CN108676871B - Diagnostic marker for type II diabetes - Google Patents
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- CN108676871B CN108676871B CN201810744848.3A CN201810744848A CN108676871B CN 108676871 B CN108676871 B CN 108676871B CN 201810744848 A CN201810744848 A CN 201810744848A CN 108676871 B CN108676871 B CN 108676871B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses an EDU99824.1 gene of an intestinal microorganism B.coprocola strain, which can be used as a molecular marker for diagnosing type II diabetes. The invention discovers that the genotypes of the 879 th site and the 880 th site of the EDU99824.1 gene have difference in intestinal B.coprocola strains of healthy people and non-drug type II diabetes patients by using a PCR (polymerase chain reaction) and Sanger sequencing method, namely whether a subject has type II diabetes can be judged by detecting the two SNP sites of the EDU99824.1 gene in a fecal genome. The diagnostic kit can be used for diagnosing type II diabetes and has wide application prospect in clinic.
Description
Technical Field
The invention belongs to the field of molecular diagnosis, relates to a molecular marker for diagnosing type II diabetes, and particularly relates to application of an SNP locus of an EDU99824.1 gene of a B.coprocola strain in a fecal genome in preparation of a product for diagnosing type II diabetes.
Background
Type two Diabetes (Diabetes mellitus type 2, T2DM) is a metabolic disease. Type ii diabetes is a long-term metabolic disease characterized by hyperglycemia, relative insulin deficiency, insulin resistance, and the like. The common symptoms are polydipsia, frequent urination and weight loss of unknown origin. Type ii diabetes mainly occurs in obese but lack of exercise, with higher risk for congenital genetic subjects.
For more than 30 years, the prevalence rate of adult diabetes in China has increased significantly. The 2010 Chinese center for disease prevention and control (CDC) and the division of Chinese medical society into endocrinology investigate the disease condition of diabetes of people aged 18 years and older in China, and show that the prevalence rate of diabetes is 9.7%. The monitoring of chronic diseases and risk factors of the chronic diseases in China in 2013 shows that the prevalence rate of diabetes of people of 18 years old and above is 10.4%.
Diagnosis of type ii diabetes includes detection of blood glucose levels and diagnosis of chronic complications specific to diabetes:
laboratory examination indexes:
the diagnosis focus of normal blood glucose and abnormal glucose metabolism is mainly determined by the relationship between blood glucose level and the chronic complications (diabetic retinopathy) and the risk of diabetes mellitus, which are specific to diabetes mellitus. The currently commonly used diagnostic criteria are fasting glucose (FPG) and 2h post-glucose load (OGTT). Some countries use HbA1c (glycated hemoglobin) as a method for screening high risk groups for diabetes and diagnosing diabetes. Compared with the OGTT test, the HbA1c is simple and easy to implement, stable in result, small in variability, free from the influence of eating time and short-term lifestyle change, and good in patient dependence. However, because the HbA1c detection is not common in China and the standardization degree of the detection method is not enough, instruments for measuring HbA1c and quality control cannot meet the requirements of the current diabetes diagnosis standard. The current diagnosis of type ii diabetes based on blood glucose testing is costly and not suitable for general screening.
Diagnosis of chronic complications: including diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, vascular disease of lower limbs, and diabetic foot disease. The subjects for chronic complication examination are generally type II diabetes late stage patients, and cannot be used for screening early stage type II diabetes.
Based on the limitations of the means for detecting type II diabetes in the prior art, finding an effective and cheap method capable of diagnosing or screening type II diabetes at an early stage is an urgent problem to be solved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a molecular marker for early diagnosis of Diabetes mellitus type 2 (T2 DM). Compared with the traditional diagnosis method of type II diabetes, the method for diagnosing type II diabetes by using the gene SNP of the bacterial strain of the intestinal microorganism B.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a product for detecting the genotype of 879 th site and/or 880 th site of EDU99824.1 gene in an enteromicroorganism B.
Further, the above-mentioned products for determining the genotype at the 879 th site and/or the 880 rd site of the EDU99824.1 gene in the b. coprocola strain of the enteric microorganism include: and detecting the genotype products at the 879 th site and/or 880 rd site of the EDU99824.1 gene by methods of PCR, real-time quantitative PCR, Taqman probe typing, gene chip, digital PCR, Sanger sequencing and the like.
