CZ308092B6 - Method of detecting Faecalibacterium prausnitzii by real-time PCR and primers for this detection method of F. prausnitzii - Google Patents

Method of detecting Faecalibacterium prausnitzii by real-time PCR and primers for this detection method of F. prausnitzii Download PDF

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CZ308092B6
CZ308092B6 CZ2018-456A CZ2018456A CZ308092B6 CZ 308092 B6 CZ308092 B6 CZ 308092B6 CZ 2018456 A CZ2018456 A CZ 2018456A CZ 308092 B6 CZ308092 B6 CZ 308092B6
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seq
primers
prausnitzii
faecalibacterium prausnitzii
real
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CZ2018-456A
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CZ2018456A3 (en
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Ivan RYCHLÍK
Marcela Faldynová
Jitka Matiašovicová
Darina Čejková
Tereza Kubasová
Miroslava Kollarčíková
Daniela KARASOVÁ
Magdaléna CRHÁNOVÁ
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Výzkumný ústav veterinárního lékařství, v. v. i.
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Priority to CZ2018-456A priority Critical patent/CZ2018456A3/en
Publication of CZ308092B6 publication Critical patent/CZ308092B6/en
Publication of CZ2018456A3 publication Critical patent/CZ2018456A3/en

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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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • 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
    • 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

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention is a method for specifically detecting Faecalibacterium prausnitzii in the digestive tract or poultry droppings by isolating DNA from the sample and subsequently subjecting the isolated DNA to an amplification reaction by real-time PCR with primersFaecF2: CGAGGGCCTGGACTTCATCAAG,FaecR2: GCCGGTGATCCACTCGTTGGAC.

Claims (5)

1. Způsob detekce bakterie Faecalibacterium prausnitzii v obsahu trávicího traktu nebo trusu drůbeže, vyznačený tím, že se ze vzorku izoluje DNA, a následně se izolovaná DNA podrobí amplifikační reakci s primery1. A method for detecting Faecalibacterium prausnitzii in gastrointestinal or poultry droppings, characterized in that DNA is isolated from the sample, and the isolated DNA is then subjected to an amplification reaction with primers. FaecF2: CGAGGGCCTGGACTTCATCAAG (SEQ ID NO. 1),FaecF2: CGAGGGCCTGGACTTCATCAAG (SEQ ID NO. 1) FaecR2: GCCGGTGATCCACTCGTTGGAC (SEQ ID NO. 2) metodou PCR v reálném čase.FaecR2: GCCGGTGATCCACTCGTTGGAC (SEQ ID NO. 2) by real-time PCR. 2. Způsob podle nároku 1, vyznačený tím, že se dále provede amplifikační reakce metodou PCR v reálném čase s primery 16S_An_Fl: TCCTACGGGAGGCAGCAG (SEQ ID NO. 3) a 16S_An_Rl: CGTATTACCGCGGCTGCT (SEQ ID NO. 4) amplifikujícími část genu pro 16S rRNA u všech eubaktérií a vypočte se podíl Faecalibacterium prausnitzii na celkovém počtu eubakterií.The method of claim 1, further comprising performing a real-time PCR amplification reaction with primers 16S_An_F1: TCCTACGGGAGGCAGCAG (SEQ ID NO. 3) and 16S_An_R1: CGTATTACCGCGGCTGCT (SEQ ID NO. 4) amplifying a portion of the 16S rRNA gene. for all eubacteria and the proportion of Faecalibacterium prausnitzii in the total number of eubacteria is calculated. 3. Způsob podle nároku 2, vyznačený tím, že se podíl Faecalibacterium prausnitzii na celkovém počtu eubakterií vypočte tak, že se nejprve odečte Ct hodnota amplifikace Faecalibacterium prausnitzii od Ct hodnoty amplifikace eubakteriální DNA (ACt), následně se na tento rozdíl umocní základ 2 (2Mt). vypočte se převrácená hodnota (l/2ACt) a výsledek se vynásobí číslem 100 (100 x 1/2Mt). čímž se získá podíl Faecalibacterium prausnitzii v celé eubakteriální populaci v procentech.Method according to claim 2, characterized in that the proportion of Faecalibacterium prausnitzii in the total number of eubacteria is calculated by first subtracting the Ct value of the Faecalibacterium prausnitzii amplification from the Ct value of the eubacterial DNA (ACt) amplification, then amplifying the base 2 ( 2 Mt ). the reciprocal (1/2 ACt ) is calculated and multiplied by 100 (100 x 1/2 Mt ). thereby obtaining a percentage of Faecalibacterium prausnitzii in the entire eubacterial population. 4. Sada primerů pro detekci bakterie Faecalibacterium prausnitzii ve vzorcích metodou real time PCR, vyznačující se tím, že obsahuje primery FaecF2: CGAGGGCCTGGACTTCATCAAG (SEQ ID NO. 1) a FaecR2: GCCGGTGATCCACTCGTTGGAC (SEQ ID NO. 2).4. A set of primers for the detection of Faecalibacterium prausnitzii in samples by real time PCR, characterized in that it comprises primers FaecF2: CGAGGGCCTGGACTTCATCAAG (SEQ ID NO. 1) and FaecR2: GCCGGTGATCCACTCGTTGGAC (SEQ ID NO. 2). 5. Sada podle nároku 4, vyznačující se tím, že dále obsahuje primery 16S_An_Fl: TCCTACGGGAGGCAGCAG (SEQ ID NO. 3) a 16S_An_Rl: CGTATTACCGCGGCTGCT (SEQ ID NO. 4).The kit of claim 4, further comprising primers 16S_An_F1: TCCTACGGGAGGCAGCAG (SEQ ID NO. 3) and 16S_An_R1: CGTATTACCGCGGCTGCT (SEQ ID NO. 4).
CZ2018-456A 2018-09-08 2018-09-08 Method of detecting Faecalibacterium prausnitzii by real-time PCR and primers for this detection method of F. prausnitzii CZ2018456A3 (en)

