A coronary heart disease intestinal archaea marker composition and its application

Abstract

The present invention relates to the technical field of coronary heart disease-related markers, and more particularly to a coronary heart disease intestinal archaea marker composition and its use. The marker composition comprises nine archaea: Candidatus Diapherotrites, Candidatus Altiarchaeales unclassified, Candidatus Diapherotrites unclassified, Hadesarchaea unclassified, Methanothermococcus, Thermococcus, Halorubrum hochstenium, Methanosarcina sp MTP4, and Methanothermococcus okinawensis. The present invention utilizes nine archaea to jointly diagnose coronary heart disease, demonstrating excellent diagnostic efficacy and providing an effective and reliable method for the diagnosis and early prevention of coronary heart disease.

Description

Technical Field

[0001] The present invention relates to the technical field of coronary heart disease-related markers, and in particular to a coronary heart disease intestinal archaea marker composition and application thereof. Background Art

[0002] Coronary atherosclerotic heart disease, often referred to as coronary heart disease, is a type of ischemic heart disease. The coronary arteries are the arteries responsible for supplying blood to the heart. When the coronary arteries are narrowed or blocked due to plaques formed by the accumulation of cholesterol and other substances, it can lead to myocardial ischemia, hypoxia or necrosis, triggering chest pain, chest tightness and other discomforts. Coronary heart disease generally has no obvious symptoms, which manifest as abnormal changes during exercise treadmill electrocardiograms. As plaques accumulate, the narrowing of the lumen prevents blood from flowing smoothly, leading to chest pain or discomfort, called angina pectoris. Severe cases of the disease can also lead to heart failure and arrhythmias. In addition, other possible symptoms include palpitations, shortness of breath, dizziness, etc.

[0003] The diagnosis of coronary heart disease is mainly based on medical history, clinical symptoms, coronary angiography, etc. under the guidance of a doctor. However, these methods have some limitations. For example, relying on medical history and clinical symptoms may lead to the risk of misdiagnosis or missed diagnosis. In addition, since the early symptoms of coronary heart disease are not obvious, many people do not know that they have coronary heart disease in the early stages. Coronary angiography, as an invasive and intrusive examination method, is not suitable for large-scale clinical screening and early screening. In addition, the development of coronary heart disease is related to multiple factors such as genetics, environment, and lifestyle. The diagnostic method of a single indicator cannot fully assess the patient's risk.

[0004] Archaea, or archaea, are a class of single-celled microorganisms that exist in extreme environments, such as high temperatures, high salt concentrations, and low oxygen levels. In recent years, a growing body of research has demonstrated a close relationship between intestinal archaea and human health, particularly the development and progression of asthma and colorectal cancer. Changes in the species and abundance of intestinal archaea can lead to imbalances in the gut microbiome, potentially causing metabolic disorders and even impacting cardiovascular health.

[0005] However, relatively little research exists on the association between intestinal archaea and coronary heart disease. Despite advances in existing technologies, there are still many challenges and limitations in diagnosing coronary heart disease. Therefore, further research into the association between intestinal archaea and coronary heart disease, and exploring the potential application of intestinal archaea in the diagnosis of coronary heart disease, is expected to provide new insights and methods for the early diagnosis and prevention of coronary heart disease.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a coronary heart disease intestinal archaea marker composition and its application. The present invention uses 9 types of archaea to jointly diagnose coronary heart disease with excellent diagnostic efficacy, providing an effective and reliable means for the diagnosis and early prevention of coronary heart disease.

[0008] In a first aspect, the present invention provides a coronary heart disease intestinal archaea marker composition, which includes 9 types of archaea: Candidatus_Diapherotrites, Candidatus_Altiarchaeales_unclassified, Candidatus_Diapherotrites_unclassified, Hadesarchaea_unclassified, Methanothermococcus, Thermococcus, Halorubrum_hochstenium, Methanosarcina_sp_MTP4 and Methanothermococcus_okinawensis.

