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in blood.
（試験例６）
分娩後の新生児の白血球数が５０００ｃｅｌｌｓ／μＬ未満又は２００００ｃｅｌｌｓ／μＬ以上の場合を胎児炎症反応症候群（ＦＩＲＳ）とし、それ以外の場合を正常（ｎｏｎ－ＦＩＲＳ）として５７例の妊婦を区分した。区分した妊婦の羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量、１６Ｓ－ｒＤＮＡ検出量並びにＩＬ－６濃度をそれぞれ測定した。結果を、表４および図１０に示す。表４には、各群における測定値の平均値と、９５％信頼区間と、Ｐ値を示し、図１０には測定値の分布を示す。なお、ＩＬ－６濃度はＦＩＲＳの指標の１つであり、ＥＬＩＳＡ－Ｋｉｔ（Ｑｕａｎｔｉｋｉｎｅ（Ｒ）ＨＳ，Ｒ＆Ｄ Ｓｙｓｔｅｍｓ Ｍｉｎｎｅａｐｏｌｉｓ，ＵＳＡ）を用い、４５０ｎｍにおける吸光度によって測定した。 (Test Example 6)
Fetal inflammatory reaction syndrome (FIRS) was defined as a postpartum neonatal leukocyte count of less than 5000 cells / μL or 20000 cells / μL or higher, and 57 pregnant women were classified as normal (non-FIRS) in other cases. The expression levels of hsa-miR-4535 and hsa-miR-1915-5p, the detected amount of 16S-rDNA, and the IL-6 concentration were measured in the amniotic fluid of the classified pregnant women, respectively. The results are shown in Table 4 and FIG. Table 4 shows the average value of the measured values in each group, the 95% confidence interval, and the P value, and FIG. 10 shows the distribution of the measured values. The IL-6 concentration is one of the indexes of FIRS, and was measured by absorbance at 450 nm using ELISA-Kit (Quantikine (R) HS, R & D Systems Minneapolis, USA).
分娩後の新生児の白血球数が５０００ｃｅｌｌｓ／μＬ未満又は２００００ｃｅｌｌｓ／μＬ以上の場合を胎児炎症反応症候群（ＦＩＲＳ）とし、それ以外の場合を正常（ｎｏｎ－ＦＩＲＳ）として５７例の妊婦を区分した。区分した妊婦の羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量、１６Ｓ－ｒＤＮＡ検出量並びにＩＬ－６濃度をそれぞれ測定した。結果を、表４および図１０に示す。表４には、各群における測定値の平均値と、９５％信頼区間と、Ｐ値を示し、図１０には測定値の分布を示す。なお、ＩＬ－６濃度はＦＩＲＳの指標の１つであり、ＥＬＩＳＡ－Ｋｉｔ（Ｑｕａｎｔｉｋｉｎｅ（Ｒ）ＨＳ，Ｒ＆Ｄ Ｓｙｓｔｅｍｓ Ｍｉｎｎｅａｐｏｌｉｓ，ＵＳＡ）を用い、４５０ｎｍにおける吸光度によって測定した。 (Test Example 6)
Fetal inflammatory reaction syndrome (FIRS) was defined as a postpartum neonatal leukocyte count of less than 5000 cells / μL or 20000 cells / μL or higher, and 57 pregnant women were classified as normal (non-FIRS) in other cases. The expression levels of hsa-miR-4535 and hsa-miR-1915-5p, the detected amount of 16S-rDNA, and the IL-6 concentration were measured in the amniotic fluid of the classified pregnant women, respectively. The results are shown in Table 4 and FIG. Table 4 shows the average value of the measured values in each group, the 95% confidence interval, and the P value, and FIG. 10 shows the distribution of the measured values. The IL-6 concentration is one of the indexes of FIRS, and was measured by absorbance at 450 nm using ELISA-Kit (Quantikine (R) HS, R & D Systems Minneapolis, USA).
