阅读说明：本技术 ***微生物在复发性流产中的应用 (Application of vaginal microorganism in recurrent abortion ) 是由 郭燕华 李平 朱小凤 于 2020-07-08 设计创作，主要内容包括：本发明公开了阴道微生物在复发性流产中的应用,所述微生物为Rhodoluna。本发明的微生物标志物预测复发性流产敏感性高,通过检测微生物标志物的丰度,可用于警示受试者,进一步通过调整饮食或医疗介入减少风险。(The invention discloses application of vaginal microorganism in recurrent abortion, wherein the microorganism is Rhodoluna. The microbial marker provided by the invention has high sensitivity in predicting recurrent abortion, can be used for warning a subject by detecting the abundance of the microbial marker, and further reduces the risk by adjusting diet or medical intervention.)

1. A microbial marker of recurrent miscarriage, which is Rhodoluna.

2. A reagent for detecting the microbial marker of claim 1.

3. Use of the microbial marker of claim 1 or the reagent of claim 2 for constructing a model for predicting the risk of recurrent miscarriage, for the manufacture of a product for diagnosing recurrent miscarriage.

4. Use according to claim 3, wherein the product comprises a chip, a kit, a strip.

5. Use according to claim 3, wherein the input variable of the model is the abundance of the microbial marker of claim 1.

6. The use according to claim 5, wherein the determination method of the abundance of the microbial marker comprises any one or more of metagenomic sequencing, 16S sequencing or qPCR quantitative detection.

7. A product for diagnosing recurrent miscarriage, which comprises the agent of claim 2.

8. The product of claim 7, wherein the reagents comprise primers, probes, antisense oligonucleotides, aptamers, or antibodies specific for detecting Rhodoluna.

Use of Rhodoluna in the preparation of a medicament or food for the prevention of recurrent abortion.

10. Use according to claim 9, wherein the medicament or food product comprises an agent that promotes increased abundance of Rhodoluna.

Technical Field

The invention belongs to the technical field of biology, and relates to application of vaginal microorganisms in recurrent abortion.

Background

Recurrent abortion (RSA) is a complex disease, meaning two or more pregnancy failures that occur within 20 weeks (Kim J, Jeon Y J, Lee B E, et al. Association of a methionine synthesis and a thymidylate synthesis with a genetic regenerative prediction loss [ J ]. Fertil Steril,2013,99(6):1674 1680.). The incidence in women of childbearing age is 1% -5%, and the probability of spontaneous abortion in RSA women after pregnancy is as high as 70% -80% (Wu M, Liu P, Cheng L.Galetin-1 reduction and changes in T regulation bacteria mass botanical efficacy rollers in tissues with unexplained recurrent spontaneous abortion [ J ]. Int J Clin ExpPathol,2015,8(2): 1973-. RSA has complex and various causes, and 50% of RSA has unknown causes except genetic factors, endocrine disorders, anatomical abnormalities, infection, autoimmunity, sperm quality, lifestyle, psychology and environment, and is called as unexplained recurrent abortion (URSA). The risk of recurrence of URSA increases with the number of abortions (Gynaeocolsists R C O. the initiation and treatment of duplicates with a reliable first and second third mistrariae (Green-top Guidelin eNO.17) [ J ]. Royal College of obstertriciae and Gynaeocolsists, 2011.), which has serious influence on families and society, but currently, there is no index for effectively predicting the occurrence of URSA clinically.

Microbiology is a discipline that studies the sum of all microorganisms in a particular environment. At present, it is widely believed that there are many symbiotic microbial flora in human body, the total content of the genome is 10 times of the total content of the genome in human body, the method for culturing the microorganisms can only detect the diversity of a small part of microorganisms existing in the human body, and most of the microorganisms cannot be identified by the traditional culture method, but are closely related to the health and diseases of human beings. The national institutes of health of the United states has incorporated human microbiome programs into the study, the main study including gastrointestinal microorganisms, oral microorganisms, nasal microorganisms, skin microorganisms, and urogenital microorganisms (Nguyen L D N, Viscomiosi E, Delhaes L. Long microorganisms: an emulsifying field of the human respiratory microorganisms [ J ]. Frontiers in Microbiology 2015,6: 89.). Currently, the research on the microbiome characteristics of patients with recurrent abortion is very limited, and the application discovers microbial flora presenting significant difference by researching the microbiome characteristics of patients with recurrent abortion and normal pregnant subjects so as to realize the diagnosis of recurrent abortion.

