KR102141246B1 - Method for the diagnosis of colon cancer using analysis of qPCR - Google Patents

Abstract

The present invention relates to a method of diagnosing colon cancer through qPCR analysis, and relates to a method of diagnosing colon cancer by measuring the amount of microorganisms contained in a fecal sample to be analyzed. The diagnostic method according to the present invention is widely used in the field of colorectal cancer diagnosis because it can predict and/or diagnose colorectal cancer with high accuracy and sensitivity using feces that can be easily and painlessly obtained from a subject to be diagnosed. In addition to this, it is expected that this will enable the early diagnosis of colorectal cancer and significantly increase the effectiveness of colorectal cancer treatment.

Description

{Method for the diagnosis of colon cancer using analysis of qPCR}

The present invention relates to a method of diagnosing colon cancer through qPCR analysis, and more particularly, to a method of diagnosing colon cancer by analyzing genes of microorganisms present in feces through qPCR analysis.

Colon cancer or colorectal cancer is a malignant tumor of the appendix, colon, and rectum that originates from the mucosa, the innermost surface of the large intestine. According to the announcement in 2006, it is the second most common cancer in Korea after gastric cancer, and the frequency of eating has been increasing rapidly in recent years. The death rate from colorectal cancer in the last 10 years is about 80%. Increasingly, the rate of ascent continues to increase. The prevalence age occurs most frequently in the 60's, and by region, it occurs slightly more often in the rectum than in the colon. Colon cancer can occur at any age, but in over 90% of people with colorectal cancer, the age is over 40, and the incidence rate doubles every 10 years. It is known that all colorectal cancers start with a benign polyp, and polyps are the abnormal growths of the epithelium in the lining of the large intestine, which is a very common disease in 15-20% of adults. In addition to colon polyps, if a family member has colon cancer, the risk of colorectal cancer increases even if they have had ulcerative colitis for a long period of time. In addition, colorectal cancer is known to be associated with food, and is representative of cancers. It is known that the westernization of diet has low fiber intake, high intake of animal fat, or excessive intake of refined sugar (sugar). .

In the case of early colorectal cancer, there are no special symptoms, but even in the absence of symptoms, anemia may occur due to loss of blood due to invisible intestinal bleeding, and sometimes anorexia and weight loss. If the cancer has progressed, there may be changes in bowel habits, such as pain in the stomach, diarrhea, or constipation. Symptoms of rectal bleeding from the anus may appear, and the blood may appear bright red or black. When colon cancer has progressed, a lump that was not normally touched in the stomach may be touched. The most noticeable symptoms are changes in bowel movements, bloody stools, pain and anemia, and it is necessary to thoroughly investigate such changes in adults over 40 years of age.

Confirmation of colorectal cancer is possible only by detecting cancer cells through biopsy through colonoscopy. Most colorectal cancers have no symptoms at an early stage, making diagnosis very difficult, and currently there is no way to predict colorectal cancer in a non-invasive way. Since conventional diagnosis methods are often found when solid cancer such as colorectal cancer has progressed, in order to prevent medical costs and death caused by colorectal cancer, colorectal cancer incidence in high-risk groups is predicted in advance by predicting colorectal cancer occurrence and causative factors. It is an efficient way to provide preventative measures.

Meanwhile, it is known that the microorganisms that symbiotic with the human body reach 100 trillion times and are 10 times more than human cells, and the number of genes in the microorganism is more than 100 times that of the human gene. Microbiota (microbiota or microbiome) refers to a microbial community including bacteria, archaea, and eukarya in a given place of residence, and the gut microbiota plays an important role in human physiology. It is known to have a great influence on human health and disease through interaction with human cells. Recently, it has been reported that colon cancer is caused by intestinal bacteria having a specific toxin such as metaloprotease. However, in the diagnosis of colorectal cancer, a method for diagnosing colorectal cancer through genetic analysis by separating only bacteria from human derivatives such as feces has not been reported.

The present inventors have studied a method for predicting or diagnosing colorectal cancer with high accuracy and sensitivity, and have developed a method for diagnosing colorectal cancer with high accuracy and sensitivity by separating bacteria from fecal matter obtained.

Domestic application patent 10-2004-0039304

The present invention was devised to solve the problems in the prior art as described above, and a method for predicting or diagnosing colon cancer by separating the gene of a microorganism from a fecal sample that can be easily obtained and analyzing it with qPCR The purpose is to provide.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention comprises the steps of: (a) extracting DNA from a subject fecal sample;

(b) selected from the group consisting of bacteria of the genus Luminococcus (Ruminococcus), Fusobacterium (Fusobacterium), Bacteroides (Bacteroides), and Catenibacterium (Catenibacterium) for the extracted DNA Performing qPCR (quantitative PCR) using primer pairs and probes specific to one or more; And

(c) quantifying the DNA through the qPCR analysis, and providing a method for providing information for the diagnosis of colorectal cancer, comprising the step of comparing the amount of DNA derived from normal feces.