表４から、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量を測定することで、ＦＩＲＳとｎｏｎ－ＦＩＲＳを、有意差をもって区別できたことが分かる。また、ＲＯＣ解析によりカットオフ値を設定して、ＦＩＲＳの診断精度を評価した。結果を表５に示す。なお、ＡＵＣはＲＯＣ曲線下の面積値であり、値が大きいほど診断精度に優れている。
From Table 4, it can be seen that FIRS and non-FIRS could be distinguished with a significant difference by measuring the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid. In addition, the cutoff value was set by ROC analysis to evaluate the diagnostic accuracy of FIRS. The results are shown in Table 5. The AUC is an area value under the ROC curve, and the larger the value, the better the diagnostic accuracy.
表５から、羊水中のｈｓａ－ｍｉＲ－４５３５及びｈｓａ－ｍｉＲ－１９１５－５ｐの発現量を測定することで、ＦＩＲＳを優れた精度で診断できたことが分かる。
From Table 5, it can be seen that FIRS could be diagnosed with excellent accuracy by measuring the expression levels of hsa-miR-4535 and hsa-miR-1915-5p in amniotic fluid.
（試験例７）
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）２８例及び非絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩ以下）１３例について、血液中及び羊水中のｈｓａ－ｍｉＲ－４５３５の発現量を、上記と同様にして測定した。血液中の発現量の測定結果を図１１に示す。また、血液（Ｂｌｏｏｄ）中及び羊水（ａｍｎｉｏｔｉｃ ｆｌｕｉｄ）中におけるｈｓａ－ｍｉＲ－４５３５の発現量の関係を図１２に示す。 (Test Example 7)
For 28 patients in the chorioamnionitis group (Blanc classification stage II or higher) and 13 cases in the non-chorionic amniotic inflammation group (Blanc classification stage I or lower), the expression levels of hsa-miR-4535 in blood and amniotic fluid are as described above. It was measured in the same manner. The measurement result of the expression level in blood is shown in FIG. In addition, the relationship between the expression levels of hsa-miR-4535 in blood and amniotic fluid is shown in FIG.
絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩＩ以上）２８例及び非絨毛膜羊膜炎群（Ｂｌａｎｃ分類ステージＩ以下）１３例について、血液中及び羊水中のｈｓａ－ｍｉＲ－４５３５の発現量を、上記と同様にして測定した。血液中の発現量の測定結果を図１１に示す。また、血液（Ｂｌｏｏｄ）中及び羊水（ａｍｎｉｏｔｉｃ ｆｌｕｉｄ）中におけるｈｓａ－ｍｉＲ－４５３５の発現量の関係を図１２に示す。 (Test Example 7)
For 28 patients in the chorioamnionitis group (Blanc classification stage II or higher) and 13 cases in the non-chorionic amniotic inflammation group (Blanc classification stage I or lower), the expression levels of hsa-miR-4535 in blood and amniotic fluid are as described above. It was measured in the same manner. The measurement result of the expression level in blood is shown in FIG. In addition, the relationship between the expression levels of hsa-miR-4535 in blood and amniotic fluid is shown in FIG.
Ｄｕｎｎ’ｓ ｍｕｌｔｉｐｌｅ ｃｏｍｐａｒｉｓｏｎｓ ｔｅｓｔの結果、ステージＩＩＩとステージＩＩの間に有意差（ｐ＝０．０００８）、ステージＩＩＩとステージ０－Ｉの間に有意差（ｐ＝０．０３６７）が認められた。また、血清中と羊水中の発現量に相関が認められた。
As a result of Dunn's multiple comparisons test, a significant difference (p = 0.0008) was observed between stage III and stage II, and a significant difference (p = 0.0367) was observed between stage III and stage 0-I. .. In addition, a correlation was observed between the expression levels in serum and amniotic fluid.
血液中のｈｓａ－ｍｉＲ－４５３５の発現量を用いて、子宮内感染の検出を行った。絨毛膜羊膜炎のステージＩＩＩとステージ０－Ｉ間のＲＯＣ曲線を図１３Ａ、ステージＩＩＩとステージ０－ＩＩ間のＲＯＣ曲線を図１３Ｂに示す。Ｙｏｕｄｅｎ Ｉｎｄｅｘ、感度、特異度を表６に示す。
Intrauterine infection was detected using the expression level of hsa-miR-4535 in blood. The ROC curve between stage III and stage 0-I of chorioamnionitis is shown in FIG. 13A, and the ROC curve between stage III and stage 0-II is shown in FIG. 13B. Table 6 shows Youden Index, sensitivity, and specificity.