[0009] The present invention specifically screened the intestinal flora of patients with coronary heart disease and healthy controls through discovery cohorts and validation cohorts, and for the first time found that there were significant differences in the archaeal structure of patients with coronary heart disease compared with healthy controls. Among them, 9 enriched archaea included 4 methanogens, 2 halophilic archaea, 2 thermococci and 1 Heimdall archaea. The diagnostic performance of the 9 enriched archaea was further evaluated by the receiver operating characteristic curve (ROC). The AUC values ​​of the discovery cohort and validation cohort were found to be 0.73 and 0.75, respectively, indicating that the 9 archaeal species biomarkers proposed in the present invention have good diagnostic value for assessing the risk of individual coronary heart disease.

[0010] Therefore, the nine archaeal species biomarkers of the present invention provide an effective and reliable means for the diagnosis and early prevention of coronary heart disease, which is not only beneficial for the diagnosis and typing of coronary heart disease, but also improves the knowledge and understanding of the clinical pathology of coronary heart disease, and has potential value in the treatment and prevention of coronary heart disease.

[0011] In the second aspect, the present invention also discloses the use of the above-mentioned coronary heart disease intestinal archaea marker composition in the preparation of a product for diagnosing coronary heart disease, which should also fall within the scope of protection of the present invention. Specifically, the product is tested by measuring the relative abundance of 9 archaea in the test sample of the subject. The greater the difference in expression abundance of the 9 archaea compared with the healthy control group, the higher the individual's risk of coronary heart disease. At the same time, ROC curve statistical analysis is performed to evaluate whether the subject has coronary heart disease by using the ROC curve and the area under the curve AUC.

[0012] Specifically, archaea, as a type of microorganism, mainly exist in the intestines. Therefore, the test samples provided by the subjects are specifically feces or intestinal contents. Archaea can exist stably in the intestines, so they have high feasibility as biomarkers and are expected to become a powerful tool for the diagnosis of coronary heart disease.

[0013] As a preferred embodiment of the present technical solution, the product is a detection chip, a detection test paper or a detection kit.

[0014] As a preferred embodiment of the present technical solution, the product further comprises a carrier recording the following judgment criteria: the lower the content of the nine types of archaea in the test sample, the higher the risk of coronary heart disease.

[0015] As a preferred embodiment of the present technical solution, the product also includes reagents required for extracting DNA from the sample to be tested and measuring the relative abundance of 9 types of archaea.

[0016] In addition, the product of the present invention can also be a device for assessing the risk of developing coronary heart disease, which includes a detection unit and a data analysis unit. The detection unit includes any reagent material that can obtain information about the characteristic bacteria in the intestinal flora of the patient's sample to be tested, specifically including reagent materials for detecting DNA data from fecal samples. The data analysis unit is mainly used to analyze and process the detection results of the detection unit. Specifically, the analysis and processing process includes:

[0017] The microbial community DNA extracted from fecal samples is used to construct a DNA library through steps such as DNA fragment breakage, end repair, adapter ligation, and library amplification;

[0018] After the DNA library passed the test, the quality control software trimmomatic-0.33 was used to remove the adapter sequence and paired reads with a length of less than 50 based on the Illu mina Hiseq PE150 platform, and the original metagenomic sequencing data was obtained after filtering;

[0019] Fastq software (Vension 0.19.7) was used to control the quality of the raw sequencing data. After quality control and filtering, high-quality, usable metagenomic sequencing data were obtained. Metaphlan3 (version 3.0.13) software was used to align the sample sequencing data with the species marker gene database mpa_v30_CHOCOPhlAn_201901. Based on the alignment results, the species and relative abundance information of archaea and bacteria at different taxonomic levels (including kingdom, phylum, class, order, family, genus, and species) and subsequent biological information were obtained.

[0020] The intestinal flora characteristic data was determined based on the relative abundance information of differential bacteria.

[0021] In a third aspect, the present invention also discloses the application of the above-mentioned intestinal archaea marker composition for coronary heart disease in the preparation of products for prognosis assessment of coronary heart disease, that is, the 9 archaeal species biomarkers of the present invention can also be used to prepare coronary heart disease prognosis assessment detection tools to effectively evaluate the patient's response to treatment and the risk of future disease.

