HRP20211757T1 - Postupak kvantificiranja članova filogrupe i i filogrupe ii bakterije faecalibacterium prausnitzii i njihova upotreba kao bioloških biljega - Google Patents

Postupak kvantificiranja članova filogrupe i i filogrupe ii bakterije faecalibacterium prausnitzii i njihova upotreba kao bioloških biljega Download PDF

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HRP20211757T1
HRP20211757T1 HRP20211757TT HRP20211757T HRP20211757T1 HR P20211757 T1 HRP20211757 T1 HR P20211757T1 HR P20211757T T HRP20211757T T HR P20211757TT HR P20211757 T HRP20211757 T HR P20211757T HR P20211757 T1 HRP20211757 T1 HR P20211757T1
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Mireia LÓPEZ SILES
Librado Jesús GARCÍA GIL
Xavier ALDEGUER MANTÉ
Margarita MARTÍNEZ MEDINA
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Universitat De Girona
Fundacio Institut D'investigació Biomèdica De Girona Dr. Josep Trueta
Goodgut S.L.
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Claims (17)

1. In vitro postupak određivanja zastupljenosti članova filogrupe I bakterije Faecalibacterium prausnitzii (PHGI) i/ili članova filogrupe II bakterije Faecalibacterium prausnitzii (PHGII) kvantificiranjem gena za 16S rRNA specifičnog za filogrupu u crijevnom uzorku iz subjekta; pri čemu se određivanje zastupljenosti PHGI sastoji u hibridizaciji sonde specifične za PHGI koja sadrži slijed SEQ ID NO: 3; i pri čemu se određivanje zastupljenosti PHGII sastoji u hibridizaciji sonde specifične za PHGII koja sadrži slijed SEQ ID NO: 4.
2. Postupak detektiranja crijevne bolesti kod ljudskog subjekta, koji se sastoji u sljedećim koracima: a. određivanja zastupljenosti članova filogrupe II bakterije Faecalibacterium prausnitzii (PHGII) u crijevnom uzorku iz navedenog subjekta postupkom u skladu s patentnim zahtjevom 1; i b. uspoređivanja zastupljenosti PHGII, i/ili matematičke kombinacije sa zastupljenosti članova filogrupe I bakterije Faecalibacterium prausnitzii (PHGI) koju se određuje postupkom u skladu s patentnim zahtjevom 1, i/ili izborno matematičke kombinacije bilo koje od tih s ukupnom zastupljenosti F. prausnitzii (FT) i/ili zastupljenosti E. coli (EC), u uzorku iz subjekta s odgovarajućim vrijednostima u referentnom uzorku; pri čemu značajno odstupanje u uzorku iz subjekta vrijednosti u odnosu na navedeni referentni uzorak ukazuje na crijevnu bolest; pri čemu se navedenu crijevnu bolest bira iz skupine koju čine upalna crijevna bolest (IBD), sindrom iritabilnih crijeva (IBS) i kolorektalni rak (CRC); i pri čemu je navedeni referentni uzorak po mogućnosti uzorak iz zdravog subjekta i/ili uzorak iz pacijenta čija je crijevna bolest u povlačenju.
3. Postupak u skladu s patentnim zahtjevom 2, pri čemu je navedeni postupak postupak probiranja i/ili dijagnosticiranja crijevne bolesti kod ljudskog subjekta, a navedeni referentni uzorak je uzorak iz zdravog subjekta i/ili uzorak iz subjekta čija je crijevna bolest u povlačenju.
4. Postupak u skladu s patentnim zahtjevom 2, pri čemu je navedeni postupak postupak praćenja aktivnosti i/ili napredovanja crijevne bolesti kod ljudskog subjekta, a navedeni referentni uzorak je raniji uzorak iz istog subjekta.
5. Postupak u skladu s bilo kojim od patentnih zahtjeva 2 do 4, pri čemu je navedena crijevna bolest IBD, po mogućnosti ulcerozni kolitis (UC) ili Crohnova bolest (CD), poželjnije CD.
