EP1836318A2 - Procédé et nécessaire de détection de mycobacterium avium subsp. paratuberculosis (map) dans des échantillons d'excréments, de tissus corporels, ou de lait - Google Patents

Procédé et nécessaire de détection de mycobacterium avium subsp. paratuberculosis (map) dans des échantillons d'excréments, de tissus corporels, ou de lait

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Publication number
EP1836318A2
EP1836318A2 EP06700416A EP06700416A EP1836318A2 EP 1836318 A2 EP1836318 A2 EP 1836318A2 EP 06700416 A EP06700416 A EP 06700416A EP 06700416 A EP06700416 A EP 06700416A EP 1836318 A2 EP1836318 A2 EP 1836318A2
Authority
EP
European Patent Office
Prior art keywords
dna
map
pcr
seq
control
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP06700416A
Other languages
German (de)
English (en)
Inventor
Roger Stephan
Taurai Tasara
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zurich Universitaet Institut fuer Medizinische Virologie
Original Assignee
Zurich Universitaet Institut fuer Medizinische Virologie
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 Zurich Universitaet Institut fuer Medizinische Virologie filed Critical Zurich Universitaet Institut fuer Medizinische Virologie
Publication of EP1836318A2 publication Critical patent/EP1836318A2/fr
Withdrawn legal-status Critical Current

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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