[0022] Fourthly, the present invention also discloses the use of the above-mentioned coronary heart disease intestinal archaea marker composition in the preparation of products for monitoring the progression of coronary heart disease, that is, the 9 archaeal species biomarkers of the present invention can also be used to prepare detection tools for coronary heart disease monitoring, so as to assist in identifying the progression of the disease or possible future progression, thereby achieving precise treatment of the disease.

[0023] In a fifth aspect, the present invention also discloses the application of the above-mentioned coronary heart disease intestinal archaea marker composition in the screening of drugs for the prevention or treatment of coronary heart disease. Specifically, the drug screening includes determining whether the drug is selected by judging the degree of targeting promotion of the drug on 9 types of archaea.

[0024] The coronary heart disease intestinal archaea marker composition of the present invention has at least the following beneficial effects:

[0025] 1. The present invention specifically screened the intestinal flora of patients with coronary heart disease and healthy controls through discovery cohorts and validation cohorts, and found for the first time that there were significant differences in the archaeal structure of patients with coronary heart disease compared with healthy controls. Among them, 9 enriched archaea included 4 methanogens, 2 halophilic archaea, 2 thermococci, and 1 Heimdall archaea. The diagnostic performance of the 9 enriched archaea was further evaluated by the receiver operating characteristic curve (ROC). The AUC values ​​of the discovery cohort and validation cohort were 0.73 and 0.75, respectively, indicating that the 9 archaeal species biomarkers proposed by the present invention have good diagnostic value for coronary heart disease. Therefore, the 9 archaeal species biomarkers of the present invention provide an effective and reliable means for the diagnosis and early prevention of coronary heart disease, which is not only beneficial for the diagnosis and typing of coronary heart disease, but also improves the understanding and understanding of the clinical pathology of coronary heart disease, and has potential value in the treatment and prevention of coronary heart disease.

[0026] 2. The present invention demonstrates superior diagnostic efficacy for coronary artery disease using a combination of multiple archaea, surpassing conventional diagnostic methods. The importance of archaea as biomarkers for coronary artery disease lies in their ability to provide more comprehensive and personalized information, rather than simply measuring a single indicator. Therefore, by studying the species, abundance, and function of archaea, we can better understand the pathogenesis of coronary artery disease and provide a basis for personalized treatment and prevention. Furthermore, as a class of microorganisms, archaea are relatively stable in the intestine, making them highly viable as biomarkers and promising tools for the diagnosis of coronary artery disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Analysis of bacterial species diversity between the control group and the coronary heart disease group in the discovery cohort and validation cohort of this invention;

[0029] Figure 2 Species rarefaction curves for the discovery cohort (a) and validation cohort (b) of the present invention;

[0030] Figure 3 This is the distribution map of intestinal archaea in healthy people of the present invention;

[0031] Figure 4 The effects of the confounding factors of the present invention on intestinal archaea in healthy people;

[0032] Figure 5 The changes in intestinal archaea of ​​patients with coronary heart disease in the present invention;

[0033] Figure 6 is a bar graph of the abundance of different archaeal species among the groups of the present invention;

[0034] Figure 7 This is the test effect of the intestinal archaea of ​​the present invention as a diagnostic marker for coronary heart disease. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0037] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] Example

[0039] 1. Study population and sample collection

[0040] This study includes a discovery cohort and a validation cohort:

[0041] Discovery Queue:

[0042] Gut microbiota metagenomic sequencing data of 385 individuals from CHD patients and healthy controls in Guangzhou, China were downloaded from a public database (https: / / www.ebi.ac.uk / ena / browser / view / PRJEB 21528). Samples with a large number of unknown sequences were removed, resulting in 370 samples as the discovery cohort, including 167 samples from the healthy control group and 203 samples from the CHD group.

[0043] The inclusion and exclusion criteria for the subjects were as follows: patients diagnosed with coronary heart disease were found to have a stenosis of ≥50% in one or more vessels by percutaneous coronary angiography; healthy controls were subjects who had negative results on coronary CT angiography or percutaneous coronary angiography and had no obvious clinical symptoms and signs related to cardiovascular disease.

