WO1998039473A1 - In vitro analysis for detection of tumours of the intestine - Google Patents
In vitro analysis for detection of tumours of the intestine Download PDFInfo
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- 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.
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Abstract
Description
In vitro Nachweis zur Erkennung von Darmtumoren In vitro detection for the detection of intestinal tumors
Die Erfindung betrifft die Verwendung eines in vitro - Nachweises von intraepithelialen Darmbakterien, Bestandteilen derselben und Reaktionsprodukten des Wirtes auf diese, zur Erkennung von Darmtumoren und deren Vorstufen.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.
Maligne Tumoren sind wegen ihrer schlechten Prognose eine ernste Bedrohung der menschlichen Gesundheit. Die Therapiemöglichkeiten der Mehrzahl bösartiger Tumoren sind zur Zeit noch beschränkt und vom Krankheits Stadium abhängig. Eine Früherkennung der Krankheit, sowie deren Vorstufen gehört daher zu den vorrangigen Zielen der klinisch wissenschaftlichen Forschung. Die Karzinogenese wird als eine Wechselwirkung endogener (genetischer) und exogener Faktoren betrachtet. Während genetische Veränderungen der Zelle bisher wenig Möglichkeiten zur therapeutischen Intervention boten, erhofft man vom Verständnis der exogenen Karzinogene einen unmittelbaren Nutzen. Am Anfang galt die Aufmerksamkeit vor allem chemischen und radioaktiven Substanzen, da die Wirkung dieser Karzinogene in Zellkulturen und Tierexperimenten leicht zu verfolgen ist. Mit der Entdeckung karzinogener Viren rückten die infektiösen Ursachen der Tumorentstehung in den Vordergrund des Interesses. Viren sind nicht die einzigen Erreger mit karzinogener Potenz. Eine Verbindung zwischen bestimmtem Karzinomarten und einer chronischen Infektion mit Schistosoma oder Leberegel ist seit langem bekannt.Malignant tumors are a serious threat to human health because of their poor prognosis. The treatment options for the majority of malignant tumors are currently limited and depend on the stage of the disease. Early detection of the disease and its preliminary stages is therefore one of the primary goals of clinical scientific research. Carcinogenesis is viewed as an interaction of endogenous (genetic) and exogenous factors. While genetic changes in the cell have so far offered few opportunities for therapeutic intervention, an understanding of exogenous carcinogens is hoped for an immediate benefit. In the beginning, the main focus was on chemical and radioactive substances, as the effects of these carcinogens in cell cultures and animal experiments are easy to follow. With the discovery of carcinogenic viruses, the infectious causes of tumor development came to the fore. Viruses are not the only pathogens with carcinogenic potency. A connection between certain types of carcinoma and a chronic infection with schistosoma or liver fluke has long been known.
Die Rolle der Bakterien wurde dagegen erst kürzlich aufgedeckt. So wurde Helicobacter pylori als erstes Bakterium dem Karzinogen der Gruppe I zugeordnet (WHO/IARC Kongreß in Lyon, 1994 und Logan RPH, Helicobacter pylori and gastric cancer, Lancet 1994, 334, 1078-79).The role of bacteria, however, has only recently been revealed. 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).