Definitions

  • MAP Mycobacterium avium subsp. paratuberculosis
  • the invention relates to a method and a kit for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in samples of faeces, body tissues or milk.
  • MAP Mycobacterium avium subsp. paratuberculosis
  • the invention relates to the study of sample material from ruminants, and in particular to the study of milk or milk products made therefrom.
  • the invention should also include the study of sample material from other animal sources as well as humans.
  • Suitable tissues which can be tested in addition to faeces or milk in the context of MAP detection are e.g. Blood, lymphoid tissue and muscle tissue, to name just a few examples.
  • MAP paratuberculosis
  • ruminants especially cows.
  • MAP can be isolated from milk from clinically and subclinically paratuberculosis patients. It has also been found that MAP has a higher thermoresistance compared to other mycobacteria, so that the usual milk pasteurization may not lead to the destruction of MAP. Finally, it has been shown that MAP also comes from the intestinal tissue as well as the blood of Crohn's disease patients so that a possible causal relationship between MAP and Crohn's disease can not be ruled out.
  • a suitable primer pair represents only a part of a functioning detection system.
  • the samples usually analyzed, such as feces or blood, but in particular also milk samples are generally relatively difficult to assay by means of PCR, since a whole Range of substances that have inhibitory properties.
  • the object of the invention was therefore to provide a method and a kit for detecting MAP possibly contained in samples, in which all steps, or components, are optimally matched to one another.
  • the object is achieved by a method according to claim 1 and a kit according to claim 20.
  • the materials investigated by the method or kit according to the invention may be samples of excrement, body tissue or milk, which may originate in particular from ruminants but also from other animals or from humans.
  • suitable tissues which can be examined in addition to faeces or milk in the context of MAP detection are e.g. Blood, lymph tissue and muscle tissue.
  • a DNA extract is obtained from the sample to be examined, which can be further processed by means of PCR.
  • the milk is centrifuged and the bacteria contained are pelleted.
  • sample materials may need to first Suspension are prepared with subsequent separation of coarse particles, before by centrifugation a bacterial pellet can be obtained.
  • the recovery of the contained bacteria from the different sample materials represents a routine measure for a person skilled in the art.
  • bacterial pellet obtained bacterial pellet under lysis conditions.
  • the conditions are chosen so that mycobacteria lyse and generally agree with samples of different origin.
  • the DNA is then further extracted and purified by means of, in particular, alcoholic precipitation, if appropriate after prior precipitation of the proteins.
  • the milk, or milk product sample was processed using a "high pure template preparation kit Cat No. 1796 828" from Roche Diagnostics in accordance with the prescribed protocol.
  • a "high pure template preparation kit Cat No. 1796 828” from Roche Diagnostics in accordance with the prescribed protocol.
  • an initially enzymatic treatment of the pelleted bacteria was assisted by mechanical treatment of the cells.
  • the lysate was then loaded onto a DNA-binding column and the column subsequently eluted as prescribed in the kit.
  • the eluate was then further processed as a DNA extract in the PCR.
  • the recovered DNA extract is worked up in the presence of MAP specific primers.
  • primers suitable for e.g. recognize the f57 sequence in MAP are particularly preferred.
  • primer pair having the following sequences:
  • SEQ ID NO 2 TTG GAC GAT CCGAAT ATG Tand SEQ ID NO 3: AGT GGGAGG CGTACC A
  • the mentioned primers correspond to sections 126-144 (SEQ ID NO 2) and 365-380 (SEQ ID NO 3) of the MAP f57 sequence. Details of the primers used are summarized in Table Ia.
  • Target sequence primer sequence (5f-3i) position amplicon (oligonucleotides) (bp)
  • MAPf57 MAPfSVpI (SEQ ID TTG GAC GAT CCG AAT ATG T 126-144 254
  • MAPf57p2 (SEQ ID AGT GGG AGG CGT ACC A 365-380
  • milk is a critical sample material because of the PCR inhibitors it contains. Even in the above-described particularly preferred methods for obtaining a DNA extract, it can not be ruled out that substances which inhibit the PCR may still be present in the PCR mixture.
  • the invention therefore further provides that the PCR is carried out in the presence of an internal amplification control.
  • Suitable amplification controls can be any desired target DNA sequences (control DNA) which can be standardized with appropriately designed primers (control primers) and reproducibly amplified under similar conditions as the MAP sequences.
  • the internal amplification control is carried out in the same approach in which the detection of the MAP sequence is carried out. In this case, care must be taken that the amplicon of the control DNA and that of the MAP sequence can be distinguished from each other.
  • the control DNA used in the invention preferably PuC 19 plasmid DNA. Details of this plasmid, in particular to the sequence are for example from “Yanisch-Perron et al.” in “Gene 33 (1), 103-119 (1985)". PuC19 can be obtained under No. ATCC 37254 / L09137 or from New England Biolabs (Cat. No. N3041S).
  • Suitable control primers may be e.g. have the following sequences:
  • Target sequence oligonucleotide sequence (5f-3 Q position amplicon (bp)
  • PuC19 plasmid DNA PuC 19fw (SEQ ID CGG AGA CGG TCA CAG CT 49-65 400
  • An advantageous embodiment of the invention therefore provides that the workup of the DNA extract by means of real-time PCR.
  • Real-time PCR methods have been known for some time and essentially differ from conventional PCR methods in that the PCR approach additionally contains probes which generate a changing signal which correlates with the increase of amplified DNA. Furthermore, the PCR apparatus (cycler) used has a measuring device which detects the signals generated during the PCR in the batches, so that directly, i. can still be determined during the measurement, whether an amplification of the target sequence has taken place.
  • Suitable for real-time PCR probe and cycler systems with appropriate measuring devices are offered by different manufacturers known in the art.
  • Sybr-Green is a dye that intercalates into double-stranded DNA and produces a fluorescence signal only in the intercalated state, which can be measured in a suitable cycler. The signal is the stronger, the more double-stranded DNA is present, in which the dye can be stored.
  • a problem with the Sybr-Green system mentioned is that only non-specific detection of double-stranded DNA is possible here.
  • the invention therefore particularly preferably provides that specific probes are used to detect the amplicons generated during the PCR specifically generate a signal only upon amplification of MAP sequences and / or the internal control DNA sequence. It is understood that the signals generated while working up the amplification control in a common approach of the signals generated in amplification of the MAP sequence must be differentiable, which is not a problem by appropriate marker selection.
  • probes typically have a portion of DNA chosen to specifically hybridize to a portion of the target DNA.
  • a marker or a marker system is coupled which generates a different signal upon hybridization than in the non-hybridized state.
  • Suitable probes have become known, for example, in the art by the term "TAQMAN probes".
  • MAP probes having the following sequences have been found to be particularly suitable:
  • SEQ ID NO 8 GCA AGG CGA TTA AGT TGG GTA AC SEQ ID NO 9: CAG GGT TTT CCC AGT CAC GAC
  • the invention further relates to a kit in which the reagents necessary for carrying out the method are contained.
  • the kit according to the invention contains at least reagents for obtaining a DNA extract from mycobacteria cells possibly contained in sample material, in particular in milk or milk products, and reagents for real-time PCR, containing a primer pair specific for a MAP sequence, an internal amplification control and with the MAP sequence and the internal amplification control hybridizing probes, each having different detectable markers.
  • a positive control for the PCR may be included. It may be e.g. the sequence f57 (SEQ ID NO 1) from MAP already mentioned in a suitable vector, which is processed in a separate batch in the PCR to ensure that the conditions chosen for the PCR for the amplification and detection of MAP Sequences are suitable.
  • the sample After addition of 60 ⁇ l Proteinase K, the sample is incubated for 2 hours at 55 0 C and periodically mixed. The incubated sample is then heated followed by the addition of 300 ul buffer (Roche kit), and addition of glass beads in a Ribolyer (Hybaid, Ashford) spent ( ⁇ .Smsec "1 for 45s). The sample is immediately for 10 minutes at 7O 0 C in order to possibly destroying upcoming DNAses. The sample is then cooled at room temperature for 1 minute, 150 ⁇ l of isopropanol (2-propanol, Fluka) are added, centrifuged briefly and loaded onto the DNA binding column of the kit. The DNA templates are then washed out with 100 ⁇ l of the elution buffer (Kit). 5 ⁇ l aliquots are then used in the PCR.
  • Real-time PCR is performed in 20 ⁇ l glass capillaries using Light Cycler 2.0 instrument (Roche Diagnostics).
  • the reaction mixture consists of Ix LightCycler-Faststart DNA master plus TM hybridization probes mix (Roche Diagnostics), 800 nM of each primer (MAPf57pl / SEQ NO ID 2, MAPf57p2 / SEQ ID ID 3, PuC400fw / SEQ ID NO 4 and puC400rv / SEQ NO ID 5), 200 nM of each probe and 2000 copies of PuC19 plasmid DNA used as an internal amplification control.
  • the amplification starts with an initial pre-incubation at 95 0 C for 10 minutes, followed by 45 cycles (95 0 C for 5 1os 56 ° C for 20s and 72 0 C for 18s).
  • the DNA was extracted from MAP artificially contaminated milk samples and purified and the respective DNA extracts were processed by means of real-time PCR.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic 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)