Further, the product for diagnosing type II diabetes by PCR comprises a primer for amplifying the EDU99824.1 gene; the product for typing and diagnosing the type II diabetes mellitus by using the Taqman probe comprises a primer for amplifying an EDU99824.1 gene; the product for diagnosing the type II diabetes by using the real-time quantitative PCR comprises a primer for amplifying an EDU99824.1 gene; the product for diagnosing the type II diabetes by using the gene chip comprises a primer for amplifying an EDU99824.1 gene; the method using digital PCR comprises a primer for amplifying the EDU99824.1 gene; the method of sequencing by Sanger included amplification of primers for the EDU99824.1 gene.
In a specific embodiment of the present invention, the primers for amplifying the EDU99824.1 gene include a primer for specifically amplifying the EDU99824.1 gene and/or a primer for non-specifically amplifying the DU99824.1 gene; wherein the primer sequence of the specific amplification EDU99824.1 gene is shown as SEQ ID NO.1-SEQ ID NO.6, and the primer sequence of the non-specific amplification EDU99824.1 gene is shown as SEQ ID NO.7-SEQ ID NO. 10.
The present invention also provides a tool for diagnosing type two diabetes mellitus by detecting the genotype of 879 th site and/or 880 rd site of EDU99824.1 gene in coprocola strain in a sample.
Further, the diagnostic tool comprises a chip, a kit or a high throughput sequencing platform.
The high-throughput sequencing platform is a special diagnostic tool, and with the development of a high-throughput sequencing technology, the construction of a gene expression profile of a human intestinal microorganism becomes very convenient work. By comparing the gene expression maps of the intestinal microorganisms of patients with diseases and normal people, which gene abnormality is related to the diseases is easily analyzed. Therefore, the application of the SNP site of the EDU99824.1 gene of the B.coprocolla strain and the SNP of the EDU99824.1 gene of the B.coprocolla strain which are related to type II diabetes are also known in high-throughput sequencing and are also in the protection scope of the invention.
Further, the chip comprises a gene chip; the gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting genotypes of 879 th site and/or 880 th site of EDU99824.1 gene; the gene chip can be used for detecting a plurality of genes (for example, a plurality of intestinal microorganism genes related to type II diabetes) including the EDU99824.1 gene. By simultaneously detecting a plurality of markers related to the type II diabetes, the accuracy of the type II diabetes can be greatly improved.
Further, the kit comprises a gene detection kit; the gene detection kit comprises a reagent for detecting the genotype of 879 th site and/or 880 rd site of EDU99824.1 gene.
Further, the reagents include reagents required for detecting the genotype of the 879 th site and/or 880 rd site of the EDU99824.1 gene by using PCR, Taqman probe typing, real-time quantitative PCR, gene chip, and digital PCR.
Preferably, the reagents comprise primers and/or probes for the EDU99824.1 gene. Primers and probes for detecting the SNP sites of the EDU99824.1 gene can be easily designed according to the nucleotide sequence of the EDU99824.1 gene and specific SNP site information.
The probes hybridizing to the nucleic acid sequences of the EDU99824.1 gene and the EDV01869.1 gene may be DNA, RNA, DNA-RNA chimeras, PNA or other derivatives. Therefore, the length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene.
Further, the high-throughput sequencing platform comprises a reagent for detecting the genotype of 879 th site and/or 880 th site of EDU99824.1 gene.
Further, the reagent for detecting the genotype of the 879 th site and/or 880 th site of EDU99824.1 in the B.coprocola strain comprises a primer and/or a probe aiming at the EDU99824.1 gene.
Further, the primers for amplifying the EDU99824.1 gene include a primer for specifically amplifying the EDU99824.1 gene and/or a primer for non-specifically amplifying the DU99824.1 gene; wherein the primer sequence of the specific amplification EDU99824.1 gene is shown as SEQ ID NO.1-SEQ ID NO.6, and the primer sequence of the non-specific amplification EDU99824.1 gene is shown as SEQ ID NO.7-SEQ ID NO. 10.
The EDU99824.1 gene for diagnosing type II diabetes is derived from the genome DNA of intestinal microorganisms of a subject. In a specific embodiment of the present invention, the source of the EDU99824.1 gene for diagnosing type ii diabetes is fecal sample genomic DNA.
