KR20170085995A - Method for Gut Microbiota Analysis Using Real-time PCR - Google Patents

Abstract

The present invention relates to a method for intestinal bacteria analysis for analyzing intestinal health information of a subject, comprising the steps of: collecting a sample of a flesh in a specimen kit comprising a buffer, treating the specimen with a low temperature, extracting DNA from the specimen, And Lactobacillus using the extracted DNA quantitation (Lactobacillus spp.), Bifidobacterium (Bifidobacterium spp . ), Clostridium spp ., And Bacteroides spp. In the intestinal bacterium. The present invention also relates to a method for analyzing intestinal bacteria.

Description

Intestinal bacteria analysis method using real-time PCR {Method for Gut Microbiota Analysis Using Real-time PCR}

The present invention comprises the steps of collecting a collection sample in a collection kit containing a buffer; low-temperature processing of the collection sample; extracting DNA from the collection sample; And the intestinal distribution of Lactobacillus spp., Bifidobacterium spp., Clostridium spp., and Bacteroides spp. through the extracted DNA quantitative analysis. It relates to a method for analyzing intestinal bacteria comprising the step of.

The number of bacteria living in the human body is known to be over 100 trillion. This is about 10 times more than the number of cells that make up the human body. In particular, bacteria living in the intestine of the human body develop their composition and function from birth and form a complex network by interacting with the individual's genome, nutrients, and life style. The intestinal bacteria thus formed regulate a number of metabolic functions for metabolism, signal transduction, and immune responses, which are involved in the intestine, liver, muscle, and brain.

In the intestine of the human body, there are beneficial bacteria that are beneficial to the human body, harmful groups that are harmful to the human body, and intermediate bacteria that live in the human body but become beneficial bacteria when the beneficial bacteria are in a dominant environment, and intermediate bacteria that become harmful bacteria when harmful bacteria multiply. , Ultimately, it is involved in the health of the human body. Beneficial bacteria are bacteria that are beneficial to the human body and are known to help inhibit the growth of harmful bacteria and form a healthy immune system, for example. On the other hand, harmful bacteria are bacteria that are harmful to the human body.For example, it is known that harmful bacteria are increased in irritable growth syndrome and atopy.Other immune diseases such as obesity, inflammatory growth disease, colon polyps, allergies or asthma, autism, depression, hypertension and It has been reported to be associated with various diseases such as circulatory system diseases.

On the other hand, in relation to the improvement of the intestinal health of the human body, a pharmaceutical or food composition for preventing (or treating) intestinal diseases or improving intestinal function has been proposed. For example, Korean Patent Publication No. 10-2015-0106075 discloses a health food and pharmaceutical composition for preventing or improving intestinal diseases containing a Bacillus subtilis strain or a culture solution thereof as an active ingredient. Registered Patent No. 10-0811683 discloses a composition for improving intestinal function comprising hemp extract as an active ingredient and a functional health food containing the same. In addition, techniques for promoting the growth of beneficial bacteria in the intestine and inhibiting the growth of harmful bacteria in the intestine have been attempted. For example, Korean Patent Registration No. 10-1395875 discloses probiotics containing persimmon leaf extract for promoting the proliferation of beneficial bacteria in the intestine and inhibiting the growth of harmful bacteria in the intestine.

However, with regard to intestinal health, most of the prior art, including the above patent documents, aims to improve intestinal health through prevention (or treatment) of intestinal diseases, proliferation of beneficial bacteria in the intestine, and inhibition of growth of harmful bacteria in the intestine. There is a lack of research on the types of bacteria to be analyzed for management and the relative distribution of the bacteria.

Therefore, scientific and objective research on intestinal bacteria is still needed as a method for analyzing the subject's intestinal health information.

In order to solve the above problem, the present inventors have studied a method of analyzing the distribution of bacteria in the intestine for specific bacteria. As a result, the specificity and sensitivity of quantitative analysis for Lactobacillus spp., Bifidobacterium spp., Clostridium spp. and Bacteroides spp. ).

