阅读说明：本技术 肠道菌群丰度检测在肝纤维化、肝硬化中的应用 (Application of intestinal flora abundance detection in hepatic fibrosis and liver cirrhosis ) 是由 沈悦 吴盛迪 蒋炜 于 2021-09-29 设计创作，主要内容包括：本发明涉及一种肠道菌群丰度检测在肝纤维化、肝硬化中的应用,属于生物医药技术领域。本发明通过收集慢性乙型肝炎病人的粪便标本,进行宏基因组菌群测序,获得相应属水平Streptococcus、Lactococcus、Veillonella、Faecalibacterium的丰度值；根据模型公式计算丰度值得分,根据得分评估患者是否有肝纤维化、肝硬化的风险。以菌群丰度特征为基础,通过对乙肝病人进行特异的肠道微生物群组合丰度的检测,获得对肝脏纤维化严重程度的评估结果和早期肝脏相关事件的预测,为对患者制定进一步优化的监测和干预策略提供参考；也避免了多次肝活检给患者带来的痛苦。(The invention relates to application of intestinal flora abundance detection in hepatic fibrosis and liver cirrhosis, and belongs to the technical field of biological medicines. The invention collects the stool sample of the chronic hepatitis B patient, carries on the sequencing of the metagenome flora, obtains the abundance value of the corresponding level Streptococcus, Lactococcus, Veillonella, Faecalibacterium; and calculating the abundance value score according to a model formula, and evaluating whether the patient has the risk of hepatic fibrosis and liver cirrhosis according to the score. Based on the flora abundance characteristics, the evaluation result of the liver fibrosis severity and the prediction of early liver related events are obtained by detecting the specific intestinal microbiota combination abundance of hepatitis B patients, and reference is provided for the further optimized monitoring and intervention strategy made by the patients; and the pain of the patient caused by multiple times of liver biopsy is also avoided.)

1. The application of the detection of the abundance of the intestinal flora in the liver fibrosis and the liver cirrhosis is characterized in that the detection of the abundance of the intestinal flora is to collect an excrement sample of a chronic hepatitis B patient and carry out metagenomic flora sequencing to obtain abundance values of the corresponding genera of Streptococcus, Lactococcus, Veillonella and Faecalibacterium; calculating a score according to a model formula: 5.090 Streptomyces +3.331 ^ 10^5Lactococcus +1.692 ^ Veillonella +7.139 ^ Fae calibacterium-0.944;

if the score is larger than 0.658, the patient is indicated to have the risk of liver fibrosis and liver cirrhosis.

2. The use of the detection of abundance of intestinal flora according to claim 1 in the preparation of a diagnostic kit for hepatitis b, liver fibrosis and/or cirrhosis.

3. The use of the detection of abundance of intestinal flora according to claim 1 in the preparation of a prognosis kit for hepatitis b, liver fibrosis and/or cirrhosis.

Technical Field

The invention relates to application of intestinal flora abundance detection in hepatic fibrosis and liver cirrhosis, and belongs to the technical field of biological medicines.

Background

Chronic hepatitis b is still the main cause of chronic liver disease and liver related fate events (including liver cirrhosis decompensation, hepatocellular carcinoma and death) in China. About 20% to 30% of chronic hepatitis B patients develop liver-related outcome events, which is further elevated in hepatitis B-compensated cirrhosis patients. Therefore, it is very important to further develop an optimized monitoring and intervention strategy for evaluating the severity of liver fibrosis and detecting early liver-related events of hepatitis B patients.

In clinical practice, the pathological outcome of liver biopsies is currently considered to be the gold standard for assessing the severity of liver fibrosis in patients with hepatitis B. Liver biopsy, however, is an invasive test procedure, and the patient is at risk of bleeding during the procedure. Liver biopsy procedures also suffer from sampling errors and subjective interpretation of results. Also, in clinical practice, sequential or multiple biopsies are difficult to achieve in order to predict the risk of liver-related outcome events or the efficacy of antiviral therapy.

In early embryology, the liver develops from the foregut origin, anatomically and functionally complementing the gut, constituting the "gut-liver axis". In chronic infection with hepatitis b virus, gut microbes are able to activate systemic immunity. In addition, the permeability of the intestinal tract of a hepatitis B virus infected patient is changed, bacterial translocation and endotoxin load in the portal vein are increased, and immune-mediated liver injury is promoted to further cause chronic inflammation to cause liver lesion, so that the effect of the homeostatic imbalance of the intestinal flora in the chronic hepatitis B virus infection also becomes a research hotspot at home and abroad. However, no research is currently carried out on the combination of the intestinal flora and the noninvasive diagnosis of hepatitis B, so that the noninvasive diagnosis of hepatic fibrosis and cirrhosis of hepatitis B based on the abundance characteristics of the intestinal flora has certain originality.

Disclosure of Invention

The invention aims to solve the technical problem of how to perform noninvasive detection of hepatitis B, hepatic fibrosis and cirrhosis on the basis of the abundance characteristics of intestinal flora.