The reference gene sequence of the EDU99824.1 gene provided by NCBI is shown in SEQ ID NO. 11.
In the context of the present invention, "diagnosing type two diabetes" includes both determining whether a subject has suffered from type two diabetes and determining whether a subject is at risk of suffering from type two diabetes.
The invention has the advantages and beneficial effects that:
the gene of the B.coprocola bacterium is specifically amplified by adopting a PCR method from the genome DNA of the intestinal microorganism for the first time, the correlation between the SNP locus genotype of the EDU99824.1 gene of the intestinal microorganism B.coprocola bacterium and type II diabetes is found for the first time, and whether the subject suffers from type II diabetes or not or whether the risk of suffering from type II diabetes exists in the subject can be judged by detecting the SNP locus genotype specific to the EDU99824.1 gene in the subject, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject.
Compared with the traditional detection means, the invention provides a new method for diagnosing type II diabetes by aiming at the diagnosis of the intestinal microorganism gene and also provides a new reference value for clinically treating type II diabetes.
Drawings
FIG. 1 is a diagram showing the result of comparison of the mutation regions of EDU99824.1 gene;
FIG. 2 shows a graph of the results of a partial gene evolutionary tree analysis of EDU99824.1, in which A: a clustering result graph; b: analysis of mutation sites.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under the conditions as usual or as suggested in the specification.
Example screening of SNP site of EDU99824.1 Gene in Enterobacter microorganism B. coprocolla associated with type II diabetes
1. Study object
Type ii diabetes group: 74 initial patients with type II diabetes mellitus who were collected in the endocrinology department of hospitals were randomly selected and included in the standard: meets the diabetes diagnosis standard of WHO 1999.
Normal group: 77 healthy volunteers were selected.
All subjects were informed of the study and signed an informed consent.
2. Extraction of fecal genomic DNA
200g of a fecal sample stored in a refrigerator at-80 ℃ is taken, and the genomic DNA of the fecal sample is extracted by using a Tiangen fecal genomic DNA extraction kit according to the operation flow of the Tiangen fecal genomic DNA extraction instruction manual.
3. DNA purity detection
DNA purity: the OD260/OD280 ratio is an index for the degree of protein contamination in the DNA sample. The OD260/OD280 of the high-quality DNA sample is about 1.8.
4. PCR amplification of target genes
(1) Primer design
PCR amplification products were designed based on the coding sequence of EDU99824.1 gene in Genbank and synthesized by Biotech, Inc., Yihui, Beijing. The specific primer sequences of the synthesized primer capable of specifically amplifying the target gene (secondary PCR primer) are as follows:
first pair of specific amplification primers:
forward primer 5'-ACACTCTCCAAAGCCTCTGC-3' (SEQ ID NO.1),
reverse primer 5'-AAAGGCATGTGCTGTTGCTG-3' (SEQ ID NO.2)
A second pair of specific amplification primers:
forward primer 5'-CCCCTAATCCAGCAACAGCA-3' (SEQ ID NO.3)
Reverse primer 5'-GCAAGCGAGAAAAATGGGCA-3' (SEQ ID NO.4)
A third pair of specific amplification primers:
forward primer 5'-ATACAGAATCACGTCCCGGC-3' (SEQ ID NO.5)
Reverse primer 5'-TGCAGTTTGATGGTCAGCCT-3' (SEQID NO.6)
The specific primer sequences of the synthesized target gene (primary PCR primer) capable of non-specifically amplifying are as follows:
first pair of non-specific amplification primers:
forward primer 5'-CTTTCGGGTCATTATCACGTACAAG-3' (SEQ ID NO.7)
Reverse primer 5'-TAGCTTAGACCGTATAAATTCGGGG-3' (SEQ ID NO.8)
Second pair of non-specific amplification primers:
forward primer 5'-TCGTATTTTGCACATCCTCTCCTAT-3' (SEQ ID NO.9)
Reverse primer 5'-TCAGATAAAGATTGTTGCACGTCAC-3' (SEQ ID NO.10)
(2) The amplification mode adopts common PCR, a secondary amplification mode is applied, two pairs of pre-designed non-specific amplification primers are selected to carry out primary PCR (20 mu l system), the reaction system is shown in Table 1, and the reaction conditions are as follows: 3min at 94 ℃ (30 s at 94 ℃, 30s at 58 ℃, 2min at 72 ℃) by 40 cycles, 5min at 72 ℃. Three pairs of pre-designed specific amplification primers were selected for secondary PCR (30. mu.l system), the reaction system is shown in Table 2, and the reaction conditions: 3min at 94 ℃ (30 s at 94 ℃, 30s at 55 ℃, 2min at 72 ℃) by 40 cycles, 5min at 72 ℃.