The present invention provides a method for analyzing intestinal bacteria for analyzing intestinal health information of a subject, comprising: collecting a collection sample in a collection kit including a buffer; low-temperature processing the collection sample; extracting DNA from the collection sample; And the intestinal distribution of Lactobacillus spp., Bifidobacterium spp., Clostridium spp., and Bacteroides spp. through the extracted DNA quantitative analysis. An object of the present invention is to provide a method for analyzing intestinal bacteria comprising the step of:

The low-temperature treatment may be performed at -75°C to -85°C.

The step of extracting the DNA may include adding 2 ml of InhibitEX buffer solution containing 0.15 to 0.25 g of beads to a collection kit; Homogenizing the buffer solution; Separating the supernatant in which DNA is present; It may include the step of eluting the supernatant.

The DNA quantitative analysis may be performed using real-time PCR.

The annealing temperature of the real-time PCR may be 55° C. to 60° C., and the template DNA may be 10 ng/ul to 15 ng/ul.

The real-time gene amplification technique (Real-time PCR) may use a primer pair of SEQ ID NO: 1 to SEQ ID NO: 9.

The intestinal bacteria may be Lactobacillus spp., Bifidobacterium spp., Clostridium spp., and Bacteroides spp..

The intestinal health information of the subject may include a possibility of developing a disease related to the intestine.

The present invention improves specificity and sensitivity during quantitative analysis of Lactobacillus spp., Bifidobacterium spp., Clostridium spp., and Bacteroides spp. Intestinal bacteria analysis is possible to analyze intestinal health information.

Figure 1 shows the results of Lactobacillus spp., Bifidobacterium spp., Clostridium spp., and Bacteroides spp. analysis using Real-time qPCR.
Fig. 2 is a chart for analyzing the number of bacteria schematically showing the results of analysis of intestinal bacteria.
3 is a polygonal analysis chart schematically showing the analysis results of intestinal bacteria.
4 is an example of a ranking analysis chart of intestinal bacteria.

The present invention is a method for analyzing intestinal bacteria for analyzing intestinal health information of a subject, comprising the steps of: collecting a collection sample in a collection kit including a buffer; Cold-treating the collected sample; Extracting DNA from the stool sample; And the intestinal distribution of Lactobacillus spp., Bifidobacterium spp., Clostridium spp., and Bacteroides spp. through the extracted DNA quantitative analysis. It relates to a method for analyzing intestinal bacteria comprising the step of.

The collection kit of the present invention may be composed of one or more other component compositions, solutions, or devices suitable for the analysis method, and specifically, may be a collection tube containing an InhibitEX buffer.

InhibitEX buffer efficiently removes PCR interference elements present in the fecal sample in the fecal tube, thereby efficiently performing PCR on the DNA of microorganisms present in the fecal sample. In addition, when receiving feces from the collection kit, 4 ml of inhibitEX buffer is added in advance to reduce DNA damage.

In addition, the collection kit may be autoclaved at 121° C. for 15 minutes to prevent contamination by microorganisms or environmental factors.

Real-time PCR kits may include test tubes or other suitable containers, reaction buffers (various in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase, sterile water, and the like.

In addition, the present invention provides a method for quantitative analysis of intestinal bacteria comprising the step of extracting DNA from a fecal sample and performing real-time polymerase chain reaction (PCR) using the primers of the present invention.

Provides a method of quantitatively analyzing specific microorganisms present in fecal samples by substituting Ct (Cycle threshold) values obtained by performing real-time polymerase chain reaction into a standard calibration curve.

In the real-time PCR method, the amount of the initial sample at which exponential amplification occurs is expressed as the number of cycles at which an exponential increase of the fluorescent substance begins to be detected (Cycle threshold, hereinafter referred to as'Ct'), enabling more accurate quantitative analysis and reaction. It is a method that can be analyzed in real time.

This method eliminates the step of measuring the intensity with an image analyzer through electrophoresis, and is a method that enables quick and simple diagnosis by automating and quantifying the amplification degree of the amplified product. For example, among the real-time PCR methods using fluorescence, a fluorescent dye is inserted into the double-stranded DNA amplified by PCR to display fluorescence, and a fluorescence intercalation method can be used. The amount of production can be measured.

The present invention is a key technology for the management of intestinal bacteria in fecal samples, and its necessity and importance are great, and in the present invention, a quantitative real-time PCR analysis method capable of quantitatively detecting microorganisms present in fecal samples has been established.

