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WO1998039473A1 - Analyse in vitro permettant de detecter des tumeurs de l'intestin - Google Patents

Analyse in vitro permettant de detecter des tumeurs de l'intestin Download PDF

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Publication number
WO1998039473A1
WO1998039473A1 PCT/EP1998/001223 EP9801223W WO9839473A1 WO 1998039473 A1 WO1998039473 A1 WO 1998039473A1 EP 9801223 W EP9801223 W EP 9801223W WO 9839473 A1 WO9839473 A1 WO 9839473A1
Authority
WO
WIPO (PCT)
Prior art keywords
use according
process step
detection
physiological saline
sds
Prior art date
Application number
PCT/EP1998/001223
Other languages
German (de)
English (en)
Inventor
Herbert Lochs
Alexander Swidsinski
Original Assignee
Schering Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Schering Aktiengesellschaft filed Critical Schering Aktiengesellschaft
Priority to AU67275/98A priority Critical patent/AU721826B2/en
Priority to CA002293704A priority patent/CA2293704A1/fr
Priority to EP98912439A priority patent/EP0977888A1/fr
Publication of WO1998039473A1 publication Critical patent/WO1998039473A1/fr

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Classifications

    • 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
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention relates to the use of in vitro detection of intraepithelial intestinal bacteria, constituents thereof and reaction products of the host thereon, for the detection of intestinal tumors and their precursors.
  • Helicobacter pylori was the first bacterium to be assigned to group I carcinogen (WHO / IARC Congress in Lyon, 1994 and Logan RPH, Helicobacter pylori and gastric cancer, Lancet 1994, 334, 1078-79).
  • Intestinal bacteria constituents of the same and reaction products of the host to them, for the detection of intestinal tumors and precursors of intestinal tumors.
  • the use is characterized in that the following steps are carried out for the in vitro detection: a) shake biopsy samples in an aqueous solution and then take them up again in aqueous solution, b) process step a) is repeated several times, the supernatants are discarded, c) the residues transferred to an SDS-containing solution at room temperature and carefully or slowly shaken or rotated to dissolve the tissue and the DNA fraction, d) the DNA was purified with phenol and glass beads and then resuspended in water, e) the DNA thus obtained was used with universal 16 sRNA primers amplified and then the strength of the PCR product of the sample with the strength of
  • the proof can be modified in many ways. Species-specific primers can be used for the PCR. Virulence factors can be specifically detected in the biopsy sample or even in stool samples. In situ hybridization can also be carried out. Furthermore, immunohistochemical and electron microscopic detection of bacteria in the mucosa can be carried out. Nuclear medical methods such as scintigraphy or breath tests are also conceivable.
  • the aqueous solution used in method step a) of the detection is preferably a physiological saline solution.
  • the SDS-containing solution used in method step c) of the detection is preferably a 0.5-5% SDS / physiological saline solution.
  • the time for shaking the biopsy samples in method step a) of the detection is preferably 10-120 s, particularly preferably 30-60 s.
  • the biopsy samples are preferably taken in step a) of the detection in 100-1000 ⁇ ⁇ physiological saline, particularly preferably in 500 l.
  • Process step a) of the detection is preferably repeated 5-20 times, particularly preferably 7-10 times.
  • the fractions in method step c) of the detection are preferably added to a volume of 100-500 l of 0.5-5% SDS / physiological saline solution, with a 1% solution being particularly preferred.
  • the preferred time of shaking and rotating in process step c) of the detection is 10-24 hours, particularly preferably 12 hours.
  • process step d) of the detection it is preferably resuspended in a volume of 10-50 ⁇ l of water, particularly preferably in a volume of 20 ⁇ l of water.
  • the use relates in particular to the detection of E. coli bacteria in colon biopsies.
  • the in vitro detection is also used in the examination of samples from other body compartments such as lymph and body fluids such as blood, fluids of the gastrointestinal tract and fluids from neighboring tissues of the stomach and intestine.
  • the host's reaction products to the infestation with E. coli or other bacteria characteristic of the tumor can be detected by means of customary analytical methods, for example an immunoassay.
  • the use of the in vitro detection according to the invention relates to E. coli, constituents of E. coli, reaction products of the host to E. coli and related bacteria in the intestinal mucosa or other compartments of the body outside the gastrointestinal lumen.
  • the following exemplary embodiments explain the invention, without restricting it to the examples.
  • Tissues from biopsies were placed in physiological saline and held at 4 ° C for two hours until further use. Each sample was crushed vigorously with a chopper (vortex stick) in a 1.5 ml plastic tube for 1 minute and then added to 500 ⁇ l of fresh physiological saline. This
  • the 5 'end of the 16S rRNA genes (600 bp) was amplified by PCR using a universal primer for bacteria:
  • pre- and post-PCR stages were carried out in different setups and parallel control experiments with the omission of template DNA.
  • the amount of bacteria in the samples was determined by means of electrophoretic comparison of the PCR
  • amplified 16S rRNA sequences were cloned into a pCMV-LIC vector according to a ligation-independent cloning protocol. Between 40 and 200 clones were sequenced in a sequencer for each biopsy. The bacterial sequences were analyzed and compared as previously described with 16S rRNA primary structures from databases.
  • Biopsies were taken from humans to demonstrate the feasibility of the method according to the invention.
  • 3rd carcinoma group 18 people with carcinomas, 11 of whom had the condition after
  • biopsies were taken from each examined. These biopsies were examined with the method according to the invention for intraepithelial and related E. coli bacteria.
  • biopsies were taken from both the suspected tumor and the normal mucosa.
  • the sex, the average age and the age structure of the examined in the three groups is as follows:
  • Age structure 25-60 39-85 46-67 The bacterial concentration was determined quantitatively in 103, 56 and 47 biopsies from investigators from the normal, adenoma and carcinoma groups.
  • the following table shows the results of the quantitative PCR and the results of the sequence analysis of cloned PCR products.
  • Mucosa as well as in biopsies of tumor tissue of those examined with colon carcinoma and colon adenoma intraepithelial bacteria are present.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)