Die Beziehung zwischen einer permanenten Besiedelung des Magens durch Helicobacter pylori und dem Magenkarzinom gilt als gesichert. Kolorektale Karzinome sind gleich dem Magenkrebs eine der vorherrschenden Geschwulstkrankheiten weltweit. Die epidemiologischen Besonderheiten der Ausbreitung kolorektaler Karzinome haben schon in den siebziger Jahren zu der Vermutung geführt, das gastrointestinale Bakterien ursächlich mit den kolorektalen Karzinomen zusammenhängen (Aries VC, Crowther JS, Drasar MJ, Hill MJ and Williams REO, Bacteria and the etiology of cancer of the large bowel, GUT 10, 334, 1969). Trotz einer intensiven und zielgerichteten Forschung konnte die postulierte Beziehung zwischen Magendarmbakterien und Karzinogenese bisher nur für Helicobacter pylori gesichert werden. Der Grund dafür ist eine extreme Komplexität der Darmflora. Diese weist im Koloninhalt Bakteriendichten bis zu lO^- g Trockengewicht auf. Die Aufmerksamkeit galt jedoch bisher überwiegend intraluminalen Bakterien. Ob ein Pathogen, das die Dickdarmmukosa permanent besiedelt bei der Entstehung kolorektaler Karzinome eine Rolle spielt, ist nicht bekannt. Dies konnte mit den klassischen bekannten bakteriologischen Methoden nicht beantwortet werden.The relationship between permanent gastric colonization by Helicobacter pylori and gastric carcinoma is considered established. Colorectal cancer, like gastric cancer, is one of the most prevalent tumors worldwide. The epidemiological peculiarities of the spread of colorectal cancer have led to the assumption in the 1970s that gastrointestinal bacteria are causally related to colorectal cancer (Aries VC, Crowther JS, Drasar MJ, Hill MJ and Williams REO, Bacteria and the etiology of cancer of the large bowel, GUT 10, 334, 1969). Despite intensive and targeted research, the postulated relationship between gastrointestinal bacteria and carcinogenesis has so far only been confirmed for Helicobacter pylori. The reason for this is an extreme complexity of the intestinal flora. This contains bacterial densities of up to 10 ^ g dry weight in the colon content. However, attention has so far been focused mainly on intraluminal bacteria. If a Pathogen that permanently colonizes the large mucosa in the development of colorectal cancer is not known. This could not be answered with the classic known bacteriological methods.
Es wird nun die Verwendung eines in vitro - Nachweises von intraepithelialenThere is now use of in vitro detection of intraepithelial
Darmbakterien, Bestandteilen derselben und Reaktionsprodukten des Wirtes auf diese, zur Erkenung von Darmtumoren und Vorstufen von Darmtumoren beschrieben.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.
Die Verwendung ist dadurch gekennzeichnet, daß man für den in vitro Nachweis folgende Schritte durchführt: a) Biopsieproben in einer wässrigen Lösung schüttelt und diese anschließend erneut in wässriger Lösung aufnimmt, b) Verfahrensschritt a) mehrmals wiederholt, die Überstände verwirft, c) die Rückstände bei Raumtemperatur in eine SDS - haltigen Lösung überführt und zur Auflösung des Gewebes und der DNA-Fraktion, vorsichtig und langsam schüttelt oder rotiert, d) die DNA mit Phenol und Glaskügelchen aufreinigt und anschließend in Wasser resuspendiert, e) die so erhaltene DNA mit universellen 16 sRNA Primern amplifiziert und anschließend die Stärke des PCR-Produktes der Probe mit der Stärke vonThe 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
PCR-Produkten standardisierter Bakterienlösungen bestimmter Dichte vergleicht, f) positive PCR-Produkte kloniert und sequenziert und schließlich g) die in der Biopsie gefundenen DNA-Sequenzen mit den bekannten bakteriellen DNA-Sequenzen aus DNA-Datenbanken vergleicht.Compares PCR products of standardized bacterial solutions of specific density, f) clones and sequences positive PCR products and finally g) compares the DNA sequences found in the biopsy with the known bacterial DNA sequences from DNA databases.
Der Nachweis kann vielfältig modifiziert werden. Es können speziesspezifische Primer für die PCR angewandt werden. Es können Virulenzfaktoren gezielt in der Biopsieprobe oder sogar in Stuhlproben nachgewiesen werden. Es kann ferner eine in situ Hybridisierung durchgeführt werden. Ferner kann ein immunhistochemischer und elektronenmikroskopischer Nachweis von Bakterien in der Mukosa durchgeführt werden. Ebenfalls denkbar sind nuklearmedizinische Methoden wie Szintigraphie oder Atemtest.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.
Die in Verfahrensschritt a) des Nachweises verwendete wässrige Lösung ist vorzugsweise eine physiologische Kochsalzlösung. Die in Verfahrensschritt c) des Nachweises verwendete SDS - haltige Lösung ist vorzugsweise eine 0,5 - 5 %ige SDS/ physiologische Kochsalzlösung.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.
Vorzugsweise beträgt die Zeit für das Schütteln der Biopsieproben in Verfahrensstufe a) des Nachweises 10-120 s, besonders bevorzugt 30-60 s.The time for shaking the biopsy samples in method step a) of the detection is preferably 10-120 s, particularly preferably 30-60 s.