In the present invention, the low-temperature treatment may be carried out at -75°C to -85°C.

This is a measure to prevent changes in physical properties.

In the present invention, the step of extracting DNA includes adding 2 ml of InhibitEX buffer solution containing 0.2 g of beads to a collection kit; Homogenizing the buffer solution; Separating the supernatant in which DNA is present; It may include the step of eluting the supernatant.

Beads are homogenized using a buffer to extract DNA from fecal-living bacteria. Beads act as an enhencer to disrupt the cell walls of gram-positive or gram-negative bacteria.

In the present invention, the annealing temperature of real-time PCR is 55° C. to 60° C., and the template DNA may be 10 ng/ul to 15 ng/ul.

The setting of an appropriate bonding temperature in the PCR process is an important factor in determining the accuracy of the reaction. If the binding temperature is set too high, the primer binds too weakly to the template DNA and the amplified DNA product is very small. If the temperature is set too low, the primer binds non-specifically, so non-specific DNA other than the template DNA may be amplified. have.

In the present invention, the real-time gene amplification technique (Real-time PCR) may use a primer pair of SEQ ID NO: 1 to SEQ ID NO: 9.

Primers are short 18-24mer gene sequences made by chemically synthesizing specific gene sites of bacteria, and have a base structure opposite to that of the bacteria's genes, and are important samples for replication and amplification by targeting the template DNA. The nucleotide sequence and concentration of the primer can be seen as the factors that have the greatest influence on the overall analysis result.

The primer sequence for the bacteria to be analyzed in the present invention is as follows.

<Table>. Bacterial primer sequence (5' -> 3')

The primer sequence is 18 to 24 nt in length, the difference between the Tm values of the two primers is within 5°C and the G+C value is 40 to 60%, so that there is no complementary binding between the 3'of the two primers. Due to the primer set, it is possible to accurately quantitatively analyze four kinds of bacteria simultaneously or separately without mutual interference of the quantitative analysis values of the bacteria to be analyzed.

In the present invention, the enteric bacteria may be Lactobacillus spp., Bifidobacterium spp., Clostridium spp., and Bacteroides spp..

Intestinal bacteria analysis The above-described intestinal bacteria are routinely analyzed for a subject. In the present invention, the subject of intestinal bacteria analysis is a specific individual and is a subject of intestinal bacterial environment management (hereinafter, referred to as'subject'). In order to analyze the intestinal bacteria of the subject, the bacteria living in the intestine are separated into beneficial bacteria, intermediate bacteria and harmful bacteria, and the number of each of the beneficial bacteria, intermediate bacteria and harmful bacteria is quantitatively analyzed. For this, it is essential to select beneficial bacteria, harmful bacteria, and intermediate bacteria that are indicators among bacteria present in the intestine.

The selection of the above beneficial bacteria and harmful bacteria and intermediate bacteria is based on the fact that the correlation between various diseases such as irritable bowel syndrome, inflammatory bowel disease, colon polyps, allergies, asthma and gastritis caused by Helicobacter bacteria has been found through research. It was selected as.

Beneficial bacteria were selected from Lactobacillus spp. and Bifidobacterium spp. This fungus helps the absorption of nutrients by preventing the invasion of harmful bacteria in the intestine or inhibiting growth and strengthening the intestinal wall. In addition, it is known to help digestion by secreting enzymes so that food that cannot be decomposed in the stomach is easily absorbed by the human body, and protects the body from diseases by forming a healthy immune system by preventing immune cells from reacting excessively.

Bacteroides spp. was selected as the intermediate bacteria. This fungus lives in the body but does not perform harmful or beneficial functions, and is a neutral fungus with no distinct function. However, if beneficial bacteria are in a predominant environment, they become beneficial bacteria, and when harmful bacteria multiply, they become harmful bacteria, and the intestinal environment in which intermediate bacteria do not change into harmful bacteria must be maintained.

Clostridium spp. was selected as the harmful bacteria. This fungus is harmful to the human body and is not beneficial to health because it is known to make toxins or waste products or cause diarrhea or infection to reduce immunity to make it easier to get sick and to promote cancer or aging.

