KR102533533B1 - Information provision method for the treatment of alcoholic cirrhosis using Veillonella dispar strain - Google Patents

Abstract

Veillonella dispar Relates to a method for providing information for diagnosing alcoholic cirrhosis using a strain, and more specifically, to a method for diagnosing alcoholic cirrhosis using qPCR analysis.

Description

Information provision method for the treatment of alcoholic cirrhosis using Veillonella dispar strain}

The present invention relates to a method for providing information for diagnosing alcoholic cirrhosis using a Veillonella dispar strain, and more specifically, to a method for diagnosing alcoholic cirrhosis using qPCR analysis.

Microbiome refers to microorganisms that exist in a specific environment and genetic information of these microorganisms, and microorganisms that affect human health and their genetic information are referred to as human microbiome. The intestine is the most abundant part of the human microbiome. The gut microbiome is composed of complex and diverse communities. Beneficial bacteria, non-beneficial bacteria and harmful bacteria exist in the intestine, and the ideal intestinal environment is a state in which a balance with harmful bacteria is maintained in an environment where beneficial bacteria dominate. However, Western-style eating habits and frequent drinking habits of modern people inhibit the growth of beneficial bacteria and promote the growth of harmful bacteria, breaking the balance of the intestinal environment. In particular, when harmful bacteria increase, the immune function to defend against intestinal infections is weakened, which affects the development of inflammatory bowel disease, obesity, and diabetes [Chassaing B. et al. Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome. Nature. 2015; 519: 92-6]. In addition, there are studies showing that changes in intestinal bacteria increase the risk of liver diseases such as fatty liver and cirrhosis, heart disease, and gastrointestinal cancer [Qin N. et al. Alterations of the human gut microbiome in liver cirrhosis. Nature. 2014; 513(7516):59-64].

Continuous drinking, which affects the intestinal microbial environment among modern lifestyles, causes fatty liver, alcoholic hepatitis, and cirrhosis, which is called alcoholic liver disease. Double alcoholic liver cirrhosis refers to a state in which alcoholic fatty liver or alcoholic hepatitis becomes chronic and the liver becomes hard or shrinks in size. According to the statistics on causes of death announced by the National Statistical Office of Korea, the number of deaths from alcoholic liver disease in 2018 was 3,818, of which 64% were due to alcoholic liver cirrhosis [Statistics on causes of death, 2018, Statistics Korea]. The occurrence of alcoholic liver disease is influenced by genetic factors such as genetic susceptibility to alcoholic liver damage and environmental factors such as gender and nutritional status. Alcoholic liver disease is caused by functional changes in the liver and intestines due to increased intestinal permeability or liver sensitivity.

The liver affects changes in the microbiome through blood circulation of metabolites produced by the intestinal microbiome. In the case of mice, continuous alcohol supply not only causes fatty liver and cirrhosis, but also affects the composition of the intestinal microbiome [Yan AW et al. Enteric dysbiosis associated with a mouse model of alcoholic liver disease. Hepatology. 2011; 53:96-105].

Diagnosis of alcoholic liver disease is based on biochemical indicators such as serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma glutamyl transpeptidase (GGT). Diagnosis can be made by liver biopsy or, alternatively, by liver tissue biopsy. However, biopsy may cause side effects such as pain and infection, so a safer and more accurate diagnosis is required.

Korean Patent Publication No. 10-2017-0034520 Korean Patent Publication No. 10-2015-0100722

Therefore, in consideration of the above situation, the present invention extracts a gene from blood, which is a sample derived from a subject, and performs qPCR on it, comparing it to a sample derived from a normal person, in a sample derived from a patient with alcoholic cirrhosis. The purpose is to propose a method for diagnosing alcoholic liver cirrhosis by comparing the increase and decrease in the content of Veillonella dispar strains.

In addition, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

According to one embodiment of the present invention in order to achieve the above object, from a biological sample isolated from the subject, Veillonella dispar ( Veillonella dispar ) For diagnosing alcoholic cirrhosis, including the step of measuring the content of the strain A method of providing information is provided.

According to one embodiment of the present invention, the method further comprises the steps of measuring the content of the Veillonella dispar strain from a biological sample isolated from the control group and comparing the strain content of the individual and the control group may include

According to one embodiment of the present invention, the Veillonella dispar ( Veillonella dispar ) The content of the strain may be measured by qPCR of the Veillonella dispar strain gene.

According to another embodiment of the present invention, a formulation capable of measuring the content of Veillonella dispar strain is provided.

According to one embodiment of the present invention, the Veillonella dispar ( Veillonella dispar ) The content of the strain may be measured by qPCR of the Veillonella dispar strain gene.

According to another embodiment of the present invention, a kit for diagnosing alcoholic cirrhosis comprising the above composition is provided.

The method for providing information for diagnosing alcoholic cirrhosis according to the present invention uses a blood sample that can be easily obtained from a subject to be diagnosed, and predicts or predicts alcoholic cirrhosis through qPCR analysis of Veillonella dispar strains. Because it can be diagnosed, it has the effect of diagnosing and predicting the risk group of alcoholic cirrhosis at an early stage, so there is an advantage that it can be used for prevention and diagnosis of alcoholic cirrhosis.