In order to solve the problems, the technical scheme adopted by the invention is to provide an application of detecting the abundance of intestinal flora in hepatic fibrosis and cirrhosis, wherein the detection of the abundance of the intestinal flora is to collect stool samples of patients with chronic hepatitis B and perform metagenomic flora sequencing to obtain abundance values of corresponding genera of Streptococcus, Lactococcus, Veillonella and Faecalibacterium; calculating a score according to a model formula:

5.090*Streptococcus+3.331*10^5Lactococcus+1.692*Veillonella+7.139*Faecalibacterium-0.944；

if the score is larger than 0.658, the patient is indicated to have the risk of liver fibrosis and liver cirrhosis.

The invention provides application of detecting the abundance of intestinal flora in preparing a diagnostic kit for hepatitis B, hepatic fibrosis and/or cirrhosis.

The invention provides application of detecting the abundance of intestinal flora in preparing a hepatitis B liver fibrosis and/or cirrhosis prognosis kit.

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

the invention extracts and sequences DNA of microbial flora in human excrement of hepatitis B patients to obtain the characteristics of microbial diversity, species and abundance, and predicts hepatic fibrosis and cirrhosis based on the abundance characteristics of the microbes. Based on the abundance characteristics of flora, establishing the intestinal microbiota combination for noninvasive detection of hepatic fibrosis and cirrhosis of chronic viral hepatitis B by combining the sequencing and noninvasive detection of the intestinal flora of patients with chronic hepatitis B; the evaluation result of the liver fibrosis severity and the prediction of early liver related events are obtained by detecting the specific intestinal microbiota combination abundance of the hepatitis B patients and are used as references for establishing further optimized monitoring and intervention strategies for the patients; and the pain of the patient caused by multiple times of liver biopsy is also avoided.

Drawings

FIG. 1 is a detection ROC curve of the horizontal intestinal flora of an individual genus.

FIG. 2 is a joint detection ROC curve; detection threshold 0.658, area under ROC curve: 0.788

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:

the invention provides an application of detecting the abundance of intestinal flora in hepatic fibrosis and cirrhosis, wherein the detection of the abundance of the intestinal flora is to collect excrement samples of patients with chronic hepatitis B and carry out metagenomic flora sequencing to obtain abundance values of corresponding levels of Streptococcus, Lactococcus, Veillonella and Faecalibacterium; calculating a score according to a model formula:

5.090*Streptococcus+3.331*10^5Lactococcus+1.692*Veillonella+7.139*Faecalibacterium-0.944；

if the score is larger than 0.658, the patient is indicated to have the risk of liver fibrosis and liver cirrhosis.

The invention provides application of detecting the abundance of intestinal flora in preparing a diagnostic kit for hepatitis B, hepatic fibrosis and/or cirrhosis.

The invention provides application of detecting the abundance of intestinal flora in preparing a hepatitis B liver fibrosis and/or cirrhosis prognosis kit.

The invention relates to a method for detecting the correlation of the abundance of intestinal flora and the specificity of hepatitis B liver fibrosis patients and hepatitis B cirrhosis patients, which comprises the following steps:

1. extracting DNA of microbial flora in feces of patients with hepatitis B (including hepatitis B liver fibrosis patients and hepatitis B cirrhosis patients) and sequencing.

2. By LEfSe analysis (LDA Effect Size analysis), intestinal flora with obvious difference in hepatitis B liver fibrosis and hepatitis B cirrhosis patients belonging to genus (genus) level is obtained through comparative analysis and is used as a detection index for predicting hepatitis B cirrhosis.

3. And respectively drawing ROC diagnosis curves of the differential bacteria to obtain a flora combination reaching the highest diagnosis efficiency.

In clinical practice, stool specimens of patients with chronic hepatitis B are easily obtained; meanwhile, with the continuous development of sequencing technology, the abundance of the flora can be rapidly and accurately quantified by metagenome sequencing at present. By combining the metagenome sequencing of excrement samples of chronic hepatitis B patients and LEfSe analysis based on flora abundance, the differential flora of the patients with different disease courses of the chronic hepatitis B can be determined, so that a model for diagnosing liver cirrhosis is constructed, and noninvasive diagnosis of the chronic hepatitis B is promoted.

Examples

FIG. 1 shows a diagnostic ROC curve for a horizontal intestinal flora;

FIG. 2 shows the combined diagnostic ROC curve: where the threshold is 0.658, the area under the ROC curve: 0.788;

in clinical practice, stool samples of patients suffering from bradykinin are collected and subjected to metagenome flora sequencing to obtain abundance values of corresponding levels of Streptococcus, Lactococcus, Veillonella and Faecalibacterium.

Calculating a score according to a joint diagnosis model formula: 5.090 Streptococcus +3.331 ^ 10^5Lactococcus +1.692 ^ Veillonella +7.139 ^ 0.944. If the combined diagnosis model index is larger than 0.658, the patient has the risk of liver fibrosis and liver cirrhosis.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.