The correlation between the action of each of these bacteria and disease was revealed through various studies, and the correlation shown in the table below was confirmed. By selecting these bacteria, it is possible to grasp various health conditions in the large intestine. Health conditions may be irritable bowel syndrome, inflammatory bowel disease, colon polyps, allergies, asthma, atopy, and intestinal health related to Helicobacter bacteria.

By providing the primers optimized for the bacteria, it is possible to quantitatively analyze 4 kinds of bacteria by real-time PCR without mutual interference. Intestinal bacterial analysis is performed through genetic analysis by extracting DNA from the subject's feces. The number of the selected beneficial bacteria, harmful bacteria, and intermediate bacteria is quantitatively analyzed. DNA is extracted from the collected sample and the extracted DNA is amplified using real time qPCR to detect intestinal bacteria while simultaneously measuring the amount of bacteria.

In other words, the Ct (threshold cycle) obtained by creating a primer set that can amplify a specific part of the bacteria based on the nucleotide sequences of the selected beneficial bacteria, harmful bacteria, and intermediate bacteria, and performing real-time gene amplification with the primer set. ) To get the correct result.

Create a result sheet based on the analyzed results. As a result sheet, one or more analysis charts selected from the following (a) to (d) are prepared.

(a) Count analysis chart

Among the bacteria distributed in the intestine of the subject, the analyzed values of the selected beneficial bacteria, harmful bacteria, and intermediate bacteria are shown in a chart. As shown in Fig. 2, it is possible to check the absolute value of each bacteria at a glance by displaying it as a bar graph.

(b) polygon analysis chart

This result shows the cumulative result value of each bacteria in a picture. As shown in FIG. 3, by showing a polygonal shape, it is possible to check at a glance the balance of each bacteria according to the result of analyzing the intestinal bacteria of the subject.

(c) Ranking analysis chart

Based on the statistical data of Korean intestinal bacteria analysis, the results of analyzing the intestinal bacteria of the subject are substituted into the statistical data to show what percentage of the current subject's location belongs to the top percentage. As shown in Fig. 4, the selected beneficial bacteria, harmful bacteria, and intermediate bacteria are each graphed to show the results. Through this ranking analysis chart, it is possible to accurately grasp the position of the subject compared to statistics. In general, if it is in the top 30% or less, it is suggested that it is a balanced intestinal environment, and above that, it is suggested that it is an unbalanced intestinal environment.

At this time, in the above graph, a specific criterion is set and colors such as blue zone (upper 30%), green zone (upper 31-50%), yellow zone (upper 50-80%), and red zone (upper 81-100%). It can be presented so that you can see whether the intestinal bacterial environment is in a balanced state (blue zone) or in an unstable state (green zone, yellow zone, red zone).

(d) Monitoring analysis chart

This chart provides changes in beneficial bacteria, harmful bacteria, and intermediate bacteria when the analysis of intestinal bacteria for a subject is performed multiple times, that is, at least two times at a time difference. The results are shown by showing the increase or decrease for each bacteria by the analyzed order, such as the 1st, 2nd, 3rd, etc. as a bar graph.

It is desirable to monitor the change of intestinal bacteria through regular intestinal bacteria analysis every 3 to 6 months to help health through continuous monitoring.However, it is desirable to check the change in the distribution of intestinal bacteria through a monitoring analysis chart. Make it possible to monitor.

Based on the analyzed result, it is possible to provide expert advice on a comprehensive intestinal health condition and management plan. By providing comprehensive advice on the possibility of occurrence and prevention of various diseases according to the distribution of beneficial bacteria, harmful bacteria, and intermediate bacteria, each individual can manage their health according to the advice. For example, it may include advice on a desirable dietary direction according to the distribution of bacteria in the intestine or guidance on a later retest schedule.

Example 1 ( Feces DNA extraction method)

Take out the sample stored at -80°C in a deep freezer and store at 4°C for 20 minutes. While vortexing the sample for 1 minute (Vortex Genie2 No.G560E), it was confirmed that the sample was melted. At this time, 2 ml of InhibitEX buffer is added after visually checking the amount of fecal sample.