1 is a comparison diagram of gene content of Veillonella dispar strains isolated from blood in a normal control group and alcoholic cirrhosis patients.
Figure 2 is made Veillonella dispar ( Veillonella dispar ) It is a primer verification diagram.
3 is a model diagram of alcoholic liver cirrhosis diagnosis with the content of Veillonella dispar strain isolated from blood as a variable.

Hereinafter, the present invention will be described in detail. Prior to this, terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventor appropriately uses the concept of terms in order to describe his/her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical spirit of the present invention based on the principle that it can be defined in the following way. Therefore, the configuration described in the embodiments described in this specification is only one of the most preferred embodiments of the present invention and does not represent all of the technical ideas of the present invention, so various equivalents and equivalents that can replace them at the time of this application It should be understood that variations may exist.

One embodiment of the present invention, from a biological sample isolated from the subject, Veillonella dispar ( Veillonella dispar ) Provides an information providing method for diagnosing alcoholic cirrhosis, comprising measuring the content of the strain.

At this time, the method may further include measuring the content of Veillonella dispar strain from the biological sample separated from the control group and comparing the strain content of the individual and the control group.

In addition, the Veillonella dispar strain content may be measured by qPCR of the Veillonella dispar strain gene.

The term used throughout the present specification, alcoholic cirrhosis is a type of alcoholic liver disease caused by excessive drinking, and includes ascites, esophageal varices (pressure in the esophageal blood vessels increases and the number and size of the esophageal veins increase, and accordingly the veins become lumpy). It refers to a disease in which hepatic coma (consciousness is blurred or behaves like another person), etc., and the onset of alcoholic cirrhosis differs depending on the hereditary characteristics and gender, and generally It can develop if you consume more than 80g of alcohol per day for 10 to 20 years. In the present application, the "alcoholic cirrhosis" may be interchangeably named with "cirrhosis of the liver".

On the other hand, the term used throughout the present specification, Veillonella dispar ( Veillonella dispar ) The strain was provided and used by Cheonlab Co., Ltd.

In addition, the term qPCR used throughout the present specification is a real-time polymerase chain reaction (real-time PCR, quantitative real time polymerase chain reaction, qPCR), based on polymerase chain reaction in molecular biology. as an experimental method. Real-time polymerase chain reaction simultaneously amplifies and quantifies target DNA molecules, and for one or more specific sequences in a DNA sample, real-time polymerase chain reaction enables detection and quantification. Quantification can count absolute copy number or relative quantity.

The term "bioinformatics" used throughout the present specification is a discipline that mainly deals with biological problems at the molecular level using applied mathematics, information science, statistics, computer science, artificial intelligence, chemistry, and biochemistry. Research fields in computational biology often overlap with systems biology. Major research areas are diverse, including sequence alignment, gene search, gene combination, protein structure alignment, protein structure prediction, gene expression prediction, protein interactions, and evolution models. As a sub-field of bioinformatics, genomics (personal genomics, functional genomics, comparative genomics), variant genomics, proteomics, interactomics and multiomics, etc. are included.

As used throughout the present specification, the term "sample" means a material derived from an individual who has or is likely to develop alcoholic cirrhosis, and specifically, tissue, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, urine and the like, but is not limited thereto. In addition, genetic samples can be obtained from these samples, and the genetic samples may include nucleic acids, for example, DNA, mRNA, or cDNA synthesized from mRNA, from which Veillonella dispar strains As long as the content of can be confirmed, the type is not limited thereto.

According to one embodiment of the present application, the subject may be a human, specifically Korean or Asian, but is not limited thereto.

As used throughout the present specification, the term "control group" may mean a general individual or non-patient group without alcoholic cirrhosis.

The term “diagnosis” used throughout the present specification means determining whether alcoholic cirrhosis has already occurred in a specific individual.

The term "prediction" used throughout the present specification refers to determining whether a specific individual has a possibility of developing alcoholic cirrhosis, and if there is a possibility of developing alcoholic cirrhosis, whether the possibility of developing alcoholic cirrhosis is relatively higher than that of an unspecified number of people. it means.

Another embodiment of the present invention provides a composition for diagnosing alcoholic cirrhosis, including an agent capable of measuring the content of Veillonella dispar strain.

The Veillonella dispar The content of the strain may be measured by qPCR of the gene of the Veillonella dispar strain.

According to one embodiment of the present application, the composition for diagnosing alcoholic cirrhosis may diagnose the onset of alcoholic cirrhosis, predict the risk of developing alcoholic cirrhosis, or predict the prognosis of alcoholic cirrhosis, but is not limited thereto.

Meanwhile, another embodiment of the present invention provides a kit for diagnosing alcoholic cirrhosis comprising the above composition.