In addition, the present invention (a) extracting the DNA from the subject fecal sample;

(b) selected from the group consisting of bacteria of the genus Luminococcus (Ruminococcus), Fusobacterium (Fusobacterium), Bacteroides (Bacteroides), and Catenibacterium (Catenibacterium) for the extracted DNA Performing qPCR (quantitative PCR) using primer pairs and probes specific to one or more; And

(c) quantifying DNA through the qPCR analysis, and comparing the amount of DNA derived from normal feces, to provide a diagnostic method for diagnosis of colorectal cancer.

In addition, the present invention (a) extracting the DNA from the subject fecal sample;

(b) selected from the group consisting of bacteria of the genus Luminococcus (Ruminococcus), Fusobacterium (Fusobacterium), Bacteroides (Bacteroides), and Catenibacterium (Catenibacterium) for the extracted DNA Performing qPCR (quantitative PCR) using primer pairs and probes specific to one or more; And

(c) quantifying DNA through the qPCR analysis, and comparing the amount of DNA from normal feces, to provide a method for predicting the risk of developing colon cancer.

As an embodiment of the present invention, the DNA of step (a) may be DNA isolated from bacterial-derived extracellular vesicles in feces.

As another embodiment of the present invention, the primer pair of step (b) may be any one or more of the following:

A primer pair consisting of SEQ ID NOs: 3 and 4 capable of specifically amplifying the 16S rDNA of a bacterium of the genus Luminococcus;

A primer pair consisting of SEQ ID NOs: 6 and 7 capable of specifically amplifying 16S rDNA of a bacterium of the genus Fuzobacterium;

A primer pair consisting of SEQ ID NOs: 9 and 10 capable of specifically amplifying 16S rDNA of bacteria of the genus Bacteroides; And

Primer pair consisting of SEQ ID NOs: 15 and 16 capable of specifically amplifying 16S rDNA of bacteria of the genus Catenibacterium.

As another embodiment of the present invention, the probe of step (b) may be any one or more of the following:

A probe represented by SEQ ID NO: 5 capable of specifically binding to 16S rDNA of a bacterium of the genus Luminococcus;

A probe represented by SEQ ID NO: 8 capable of specifically binding to 16S rDNA of a bacterium of the genus Fuzobacterium;

A probe represented by SEQ ID NO: 11 capable of specifically binding to 16S rDNA of bacteria of the genus Bacteroides; And

A probe represented by SEQ ID NO: 17 that can specifically bind to 16S rDNA of bacteria of the genus Catenibacterium.

As another embodiment of the present invention, the step (c) comprises a primer pair specific for a bacterial universal 16S rDNA (bacterial 16S rDNA) or a human 18S rDNA and the gene amount quantified through qPCR in the step (b). As a result of performing qPCR using a probe, the method may further include a step of normalizing to a quantified gene amount.

As another embodiment of the present invention, the primer pair specifically amplifies a human 18S rDNA or a primer pair consisting of SEQ ID NOs: 18 and 19 that can specifically amplify a bacterial 16S rDNA (bacterial universal 16S rDNA). It may be a primer pair consisting of SEQ ID NO: 21 and 22.

As another embodiment of the present invention, the probe is a probe capable of specifically binding to bacterial universal 16S rDNA (bacterial universal 16S rDNA), or a sequence capable of specifically binding to human 18S rDNA. It may be a probe represented by the number 23.

As another embodiment of the present invention, when corrected with 16S rDNA of bacteria in step (c),

When the gene amount of bacteria in the genus Fusobacterium or Bacteroides is increased compared to normal people, it can be diagnosed as colon cancer.

As another embodiment of the present invention, when the step (c) is corrected with human 18S rDNA,

When the gene amount of bacteria in the genus Fusobacterium or Bacteroides is increased compared to the normal person; or

It can be diagnosed as colorectal cancer when the gene amount of bacteria in the genus Luminococcus or Catenibacterium is reduced compared to normal persons.

The diagnostic method according to the present invention is widely used in the field of colorectal cancer diagnosis because it can predict and/or diagnose colorectal cancer with high accuracy and sensitivity using feces that can be easily and painlessly obtained from a subject to be diagnosed. In addition to this, it is expected that this will enable the early diagnosis of colorectal cancer and significantly increase the effectiveness of colorectal cancer treatment.