Vortexing (Benchmark BenchMixer, Multi-Tube Vortexer BV1010) at 2,500 rpm for 10 minutes to confirm that the sample is homogenized.

After storing the sample at 95°C (Biofree, Drying oven) for 10 minutes, centrifuge for 10 minutes. Separate 200 µl of the supernatant obtained through the centrifugation in a 1.5 ml microtube, and add 15 µl proteinase K. Add 200 µl of AL buffer and spin down after vortexing (Vortex Genie2 No.G560E) for 15 seconds. After storing the sample at 70°C (Biofree, Drying oven) for 10 minutes, add 200 μl of 100% ethanol to the sample, vortexing (Vortex Genie2 No.G560E) and spin down.

Transfer to a new 2 ml QIAamp spin column cap and centrifuge (VISION, VS-15000CFNII) for 2 minutes at 12,000 rpm. After inserting the column into a new 2ml collection tube, add 500 µl AW1 buffer and centrifuge at 12,000 rpm for 2 minutes (VISION, VS-15000CFNII) to discard the filtered liquid. After inserting the column into a new 2ml collection tube, add 500 µl AW2 buffer and centrifuge (VISION, VS-15000CFNII) at 12,000 rpm for 4 minutes to discard the filtered liquid. Place the column in a new 2ml collection tube and centrifuge for 4 minutes (VISION, VS-15000CFNII) to discard the filtered liquid. Insert the column into a new 1.5 ml microtube and add 200 µl of ATE buffer to the membrane. After storing for 5 minutes at room temperature, centrifuge (VISION, VS-15000CFNII) for 2 minutes at 12,000 rpm.

The eluted solution is transferred to the membrane and centrifuged at 12,000 rpm for 2 minutes (VISION, VS-15000CFNII), and then DNA is extracted.

Example 2 (real time PCR )

Take out the primer and DNA to be analyzed at -20℃ and melt it at 4℃. Put the second distilled water in a 15 ml conical tube. Label the DNA mixture and Taq mixture in a 1.7 ml tube. Prepare the Taq mixture and DNA mixture accordingly. Taq mixture is prepared by calculating 2 (number of repetitions) x (number of DNA + NC + spare) including negative control (hereinafter, NC, control). Dispense 14.4 µl of taq mixture into PCR plate (96-well, 0.1 ml, APPLIED BIOSYSTEMS) or strip (MicroAmp ^ⓡ FAST Reaction Tubes, 8 Tubes/Strip) to suit the plate setting. Dispense 5.6 µl of DNA mixture and DW according to the plate setting on the PCR plate or strip where the taq mixture was dispensed. ^{MicroAmp TM} when dispensed on PCR plate Adheres well to Optical Adhesive Film PCR Compatible, DNA / RNA / RNase Free , and if the busy Strip to close the ^ⓡ MicroAmp Optical 8-Cap Strip, and spun down. PCR plate (96-well, 0.1 ml, APPLIED BIOSYSTEMS) or strip (MicroAmp ^ⓡ FAST Reaction Tubes, 8 Tubes/Strip) dispensed in Real-time qPCR (Step One Plus ^{TM, APPLIEDBIOSYSTEMS) and conduct PCR.}

Experimental Example 1 .

To determine the amount of InhibitEX buffer required to extract DNA of an appropriate concentration, set the amount of InhibitEX buffer to 2 ml and 4 ml in the collection tube containing fecal samples, homogenize for DNA extraction, and determine the amount of DNA in 200 ul of the elution buffer. It was measured.

As a result, the DNA concentration confirmed as a result of attempting to extract DNA with 2 ml buffer was 43.9 ng/ul, and the confirmed DNA concentration as a result of attempting to extract DNA with 4 ml buffer was 7.35 ng/ul. Eventually, when DNA was extracted by adding 2ml Inhibit buffer than when 4ml Inhibit buffer was added, it was confirmed that about 6 times more DNA was detected.

<Table> Experimental result of volume measurement of InhibitEx buffer for DNA extraction

Experimental Example 2 .

In order to set the appropriate concentration of the beads used for DNA extraction, the concentrations of the beads were respectively 0g/0.1g/0.2g, and the degree to which DNA was extracted was compared.

