WO2023234577A1 - 프로바이오틱스 조성물 내 균종 판별을 위한 프라이머 세트 및 이를 이용한 균종 판별 방법 - Google Patents
프로바이오틱스 조성물 내 균종 판별을 위한 프라이머 세트 및 이를 이용한 균종 판별 방법 Download PDFInfo
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- WO2023234577A1 WO2023234577A1 PCT/KR2023/006187 KR2023006187W WO2023234577A1 WO 2023234577 A1 WO2023234577 A1 WO 2023234577A1 KR 2023006187 W KR2023006187 W KR 2023006187W WO 2023234577 A1 WO2023234577 A1 WO 2023234577A1
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- bacterial species
- species
- primer set
- probiotic
- probiotic composition
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- 239000006041 probiotic Substances 0.000 title claims abstract description 78
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 78
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 45
- 239000000203 mixture Substances 0.000 title claims abstract description 35
- 241000894007 species Species 0.000 claims abstract description 133
- 230000000529 probiotic effect Effects 0.000 claims abstract description 62
- 108020004414 DNA Proteins 0.000 claims description 14
- 230000003321 amplification Effects 0.000 claims description 8
- 230000002538 fungal effect Effects 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 238000012163 sequencing technique Methods 0.000 claims description 6
- 108091023242 Internal transcribed spacer Proteins 0.000 claims description 4
- 241000194032 Enterococcus faecalis Species 0.000 claims description 3
- 240000002605 Lactobacillus helveticus Species 0.000 claims description 3
- 235000013967 Lactobacillus helveticus Nutrition 0.000 claims description 3
- 229940032049 enterococcus faecalis Drugs 0.000 claims description 3
- 229940054346 lactobacillus helveticus Drugs 0.000 claims description 3
- 241000193749 Bacillus coagulans Species 0.000 claims description 2
- 241001134770 Bifidobacterium animalis Species 0.000 claims description 2
- 241000186016 Bifidobacterium bifidum Species 0.000 claims description 2
- 241000186012 Bifidobacterium breve Species 0.000 claims description 2
- 241001608472 Bifidobacterium longum Species 0.000 claims description 2
- 241000194031 Enterococcus faecium Species 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000186660 Lactobacillus Species 0.000 claims description 2
- 240000001046 Lactobacillus acidophilus Species 0.000 claims description 2
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 2
- 241000186606 Lactobacillus gasseri Species 0.000 claims description 2
- 241000194035 Lactococcus lactis Species 0.000 claims description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 2
- 241000607142 Salmonella Species 0.000 claims description 2
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 2
- 241000194020 Streptococcus thermophilus Species 0.000 claims description 2
- 229940054340 bacillus coagulans Drugs 0.000 claims description 2
- 229940002008 bifidobacterium bifidum Drugs 0.000 claims description 2
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- 229940039696 lactobacillus Drugs 0.000 claims description 2
- 229940039695 lactobacillus acidophilus Drugs 0.000 claims description 2
- 238000012408 PCR amplification Methods 0.000 claims 1
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- 238000004458 analytical method Methods 0.000 abstract description 25
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- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 241000218492 Lactobacillus crispatus Species 0.000 description 1
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- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
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Classifications
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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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- Probiotics are known to have beneficial health effects on the host when consumed in appropriate amounts. Scientific evidence continues to accumulate about the beneficial effects of probiotics on human health in various aspects such as alleviating immune disorders, inflammatory bowel disease, type 2 diabetes, and arteriosclerosis.
- Two substantially complementary strands may be, for example, completely complementary or may contain from one to a number of mismatches, for example, sufficiently allowing for differences between the paired and unpaired sequences. You can. Accordingly, a “substantially complementary” sequence can mean a sequence that has a base pair complementarity of more than 100, 95, 90, 80, 75, 70, 60, 50%, or any of the above numbers in the double-stranded region. .
- the fungal species may be probiotic species and pathogenic bacterial species.
- the probiotic species may be a bacterial species recognized and notified as a health functional food by the Ministry of Food and Drug Safety and a bacterial species that can be used in food.
- detection of the amplification product in step 3) may be performed through sequencing, such as Next Generation Sequencing (NGS).
- NGS Next Generation Sequencing
- sequencing can be performed using small NGS equipment such as iSeq100, thus lowering sequencing costs and improving speed.
- NCBI National Center for Biological Information
- ANI Average Nucleotide Identity
- pyani a program to obtain ANI
- the maximum value of ANI for each strain between species was calculated for the 160bp region of the ITS sequence located after the primer region, and the ANI standard for species discrimination was set. Specifically, the maximum ANI values between species are shown in [Table 5] below.
- Amplicon PCR was performed on the extracted sample DNA using the fusion primer set of ⁇ Example 1> above as follows. Add 10 mM Tris pH 8.5 to the extracted sample DNA, dilute the DNA concentration to 12 ng/ ⁇ L, and prepare a PCR reaction solution using the fusion primer prepared in ⁇ Example 1> as shown in [Table 8] below. , PCR was performed under the conditions shown in [Table 9] below. Next, purification of the PCR-completed sample was performed using HiAccuBead according to the manufacturer's procedures.
- the primer set is used to determine the bacterial species in the probiotic composition. It can be useful for discrimination.
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Chemical Kinetics & Catalysis (AREA)
Abstract
Description
Claims (9)
- 서열번호 1 내지 9로 표시되는 프라이머를 포함하는 프로바이오틱스 조성물 내 균종 판별용 프라이머 세트.
