阅读说明：本技术 多囊卵巢综合征肠道菌群生物标志物筛选及其应用 (Screening and application of polycystic ovarian syndrome intestinal flora biomarker ) 是由 徐建华 汪湾 刘冬冬 于 2020-11-05 设计创作，主要内容包括：本发明公开了一种基于肠道菌群的多囊卵巢综合征(PCOS)生物标志物组合,包括多囊卵巢综合征肠道菌群检测、多囊卵巢综合征患病风险预测、多囊卵巢综合征预后监测中的应用。基于肠道菌群的多囊卵巢综合征生物标志物组合其特征在于,包括：1)生物标志物1：Ruminococcus-bromii；生物标志物2：Gordonibacter-pamelaeae；生物标志物3：Eesulfovibrio-piger；生物标志物4：Eubacterium-ventriosum；生物标志物5：Prevotella-stercorea。2)基于肠道菌群的多囊卵巢综合征生物标志物组合诊断模型,具有灵敏性高、特异性强、结果判定简单的优势。(The invention discloses a polycystic ovarian syndrome (PCOS) biomarker combination based on intestinal flora, which comprises applications in polycystic ovarian syndrome intestinal flora detection, polycystic ovarian syndrome risk prediction and polycystic ovarian syndrome prognosis monitoring. The intestinal flora-based polycystic ovarian syndrome biomarker combination is characterized by comprising the following components in parts by weight: 1) biomarker 1: ruminococcus _ bromi; biomarker 2: gordonibacter _ pamelaeae; biomarker 3: eesulfovibrio _ piger; biomarker 4: eubacterium _ ventriosum; biomarker 5: prevotella _ sterorea. 2) The polycystic ovary syndrome biomarker combined diagnosis model based on the intestinal flora has the advantages of high sensitivity, strong specificity and simple result judgment.)

1. An intestinal flora-based polycystic ovarian syndrome biomarker panel for providing relative abundance information, comprising a plurality of:

biomarker 1: ruminococcus _ bromi;

biomarker 2: gordonibacter _ pamelaeae;

biomarker 3: eesulfovibrio _ piger;

biomarker 4: eubacterium _ ventriosum;

biomarker 5: prevotella _ sterorea.

2. The intestinal flora-based polycystic ovarian syndrome biomarker panel of claim 1, wherein the relative abundance information provided by the biomarker panel is used to compare to a reference value.

3. The intestinal flora-based polycystic ovarian syndrome biomarker panel of claim 1, wherein the relative abundance information of the biomarkers 1-5 is provided based on gene sequences for which abundance calculations can be performed.

4. Use of the intestinal flora-based polycystic ovary syndrome biomarker combination as defined in claim 1 as a detection target or a detection target in preparation of a detection kit.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an intestinal flora marker and application thereof in detection of intestinal flora of polycystic ovarian syndrome, prediction of polycystic ovarian syndrome suffering risk and prognosis monitoring of polycystic ovarian syndrome.

Background

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of childbearing age, and is characterized mainly by irregular menstrual cycle, hyperandrogenism, anovulation and polycystic ovary morphology. Many studies have shown that cardiovascular and cerebrovascular and metabolic related diseases, type II diabetes, and even endometrial cancer can result if patients with PCOS are not treated effectively, severely affecting the quality of life of the patients. Recent epidemiological surveys in China show that the incidence of PCOS in 19-45 year old women is 5-10%, while the incidence of PCOS in foreign countries can be as high as 10-22%.

The relationship between the microbial community in human intestinal tract and host is very close, and according to research, the imbalance of the intestinal flora is closely related to various clinical diseases. In recent years, more and more studies have shown that gut flora dysregulation is closely related to the occurrence and development of PCOS. At present, the diagnosis of PCOS is an exclusive diagnosis, mainly relying on clinical symptoms and ultrasound detection. The clinical manifestations of patients have large heterogeneity, and are easily confused with other diseases, and many factors such as lack of experience of sonographers are not favorable for accurate clinical diagnosis and guidance of related treatments. Therefore, the search for the more stable PCOS intestinal flora biomarker has important clinical application value.

Disclosure of Invention

The invention aims to provide a detection method of PCOS from the perspective of intestinal flora, and provides a reference for auxiliary diagnosis and curative effect monitoring of PCOS.

The invention is realized by adopting the following technical scheme:

a gut flora-based PCOS biomarker combination for providing relative abundance information, comprising a plurality of:

biomarker 1: ruminococcus _ bromi;

biomarker 2: gordonibacter _ pamelaeae;

biomarker 3: eesulfovibrio _ piger;

biomarker 4: eubacterium _ ventriosum;

biomarker 5: prevotella _ sterorea.