6. Postupak postavljanja diferencijalne dijagnoze fenotipova upalne crijevne bolesti (IBD) kod ljudskog subjekta, koji se sastoji u sljedećim koracima: a. određivanja zastupljenosti članova filogrupe I bakterije Faecalibacterium prausnitzii (PHGI) i/ili članova filogrupe II bakterije Faecalibacterium prausnitzii (PHGII) u crijevnom uzorku iz navedenog subjekta postupkom u skladu s patentnim zahtjevom 1; i b. uspoređivanja zastupljenosti PHGI i/ili PHGII, i/ili njihove matematičke kombinacije, i/ili izborno matematička kombinacija bilo koje od tih s ukupnom zastupljenosti F. prausnitzii (FT) i/ili zastupljenosti E. coli (EC), u uzorku iz subjekta s odgovarajućim vrijednostima u referentnom uzorku fenotipova IBD s kojima treba provesti usporedbu radi određivanja fenotipa IBD od kojeg boluje subjekt; pri čemu uzorak iz subjekta koji pokazuje vrijednosti značajno slične onima iz navedenih fenotipova IBD ukazuje da subjekt boluje od navedenog fenotipa IBD; i – pri čemu se navedene fenotipove IBD bira iz skupine koju čine: fenotipovi CD koje određuje jedan ili više, po mogućnosti svi, od sljedećih parametara: ◦ lokacija bolesti; ◦ dob prilikom dijagnosticiranja; i ◦ ponašanje; – fenotipovi UC koje određuje jedan ili više, po mogućnosti svi, od sljedećih parametara: ◦ lokacija ili opseg bolesti; i ◦ težina.
7. Postupak u skladu s patentnim zahtjevom 6, pri čemu se navedene fenotipove IBD bira iz skupine koju čine: – fenotipovi CD koje čine ilealni CD (I-CD), ileokolonski CD (IC-CD) i kolonski CD (C-CD); i – fenotipovi UC koje čine ulcerozni proktitis (UC-E1), distalni kolitis (UC-E2) i opsežni UC ili pankolitis (UC-E3).
8. Postupak dijagnosticiranja C-CD kod ljudskog subjekta koji boluje od IBD kod kojeg je zahvaćeno debelo crijevo, koji se sastoji u sljedećim koracima: i. određivanja zastupljenosti ciljnog mikroorganizma u crijevnom uzorku iz navedenog subjekta, pri čemu se navedeni ciljni mikroorganizam bira iz skupine koju čine članovi filogrupe I bakterije Faecalibacterium prausnitzii (PHGI) i članovi filogrupe II bakterije Faecalibacterium prausnitzii (PHGII); i ii. uspoređivanja razina zastupljenosti u uzorku iz subjekta s razinama u referentnom uzorku, pri čemu značajno smanjenje razina zastupljenosti u uzorku iz subjekta u odnosu na navedeni referentni uzorak ukazuje na C-CD; pri čemu je po mogućnosti navedeni ciljni mikroorganizam PHGII.
9. Postupak u skladu s bilo kojim od patentnih zahtjeva 1 do 8, pri čemu se određivanje zastupljenosti PHGI i/ili PHGII, te izborno određivanje zastupljenosti FT i/ili EC, provodi molekulskim postupkom kojeg se bira iz skupine koju čine kvantitativna lančana reakcija polimerazom (qPCR), PCR-pirosekvenciranje, fluorescentna hibridizacija in situ (FISH), mikrorešetke, te PCR-ELISA, gdje se po mogućnosti kvantificiranje provodi qPCR-om.
10. Postupak u skladu s bilo kojim od patentnih zahtjeva 1 do 9, pri čemu se određivanje zastupljenosti PHGI provodi sondom koju čini oligonukleotidni slijed SEQ ID NO: 3; i/ili pri čemu se određivanje zastupljenosti PHGII provodi sondom koju čini oligonukleotidni slijed SEQ ID NO: 4.
11. Postupak u skladu s bilo kojim od patentnih zahtjeva 1 do 10, pri čemu se određivanje zastupljenosti PHGI provodi qPCR-om, uz početnice koje čine oligonukleotidni sljedovi SEQ ID NO: 1 i SEQ ID NO: 2, ili slijed koji im je istovjetan najmanje 75 %; i sondu koju čini oligonukleotidni slijed SEQ ID NO: 3; i/ili pri čemu se određivanje zastupljenosti PHGII provodi qPCR-om, uz početnice koje čine oligonukleotidni sljedovi SEQ ID NO: 1 i SEQ ID NO: 2, ili slijed koji im je istovjetan najmanje 75 %; i sondu koju čini oligonukleotidni slijed SEQ ID NO: 4.