TABLE 1 one-time PCR reaction System
TABLE 2 Secondary PCR reaction System
| Reagent | Volume of |
| Forward primer | 0.6μl |
| Reverse primer | 0.6μl |
| Tiangen 2 Xpfu PCR MasterMix | 15μl |
| Form panel | 1μl |
| ddH2O | Make up to 30. mu.l |
5. Detecting a target strip
The band of interest was detected by the method of ordinary agarose gel electrophoresis (electrophoresis conditions: 1% gel; 1 XTAE electrophoresis buffer; 90v, 30 min).
6. Sequencing of PCR products
The PCR products were sequenced by Sanger.
7. Analysis of sequencing results
The obtained sequencing results were subjected to evolutionary tree analysis using MEGA7 and RAxML, and SNPs in the sequence were counted using SeqMan.
8. Results
As a result, as shown in fig. 1 and fig. 2, compared with the reference gene sequence (Ref) of EDU99824.1 gene provided by NCBI, 40 healthy persons (HC) mutated at the 879 th site (a site in fig. 1) of EDU99824.1 gene from a base to G base, but the encoded amino acid was unchanged and still glutamic acid, which is synonymous mutation; in the 39 type II diabetics (T2D), mutation occurred at the 880 rd site (a +1 site in FIG. 1) of EDU99824.1 gene from C base to T base, and the encoded amino acid from proline to serine, which is missense mutation.
In FIG. 2A, the triangles represent healthy persons, the dots represent diabetic patients, the clustering results are clearly divided into two categories, the first category is mainly diabetic patients, the healthy persons are concentrated in the second category, FIG. 2B is the analysis of the corresponding mutation sites, the first category is subjected to mutation at 880 th site, the second category is subjected to mutation at 879 th site or not, the OR (odds ratio) value is 3.65, and the chi-square test p is 3.8 × 10-4Coprocola bacteria EDU99824.1 gene 879 site genotype and 880 site genotype are shown to have significant difference between diabetic patients and healthy people.
The above description of the examples is only suitable for understanding the method of the invention and its core idea. It should be noted that it would be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the principles of the invention, and these changes and modifications are also within the scope of the invention as defined in the appended claims.
Sequence listing
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<120> diagnostic marker for type II diabetes
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aaaggcatgt gctgttgctg 20
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cccctaatcc agcaacagca 20
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gcaagcgaga aaaatgggca 20
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atacagaatc acgtcccggc 20
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tgcagtttga tggtcagcct 20
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tagcttagac cgtataaatt cgggg 25
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tcagataaag attgttgcac gtcac 25
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atgaaaaaca gaagtatttt attagggtgt ttgggagcta tgctttgtat tacaagtatg 60
gctcagacta cagaagattt tccaaaaaaa cagaaacctt ggtctgtacg aatggccgaa 120
tcggaaatgg tacgtaatcc agaatcttgg caagtagatt ttcagccttc tttaaaatgg 180
gattattgcc atggattaga attgggggct atgatggatg tgtatgatcg atatggcgat 240
tcgaaatttt atgattatgc gctggcgtat gcagatacaa tggtaaatga ggatgggaca 300
ataaagaaat ataagttgac agattatagc ttagaccgta taaattcggg gaaaatgctt 360
ttccggattt atgagcagac taaggatgaa aaatataaaa aggctttaaa tctgttgaga 420
ctgcagtttg atggtcagcc tcgtaatgaa gatggcggtt tttggcataa aaaagtttat 480
cctaatcaaa tgtggctgga tgggctttat atggggacgc cgtttcttgc tgaatatgct 540
tatagaaata atgatccgca tgcctatcag gaagtgataa atcagataaa gattgttgca 600