1 is a view showing a result of comparing the amount of genes by separating bacteria and bacterial-derived vesicles (EV) from human feces according to an embodiment of the present invention.
2 is a view showing the results of evaluating analytical performance of the Ruminococcus genus, Fusobacterium genus, Bacteroides genus, Acinetobacter genus, Catenibacterium genus, bacterial universal 16S rDNA sequence and 18S rDNA specific gene primer according to an embodiment of the present invention.
Figure 3 is a colon cancer patient and normal control feces according to an embodiment of the present invention by separating bacteria from Ruminococcus genus, Fusobacterium genus, Bacteroides genus, Acinetobacter genus, Catenibacterium genus bacterial universal 16S rDNA and human cells It is a diagram showing the results confirmed by correcting with the 18S rDNA gene.

The present invention comprises the steps of: (a) extracting DNA from a subject's fecal sample;

(b) selected from the group consisting of bacteria of the genus Luminococcus (Ruminococcus), Fusobacterium (Fusobacterium), Bacteroides (Bacteroides), and Catenibacterium (Catenibacterium) for the extracted DNA Performing qPCR (quantitative PCR) using primer pairs and probes specific to one or more; And

(c) quantifying the DNA through the qPCR analysis, and providing a method for providing information for the diagnosis of colorectal cancer, comprising the step of comparing the amount of DNA derived from normal feces.

In addition, the present invention (a) extracting the DNA from the subject fecal sample;

(b) selected from the group consisting of bacteria of the genus Luminococcus (Ruminococcus), Fusobacterium (Fusobacterium), Bacteroides (Bacteroides), and Catenibacterium (Catenibacterium) for the extracted DNA Performing qPCR (quantitative PCR) using primer pairs and probes specific to one or more; And

(c) quantifying DNA through the qPCR analysis, and comparing the amount of DNA derived from normal feces, to provide a diagnostic method for diagnosis of colorectal cancer.

In addition, the present invention (a) extracting the DNA from the subject fecal sample;

(b) selected from the group consisting of bacteria of the genus Luminococcus (Ruminococcus), Fusobacterium (Fusobacterium), Bacteroides (Bacteroides), and Catenibacterium (Catenibacterium) for the extracted DNA Performing qPCR (quantitative PCR) using primer pairs and probes specific to one or more; And

(c) quantifying DNA through the qPCR analysis, and comparing the amount of DNA from normal feces, to provide a method for predicting the risk of developing colon cancer.

In the present invention, the DNA of step (a) may be DNA isolated from bacterial-derived extracellular vesicles in feces.

In the present invention, the primer pair of step (b) may be any one or more of the following:

A primer pair consisting of SEQ ID NOs: 3 and 4 capable of specifically amplifying the 16S rDNA of a bacterium of the genus Luminococcus;

A primer pair consisting of SEQ ID NOs: 6 and 7 capable of specifically amplifying 16S rDNA of a bacterium of the genus Fuzobacterium;

A primer pair consisting of SEQ ID NOs: 9 and 10 capable of specifically amplifying 16S rDNA of bacteria of the genus Bacteroides; And

Primer pair consisting of SEQ ID NOs: 15 and 16 capable of specifically amplifying 16S rDNA of bacteria of the genus Catenibacterium.

In the present invention, "primer (primer)" is a nucleic acid sequence having a short free 3-terminal hydroxyl group (free 3'hydroxyl group) that can form a base pair (base pair) that acts as a complementary template (template) A short nucleic acid sequence as a starting point for copying. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures and four different nucleoside triphosphates. PCR conditions, sense and antisense primer lengths can be modified based on those known in the art. The primer pair is composed of one pair of a forward alignment primer and a reverse alignment primer.

In the present invention, the probe of step (b) may be any one or more of the following:

A probe represented by SEQ ID NO: 5 capable of specifically binding to 16S rDNA of a bacterium of the genus Luminococcus;

A probe represented by SEQ ID NO: 8 capable of specifically binding to 16S rDNA of a bacterium of the genus Fuzobacterium;

A probe represented by SEQ ID NO: 11 capable of specifically binding to 16S rDNA of bacteria of the genus Bacteroides; And

A probe represented by SEQ ID NO: 17 that can specifically bind to 16S rDNA of bacteria of the genus Catenibacterium.

In the present invention, "probe (probe)" refers to a DNA or RNA fragment that is a fragment (or fragment) complementary to a specific nucleotide sequence of DNA or RNA, having a terminal base labeled with a radioactive element or fluorescence. It usually varies in length from 100-1000 bp and has complementary sequences to find specific nucleotide sequences in DNA or RNA samples in the molecular biology domain. Probe is used to identify the gene sequence you are looking for in a single strand of DNA or RNA through complementary binding to a target gene.