12. Postupak u skladu s bilo kojim od patentnih zahtjeva 1 do 11, pri čemu se DNA dobiva iz crijevnog uzorka prije određivanja zastupljenosti PHGI i/ili PHGII, te izborno FT i/ili EC.
13. Postupak u skladu s bilo kojim od patentnih zahtjeva 1 do 12, pri čemu se navedeni crijevni uzorak bira iz skupine koju čine biopsija sluznice i uzorak stolice, a po mogućnosti je uzorak stolice.
14. Komplet koji sadrži: a. reagens za određivanje zastupljenosti članova filogrupe I bakterije Faecalibacterium prausnitzii (PHGI) koju čine: – par nukleinskokiselinskih početnica kojeg čine oligonukleotidni sljedovi SEQ ID NO: 1 i SEQ ID NO: 2 ili oligonukleotidni sljedovi koji su im istovjetni najmanje 75 %; i – sonda koju čini oligonukleotidni slijed SEQ ID NO: 3; i/ili b. reagens za određivanje zastupljenosti članova filogrupe II bakterije Faecalibacterium prausnitzii (PHGII) koju čine: – par nukleinskokiselinskih početnica kojeg čine oligonukleotidni sljedovi SEQ ID NO: 1 i SEQ ID NO: 2 ili oligonukleotidni sljedovi koji su im istovjetni najmanje 75 %; i – sonda koju čini oligonukleotidni slijed SEQ ID NO: 4; c. izborno, upute o upotrebi jednog ili više navedenih reagensa u određivanju zastupljenosti PHGI, i/ili PHGII, iz uzorka iz ljudskih crijeva.
15. Upotreba kompleta za detektiranje crijevne bolesti i/ili za postavljanje diferencijalne dijagnoze fenotipova IBD; pri čemu navedeni komplet sadrži: a. reagens za određivanje zastupljenosti članova filogrupe I bakterije Faecalibacterium prausnitzii (PHGI) koju čine sonda koja sadrži slijed SEQ ID NO: 3; i/ili b. reagens za određivanje zastupljenosti članova filogrupe II bakterije Faecalibacterium prausnitzii (PHGII) koju čine sonda koja sadrži slijed SEQ ID NO: 4; i c. izborno, upute o upotrebi jednog ili više navedenih reagensa u određivanju zastupljenosti PHGI, i/ili PHGII, iz uzorka iz ljudskih crijeva; pri čemu se navedenu crijevnu bolest bira iz skupine koju čine upalna crijevna bolest (IBD), sindrom iritabilnih crijeva (IBS) i kolorektalni rak (CRC).
16. Upotreba kompleta u skladu s patentnim zahtjevom 15, pri čemu: a. navedeni reagens za određivanje zastupljenost PHGI se sastoji od: – para nukleinskokiselinskih početnica kojeg čine oligonukleotidni sljedovi SEQ ID NO: 1 i SEQ ID NO: 2 ili oligonukleotidni sljedovi koji su im istovjetni najmanje 75 %; i – sonda koju čini oligonukleotidni slijed SEQ ID NO: 3; i/ili b. navedeni reagens za određivanje zastupljenost PHGII se sastoji od: – para nukleinskokiselinskih početnica kojeg čine oligonukleotidni sljedovi SEQ ID NO: 1 i SEQ ID NO: 2 ili oligonukleotidni sljedovi koji su im istovjetni najmanje 75 %; i – sonda koju čini oligonukleotidni slijed SEQ ID NO: 4.
17. Molekula nukleinske kiseline koju se bira iz skupine koju čine SEQ ID NO: 3 i SEQ ID NO: 4.
HRP20211757TT 2015-08-11 2016-08-11 Postupak kvantificiranja članova filogrupe i i filogrupe ii bakterije faecalibacterium prausnitzii i njihova upotreba kao bioloških biljega HRP20211757T1 (hr)

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EP15382427.1A EP3130680A1 (en) 2015-08-11 2015-08-11 Method for the detection, follow up and/or classification of intestinal diseases
EP16754468.3A EP3334838B1 (en) 2015-08-11 2016-08-11 Method for the quantification of faecalibacterium prausnitzii phylogroup i and/or phylogroup ii members and the use thereof as biomarkers
PCT/EP2016/069188 WO2017025617A1 (en) 2015-08-11 2016-08-11 Method for the quantification of faecalibacterium prausnitzii phylogroup i and/or phylogroup ii members and the use thereof as biomarkers

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