Vorzugsweise werden die Biopsieproben in Verfahrensstufe a) des Nachweises in 100- 1000 μ\ physiologischer Kochsalzlösung aufgenommen, besonders bevorzugt in 500 l.The biopsy samples are preferably taken in step a) of the detection in 100-1000 μ \ physiological saline, particularly preferably in 500 l.
Vorzugsweise wird der Verfahrensschritt a) des Nachweises 5-20 mal wiederholt, besonders bevorzugt 7-10 mal.Process step a) of the detection is preferably repeated 5-20 times, particularly preferably 7-10 times.
Vorzugsweise werden die Fraktionen in Verfahrensschritt c) des Nachweises in ein Volumen von 100-500 l 0,5-5%ige SDS/ physiologische Kochsalzlösung gegeben, wobei besonders bevorzugt eine l%ige Lösung ist.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.
Die bevorzugte Zeit des Schütteins und Rotierens in Verfahrensstufe c) des Nachweises beträgt 10-24 Stunden, besonders bevorzugt 12 Stunden.The preferred time of shaking and rotating in process step c) of the detection is 10-24 hours, particularly preferably 12 hours.
Vorzugsweise wird in Verfahrensstufe d) des Nachweises in einem Volumen von 10-50 μl Wasser resuspendiert, besonders bevorzugt in einem Volumen von 20 μ\ Wasser.In process step d) of the detection, it is preferably resuspended in a volume of 10-50 μl of water, particularly preferably in a volume of 20 μl of water.
Die Verwendung betrifft insbesondere den Nachweis von E. coli Bakterien in Dickdarmbiopsien.The use relates in particular to the detection of E. coli bacteria in colon biopsies.
Der in vitro Nachweis kommt auch bei der Untersuchung von Proben anderer Körperkompartimenten wie Lymphe und Körperflüssigkeiten wie Blut, Flüssigkeiten des Magen-Darm-Traktes und Flüssigkeiten benachbarter Gewebe des Magen und des Darmes zur Anwendung. Reaktionsprodukte des Wirtes auf den Befall mit E. coli oder anderen für den Tumorbefall charakteristischen Bakterien können mittels gebräuchlicher analytischer Methoden, zum Beispiel einem Immunoassay, nachgewiesen werden.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.
Die erfindungsgemäße Verwendung des in vitro Nachweises bezieht sich auf E. coli, Bestandteile von E. coli, Reaktionsprodukte des Wirtes auf E. coli und verwandte Bakterien in der Darmmucosa oder anderen Kompartimenten des Körpers außerhalb des Magen-Darmlumens. Die folgenden Ausführungsbeispiele erläutern die Erfindungsgemäße, ohne diese auf die Beispiele einzuschränken.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.
Beispiel 1example 1
Gewebe von Biopsien wurden in physiologische Kochsalzlösung gegeben und zwei Stunden bei 4°C bis zur weiteren Verwendung gehalten. Jede Probe wurde kräftig mit einem Zerkleinerer (Vortexstab) 1 Minute lang in einem 1 ,5 ml Plastik - Tube zerkleinert und anschließend in 500 μl frische physiologische Kochsalzlösung gegeben. DieserTissues 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 μl of fresh physiological saline. This
Vorgang wurde 8mal wiederholt. Danach wurde die Probe bei Raumtemperatur in einer 1 %igen SDS - Kochsalzlösung inkubiert und über Nacht abzentrifugiert. Aus dem Überstand wird DNA mit Phenol extrahiert, unter Verwendung eines GeneClean Kits weiter aufgereinigt uns schließlich in 30 μl Wasser, das für die Hochdruckflüssigkeitschromatographie (HPLC) verwendet wird, resuspendiert.The process was repeated 8 times. The sample was then incubated at room temperature in a 1% SDS saline solution and centrifuged off overnight. DNA is extracted from the supernatant with phenol, further purified using a GeneClean kit and finally resuspended in 30 μl of water which is used for high pressure liquid chromatography (HPLC).