As another embodiment of the present invention, the step (c) comprises a primer pair specific for a bacterial universal 16S rDNA (bacterial 16S rDNA) or a human 18S rDNA and the gene amount quantified through qPCR in the step (b). As a result of performing qPCR using a probe, the method may further include a step of normalizing to a quantified gene amount, wherein the primer pair can specifically amplify bacterial universal 16S rDNA (bacterial universal 16S rDNA). It may be a primer pair consisting of SEQ ID NOs: 18 and 19 or a primer pair consisting of SEQ ID NOs: 21 and 22 that can specifically amplify human 18S rDNA, and the probe is specific for bacterial universal 16S rDNA (bacterial bacterial universal 16S rDNA). It may be a probe represented by SEQ ID NO: 20 capable of binding to or a probe represented by SEQ ID NO: 23 capable of specifically binding to human 18S rDNA.

In the present invention, when corrected with 16S rDNA of the bacteria in the step (c), when the gene amount of bacteria in the genus Fusobacterium or Bacteroides is increased compared to normal people, it becomes colon cancer. Can be diagnosed, and when corrected with human 18S rDNA in step (c), when the gene amount of bacteria in the genus Fusobacterium or Bacteroides is increased compared to normal humans; Alternatively, it can be diagnosed as colorectal cancer when the gene amount of bacteria in the genus Luminococcus or Catenibacterium is reduced compared to normal persons.

As used in the present invention, the term "diagnosis of colorectal cancer" means to determine whether a patient has a possibility of developing colorectal cancer, a relatively high probability of developing colorectal cancer, or whether colorectal cancer has already occurred. . The method of the present invention can be used to delay or prevent the onset of disease through special and appropriate management as a patient at high risk of developing colon cancer for any particular patient. In addition, the method of the present invention can be used clinically to determine treatment by early diagnosis of colorectal cancer and selecting the most appropriate treatment method.

In the present invention, “Polymerase Chain Reaction (PCR)” is a technique for rapidly amplifying a specific DNA sequence, and is used in various ways such as phylogenetic analysis, genetic testing, and DNA cloning. In particular, "quantitative PCR (qPCR)", also known as real-time polymerase chain reaction (Real-time PCR), is mainly used for screening high-speed and large-scale experimental results by identifying expression changes of multiple target genes. .

In one embodiment of the present invention, DNA is separated from fecal microorganisms (see Example 1), bacterial universal 16S rDNA sequence and Human 18S used as bacteria and correction markers of the genus Ruminococcus, Fusobacterium, Bacteroides, Acinetobacter, Catenibacterium Primers and probes specific to the rDNA gene were prepared (see Example 2).

In another embodiment of the present invention, as a result of performing an analytical performance test to determine the analytical sensitivity and specificity of the primers and probes, it was confirmed that the concentration was dependent on the amount of bacteria, and the gene content of bacteria isolated from feces was colonized. When compared to the cancer control group and the normal control group, when corrected to 16S, the Fusobacterium genus and Bacteroides genus showed a significant difference, and when corrected to 18S, the Ruminococcus genus, the Fusobacterium genus, the Bacteroides genus, and the Catenibacterium genus showed significant difference ( See Example 3).

In another embodiment of the present invention, based on the qPCR results, a colon cancer diagnosis model was prepared by using a logistic method using each microbial gene content as a variable, and using this, Ruminococcus genus, Fusobacterium genus, Bacteroides obtained from feces as an analysis sample It was confirmed that colon cancer can be diagnosed by using the amount of microorganisms of the genus and Catenibacterium as variables (see Example 4).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example One: From feces Microbial separation and gene quantification

To separate the microorganisms from the fecal sample, the large particles present in the feces were first removed using gauze, and then feces were added to a 10 ml tube. Subsequently, the pellet and the supernatant were separated by centrifugation (3,500 x g, 10 min, 4°C), and the pellet part and the supernatant part were added separately to a new 10 ml tube. After removing the bacteria and foreign substances using a 0.22㎛ filter, the supernatant is transferred to a centripreigugal filters (50 kD), centrifuged at 1500 xg, 4℃ for 15 minutes to remove substances smaller than 50 kD and concentrated to 10 ml. Ordered. Then, once again, 0.22㎛ filter was used to remove microorganisms and foreign substances, and the supernatant was discarded using a super-high-speed centrifugation method at 150,000 xg, 4℃ for 3 hours using a Type 90ti rotor, and the lumped pellet was dissolved in physiological saline (PBS) Ordered.

The extracellular vesicle (EV) separated by the above method was heated at 100° C. in a heatblock at 100° C. to allow the DNA inside to come out of the lipid and then cooled on ice. And the amount of DNA was quantified using Nanodrop. As a result, after centrifugation, 9.4 ng gDNA was extracted from 1 mL of feces with large particles removed from the pellet portion, and 3.5 ng gDNA from 1 mL of feces was extracted from extracellular vesicles extracted by ultracentrifugation. It was extracted (see Figure 1).