[0044] Exclusion criteria included peripheral arterial disease, hereditary cardiomyopathy, various infectious diseases, cancer, liver and kidney failure, peripheral neuropathy, stroke, etc. Individuals who had used antibiotics within 1 month were also excluded.

[0045] All study subjects were Han Chinese, non-consanguineous, and aged between 40 and 80. Demographic data and cardiovascular risk factors were collected through medical interviews and questionnaires.

[0046] Verification Queue:

[0047] Subjects were strictly enrolled according to the following inclusion criteria: patients diagnosed with coronary artery disease (CAD) as determined by coronary angiography (CAG) showing at least 50% stenosis of one or more major coronary arteries; controls were healthy individuals with negative computed tomography coronary angiography (CTCA) or CAG results or no prior clinical symptoms or signs of CAD. A total of 36 patients with CAD and 36 healthy controls were enrolled as the validation cohort.

[0048] Exclusion criteria included patients with gastrointestinal diseases, history of malignancy, autoimmune diseases, infectious diseases, severe renal impairment (creatinine level exceeding 3.0 mg / dL), history of gastrointestinal surgery within 1 year, or use of antibiotics for more than 3 days within 1 month.

[0049] Baseline data for patients in the CHD group were collected through interviews and physical examinations upon admission, and through the medical record system. Baseline data for the healthy control group were obtained through questionnaires. This study was approved by the hospital's institutional review board, and informed consent was obtained from all participants.

[0050] 2. Stool sample collection

[0051] Intestinal stool samples from the validation cohort were collected and stored at -20°C within 4 hours. They were then transferred to a -80°C freezer for long-term storage. Metagenomic sequencing was performed using the Illumina HiSeq Rapid SBS Kit according to the manufacturer's instructions. Fasting peripheral venous blood was collected from all participants using a vacuum collection tube the morning after admission and immediately sent to the hospital laboratory for testing.

[0052] 3. Metagenomic sequencing data analysis and species annotation

[0053] Metaphlan3 (version 3.0.13) software was used to compare metagenomic sequencing data of 370 patients downloaded from public databases with the species marker gene database mpa_v30_CHOCOPhlAn_201901 using bowtie2 (version 2.2) to obtain the relative abundance of species at each taxonomic level (kingdom, phylum, class, order, family, genus, species) of archaea and bacteria.

[0054] For the stool samples of the validation cohort, the DNA of the microbial community was extracted from the stool samples using the HiSeq DNA extraction kit according to the manufacturer's operating instructions. The DNA library was constructed through steps such as DNA fragment breakage, end repair, adapter ligation, and library amplification.

[0055] After the DNA library passed the test, the quality control software trimmomatic-0.33 was used to remove the adapter sequences and paired reads with a length of less than 50 based on the Illu mina Hiseq PE150 platform, and the original metagenomic sequencing data was obtained after filtering.

[0056] The quality of the raw sequencing data was controlled using Fastq software (Vension 0.19.7). Read sequences were discarded if there was adapter contamination in the sequence, more than 10% of uncertain bases per read, or more than 50% of low-quality bases (Phred quality < 20).

[0057] After quality control and filtering, high-quality, usable metagenomic sequencing data were obtained. Metaphlan3 (version 3.0.13) software was used to compare the sample sequencing data with the species marker gene database mpa_v30_CHOCOPhlAn_201901. Based on the comparison results, the species and abundance information of archaea and bacteria at different taxonomic levels (including kingdom, phylum, class, order, family, genus, and species) as well as subsequent biological information were obtained.

[0058] 4. Statistical analysis methods

[0059] Based on the sample-species abundance matrix of archaea and bacteria, the R package vegan was used to calculate alpha diversity indices such as Shannon and Richness, and the changing trend of alpha diversity indices with sequencing depth was evaluated. Principal coordinate analysis (PCoA) of species abundance based on Bray-Curtis distance was performed using R language to evaluate β-diversity among samples. Permutational multivariate analysis of variance (PerManova) was used to test the statistical significance of the microbial communities between the two groups. The degree to which each variable explained the microbial changes was calculated, and P values ​​were generated based on 9999 permutations.

