CN-122012699-A - Clostridium perfringens in feces as grading judgment molecular marker for fatty liver disease related to metabolic dysfunction and application thereof

Abstract

The clostridium perfringens in the feces is used as a molecular marker for grading and judging fatty liver related to metabolic dysfunction and application thereof, the clostridium perfringens contains alpha toxin CPA according to the type of main toxin produced by the clostridium perfringens, the coding gene of the alpha toxin CPA is plc genes, the molecular level detection of the clostridium perfringens in the feces sample is realized by quantitatively detecting the plc genes, in addition, the clostridium perfringens also contains a nirA gene related to nitrogen metabolism, the nirA gene participates in the metabolic process related to microbial ammonia generation, the functional state of intestinal ammonia-producing microorganisms is reflected by detecting the nirA gene expression level, the stable acquisition of the intestinal microbial molecular characteristics related to MASLD is realized, and a molecular basis is provided for analyzing the association between ammonia metabolic abnormality and MASLD occurrence development.

Inventors

• LIU ENQI
• HOU YITING
• QU PENGXIANG
• LI PENG
• ZHAO JUAN
• XU LEXUAN
• XIA CONGCONG

Assignees

• 西安交通大学

Dates

Publication Date

20260512

Application Date

20260306

Claims (5)

1. 1. The molecular marker is characterized in that the clostridium perfringens in feces is used as a grading judgment molecular marker for fatty liver disease related to metabolic dysfunction, the clostridium perfringens comprises at least A, B, C, D, E toxin types according to the main toxin types generated by the clostridium perfringens, the five toxin types comprise alpha toxin CPA, the coding genes of the alpha toxin CPA are plc genes, the plc genes are quantitatively detected to realize the molecular level detection of the clostridium perfringens in the feces samples, and furthermore, the clostridium perfringens also comprises a nirA gene related to nitrogen metabolism, the nirA gene participates in the metabolic process related to microbial ammonia production, the functional state of the intestinal ammonia-producing microorganism is reflected through the detection of the nirA gene expression level, and the molecular basis is provided for analyzing the association between ammonia metabolism abnormality and MASLD development.
2. 2. The molecular marker for classifying and judging the fatty liver disease associated with metabolic dysfunction by clostridium perfringens in feces according to claim 1, wherein the nucleotide sequence of the forward amplification primer of the plc gene is SEQ ID NO.1, the nucleotide sequence of the reverse amplification primer is SEQ ID NO.2, and the probe sequence is SEQ ID NO. 3.
3. 3. The molecular marker for classifying and judging the fatty liver disease related to metabolic dysfunction by clostridium perfringens in feces according to claim 1, wherein the nucleotide sequence of the forward amplification primer of the amino-genic factor nirA is SEQ ID NO.4, the nucleotide sequence of the reverse amplification primer is SEQ ID NO.5, the nucleotide sequence of the forward amplification primer of the internal reference gene rrs is SEQ ID NO.6, and the nucleotide sequence of the reverse amplification primer is SEQ ID NO.7.
4. 4. The use of clostridium perfringens in faeces as a marker for the fractionation determination of fatty liver disease associated with metabolic dysfunction according to claim 1 wherein said clostridium perfringens contains an amino gene nirA whose expression is positively correlated with the severity of the MASLD pathology and wherein clostridium perfringens contains a toxin CPA whose expression is positively correlated with the severity of the MASLD pathology.
5. 5. Use of clostridium perfringens in faeces as a marker for the fractionation determination of fatty liver disease associated with metabolic dysfunction according to claim 4 characterized in that said use is in particular: Firstly, using qPCR (quantitative polymerase chain reaction) to obtain a Ct value of a gene plc by adopting a TaqMan probe technology, establishing a standard curve according to a Ct value of a standard substance, and obtaining plc gene copy numbers of each sample according to the standard curve; step two, taking the obtained relative quantitative value 2 (-delta Ct) of the plc gene copy number or nirA and the state MASLD needing to be identified into variables to construct a ROC curve, and selecting the point corresponding to the maximum value of the sum of sensitivity and specificity in a coordinate correspondence table as a cut-off value cut-offpoint of the ROC curve, namely a critical point for judging whether a sample is in a first state or a second state, wherein the state MASLD needing to be identified is obtained according to liver pathological staining and NAS scoring and fibrosis stage. Step three, the cut-off value obtained by the ROC curve constructed by the state information of health and MASLD and the gene copy number/relative quantitative value is marked as a critical value (C-a), the cut-off value obtained by the ROC curve constructed by the state information of simple MASL and MASH and the gene copy number/relative quantitative value is marked as a second critical value (C-b), and the cut-off value obtained by the ROC curve constructed by the state information of liver fibrosis and the gene copy number/relative quantitative value is marked as a third critical value (C-C); And step four, marking the plc gene copy number or the nirA relative quantitative value obtained by the detection in the step one as X, and comparing the X with the critical value (C-a), the second critical value (C-b) and the third critical value (C-C) in the step three so as to output a grading diagnosis result: if X is less than or equal to C-a, judging that the patient is healthy; c-a < X is less than or equal to C-b, and is interpreted as "simple fatty liver (MASL)"; if X > C-b, then it is interpreted as "metabolic-related steatohepatitis (MASH)"; If X > C-C, then it is interpreted as "liver fibrosis is present".