The same DNA extraction experiment was performed using the same sampling sample in order to reduce the error of the result value due to other factors excluding the bead concentration.

<Table> Experimental result of mass measurement of beads for DNA extraction

As a result, the DNA concentration was the highest in the sample containing 0.2g of beads in the elution buffer (200ul) after DNA extraction.

Experimental Example 3 . annealing temperature test

When performing PCR, the annealing temperature is determined according to the Tm value of the primer. The annealing temperature test was conducted at 50°C, 55°C, and 60°C, which are mainly used temperatures.

The amplification peaks were confirmed at 50°C, 55°C, and 60°C, respectively, and the Ct (cycle threshold) value was confirmed.

(a) Check the Ct (cycle threshold) value at 50℃

(b) Checking the Ct(cycle threshold) value at 55℃

(c) Check Ct(cycle threshold) value at 60℃

As a result, the probability of amplification of non-specific DNA was high at 50°C, and although the Ct (cycle threshold) values were similarly confirmed at both 55°C and 60°C, the specificity for template DNA was high at 60°C. It has been shown that 60°C is more desirable than 55°C because the samples can be viewed simultaneously.

Experimental Example 4 .

PCR can synthesize a large amount of DNA using a very small amount of DNA. The quantity and quality of template DNA absolutely influences PCR. As the number of templates decreases, the amount of the product decreases proportionally, and the impure mold having a lower purity contains a lot of reaction inhibitors, which may lower the reaction efficiency. The sensitivity of the concentration was tested by PCR at the DNA concentration of 10 ng/ul and 50 ng/ul.

Drawing. DNA concentration test result for PCR sensitivity

As a result, when comparing the ratio of each bacteria after PCR, when PCR was performed with 50 ng/ul DNA, the sensitivity of the bacteria to each primer was low, and there was a sample in which the value of a specific bacteria could not be confirmed (blue circle). Finally, DNA at a concentration of 10 ng/ul evenly showed the sensitivity of each bacteria to all samples, and accordingly, there was no problem in confirming the diversity of various bacteria, which is the purpose of this analysis service.

<110> Bioeleven <120> Method for Gut Microbiota Analysis Using Real-time PCR <130> 16p <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus spp F <400> 1 agcagtaggg aatcttcca 19 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus spp R <400> 2 caccgctaca catggag 17 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Bifidobacterium spp. F <400> 3 gggtggtaat gccggatg 18 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Bifidobacterium spp.R <400> 4 taagcgatgg actttcacac c 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Clostridium spp. F <400> 5 cggtacctga ctaagaagc 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Clostridium spp. R <400> 6 agtttyattc ttgcgaacg 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides spp. F <400> 7 atagcctttc gaaagraaga t 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides spp. R <400> 8 ccagtatcaa ctgcaatttt a 21

Claims (7)

A method for intestinal bacteria analysis for analyzing intestinal health information of a subject,
Collecting a sample of the fetus in a debriefing kit comprising a buffer;
Cryogenic treatment of the flesh sample;
Extracting DNA from the flesh sample; And
Quantitative analysis of the extracted DNA revealed that intestinal distribution of Lactobacillus spp ., Bifidobacterium spp . , Clostridium spp . And Bacteroides spp. Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
The method according to claim 1,
Wherein the low-temperature treatment is carried out at a temperature of from -75 ° C to -85 ° C.
The method according to claim 1,
The step of extracting DNA comprises the steps of adding 1.5 to 2.5 ml of InhibitEX buffer solution containing 0.15 to 0.25 g of beads to the veneer kit; Homogenizing the buffer solution; Separating the supernatant in the presence of DNA; And a step of eluting the supernatant.
The method according to claim 1,
Wherein the DNA quantitative analysis uses real-time gene amplification technology (Real-time qPCR).
5. The method of claim 4,
Wherein the annealing temperature of the real-time PCR is 55 ° C to 60 ° C and the template DNA is 10ng / ul to 15ng / ul.
5. The method of claim 4,
Wherein the real-time PCR amplification technique uses the primer pairs of SEQ ID NO: 1 to SEQ ID NO: 9.
The method according to claim 1, wherein the intestinal health information of the subject includes the possibility of disease-related disease.

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