[0060] Linear Discriminant Analysis Effect Size (LEfSe) was used to calculate the differences in species abundance at the species level between different age groups in the control group, and to identify species with significant differences between the two groups.

[0061] Based on the species enriched in the CHD group in the heat map, a random forest model was constructed using ten-fold cross-validation, and the area under the curve (AUC) of the random forest model was plotted for each variable. The Speraman correlation algorithm was used to calculate symbiotic and coexclusive relationships within the Archaea and between the Archaea and Bacteria kingdoms, with P values ​​less than 0.05 considered significant. The Mann-Whitney U test was used to determine statistical differences in species abundance between the CHD and control groups in the discovery and validation cohorts.

[0062] 5. Analysis results

[0063] 5.1 Study Cohort Population Information

[0064] A total of 370 subjects were included in the discovery cohort, including 167 healthy controls and 203 patients with coronary heart disease.

[0065] The results showed that the two groups of subjects were similar in age, but the CHD group had more male patients than the control group. Among clinical indicators related to CHD, the CHD group had higher levels of risk factors such as fasting blood glucose and triglycerides, and lower levels of protective factors such as high-density lipoprotein cholesterol and apolipoprotein A. However, in contrast, total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B, all risk factors, were higher in the control group. The clinical data of the subjects in the discovery cohort are shown in Table 1.

[0066] According to strict inclusion and exclusion criteria, a total of 36 patients with coronary heart disease and 36 healthy control subjects were enrolled in the validation cohort.

[0067] The results showed that compared with the control group, patients in the CHD group were more likely to be male and older. Among the associated complications, patients in the CHD group were more likely to have hypertension, diabetes, and hyperlipidemia. In terms of blood test parameters, the CHD group had a higher erythrocyte sedimentation rate, while other CHD-related parameters showed no significant differences between the two groups (Table 2).

[0068] Table 1 Clinical baseline data of subjects in the discovery cohort

[0069]

[0070]

[0071]

[0072] Non-normally distributed variables are expressed as medians (interquartiles), while normally distributed variables are expressed as mean ± standard deviation. Categorical variables are expressed as number of cases / total number (%). Continuous normally distributed variables between the two groups were analyzed using the t-test, while non-normally distributed data were compared using the Mann-Whitney U test, and categorical variables were compared using the chi-square test. NA indicates missing data.

[0073] Table 2 Clinical baseline data of subjects in the validation cohort

[0074]

[0075]

[0076]

[0077] Non-normally distributed variables were expressed as medians (interquartiles), while normally distributed variables were expressed as mean ± standard deviation. Categorical variables were expressed as number of cases / total (%) or number of cases (%). Continuous normally distributed variables were analyzed between the two groups using the t-test, while non-normally distributed data were analyzed using the Mann-Whitney U test, and categorical variables were compared using the chi-square test. Previous myocardial infarction was defined as myocardial infarction occurring more than 3 months ago, and previous cerebral infarction was defined as cerebral infarction occurring more than 6 months ago.

[0078] 5.2 Compositional Characteristics of Gut Archaea in Healthy Controls

[0079] To explore the composition characteristics of intestinal archaea in healthy controls and the changes in intestinal archaea characteristics in patients with coronary heart disease, the intestinal flora metagenomic sequencing data of 167 normal people and 203 patients with coronary heart disease in the discovery cohort were analyzed.

[0080] To verify the reliability of the data, we first analyzed the α and β diversity of the intestinal bacteria in the discovery cohort and validation cohort. Consistent with related studies, the α diversity index did not change between patients with coronary heart disease and healthy people, while the β diversity analysis showed significant differences between the two groups ( Figure 1 ).

[0081] Figure 1 (a, b) show alpha diversity (a) and beta diversity (b) analyses of the control and CHD groups in the discovery cohort; (c, d) show alpha diversity (c) and beta diversity (d) analyses of the control and CHD groups in the validation cohort. Differences between groups were tested using the Mann-Whitney U test. PCoA1 and PCoA2 represent the contributions of the two species with the greatest differences between the two groups, expressed as percentages.