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CZ2018-456A CZ2018456A3 (en) 2018-09-08 2018-09-08 Method of detecting Faecalibacterium prausnitzii by real-time PCR and primers for this detection method of F. prausnitzii

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CZ2018-456A CZ2018456A3 (en) 2018-09-08 2018-09-08 Method of detecting Faecalibacterium prausnitzii by real-time PCR and primers for this detection method of F. prausnitzii

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3130680A1 (en) * 2015-08-11 2017-02-15 Universitat de Girona Method for the detection, follow up and/or classification of intestinal diseases

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3130680A1 (en) * 2015-08-11 2017-02-15 Universitat de Girona Method for the detection, follow up and/or classification of intestinal diseases

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GONG J. et al.: „16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca," FEMS Microbiology Ecology, vol. 59, no. 1, 2007, str. 147 - 157, ISSN 0168-6496 *
HOLD G. L. et al.: „Oligonucleotide probes that detect quantitatively significant groups of butyrate-producing bacteria in human feces," Applied and Environmental Microbiology, vol. 69, no. 7, 2003, str. 4320 - 4324, ISSN 0099-2240 *
LUND M. et al.: „Quantification of Faecalibacterium prausnitzii- and Subdoligranulum variabile-like bacteria in the cecum of chickens by real-time PCR," Poultry Science, vol. 89, no. 6, 2010, str. 1217 - 1224, ISSN 0032-5791 *
WISE M. G. et al.: „Quantitative analysis of the intestinal bacterial community in one- to three-week-old commercially reared broiler chickens fed conventional or antibiotic-free vegetable-based diets," Journal of Applied Microbiology, vol. 102, no. 4, 2007, str. 1138 - 1149, ISSN 1364-5072 *

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