Das 5' - Ende der 16S rRNA Gene (600 bp) wurde mittels PCR unter Verwendung eines Universal - Primers für Bakterien amplifiziert:The 5 'end of the 16S rRNA genes (600 bp) was amplified by PCR using a universal primer for bacteria:
5'-CTGGTTCCGGCGAAGAGTTTGAT(c/t)fc/a TGGCTC AG-3' 5'-CTCGCTCCGGCGAACCGC(g/t (a/g)CTGCTGGCAC-3l 5'-CTGGTTCCGGCGAAGAGTTTGAT (c / t) fc / a TGGCTC AG-3 '5'-CTCGCTCCGGCGAACCGC (g / t (a / g) CTGCTGGCAC-3 l
Die 13 zusätzlichen Nukleotide (unterstrichen) wurden für die Klonierung an das 5' - Ende gebunden.The 13 additional nucleotides (underlined) were bound to the 5 'end for cloning.
Um falsche positive PCR - Ergebnisse zu vermeiden, wurden vor- und nach-PCR - Stufen in verschiedenen Aufbauten und parallele Kontrollexperimenten unter Weglassen von template DNA durchgeführt.In order to avoid false positive PCR results, pre- and post-PCR stages were carried out in different setups and parallel control experiments with the omission of template DNA.
Die Bakterienmenge in den Proben wurde mittels elektrophoretischem Vergleich der PCRThe amount of bacteria in the samples was determined by means of electrophoretic comparison of the PCR
- Proben zu PCR - Standard - Lösungen von Bakterien mit 105, 104, 103, 102 und 10 colony forming units (cfus) pro μl nach 25 and 30 Thermozyklen gemessen. Für die Sichtbarmachung wurde das Agarosegel mit Ethidiumbromid behandelt. Lösungen mit 10 cfus/μl gaben kein PCR - Signal. Proben - PCR - Signale mit 10^ cfus/μl waren zu schwach für weitere Stufen und wurden als negativ betrachtet.- Samples for PCR standard solutions of bacteria with 10 5 , 10 4 , 10 3 , 10 2 and 10 colony forming units (cfus) per μl measured after 25 and 30 thermal cycles. The agarose gel was treated with ethidium bromide for visualization. Solutions with 10 cfus / μl gave no PCR signal. Sample PCR signals with 10 ^ cfus / μl were too weak for further steps and were considered negative.
Für die Bestimmung der Bakterienzusammensetzung in der Probe mit positivem PCR - Signal wurden amplifizierte 16S rRNA Sequenzen in ein pCMV-LIC Vektor gemäß einem ligationsunabhängigem Klonierungsprotokoll kloniert. In einem Sequencer wurden für jede Biopsie zwischen 40 bis 200 Klone sequenziert. Die Bakteriensequenzen wurden analysiert und wie zuvor beschrieben mit 16S rRNA Primärstrukturen aus Datenbanken verglichen.For the determination of the bacterial composition in the sample with a positive PCR signal, 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.
Beispiel 2Example 2
Zum Nachweis der Ausführbarkeit des erfindungsgemäßen Verfahrens wurden von Menschen Biopsien entnommen.Biopsies were taken from humans to demonstrate the feasibility of the method according to the invention.
Hierzu wurden 82 Menschen konsekutiv kolonoskopiert. Die entnommenen Dickdarmbiopsien wurden auf das Vorkommen intraepithelialer Keime untersucht. Es wurden drei Gruppen gebildet:For this, 82 people were consecutively colonoscoped. The removed colon biopsies were examined for the presence of intraepithelial germs. Three groups were formed:
1. Normalgruppe: 42 Menschen mit makroskopisch und histologisch unauffälligen kolonoskopischen Befund1st normal group: 42 people with macroscopic and histologically unremarkable colonoscopic findings
2. Adenomgruppe: 22 Menschen mit tubulovillösen Adenomen2nd adenoma group: 22 people with tubulovillous adenomas
3. Karzinomgruppe: 18 Menschen mit Karzinomen, davon 11 bei Zustand nach3rd carcinoma group: 18 people with carcinomas, 11 of whom had the condition after
Kolonresektion vor weniger als drei Jahren, wobei vier der resezierten Patienten rezidivfrei warenColon resection less than three years ago, with four of the resected patients free of recurrence
Keiner der Untersuchten hatte eine familiäre Polyposis oder bekam zwei Wochen vor der Untersuchung Antibiotika. Von jedem Untersuchten wurden 1 bis 7 Biopsien entnommen. Diese Biopsien wurden mit dem erfindungsgemäßen Verfahren auf intraepitheliale und verwandte E. coli Bakterien untersucht.None of the examined had a familial polyposis or received antibiotics two weeks before the examination. 1 to 7 biopsies were taken from each examined. These biopsies were examined with the method according to the invention for intraepithelial and related E. coli bacteria.