Subsequently, in order to confirm that the bacterial-derived DNA is present in the gene isolated from fecal bacteria, PCR was performed with the 16s rDNA primer shown in Table 1 below to confirm that the bacterial-derived gene exists in the extracted gene.

primer order Sequence number 16S rDNA 16S_V3_F 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3' One 16S_V4_R 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3 2

Example 2: Microbial gene primer making

Taqman Probe and Primer specific for each 16S rDNA sequence were prepared in order to quantify the amount of DNA of each of the biomarkers in DNA separated from feces by the method of Example 1 by quantitative PCR (qPCR) method (Taqman assay). . First, sequence information submitted to the NCBI database was collected, and then conserved regions and hypervariable regions in 16S rDNA sequences of each biomarker were identified through multiple alignment. In addition, Taqman Probe and Primer specific to each marker were produced through BLAST in a section with high diversity. The bacterial universal 16S rDNA sequence-specific Primer and Taqman Probe to be used as correction markers were produced through BLAST in a conserved region. Human 18S rDNA specific Primer and Probe to be used as another correction marker were produced by referring to literature. The prepared primer and detection probe sequences are shown in Table 2.

Ruminococcus Forward 5'-GTATGTAAAGCTCTATCAGC-3' SEQ ID NO: 3 Reverse 5'-TCCGGTACTCTAGATTGA-3' SEQ ID NO: 4 Probe 5'FAM-CCCGGGTTTTCACATCAGACTTGCCACTCCG-BHQ13' SEQ ID NO: 5 Fusobacterium Forward 5'-GAGGAACCTTACCAGCGT-3' SEQ ID NO: 6 Reverse 5'-CCCCAACTTAATGATGGTAACATAC-3' SEQ ID NO: 7 Probe 5'FAM-TTAGGAATGAGACAGAGATGTTTCAGTGTCC-BHQ13' SEQ ID NO: 8 Bacteroides Forward 5'-CGGGCTTAAATTGCAGTGGA-3' SEQ ID NO: 9 Reverse 5'-TGCAGCACCTTCACAGC-3' SEQ ID NO: 10 Probe 5'FAM-TGATGTGGAAACATGTCAGTGAGCAATCAC-BHQ13' SEQ ID NO: 11 Acinetobacter Forward 5'-CGAGGAGGAGGCTACTT-3' SEQ ID NO: 12 Reverse 5'-CGGTGCTTATTCTGCGA-3' SEQ ID NO: 13 Probe 5'FAM-TAGTTAATACCTAGAGATAGTGGACGTTAC-BHQ13' SEQ ID NO: 14 Catenibacterium Forward 5'-AGTCAACGCAATAAGTGAC-3' SEQ ID NO: 15 Reverse 5'-CACCACCTGTCTTCTCTATA-3' SEQ ID NO: 16 Probe 5'FAM-GATCTAAAAGGGATGGAGACATCCTCATAG-BHQ13' SEQ ID NO: 17 16S Forward 5'-TGTCGTCAGCTCGTGT-3' SEQ ID NO: 18 Reverse 5'-GGGTTGCGCTCGTTG-3' SEQ ID NO: 19 Probe 5'FAM-ATGTTGGGTTAAGTCCCG-BHQ13' SEQ ID NO: 20 18S Forward 5'-GCCCGAAGCGTTTACTTTGA-3' SEQ ID NO: 21 Reverse 5'-TCCATTATTCCTAGCTGCGGTATC-3' SEQ ID NO: 22 Probe 5'FAM-AAAGCAGGCCCGAGCCGCC-BHQ13' SEQ ID NO: 23

Example 3: In feces Using extracted DNA qPCR analysis

An analytical performance test was performed to examine the analytical sensitivity and specificity of the Primer and Taqman Probe prepared in Example 2. For the analytical performance test, a DNA Plasmid containing the DNA sequence of each target marker was prepared (Table 3).

Target Sequence (5'->3') Sequence number Ruminococcus GTATGTAAAGCTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCACCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAGTGGCAAGTCTGATGA SEQ ID NO: 24 Fusobacterium GAGGAACCTTACCAGCGTTTGACATCTTAGGAATGAGACAGAGATGTTTCAGTGTCCCTTCGGGGAAACCTAAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTTTCGTATGTTACCATCATTAAGTTGGGG SEQ ID NO: 25 Bacteroides CGGGCTTAAATTGCAGTGGAATGATGTGGAAACATGTCAGTGAGCAATCACCGCTGTGAAGGTGCTGCA SEQ.ID No. 26 Acinetobacter CGAGGAGGAGGCTACTTTAGTTAATACCTAGAGATAGTGGACGTTACTCGCAGAATAAGCACCG SEQ ID NO: 27 Catenibacterium AGTCAACGCAATAAGTGACCCGCCTGAGTAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCGATCTAAAAGGGATGGAGACATCCTCATAGCTATAGAAGAG SEQ.ID No. 28 16S TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC SEQ ID NO: 29 18S GCCCGAAGCGTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCCGAGCCGCCTGGATACCGCAGCTAGGAATAATGGA SEQ ID NO: 30