Disclosure of Invention

Aiming at the defects and actual needs of the prior art, the invention aims to provide a microbial marker for evaluating recurrent abortion and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a microbial marker for recurrent abortion, said microbial marker being Rhodoluna.

In a second aspect, the present invention provides a reagent for detecting a microbial marker according to the first aspect.

In a third aspect, the invention provides a use of the microbial marker of the first aspect or the reagent of the second aspect, which comprises constructing a model for predicting the risk of recurrent abortion and preparing a product for diagnosing recurrent abortion.

Preferably, the product comprises a chip, a kit and a test paper.

Preferably, the input variable of the model is the abundance of the microbial marker of the first aspect.

Preferably, the method for determining the abundance of the microbial marker comprises any one or more of metagenomic sequencing, 16S sequencing or qPCR quantitative detection.

In a fourth aspect, the invention provides a product for diagnosing recurrent spontaneous abortion, said product comprising the agent of the second aspect.

Preferably, the reagent comprises a primer, probe, antisense oligonucleotide, aptamer or antibody that detects specificity of Rhodoluna.

In a fifth aspect, the invention provides the use of Rhodoluna in the manufacture of a medicament or food product for the prevention of recurrent abortion.

Preferably, the medicament or food product comprises an agent that promotes increased abundance of Rhodoluna.

The invention has the advantages and beneficial effects that:

the invention discovers that Rhodoluna is related to recurrent abortion for the first time, and the abundance of Rhodoluna in patients with unexplained recurrent abortion is remarkably reduced, which indicates that Rhodoluna can be used as a detection target for diagnosis and prediction of recurrent abortion.

The invention provides a product for diagnosing recurrent abortion, which can diagnose in the early stage of diseases, realize early warning and improve the life quality of patients.

Drawings

FIG. 1 is a graph of the abundance of Rhodoluna in patients with unexplained recurrent abortion.

FIG. 2 is a graph of the diagnostic efficacy of Rhodoluna as a test variable.

Detailed Description

The invention firstly finds the correlation between bacteria and clinical medical indexes of the unexplained recurrent abortion by taking a normal pregnant population and a population suffering from the unexplained recurrent abortion as objects, and provides an early diagnosis technology of the unexplained recurrent abortion on the basis of the correlation. In order to evaluate whether the microbial flora can be used as a prediction factor for the unexplained recurrent abortion, the microbial flora related to the recurrent abortion is found by collecting vaginal secretions of a normal population and the unexplained recurrent abortion and comprehensively analyzing 16S rRNA sequencing, metagenome sequencing and quantitative polymerase chain reaction results aiming at a specific flora, and the invention firstly finds that the abundance of Rhodoluna presents significant difference in the recurrent abortion and the normal population by 16S rRNA sequencing, thereby indicating that Rhodoluna can be used as a biomarker for diagnosing the recurrent abortion.

In an embodiment of the invention, the present invention diagnoses recurrent miscarriage by: detecting one or more nucleic acid fragments corresponding to a species diagnostically associated with recurrent abortion in a nucleic acid sample from the individual. In a particular embodiment, a nucleic acid fragment corresponding to Rhodoluna is detected. In practicing the methods described herein, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used, and are well known.

The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "OTU" (operational taxonomic unit) herein refers to the terminal leaf (terminal leaf) in a phylogenetic tree, defined by a specific genetic sequence and the totality of sequences sharing sequence identity with that sequence at the family, genus, species or strain level. The specific genetic sequence may be a 16S sequence or a portion of a 16S sequence, or may be a function-conserved housekeeping gene that is present widely throughout the eubacteria kingdom. The OTUs have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity therebetween. OTUs are often defined by comparing sequences between organisms, sequences with less than 95% sequence identity are not considered to form part of the same OTU, however, as used herein, an OTU identifier may comprise sequences with sequence identity of 0 to 100%, 25% to 100% and 50% to 100%, preferably 70% to 100%, 75% to 100%, 77% to 100%, 80% to 100%, 81% to 100%, 82% to 100%, 83% to 100%, 84%, to 100%, more preferably 85% to 100%, 86% to 100%, 87% to 100%, 88% to 100%, 89% to 100%, 90% to 100%, 91% to 100%, 92% to 100%, 93% to 100%, 94% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, and 99% to 100%.