[0082] Figure 2The species rarefaction curves showed that the archaeal richness of both CHD patients and controls reached a plateau in both the discovery and validation cohorts, indicating that the species sequencing depth of intestinal archaea in the control group and CHD patients in the two cohorts was sufficient.

[0083] Figure 3 Figure 2 shows the distribution of intestinal archaea in healthy individuals. (a) shows the relative abundance of intestinal archaea at the phylum level in healthy individuals, (b), (c), and (d) show the relative abundance (inverse sine square root) of the top 10 most abundant archaea at the order, genus, and species levels, respectively. Arcsine represents the inverse sine function, and sqrt represents the square root. The results show that the intestinal archaea of ​​healthy individuals are rich in composition. At the phylum level, Euryarchaeota is the most abundant species, accounting for approximately 85%, followed by Candidatus_Bathyarchaeota and Crenarchaeota. The total proportion of these three phyla exceeds 90%. At the order level, the top 10 most abundant archaea accounted for approximately 75% of all orders, with Halofera cales and Methanobacteriales each exceeding 10%, while Methanosarcinales and Thermococcales each accounted for over 5% of the total intestinal archaea. At the genus level, Haloferax and Haloquadratum were the most abundant archaea, with relative abundances of 12.33% and 10.68%, respectively. At the species level, the most abundant archaea were Haloquadratum_walsbyi, Haloferax_prahovense, and Thermococcus_chitonophagus, with relative abundances of 12.33%, 6.20%, and 4.53%, respectively.

[0084] Compared with stable components such as bacteria and fungi in the intestinal microbiota, there is currently limited knowledge about the distribution of intestinal archaea in healthy people. Therefore, we also analyzed the effects of common factors affecting the composition of the intestinal microbiota, such as gender, body mass index (BMI), and age on the intestinal archaea composition in normal healthy people. Figure 4The effects of confounding factors (BMI) (a), sex (b), and age (c) on intestinal archaeal composition were evaluated by PCoA analysis. The normal BMI group (≥18.5, ≤24, n = 81) and the abnormal BMI group (<18.5, >24, n = 79), females (n = 99) and males (n = 67), and the age groups ≤60 years (n = 90) and >60 years (n = 70) were analyzed. (d) The enrichment of archaeal species at the species level was determined by the LEfSe method. Results showed that no differences in archaeal diversity were observed between the normal BMI group (≥18.5, ≤24) and the abnormal BMI group (<18.5, >24), or between males and females. However, differences in archaeal species composition were observed between the age groups ≤60 years and >60 years (P = 0.034). The linear effect discriminant analysis method showed that the number of methanogens (such as Methanoculleus_bourgensis) and halophilic archaea (such as Halodesulfurarchaeum_formicicum) increased in individuals over 60 years old, while the abundance of Methanobrevibactersmithii (M.smithii) decreased significantly.

[0085] 5.3. Changes in the composition of intestinal archaea in patients with coronary heart disease

[0086] To identify potential changes in gut archaeal composition in patients with CHD, we compared the archaeal diversity of patients with CHD and healthy controls in the discovery and validation cohorts. Figure 5 The changes in intestinal archaea in patients with coronary heart disease, including the analysis of the Inverse Simpson index and richness index of α diversity between the control group and the coronary heart disease group (a), and the analysis of β diversity (b); the analysis of the Inverse Simpson index and richness index of α diversity between the control group and the coronary heart disease group in the validation cohort (c), and the analysis of β diversity (d). The Mann-Whitney U test was used to test the differences in α diversity between the groups. PCoA1 and PCoA2 represent the two most important species components reflecting the diversity between the groups. The percentages represent their contribution to the differences between the groups. The permutational multivariate analysis of variance (permutational MANOVA) was used to compare the differences between the groups. The results showed that in the discovery cohort, the uniformity index invsimpson in α diversity was the same in the two groups (P=0.60), while there were differences in richness (P=0.04) ( Figure 5 a), whereas in the validation cohort, the invsimpson index was different between the two groups (P < 0.01), while the Richness index was the same (P = 0.07) ( Figure 5 c). For β diversity, the results of principal coordinate analysis showed that there were significant differences in species clustering between the CHD group and the healthy control group in both the discovery cohort and the validation cohort ( Figure 5 b, d).