Wenn möglich wurden Biopsien sowohl aus der tumorverdächtigen als auch aus der normalen Mukosa entnommen.If possible, biopsies were taken from both the suspected tumor and the normal mucosa.
Das Geschlecht, das Durchschnittsalter und die Altersstruktur der Untersuchten in den drei Gruppen ist wie folgt:The sex, the average age and the age structure of the examined in the three groups is as follows:
Normal Adenom KarzinomNormal adenoma carcinoma
Gesamtzahl der Untersuchten: 42 22 18Total number of subjects examined: 42 22 18
Durchschnittsalter: 45,6 67,2 60Average age: 45.6 67.2 60
Altersstruktur: 20-84 39-85 38-79Age structure: 20-84 39-85 38-79
Frauen: 25 14 8Women: 25 14 8
Durchschnittsalter: 48 68,1 64Average age: 48 68.1 64
Altersstruktur: 20-84 50-75 38-79Age structure: 20-84 50-75 38-79
Männer: 17 8 10Men: 17 8 10
Durchschnittsalter: 42,2 65,5 58Average age: 42.2 65.5 58
Altersstruktur: 25-60 39-85 46-67 Die quantitative Bestimmung der Bakterienkonzentration erfolgte in 103, 56 und 47 Biopsien von Untersuchten aus der Normal-, Adenom- und Karzinomgruppe.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.
In 37 von 42 Untersuchten der Normalgruppe wurden keine Bakterien gefunden (0 bis 100 cfu/μl). Dagegen befanden sich Bakterienkonzentrationen zwischen 10^ und 10^ cfu/ μl in mindestens einer Biopsie bei allen untersuchten der Karzinomgruppe und bei 21 von 22 Untersuchten der Adenomgruppe. Bakterien wurden ebenfalls in 23 der 44 Biopsien aus der normalen Mukosa der Untersuchten aus der Adenomgruppe und 31 der 37 Biopsien aus der Karzinomgruppe gefunden. PCR positive Biopsien aus der normalen Mukosa und aus Tumorgewebe von Untersuchten wurden wie folgt ermittelt:No bacteria were found in 37 out of 42 normal group studies (0 to 100 cfu / μl). In contrast, there were bacterial concentrations between 10 ^ and 10 ^ cfu / μl in at least one biopsy in all examined carcinoma groups and in 21 out of 22 examined adenoma group. Bacteria were also found in 23 of the 44 biopsies from the normal mucosa of those examined from the adenoma group and 31 of the 37 biopsies from the carcinoma group. PCR positive biopsies from the normal mucosa and from tumor tissue of the examined were determined as follows:
Mit Adenom Mit KarzinomWith adenoma With carcinoma
Entnahmestelle: normale Mukosa Tumor normale Mukosa Tumor Gesamtzahl der Biopsien: 44 12 37 10Collection point: normal mucosal tumor normal mucosal tumor Total number of biopsies: 44 12 37 10
PCR positive Biopsien: 23 (52%) 11 (91%) 33 (84%) 9 (90%)PCR positive biopsies: 23 (52%) 11 (91%) 33 (84%) 9 (90%)
5, 26 und 32 PCR-Produkte aus den Biopsien der Normal-, Adenom- und Karzinomgruppen wurden kloniert und sequenziert. In der Normalgruppe wurden je eine Probe pro Untersuchtem sequenziert. In den Adenom/ Karzinomgruppen wurden alle positiven PCR-Produkte aus dem Tumor kloniert und sequenziert. Von den Stufenbiopsien aus normalen Kolonabschnitten der Tumorgruppen wurde jeweils die Probe mit der maximalen Bakterienkonzentration ausgewählt, kloniert und sequenziert. 80% aller sequenzierter Klone in 17 von 21 Untersuchten aus der Adenomgruppe und 17 von 18 Untersuchten aus der Karzinomgruppe enthielten die E. coli Sequenz.5, 26 and 32 PCR products from the biopsies of the normal, adenoma and carcinoma groups were cloned and sequenced. In the normal group, one sample per patient was sequenced. All positive PCR products from the tumor were cloned and sequenced in the adenoma / carcinoma groups. The sample with the maximum bacterial concentration was selected, cloned and sequenced from the step biopsies from normal colon sections of the tumor groups. 80% of all sequenced clones in 17 out of 21 examined persons from the adenoma group and 17 out of 18 examined persons from the carcinoma group contained the E. coli sequence.