以上から、血液中のｈｓａ－ｍｉＲ－４５３５の発現量を測定することで、重症の絨毛膜羊膜炎を精度よく診断できることが分かる。
From the above, it can be seen that severe chorioamnionitis can be accurately diagnosed by measuring the expression level of hsa-miR-4535 in blood.
日本国特許出願２０２０－０１３９３０号（出願日：２０２０年１月３０日）の開示はその全体が参照により本明細書に取り込まれる。本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書に参照により取り込まれる。
The entire disclosure of Japanese Patent Application No. 2020-01393 (filed on January 30, 2020) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are to the same extent as if the individual documents, patent applications, and technical standards were specifically and individually stated to be incorporated by reference. Incorporated herein by reference.
Claims (7)
- 対象に由来する試料におけるｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡの発現量を測定することを含む子宮内感染の検出方法。 A method for detecting an intrauterine infection, which comprises measuring the expression level of at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p in a sample derived from a subject.
- 前記マイクロＲＮＡの発現量と子宮内感染の存在とを関連付けることを含む請求項１に記載の検出方法。 The detection method according to claim 1, which comprises associating the expression level of the microRNA with the presence of an intrauterine infection.
- 前記マイクロＲＮＡの発現量が、定量ＰＣＲ、次世代シーケンサー、ＤＮＡマイクロアレイ、及び酵素免疫測定法からなる群から選択される少なくとも１種の方法で測定される請求項１又は請求項２に記載の検出方法。 The detection according to claim 1 or 2, wherein the expression level of the microRNA is measured by at least one method selected from the group consisting of quantitative PCR, next-generation sequencer, DNA microarray, and enzyme immunoassay. Method.
- 前記試料は、対象の血液、羊水及び尿からなる群から選択される少なくとも１種の体液に由来する試料である請求項１から請求項３のいずれか１項に記載の検出方法。 The detection method according to any one of claims 1 to 3, wherein the sample is a sample derived from at least one body fluid selected from the group consisting of target blood, amniotic fluid, and urine.
- 対象の羊水に由来する試料における１６ＳリボソームＲＮＡ遺伝子を検出することを更に含む請求項１から請求項４のいずれか１項に記載の検出方法。 The detection method according to any one of claims 1 to 4, further comprising detecting the 16S ribosomal RNA gene in a sample derived from the target amniotic fluid.
- ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡを含む子宮内感染の診断剤。 A diagnostic agent for intrauterine infection containing at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p.
- ｍｉＲ－４５３５及びｍｉＲ－１９１５－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡの少なくとも一部と相補的な配列を有するポリヌクレオチドを含む子宮内感染の検出用キット。 A kit for detecting intrauterine infection, which comprises a polynucleotide having a sequence complementary to at least a part of at least one microRNA selected from the group consisting of miR-4535 and miR-1915-5p.
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| WO2019208671A1 (en) * | 2018-04-25 | 2019-10-31 | 東レ株式会社 | Kit, device, and method for detecting bladder cancer |
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| WO2015190591A1 (en) * | 2014-06-13 | 2015-12-17 | 東レ株式会社 | Breast cancer detection kit or device, and method for detecting breast cancer |
| WO2019208671A1 (en) * | 2018-04-25 | 2019-10-31 | 東レ株式会社 | Kit, device, and method for detecting bladder cancer |
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| ALMUGHLLIQ, F. B. ET AL.: "Proteomic content of circulating exosomes in dairy cows with or without uterine infection.", THERIOGENOLOGY, vol. 114, 2018, pages 173 - 179, XP055845726 * |
| KIYOSHIMA, C. ET AL.: "MiR4535 and 1915-5p as promising biomarkers for the diagnosis of chorioamnionitis.", J. OBSTET. GYNAECOL. RES., vol. 46, no. 8, August 2020 (2020-08-01), pages 1556 * |
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| SON, G.-H. ET AL.: "MicroRNA-548 regulates high mobility group box 1 expression in patients preterm birth and chorioamnionitis .", SCIENTIFIC REPORTS, vol. 9, 2019, pages 19746, XP055845724 * |
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