The prepared Plasmid was serial dilution (10 ² ~ 10 ⁶ ) in 10 copy units, and qPCR was performed as a template to perform analytical sensitivity test. qPCR experiment is Premix Ex Taq ^TM (Probe qPCR) (TAKARA #RR390A) and ABI Quantstudio 3 Real-Time PCR System, 96-well, 0.2mL (A28137). As a result, it was confirmed that Ruminococcus genus, Fusobacterium genus, Bacteroides genus, Acinetobacter genus, Catenibacterium genus, bacterial universal 16S rDNA sequence and 18S rDNA specific primer performance quantify the concentration dependent bacteria (see FIG. 2).

Then, bacteria were isolated from feces of 105 normal controls and 79 patients with colorectal cancer, and then DNA was extracted. Then, using the Ruminococcus, Fusobacterium, Bacteroides, Acinetobacter, Catenibacterium primers of Table 1 and the primers and probes of 18S rDNA and bacterial universal 16S rDNA sequences, which are correction markers, were subjected to qPCR, and the two groups were compared by relative quantification methods. . 4 µl of DNA template per well, Primer and Probe were reacted with a total of 20 µl using 18 pmol and 5 pmol respectively. At this time, PCR conditions were repeated 50 times for the initial 50°C 2 minutes, 95°C 10 minutes standby, and then 95°C 15 seconds, 60°C 1 minute cycle 40 times (Quantstudio standard run mode default). For relative quantification, 18S rDNA and bacterial universal 16S rDNA sequences of each sample were normalized, and the average value (±standard error) of the control group (n=105) and colorectal cancer group (n=79) was corrected for each biomarker correction value. ) Was compared through a t-test test. As a result, when the gene content of bacteria isolated from feces was compared in colorectal cancer patients and the normal control group, when corrected to 16S, Fusobacterium genus and Bacteroides genus showed significant differences, and when corrected to 18S, Ruminococcus genus, Fusobacterium genus, Bacteroides genus and Catenibacterium genus showed significant differences (see Table 4 and Figure 3). Through the above results, it was confirmed that colon cancer can be diagnosed by confirming the amount of microorganisms of the genus Ruminococcus, Fusobacterium, Bacteroides, and Catenibacterium in the fecal sample of the subject to be diagnosed.

16S correction Ruminococcus Fusobacterium Bacteroides Acinetobacter Catenibacterium Control Mean 0.7037 0.4527 0.4952 0.4481 0.5922 SD 0.0853 0.0494 0.0981 0.0557 0.1510 Colorectal cancer Mean 0.6579 0.5362 0.6014 0.4802 0.5394 SD 0.2017 0.3018 0.3279 0.3371 0.3228 F.TEST 0.0000 0.0000 0.0000 0.0000 0.0000 T.TEST 0.0612 0.0171 0.0065 0.4046 0.1811 18S correction Ruminococcus Fusobacterium Bacteroides Acinetobacter Catenibacterium Control Mean 1.3068 0.8405 0.9186 0.8322 1.0999 SD 0.1490 0.0825 0.1745 0.1012 0.2760 Colorectal cancer Mean 1.1705 0.9105 1.0240 0.7980 0.9243 SD 0.2125 0.1757 0.2266 0.1375 0.2718 F.TEST 0.0007 0.0000 0.0129 0.0035 0.8922 T.TEST 0.0000 0.0014 0.0008 0.0648 0.0000

Example 4: Development of a colon cancer diagnosis model through analysis of microbial genes in feces

Based on the results of Example 3, a model was developed for a method of diagnosing colon cancer by checking the amount of microorganisms in the genus Ruminococcus, Fusobacterium, Bacteroides, and Catenibacterium in fecal samples. To this end, based on the qPCR results of Example 3, a colon cancer diagnostic model was produced by using a logistic method using each microbial gene content as a variable (Table 5).