Description

Clostridium perfringens in feces as grading judgment molecular marker for fatty liver disease related to metabolic dysfunction and application thereof Technical Field The invention relates to the technical field of biological medicines, in particular to a method for detecting intestinal microorganisms, which is used for researching metabolic dysfunction related fatty liver diseases (metabolic dysfunction-associated steatotic LIVER DISEASE, MASLD) in a non-human primate model, by taking clostridium perfringens in feces as a metabolic dysfunction related fatty liver disease grading judgment molecular marker and application thereof. Background Metabolic dysfunction-related fatty liver disease (MASLD) is a chronic liver disease characterized mainly by liver lipid deposition, inflammatory response and fibrosis progression, and its disease spectrum encompasses simple fatty liver disease (MASL) as well as metabolic dysfunction-related steatohepatitis (metabolic dysfunction-associated steatohepatitis, MASH), and can progress further to liver fibrosis, cirrhosis and even hepatocellular carcinoma. Epidemiological studies have shown that MASLD has a prevalence of about 25% in the global population and has become one of the important risk factors for end-stage liver disease. However, effective therapeutic means for MASLD, especially for its progressive stages, are still limited at present, and thus research is being conducted around the mechanism of occurrence, hierarchical assessment and monitoring methods of MASLD. Studies have shown that liver ammonia metabolism disorders are one of the important pathological features in MASLD progression, with increasing intracellular ammonia load, exacerbating hepatocyte inflammatory response and metabolic damage, driving MASL to stage MASH. Recent studies have shown that intestinal microecological imbalance plays a key role in the development and progression of MASLD in addition to host metabolic disorders, where the "intestinal-hepatic axis" mechanism is increasingly receiving widespread attention. In MASLD and related metabolic liver disease states, the intestinal barrier function is impaired and the intestinal metabolites enter the liver via the portal system, thereby increasing the inflammatory response and metabolic burden on the liver. Ammonia, an important enterogenic metabolite, is thought to be involved in liver inflammatory response, mitochondrial dysfunction and hepatocyte damage due to its abnormal rise in vivo. Intestinal microorganisms are one of the important sources of ammonia in the body, and especially intestinal bacteria with ammonia production capacity can undergo significant changes in abundance and function under pathological conditions. Clostridium perfringens (Clostridium perfringens) is a conditional pathogenic bacterium widely existing in intestinal tracts, belongs to gram-positive anaerobic bacteria, has a thick cell wall structure and is rich in peptidoglycan layers, has stronger protein and amino acid metabolism capability, and can participate in the ammonia generation process through various metabolic pathways. Under the condition of intestinal microecological disturbance, the abnormal rise of the bacterial abundance can lead to the increase of the ammonia production level of the intestinal tract, and excessive ammonia is absorbed by the intestinal tract and enters the liver through a portal vein system, so that the detoxification burden of the liver is increased, and the abnormal accumulation of the ammonia in the liver can induce the mitochondrial dysfunction of liver cells, the enhancement of oxidative stress and the activation of inflammatory signal channels, so as to further promote the occurrence of the injury and inflammatory reaction of the liver cells. The article "The gun-liver axis and gut microbiota IN HEALTH AND LIVER disease. Nature Reviews" states that by The synergistic action of The "intestinal-hepatic axis" mechanism, sustained adverse effects on liver structure and function are produced, thus driving MASLD to progress from The simple fat deposition stage to The MASH and even to The liver fibrosis stage. However, because the cell wall of the gram-positive bacteria has compact structure and strong tolerance to chemical lysis and conventional lysis conditions, the genome DNA of the gram-positive bacteria is not easy to be fully released in a fecal sample, and a mature scheme for differential extraction of gram-positive bacteria and gram-negative bacteria in the fecal sample is not available on the market at present. In a complex fecal matrix environment, the factors further increase the difficulty of nucleic acid extraction and detection aiming at clostridium perfringens, and the problems of low DNA extraction efficiency, insufficient detection sensitivity, poor result stability and the like are easily caused. Therefore, how to efficiently extract gram-positive bacteria DNA while ensuring the integrity of nucleic acid is one of the ke