Herein, OTU represents bacteria that have or have not been previously classified under the names of genus, species and/or strain, that is, OTU or OTU identifies an order, family, genus, species or strain equivalent to bacteria, and the content of bacteria represented by OTU is analyzed by OUT clustering in the process of bioinformatics analysis.

The "V1-V9 region" of 16S rRNA refers to the first to ninth hypervariable regions of the 16S rRNA gene for genotyping of bacterial samples, as is well known to the skilled artisan. In some embodiments, the microbial marker is characterized by at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions. In some embodiments, the V3 and V4 regions are used to characterize microbial markers.

A probe for detecting a microbial marker herein is an oligonucleotide that "specifically hybridizes" to a microbial marker polynucleotide, having a sequence sufficiently complementary to allow hybridization (sometimes referred to as "substantial complementarity") to a nucleotide sequence of interest under predetermined conditions commonly used in the art. In particular, the expression includes hybridization of an oligonucleotide to a substantially complementary sequence contained within a single-stranded DNA or RNA molecule as described herein, substantially excluding hybridization of the oligonucleotide to single-stranded nucleic acids of non-complementary sequence. The specific length and sequence of the probes and primers depends on the complexity of the nucleic acid target desired as well as the reaction conditions (e.g., temperature and ionic strength). In general, hybridization conditions are known in the art as stringent hybridization conditions. "stringent" refers to conditions under which a nucleotide sequence is capable of binding to a related or non-specific sequence. For example, high temperatures and low salts increase stringency, causing dissociation of non-specific binding or low melting temperature binding. In some embodiments, the oligonucleotide complementary to the microbial marker polynucleotide is at least 95%, 96%, 97%, 98%, 99% or 100% complementary to the microbial marker polynucleotide.

As an alternative embodiment, the method for diagnosing recurrent miscarriage in a subject comprises: analyzing nucleic acid of a test sample of a subject; detecting the level of one or more microorganisms and/or OTUs in the nucleic acid of the test sample; diagnosing the subject as being at risk of developing recurrent miscarriage when the level of one or more microorganisms and/or OTUs in the test sample is lower than the control sample; preferably, the microbial marker is Rhodoluna.

In another embodiment, a method for diagnosing recurrent miscarriage in a subject comprises: obtaining a vaginal secretion sample of a subject; processing a vaginal secretion sample to obtain 16S rRNA gene sequence data; detecting the level of one or more microorganisms and/or OTU in a vaginal secretion sample comprising analyzing 16S rRNA gene sequence data with bioinformatics software; and diagnosing the subject as being at risk of developing recurrent abortion when the level of the one or more microorganisms and/or OTU in the vaginal secretion sample is lower than the control sample; preferably, the microbial marker is Rhodoluna.

As used herein, "significant difference" refers to the log of the level of one or more microorganisms or OTUs in a test sample as compared to a control sample₂The fold difference is changed by at least about 1.2 fold. The term "altering" includes increasing or decreasing the aqueous nature of the microorganism or OTU in the test sample as compared to the control sample. In some embodiments, the change in the level of one or more microorganisms or one or more OTUs between the test sample and the control sample can be about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.9 fold, and 5 fold, or more in log2 fold difference relative to the control sample.

In some embodiments, the level of one or more microorganisms or one or more OTUs in a test sample is log₂The fold difference is about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold or 5 fold or more compared to the control sample.

In some embodiments, the level of one or more microorganisms or one or more OTUs in a test sample is log₂The fold difference is about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold or 5 fold or more compared to the control sample.

The term "sample" or "test sample" as used herein refers to any liquid or solid material containing nucleic acids. In suitable embodiments, the test sample is obtained from a biological source (i.e., a "biological sample"), such as cells in culture, or is a tissue sample from an animal, and most preferably from a human. In an exemplary embodiment, the sample is vaginal discharge.

The methods and products of the present invention can be used to detect nucleic acids associated with various bacteria using biological samples obtained from individuals. The nucleic acid (DNA or RNA) may be isolated from the sample according to any method known to those skilled in the art. The biological sample may be obtained by standard procedures and may be used immediately or may be stored for later use under conditions appropriate for that type of biological sample.