In another embodiment of the present invention, the Veillonella dispar gene of the strain may be included in the marker composition for diagnosing alcoholic cirrhosis, and specifically, the Veillonella dispar strain is alcoholic. It may be included in a marker composition for diagnosing liver cirrhosis.

As used throughout the present specification, the term "marker" refers to a biomarker and can detect changes in living organisms, thereby objectively measuring the normal or pathological state of living organisms, the degree of response to drugs, and the like.

Hereinafter, the present invention is further detailed through examples, but it is obvious that the present invention is not limited by the following examples.

Example 1. Collection of samples

Twenty-five patients with alcoholic cirrhosis (alcoholic cirrhosis group) and 9 patients without cirrhosis symptoms (normal control group) were recruited. The alcohol-induced cirrhosis group was selected based on the doctor's diagnosis and blood was collected.

Example 2. DNA extraction from blood

To extract DNA from blood, 500 μL of blood was placed in a 1.5 mL tube, treated with 1 mL of RBC lysis buffer for 10 minutes, and centrifuged at 15° C. for 5 minutes at 500 X g. The supernatant was discarded from the centrifuged tube and the pellet was collected. If the pellet was not clearly visible, the above process was repeated once more.

After adding 50 μL of a buffer composed of 25 mM NaOH and 0.2 mM EDTA to the obtained pellet, it was heated at 98°C for one hour and waited until it cooled down to 15°C. 50 μL of 40 mM Tris HCL (pH 5.5) solution was added thereto and centrifuged at 4,000 rpm for 5 minutes. The amount of DNA in the supernatant obtained after this process was quantified as 100 μg/ml using a nanodrop.

Example 3. qPCR analysis using DNA extracted from blood

Primer base sequences are shown in Table 1 below.

Primer order sequence number Forward 5′-AACGCGTTGAAATTCGTCATGAAC-3′ One Reverse 5'-GTGTAACAAGGGAGTACGGACC-3' 2

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DNA was extracted from the blood of 9 normal control subjects and 25 subjects with alcoholic liver disease, qPCR was performed using the primers of the Veillonella dispar strains in Table 1, and the two groups were compared using a relative quantification method. A total of 20 μL was reacted using 2 μL of DNA template, 10 pmol of Primer, 10 μL of 2x PowerUP ^TM SYBR ^TM Green Master MIX, and 6 μL of nuclease free water per well. At this time, the PCR conditions were 40 cycles of 95 ° C for 15 seconds and 59 ° C for 20 seconds after initial reaction at 50 ° C for 2 minutes and 95 ° C for 4 minutes. During relative quantification, each sample was normalized with the bacterial universal 16S rDNA sequence, and the average values (± standard error) of the normal control group and alcoholic liver cirrhosis were compared through a t-test test. As a result, when the content of Veillonella dispar strain isolated from blood was compared between patients with alcoholic cirrhosis and normal controls, there was a difference. Through the above results, it was confirmed that alcoholic cirrhosis can be diagnosed if the amount of Veillonella dispar strain is confirmed in the blood sample of the subject to be diagnosed (see FIG. 1 and Table 2)

normal control group alcoholic cirrhosis Mean value (±standard error) 0.248 ± 0.087 0.725 ± 0.213

Example 4. Verification of the primary specificity of the production primer

Manufactured through Example 3 It was confirmed that the primer of the Veillonella dispar strain was amplified well. As a result of the confirmation, it was confirmed that the prepared primer amplified well and produced an accurate amplification product size (see FIG. 2).

Example 5. In blood Veillonella dispar Diagnosis development through strain genetic analysis

Based on the results of Example 3, Veillonella dispar strains in blood samples A method for diagnosing alcoholic cirrhosis by checking the amount was developed. To this end, based on the qPCR results of Example 3, a diagnostic model for alcoholic liver cirrhosis was constructed using the logistic method using the Veillonella dispar strain gene content as a variable. As a result, it was confirmed that the area under curve (AUC), which is an index of diagnostic usefulness, was 0.722. Through the above results, Veillonella dispar strain obtained from blood as an analysis sample using the alcoholic cirrhosis diagnostic model of the present invention It was confirmed that it can be used as a method for diagnosing alcoholic liver cirrhosis with very high diagnostic accuracy when diagnosing with quantity as a variable.

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

Measuring the content of a Veillonella dispar strain in a blood sample separated from the subject;
Measuring the content of Veillonella dispar strains from blood samples separated from the control group; and
Comparing the strain content of the subject and the control group; Including,
The content of the Veillonella dispar strain is measured by qPCR using the primer pair of SEQ ID NOs: 1 and 2 for the gene of the Veillonella dispar strain, information for diagnosing alcoholic liver cirrhosis. How to provide. delete delete Including an agent capable of measuring the content of the Veillonella dispar strain from a blood sample isolated from the subject,
The content of the Veillonella dispar strain is to measure the gene of the Veillonella dispar strain by qPCR,
The formulation is a primer of SEQ ID NO: 1 and 2, a composition for diagnosing alcoholic liver cirrhosis. delete A kit for diagnosing alcoholic liver cirrhosis, comprising the composition of claim 4.

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