The relative abundance information provided by the biomarker combinations is used to compare to a reference value.

The relative abundance information of the biomarkers 1-5 is provided based on gene sequences for which abundance calculation can be performed.

The PCOS biomarker combination based on the intestinal flora is used as a detection target or an application of a detection target in preparation of a detection kit.

The method for screening the PCOS biomarker based on the intestinal flora comprises the following steps:

1) collecting samples: collecting feces samples of PCOS patients and healthy people, rapidly transferring to-80 ℃ for storage, and extracting DNA;

2) metagenomic sequencing

3) The data analysis calculates the relative abundance of the species;

4) screening characteristic variables by using an R-bag random forest model, carrying out 1000 times of cross validation to obtain candidate bacteria, calculating PCOS (prestressed concrete cylinder operating System) morbidity risk of each individual by using species relative abundance screened by an RF (radio frequency) model, drawing an ROC (rock characteristic curve), calculating AUC (AUC) as a judgment model efficiency evaluation parameter, selecting a combination with the combination number of markers being less than 30 and judging the combination with the best efficiency, outputting an importance index of each species in the model, wherein the higher the importance index is, the higher the importance index represents that the importance of the markers for judging PCOS and non-PCOS is.

In the sample set, the sample subjects included 23 PCOS patients and 23 healthy individuals.

The method of using the above marker, i.e., the method of diagnosing whether a subject has PCOS or predicting the risk of whether a subject has PCOS, comprises:

1) collecting a sample from a subject;

2) determining relative abundance information of biomarkers in the sample obtained in step 1);

3) comparing the relative abundance information described in step 2) to a reference data set or reference value. The method can be used for disease diagnosis of polycystic ovarian syndrome, and can be used for scientific research or other non-disease diagnosis such as enrichment of personal genetic information and enrichment of genetic information base. The relative abundance information of each biomarker in the test subject is compared to a reference data set or reference value to determine whether the subject has, or is predicted to be at risk for having, PCOS.

The reference data set includes relative abundance information of biomarkers in samples from a plurality of PCOS patients and a plurality of healthy controls.

The reference value in the present invention refers to a reference value or normal value of a healthy control. It is known to those skilled in the art that when the sample volume is sufficiently large, a range of normal values (absolute values) for each biomarker in the sample can be obtained using detection and calculation methods well known in the art. When detecting the level of a biomarker using an assay, the absolute value of the level of the biomarker in the sample can be directly compared to a reference value to assess risk of disease and to diagnose or early diagnose PCOS, optionally, statistical methods can be included.

The step of comparing the relative abundance information with the reference data set in step 2) further comprises executing a multivariate statistical model to obtain the prevalence probability. The rapid and efficient detection can be realized by utilizing the multivariate statistical model. Specifically, the multivariate statistical model is a random forest model.

The prevalence probability being greater than a threshold value indicates that the subject has or is at risk of having PCOS or a related disease. Specifically, the threshold is 0.32.

The relative abundance information of the biomarkers in the step 2) is obtained by using a sequencing method, and further comprises the following steps: isolating a nucleic acid sample from the sample of the subject, constructing a DNA library based on the nucleic acid sample obtained, and sequencing the DNA library to obtain a sequencing result; and comparing the sequencing result to a reference gene set based on the sequencing result to determine relative abundance information of the biomarker.

According to an embodiment of the invention, at least one of SOAP2 and MAQ can be used for comparing a sequencing result with a reference gene set, so that the comparison efficiency can be improved, and the PCOS detection efficiency can be improved. According to the embodiment of the invention, multiple (at least two) biomarkers can be detected simultaneously, and the efficiency of PCOS detection can be improved.

The reference gene set comprises a step of performing metagenomic sequencing from a plurality of PCOS patients and a plurality of healthy control samples to obtain a non-redundant gene set, and then combining the non-redundant gene set and an intestinal microorganism gene set to obtain the reference gene set. The reference gene set in the present invention may be an existing gene set, such as an existing reference gene set of a disclosed gut microorganism; or metagenome sequencing can be carried out on a plurality of PCOS and a plurality of healthy control samples to obtain a non-redundant gene set, and then the non-redundant gene set is combined with the intestinal microorganism gene set to obtain the reference gene set, so that the obtained reference gene set has more comprehensive information and more reliable detection results.

The set of non-redundant genes is explained as generally understood by those skilled in the art, and is simply the set of genes remaining after removal of the redundant genes. Redundant genes generally refer to multiple copies of a gene that appear on a chromosome.