variable auc Fusobacterium.16s 0.568776 Bacteroides.16s 0.617842 Fusobacterium.18s 0.620735 Bacteroides.18s 0.640506 Ruminococcus.18s 0.690295 Catenibacterium.18s 0.690416 Fusobacterium.16s+Ruminococcus.18s 0.694635 Bacteroides.16s+Catenibacterium.18s 0.705365 Catenibacterium.18s+Fusobacterium.16s 0.705365 Bacteroides.18s+Catenibacterium.18s 0.708379 Bacteroides.16s+Ruminococcus.18s 0.714045 Catenibacterium.18s+Fusobacterium.18s 0.718385 Catenibacterium.18s+Ruminococcus.18s 0.733574 Fusobacterium.18s+Ruminococcus.18s 0.743098 Fusobacterium.16s+Fusobacterium.18s+Ruminococcus.18s 0.74346 Bacteroides.18s+Fusobacterium.16s+Ruminococcus.18s 0.744424 Bacteroides.18s+Catenibacterium.18s+Fusobacterium.18s 0.744665 Bacteroides.16s+Catenibacterium.18s+Ruminococcus.18s 0.745027 Catenibacterium.18s+Fusobacterium.16s+Ruminococcus.18s 0.747679 Bacteroides.16s+Fusobacterium.18s+Ruminococcus.18s 0.749247 Bacteroides.18s+Catenibacterium.18s+Ruminococcus.18s 0.760458 Bacteroides.18s+Fusobacterium.18s+Ruminococcus.18s 0.770102 Catenibacterium.18s+Fusobacterium.18s+Ruminococcus.18s 0.783725 Catenibacterium.18s+Fusobacterium.16s+Fusobacterium.18s+Ruminococcus.18s 0.783846 Bacteroides.16s+Catenibacterium.18s+Fusobacterium.18s+Ruminococcus.18s 0.785051 Bacteroides.18s+Catenibacterium.18s+Fusobacterium.18s+Ruminococcus.18s 0.798071 Bacteroides.16s+Bacteroides.18s+Catenibacterium.18s+Fusobacterium.18s+Ruminococcus.18s 0.798553 Bacteroides.18s+Catenibacterium.18s+Fusobacterium.16s+Fusobacterium.18s+Ruminococcus.18s 0.799156 Bacteroides.16s+Catenibacterium.18s+Fusobacterium.16s+Fusobacterium.18s+Ruminococcus.18s 0.800121

As a result, it was confirmed that AUC (area-under-curve), which is an index of diagnostic usefulness, ranged from 0.568776 to 0.8000241.

Through the above results, using the colon cancer diagnostic model of the present invention, if the amount of microorganisms of the genus Ruminococcus, Fusobacterium, Bacteroides, and Catenibacterium obtained from feces as an analysis sample is diagnosed as a variable, the diagnosis accuracy is very high. It was confirmed that it can be used as a cancer diagnosis method.

<110> MD Healthcare Inc. <120> Method for the diagnosis of colon cancer using analysis of qPCR <130> MP19-120 <150> KR 10-2018-0059683 <151> 2018-05-25 <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> 16S_V3_F <400> 1 tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50 <210> 2 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 16S_V4_R <400> 2 gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ruminococcus Forward primer <400> 3 gtatgtaaag ctctatcagc 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Ruminococcus Reverse primer <400> 4 tccggtactc tagattga 18 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Ruminococcus Probe: 5'is linked to a FAM and 3'is linked to a BHQ1 <400> 5 cccgggtttt cacatcagac ttgccactcc g 31 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium Forward primer <400> 6 gaggaacctt accagcgt 18 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium Reverse primer <400> 7 ccccaactta atgatggtaa catac 25 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium Probe: 5'is linked to a FAM and 3'is linked to a BHQ1 <400> 8 ttaggaatga gacagagatg tttcagtgtc c 31 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides Forward primer <400> 9 cgggcttaaa ttgcagtgga 20 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides Reverse primer <400> 10 tgcagcacct tcacagc 17 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides Probe: 5'is linked to a FAM and 3'is linked to a BHQ1 <400> 11 tgatgtggaa acatgtcagt gagcaatcac 30 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Acinetobacter Forward primer <400> 12 cgaggaggag gctactt 17 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Acinetobacter Reverse primer <400> 13 cggtgcttat tctgcga 17 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Acinetobacter Probe: 5'is linked to a FAM and 3'is linked to a BHQ1 <400> 14 tagttaatac ctagagatag tggacgttac 30 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Catenibacterium Forward primer <400> 15 agtcaacgca ataagtgac 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Catenibacterium Reverse primer <400> 16 caccacctgt cttctctata 20 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Catenibacterium Probe: 5'is linked to a FAM and 3'is linked to a BHQ1 <400> 17 gatctaaaag ggatggagac atcctcatag 30 <210> 18 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> bacterial universal 16S rDNA Forward primer <400> 18 tgtcgtcagc tcgtgt 16 <210> 19 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> bacterial universal 16S rDNA Reverse primer <400> 19 gggttgcgct cgttg 15 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> bacterial universal 16S rDNA Probe: 5'is linked to a FAM and 3' is linked to a BHQ1 <400> 20 atgttgggtt aagtcccg 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human 18S rDNA Forward primer <400> 21 gcccgaagcg tttactttga 20 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Human 18S rDNA Reverse primer <400> 22 tccattattc ctagctgcgg tatc 24 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Human 18S rDNA Probe: 5'is linked to a FAM and 3'is linked to a BHQ1 <400> 23 aaagcaggcc cgagccgcc 19 <210> 24 <211> 215 <212> DNA <213> Artificial Sequence <220> <223> Ruminococcus <400> 24 gtatgtaaag ctctatcagc agggaagaaa atgacggtac ctgactaaga agcaccggct 60 aaatacgtgc cagcagccgc ggtaatacgt atggtgcaag cgttatccgg atttactggg 120 tgtaaaggga gcgtagacgg agtggcaagt ctgatgtgaa aacccggggc tcaaccccgg 180 gactgcattg gaaactgtca atctagagta ccgga 215 <210> 25 <211> 176 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium <400> 25 gaggaacctt accagcgttt gacatcttag gaatgagaca gagatgtttc agtgtccctt 60 cggggaaacc taaagacagg tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg 120 gttaagtccc gcaacgagcg caaccccttt cgtatgttac catcattaag ttgggg 176 <210> 26 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> Bacteroides <400> 26 cgggcttaaa ttgcagtgga atgatgtgga aacatgtcag tgagcaatca ccgctgtgaa 60 ggtgctgca 69 <210> 27 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Acinetobacter <400> 27 cgaggaggag gctactttag ttaataccta gagatagtgg acgttactcg cagaataagc 60 accg 64 <210> 28 <211> 190 <212> DNA <213> Artificial Sequence <220> <223> Catenibacterium <400> 28 agtcaacgca ataagtgacc cgcctgagta gtacgttcgc aagaatgaaa ctcaaaggaa 60 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 120 cttaccaggt cttgacatcg atctaaaagg gatggagaca tcctcatagc tatagagaag 180 acaggtggtg 190 <210> 29 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> bacterial universal 16S rDNA <400> 29 tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aaccc 55 <210> 30 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Human 18S rDNA <400> 30 gcccgaagcg tttactttga aaaaattaga gtgttcaaag caggcccgag ccgcctggat 60 accgcagcta ggaataatgg a 81