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tggtatacaa tggctatagt tgataatctt gattttatac ctttgcatga gccgggacgt 780
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gaaggtttgt ggtatcaggt tatggataaa agtggagaac ctggaaacta tctggagtct 900
tcatgctcca gcatgttcgt ttattctctg tttaaggccg ttcgtaaggg atatatacct 960
tcatcatatt ttgctgtagc ccgtaaaggc tatgagggta ttttgaatga gtttataaaa 1020
gttgatgaga atggtcttgt ttccatcaca aaggcatgtg ctgttgctgg attaggggga 1080
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ccgaaagctg tgggaccttt tattttggcc agcttggaat gggaaggatt gcctaaggaa 1200
aaacgacgtt tcgctgagcc tagggagctg gttgttgctc aagataagac aggcgattat 1260
tctacgatag cagaggcttt ggagagtgtt cgtgctttta tggattttga tgtaaagata 1320
tatgtaaaaa agggtacgta taaagaaaaa ttgattattc cttcatggct gcaaaatgtt 1380
gaaatcatag gagaggatgt gcaaaatacg attataacta atgcagatca tgccaatatg 1440
aataatatgg gtaccttccg tacatataca gtcaaagtag aaggaaacca tataactttt 1500
cggaatataa cgatagaaaa caatgcgcct aaattaggtc aggctgtagc tttacataca 1560
gaaggagatt gtctgcgttt tattaactgc cgtttcttgg gtaatcagga tactgtctat 1620
acaggagttg caggtactcg actttatttt gaagattgtt atatcgaagg aacgaccgat 1680
ttcatatttg gaccgtcaac tgcatggttt gagaattgta cgatacacag taaggcaaat 1740
tcttatgtta cagctgcatc gactcctgaa aatatcaagt atggatatat ctttaataac 1800
tgtaagttaa cagcagaaga gggagttgat aaagtttatt taggaagacc atggcgcccg 1860
tacgcttata ctttatttat gaattgtgaa ttaggaaagc atatagtaaa agctggttgg 1920
gataattgga gaaatcctaa aaacgaaaag acagctcgtt atgcagagta taagaacacg 1980
ggagaaggag ctgatataag tcagcgtgta agttgggcac ggcaattatc tgataaagat 2040
gttaagatgc tggtgaaatc tgagttctat actttctcga atacatggga tttgtactaa 2100
Claims (12)
1. Detecting intestinal tractB.The application of the product of the genotype at the 880 th site of the coding gene of EDU99824.1 in the coprocola strain in the preparation of a tool for diagnosing type II diabetes is characterized in that the genotype at the 880 th site of the coding gene of EDU99824.1 is C/T, the site is an SNP site, and the coding gene sequence of EDU99824.1 is shown as SEQ ID NO. 11.
2. Use according to claim 1, characterized in that the product comprises: and detecting the genotype of the 880 th site of the coding gene of the EDU99824.1 by PCR, gene chip and Sanger sequencing.
3. Use according to claim 2, wherein the PCR comprises: digital PCR, real-time quantitative PCR.
4. The use of claim 2 or 3, wherein the reagents comprise primers that amplify position 880 of the gene encoding EDU 99824.1.
5. The use according to claim 4, wherein the primers comprise primers for specifically amplifying the 880 th site of the gene encoding EDU99824.1, and the sequence of the specific primers is shown as SEQ ID No.1-SEQ ID No. 6.
6. The use according to claim 5, wherein the primers further comprise a primer for non-specifically amplifying the 880 th site of the gene encoding EDU99824.1, and the sequence of the non-specific primer is shown as SEQ ID No.7-SEQ ID No. 10.
7. A tool for diagnosing type II diabetes, which comprises a reagent for detecting the genotype of the SNP site according to claim 1 in a sample, wherein the reagent comprises primers having the sequences shown as SEQ ID No.1 to SEQ ID No. 6.
8. The tool of claim 7, further comprising a chip or a high throughput sequencing platform.
9. The kit of claim 8, wherein said chip comprises a gene chip comprising a solid support and oligonucleotide probes immobilized on said solid support, said oligonucleotide probes comprising an oligonucleotide probe for detecting the genotype of 880 th site of said EDU99824.1 encoding gene.
10. The kit of claim 7, wherein the kit comprises a kit.
11. The kit of claim 10, wherein the kit comprises a genetic test kit.
12. The tool of any one of claims 7-11, wherein the sample is fecal genomic DNA.
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