In particular, the sample is a stool sample. The sequencing method is performed by a second generation sequencing method.

The sequencing method is performed by a sequencing device selected from Hiseq 2000. Therefore, the characteristics of high-throughput and deep sequencing of the sequencing devices can be utilized, so that the subsequent sequencing data can be analyzed, and particularly, the accuracy and the accuracy of statistical test are facilitated.

The invention provides application of the PCOS biomarker combination based on the intestinal flora as a detection target or a detection target in preparing a detection kit, wherein the kit is used for diagnosing whether a subject suffers from PCOS or predicting whether the subject suffers from PCOS risk.

That is, the present invention provides a kit comprising reagents for detecting biomarkers, with which the relative abundance of these markers in the gut flora can be determined, and thus, from the obtained relative abundance values, whether a subject suffers from or is susceptible to PCOS, and the efficiency of treatment for monitoring patients with PCOS can be determined.

Compared with the prior art, the invention has the following beneficial technical effects:

the present invention is based on the discovery and recognition of the following facts and problems:

feces are metabolites of the human body, and include not only metabolites of the human body but also intestinal microorganisms closely related to changes in metabolism and immunity of our body and metabolic functions. The research on the feces shows that obvious difference exists in the composition of the intestinal flora of PCOS patients and healthy people, and the PCOS can be accurately evaluated for the risk of diseases and diagnosed at early stage. According to the invention, based on comparison and analysis of the PCOS patient and healthy population intestinal flora, a plurality of related intestinal microorganisms are obtained, and the PCOS patient can be accurately subjected to disease risk assessment and early diagnosis by combining high-quality PCOS population and healthy population species relative abundance data as a training set. Compared with the conventional diagnosis method, the method has the characteristics of convenience and rapidness.

The PCOS-related biomarkers proposed by the present invention are valuable for early diagnosis. First, the markers of the present invention have high specificity and sensitivity. Second, analysis of stool ensures accuracy, safety, affordability, and patient compliance. And samples of stool are transportable. Polymerase Chain Reaction (PCR) -based assays are comfortable and non-invasive, so one would be more likely to participate in a given screening procedure. Third, the markers of the present invention can also be used as a tool for therapy monitoring of PCOS patients to detect response to therapy. For the reason of abundance measurement, the combination of 5 markers of the present invention is particularly suitable for the case of measuring abundance based on the marker gene alignment method.

Drawings

FIG. 1 analysis of LEfSe from PCOS patients and healthy controls at the level according to one embodiment of the present invention.

Figure 2 Receiver Operating Characteristics (ROC) Curve and Area under the Curve (AUC) based on a random forest model (5 gut markers) consisting of PCOS patients and healthy controls according to an embodiment of the present invention.

Detailed Description

Example 1

1.1 sample Collection

23 patients with polycystic ovarian syndrome and 23 healthy female controls who visit the gynecologic outpatient service in the traditional Chinese medicine institute in Guangdong province in 2018, 3-2019 and 5 are selected, feces samples are collected and then are rapidly transferred to-80 ℃ for storage, and DNA is extracted.

1.2 metagenomic sequencing and Assembly

The extracted DNA samples were used to construct a sequencing library for bidirectional (Paired-end) metagenomic sequencing (insert 350bp, read length 100bp) on the Illumina HiSeq2000 sequencing platform. The data generated by sequencing was filtered (quality-controlled) to remove adapter contaminating sequences, low quality sequences and host genome contaminating sequences, resulting in high quality sequencing fragments (reads).

1.3 analysis of marker strains between groups

Linear discriminant analysis was performed on the basis of Kruskal-Wallis rank sum test, plotted by LEfSe analysis software, as shown in figure one: when LDA > 2, there are four species that have the greatest impact on PCOS and health control groups. Wherein the abundance of Bacteroides vulgatus and Escherichia coli in PCOS is significantly higher than that of the healthy control group, and the relative abundance of Enterobacter and unclassified bacteria is significantly lower than that of the healthy control group.

1.4 screening of potential biomarkers for PCOS development Using random forest (ROC/AUC)

Screening characteristic variables by using an R-bag random forest model, carrying out cross validation for 1000 times to obtain 5 bacteria, calculating PCOS (PCOS) morbidity risk of each individual by using species relative abundance screened by an RF (radio frequency) model, drawing an ROC (ROC) curve, and calculating AUC (AUC) as a judgment model efficiency evaluation parameter. When 5 species were diagnosed, as shown in FIG. two: the AUC is 83.29%, and the combination has better diagnostic efficacy.