Claims (9)

(a) extracting DNA from a subject's fecal sample;
(b) qPCR using primer pairs and probes specific for 16S rDNA of bacteria of the genus Fusobacterium against the extracted DNA, and primer pairs and probes specific for 16S rDNA of bacteria of the genus Bacteroides performing (quantitative PCR); And
(c) quantifying DNA through the qPCR analysis, and comparing the amount of DNA derived from normal feces, as a method of providing information for the diagnosis of colon cancer,
The method of providing information for the diagnosis of colorectal cancer, characterized in that when the gene amount of bacteria of the genus Peugeot bacterium and Bacteroides is increased compared to normal persons.
According to claim 1,
The DNA of the step (a) is characterized in that the DNA isolated from bacterial-derived extracellular vesicles in feces, a method for providing information for the diagnosis of colorectal cancer.
According to claim 1,
The primer pair of step (b) is characterized in that the following primer pair, information providing method for the diagnosis of colorectal cancer:
A primer pair consisting of SEQ ID NOs: 6 and 7 capable of specifically amplifying 16S rDNA of bacteria of the genus Fuzobacterium; And
Primer pair consisting of SEQ ID NOs: 9 and 10 capable of specifically amplifying 16S rDNA of bacteria of the genus Bacteroides.
According to claim 1,
Probe of step (b) is characterized in that the following probes, information providing method for the diagnosis of colorectal cancer:
A probe represented by SEQ ID NO: 8 capable of specifically binding to 16S rDNA of a bacterium of the genus Fuzobacterium; And
Probe represented by SEQ ID NO: 11 capable of specifically binding to 16S rDNA of bacteria of the genus Bacteroides.
According to claim 1,
In the step (c), qPCR was performed using a primer pair and a probe specific for a bacterial 16S rDNA (bacterial universal 16S rDNA) or a human 18S rDNA in the amount of gene quantified through qPCR in the step (b). A method for providing information for the diagnosis of colorectal cancer, characterized in that it further comprises the step of correcting (normalization) with the quantified gene amount.
The method of claim 5,
The primer pair consists of SEQ ID NOs: 18 and 19 capable of specifically amplifying bacterial universal 16S rDNA (bacterial universal 16S rDNA), or consisting of SEQ ID NOs: 21 and 22 capable of specifically amplifying human 18S rDNA. Method of providing information for the diagnosis of colorectal cancer, characterized in that the primer pair.
The method of claim 5,
The probe is characterized by being a probe represented by SEQ ID NO: 20 capable of specifically binding to bacterial universal 16S rDNA (bacterial universal 16S rDNA) or a probe represented by SEQ ID NO: 23 capable of specifically binding to human 18S rDNA. The method of providing information for diagnosis of colorectal cancer.
delete delete

Images