- 제 1항에 있어서, 상기 프라이머는 5' 말단에 어댑터(adaptor) 서열을 더 포함하는 것을 특징으로 하는, 프로바이오틱스 조성물 내 균종 판별용 프라이머 세트.
- 제 2항에 있어서, 상기 프라이머는 서열번호 10 내지 18로 표시되는 것을 특징으로 하는, 프로바이오틱스 조성물 내 균종 판별용 프라이머 세트.
- 제 1항에 있어서, 상기 프라이머 세트는 균종의 ITS(internal transcribed spacer) 영역을 타겟으로 하는 것을 특징으로 하는, 프로바이오틱스 조성물 내 균종 판별용 프라이머 세트.
- 제 1항에 있어서, 상기 균종은 락토바실러스 에시도필러스(Lactobacillus acidophilus), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 불가리쿠스(Lactobacillus delbruecki ssp. Bulgaricus), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락티카제이바실러스 카제이 (Lacticaseibacillus casei), 락티카제이바실러스 파라카제이 (Lacticaseibacillus paracasei), 락티카제이바실러스 람노서스(Lacticaseibacillus rhamnosus), 리모시락토바실러스 퍼멘텀(Limosilactobacillus fermentum), 리모시락토바실러스 루테리(Limosilactobacillus reuteri), 락티플란티바실러스 플란타럼(Lactiplantibacillus plantarum), 리기락토바실러스 살리바리우스(Ligilactobacillus salivarius), 락토코커스 락티스(Lactococcus lactis), 엔테로코커스 패시움(Enterococcus faecium), 엔테로코커스 패칼리스(Enterococcus faecalis), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 비피도박테리움 비피둠(Bifidobacterium bifidum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 롱검(Bifidobacterium longum), 비피도박테리움 아니말리스 락티스(Bifidobacterium animalis ssp. lactis), 라티락토바실러스 커바투스(Latilactobacillus curvatus), 바실러스 코아귤런스(Bacillus coagulans), 대장균(Escherichia coli), 살모넬라(Salmonella spp.), 황색포도상구균(Staphylococcus aureus) 및 녹농균(Pseudomonas aeruginosa)인 것을 특징으로 하는, 프로바이오틱스 조성물 내 균종 판별용 프라이머 세트.
- 제 1항 내지 제 5항 중 어느 한 항의 프라이머 세트를 포함하는 프로바이오틱스 조성물 내 균종 판별용 조성물.
- 1) 프로바이오틱스 조성물로부터 DNA를 분리하는 단계;2) 상기 단계 1)에서 분리한 DNA를 주형으로 제 1항 내지 제 5항 중 어느 한 항의 프라이머 세트를 이용하여 PCR을 수행하여 표적 서열을 증폭하는 단계; 및3) 상기 단계 2)의 증폭 산물을 검출하는 단계를 포함하는 프로바이오틱스 조성물 내 균종 판별 방법.
- 제 7항에 있어서, 상기 단계 2)의 증폭 산물을 주형으로 인덱스 PCR(index PCR)을 수행하는 것을 더 포함하는 것을 특징으로 하는, 프로바이오틱스 조성물 내 균종 판별 방법.
- 제 7항에 있어서, 상기 PCR 증폭 산물의 검출은 시퀀싱을 통해 수행되는 것을 특징으로 하는, 프로바이오틱스 조성물 내 균종 판별 방법.
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CN202380012251.9A CN117500944A (zh) | 2022-05-31 | 2023-05-08 | 用于鉴定益生菌组合物中的菌种的引物组以及利用其的菌种的鉴定方法 |
EP23816241.6A EP4343005A1 (en) | 2022-05-31 | 2023-05-08 | Primer set for identifying bacterial species in probiotics composition and method for identifying bacterial species using same |
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KR20040077544A (ko) * | 2003-02-28 | 2004-09-04 | 일동제약주식회사 | 락토바실러스 속 균종의 동정방법 |
KR101695059B1 (ko) * | 2014-04-07 | 2017-01-23 | 건국대학교 산학협력단 | 케피어 발효유 내 미생물 그룹별 정량적인 실시간 중합효소 연쇄반응 분석용 조성물 및 그 분석방법 |
KR20200027900A (ko) | 2018-09-05 | 2020-03-13 | 주식회사 천랩 | 시료 미생물의 동정 및 분류 방법 |
KR20200029689A (ko) | 2018-09-10 | 2020-03-19 | 주식회사 조앤김지노믹스 | 유산균 동정용 참조서열 제조방법 및 이를 이용한 유산균 동정방법 |
KR102334331B1 (ko) * | 2020-07-01 | 2021-12-02 | 경희대학교 산학협력단 | 비피도박테리움 균주 검출용 실시간 pcr 프라이머 및 이의 용도 |
KR20220010100A (ko) * | 2020-07-17 | 2022-01-25 | 경북대학교 산학협력단 | 프로바이오틱스 미생물을 종 수준에서 판별하기 위한 조성물 |
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2022
- 2022-09-14 KR KR1020220115369A patent/KR20230167285A/ko unknown
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2023
- 2023-05-08 EP EP23816241.6A patent/EP4343005A1/en active Pending
- 2023-05-08 WO PCT/KR2023/006187 patent/WO2023234577A1/ko active Application Filing
- 2023-05-08 CN CN202380012251.9A patent/CN117500944A/zh active Pending
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