[0087] To further clarify the archaeal dysbiosis observed in the CHD group, species abundance difference analysis was also performed. Figure 6 The following is a bar chart showing the abundance of differentially expressed archaeal species between the control and CHD groups in cohort (a) and validation cohort (b). Differences in abundance between groups were detected at the phylum, genus, and species levels using the Mann-Whitney U test (*p < 0.05, **p < 0.01). As shown in the figure, Candidatus_Diapherotrites at the phylum level, Candidatus_Altiarchaeales_unclassified, Candidatus_Diapherotrites_unclassified, Hadesarchaea_unclassified, Methanothermococcus, and Thermococcus at the genus level, and Halorubrum_hochstenium, Methanosarcina_sp_MTP4, and Methanothermococcus_okinawensis at the species level were all reduced in the CHD group. Similar changes were observed in the independent validation cohort. These results suggest that the composition of the intestinal archaea in patients with CHD is altered.

[0088] 5.4. Validation of intestinal archaea as a diagnostic marker for coronary heart disease

[0089] Figure 7 (a) Heat map showing the changes in species-normalized relative abundance of intestinal archaea in patients with CHD compared with healthy controls in the discovery cohort, and the diagnostic performance of archaea species enriched in the CHD group in the discovery cohort for classifying CHD and controls in the discovery cohort (b) and validation cohort (c).

[0090] Depend on Figure 7aIt can be seen that in the discovery cohort, 22 archaea with different species abundance between the CHD group and the control group were identified at the species level (9 enriched and 13 sparse), among which the 9 enriched archaea included 4 methanogens (Arc lgroup_archaeon_U1lsi0528_Bin055, Candidatus_Methanopered ens_sp_BLZ1, Methanobacterium_paludis, Candidatus_Methanomassilicocc us_intestinalis), 2 halophilic archaea (Candidatus_Haloredivivus_sp_G17, Halo ferax_prahovense), 2 Pyrococcales (Thermococcales_archaeon_44_46, Pyrococ cus_sp_ST04) and 1 Heimdallarchaeota (Candidatus_Heimdallarchaeota_archaeon_B3_JM_08).

[0091] Based on the above-mentioned archaeal species enriched in the coronary heart disease group, the diagnostic performance of these enriched archaea was evaluated by the receiver operating characteristic curve (ROC).

[0092] The results showed that the area under the curve (AUC) value in the cohort was 0.73 ( Figure 7 b), and in the validation cohort, its value was 0.75 ( Figure 7 c), indicating that the above nine archaea enriched in the CHD group can be used as biomarkers to distinguish CHD patients from healthy controls, and have potential application value in the diagnosis of CHD.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a reagent for detecting the abundance of a composition of intestinal archaeal markers for coronary heart disease in the preparation of a product for diagnosing coronary heart disease, characterized in that: The product diagnoses and assesses whether a subject has coronary heart disease by measuring the abundance of nine types of archaea in a test sample from the subject; The nine species of archaea are Arc l group_archaeon_U1lsi0528_Bin055, Candidatus_Methanoperedens_sp_BLZ1, Methanobacterium_paludis, Candidatus_Methanomassilicoccus_intestinalis, Candid atus_Haloredivivus_sp_G17, Haloferax_prahovense, Thermococcales_archaeon_44_46, Pyrococcus_sp_ST04 and Candidatus_Heimdallarchaeota_archaeon_B3_JM_08; The sample to be tested is feces.

2. The use according to claim 1, characterized in that The product is a detection chip, a detection test paper or a detection kit.

3. The use according to claim 1, characterized in that The product also includes reagents required for extracting DNA from the sample to be tested and measuring the abundance of the nine archaea.

Images