In der folgenden Tabelle sind die Ergebnisse der quantitativen PCR und die Resultate der Sequenzanalyse klonierter PCR Produkte aufgeführt. The following table shows the results of the quantitative PCR and the results of the sequence analysis of cloned PCR products.
* Bakteroide, Clostridien, Pseudomonas, E. coli, keine davon mehr als 20% aller Klone ** Bei Dominanz finden sich in mehr als 80% aller Klone ein und dieselbe Sequenz* Bacteroids, clostridia, Pseudomonas, E. coli, none of them more than 20% of all clones ** If dominance is found in more than 80% of all clones, the same sequence
Aus den Ergebnissen ist ersichtlich, daß sowohl in Biopsien normal aussehenderFrom the results it can be seen that both look more normal in biopsies
Schleimhaut als auch in Biopsien von Tumorgewebe der Untersuchten mit Kolonkarzinom und Kolonadenom intraepitheliale Bakterien vorhanden sind. Mucosa as well as in biopsies of tumor tissue of those examined with colon carcinoma and colon adenoma intraepithelial bacteria are present.
Claims
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002293704A CA2293704A1 (en) | 1997-03-06 | 1998-03-04 | In vitro analysis for detection of tumours of the intestine |
| EP98912439A EP0977888A1 (en) | 1997-03-06 | 1998-03-04 | $i(IN VITRO) ANALYSIS FOR DETECTION OF TUMOURS OF THE INTESTINE |
| AU67275/98A AU721826B2 (en) | 1997-03-06 | 1998-03-04 | In vitro determination for detection of tumors of the intestine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19711111A DE19711111C2 (en) | 1997-03-06 | 1997-03-06 | In vitro detection for the detection of intestinal tumors |
| DE19711111.4 | 1997-03-06 |
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| Publication Number | Publication Date |
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| WO1998039473A1 true WO1998039473A1 (en) | 1998-09-11 |
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| PCT/EP1998/001223 Ceased WO1998039473A1 (en) | 1997-03-06 | 1998-03-04 | In vitro analysis for detection of tumours of the intestine |
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| Country | Link |
|---|---|
| EP (1) | EP0977888A1 (en) |
| AU (1) | AU721826B2 (en) |
| CA (1) | CA2293704A1 (en) |
| DE (1) | DE19711111C2 (en) |
| WO (1) | WO1998039473A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001079545A1 (en) * | 2000-04-18 | 2001-10-25 | National University Of Singapore | Molecular markers |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006015616A1 (en) | 2004-08-13 | 2006-02-16 | Indivumed Gmbh | Use of transthyretin as a biomarker for colorectal adenoma and/or carcinoma; method for detection and test system |
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| GB2290866B (en) * | 1994-06-29 | 1998-10-07 | Reckitt & Colmann Prod Ltd | Detection of Helicobacter pylori infection using antibodies to carbonic anhydrase |
-
1997
- 1997-03-06 DE DE19711111A patent/DE19711111C2/en not_active Expired - Lifetime
-
1998
- 1998-03-04 AU AU67275/98A patent/AU721826B2/en not_active Ceased
- 1998-03-04 EP EP98912439A patent/EP0977888A1/en not_active Withdrawn
- 1998-03-04 WO PCT/EP1998/001223 patent/WO1998039473A1/en not_active Ceased
- 1998-03-04 CA CA002293704A patent/CA2293704A1/en not_active Abandoned
Non-Patent Citations (6)
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001079545A1 (en) * | 2000-04-18 | 2001-10-25 | National University Of Singapore | Molecular markers |
| US7101668B2 (en) | 2000-04-18 | 2006-09-05 | National University Of Singapore | Molecular markers |
Also Published As
| Publication number | Publication date |
|---|---|
| DE19711111C2 (en) | 1999-09-02 |
| EP0977888A1 (en) | 2000-02-09 |
| DE19711111A1 (en) | 1998-09-17 |
| AU721826B2 (en) | 2000-07-13 |
| CA2293704A1 (en) | 1998-09-11 |
| AU6727598A (en) | 1998-09-22 |
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