Abstract

Utilisation d'une analyse in vitro de bactéries intestinales intraépithéliales, de fragments desdites bactéries et de produits de réaction de l'hôte sur ces bactéries, permettant de détecter les tumeurs de l'intestin et les stades précancéreux desdites tumeurs.
PCT/EP1998/001223 1997-03-06 1998-03-04 Analyse in vitro permettant de detecter des tumeurs de l'intestin WO1998039473A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU67275/98A AU721826B2 (en) 1997-03-06 1998-03-04 In vitro determination for detection of tumors of the intestine
CA002293704A CA2293704A1 (fr) 1997-03-06 1998-03-04 Analyse in vitro permettant de detecter des tumeurs de l'intestin
EP98912439A EP0977888A1 (fr) 1997-03-06 1998-03-04 ANALYSE $i(IN VITRO) PERMETTANT DE DETECTER DES TUMEURS DE L'INTESTIN

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19711111.4 1997-03-06
DE19711111A DE19711111C2 (de) 1997-03-06 1997-03-06 In vitro Nachweis zur Erkennung von Darmtumoren

Publications (1)

Publication Number Publication Date
WO1998039473A1 true WO1998039473A1 (fr) 1998-09-11

Family

ID=7823689

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/001223 WO1998039473A1 (fr) 1997-03-06 1998-03-04 Analyse in vitro permettant de detecter des tumeurs de l'intestin

Country Status (5)

Country Link
EP (1) EP0977888A1 (fr)
AU (1) AU721826B2 (fr)
CA (1) CA2293704A1 (fr)
DE (1) DE19711111C2 (fr)
WO (1) WO1998039473A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079545A1 (fr) * 2000-04-18 2001-10-25 National University Of Singapore Marqueurs moleculaires

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2295907T3 (es) 2004-08-13 2008-04-16 Indivumed Gmbh Uso de transtiretina como biomarcador para ademona colorrectal; metodo para la deteccion y sistema de ensayo.

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2290866B (en) * 1994-06-29 1998-10-07 Reckitt & Colmann Prod Ltd Detection of Helicobacter pylori infection using antibodies to carbonic anhydrase

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ARIES ET AL.: "Bacteria and the aetiology of cancer of the large bowel", GUT, vol. 10, 1969, pages 334 - 335, XP002070757 *
HILL M J: "Bacteria, bile acids and large bowel cancer.", BIOCHEMICAL SOCIETY TRANSACTIONS, (1983 JUN) 11 (3) 256-8., XP002070759 *
HILL M J: "BILE BACTERIA AND BOWEL CANCER.", GUT 24 (10). 1983. 871-875. CODEN: GUTTAK ISSN: 0017-5749, XP002070758 *
KANAZAWA ET AL.: "Factors influencing the development of sigmoid colon cancer", CANCER, vol. 77, no. S8, April 1996 (1996-04-01), pages 1701 - 1706, XP002070755 *
R. LOGAN: "H. pylori and gastric cancer", LANCET, vol. 344, 1994, pages 1078 - 1079, XP002070756 *
ZIMBALIST ET AL.: "Genetic and environmental factors in colorectal carcinogenesis", DIGESTIVE DISEASES, vol. 13, 1995, pages 365 - 378, XP002070760 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079545A1 (fr) * 2000-04-18 2001-10-25 National University Of Singapore Marqueurs moleculaires
US7101668B2 (en) 2000-04-18 2006-09-05 National University Of Singapore Molecular markers

Also Published As

Publication number Publication date
AU721826B2 (en) 2000-07-13
DE19711111A1 (de) 1998-09-17
EP0977888A1 (fr) 2000-02-09
DE19711111C2 (de) 1999-09-02
CA2293704A1 (fr) 1998-09-11
AU6727598A (en) 1998-09-22

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