Abstract

L'invention concerne un procédé pour détecter Mycobacterium avium subsp. paratuberculosis (MAP) dans des échantillons d'excréments, de tissus corporels, ou de lait. Ce procédé consiste : à isoler un extrait d'ADN dans les échantillons ; à traiter l'extrait d'ADN obtenu en présence d'amorces spécifiques à MAP (amorces MAP), au moyen de la réaction en chaîne de la polymérase (PCR), et ; à vérifier si les séquences génétiques spécifiques à MAP ont été amplifiées pendant la PCR. La présente invention se rapporte en outre à un nécessaire servant à la mise en oeuvre de ce procédé.
EP06700416A 2005-01-12 2006-01-06 Procédé et nécessaire de détection de mycobacterium avium subsp. paratuberculosis (map) dans des échantillons d'excréments, de tissus corporels, ou de lait Withdrawn EP1836318A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005001788A DE102005001788A1 (de) 2005-01-12 2005-01-12 Verfahren und Kit zur Detektion von Mycobakterium avium subsp. paratuberculosis (MAP) in Proben von Kot, Körpergewebe oder Milch
PCT/EP2006/000074 WO2006074878A2 (fr) 2005-01-12 2006-01-06 Procede et necessaire de detection de mycobacterium avium subsp. paratuberculosis (map) dans des echantillons d'excrements, de tissus corporels, ou de lait

Publications (1)

Publication Number Publication Date
EP1836318A2 true EP1836318A2 (fr) 2007-09-26

Family

ID=36643059

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06700416A Withdrawn EP1836318A2 (fr) 2005-01-12 2006-01-06 Procédé et nécessaire de détection de mycobacterium avium subsp. paratuberculosis (map) dans des échantillons d'excréments, de tissus corporels, ou de lait

Country Status (6)

Country Link
US (1) US20080145848A1 (fr)
EP (1) EP1836318A2 (fr)
AU (1) AU2006205914A1 (fr)
CA (1) CA2592859A1 (fr)
DE (1) DE102005001788A1 (fr)
WO (1) WO2006074878A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2270202A1 (fr) * 2009-07-03 2011-01-05 John Ikonomopoulos Détection mycobactérienne

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007015775A1 (de) 2007-03-30 2008-10-09 Justus-Liebig-Universität Giessen Verfahren zum Nachweis von Paratuberkolose
WO2014113785A1 (fr) 2013-01-18 2014-07-24 Biomeme Incorporated Dispositif analytique
MX372822B (es) 2013-11-01 2020-07-03 Biomeme Inc Dispositivo de preparación y extracción de muestras.
CN111356768A (zh) 2017-09-15 2020-06-30 生米公司 用于自动化样品处理的方法和系统
EP3724352A4 (fr) 2017-12-15 2021-09-01 Biomeme, Inc. Dispositifs et procédés portables pour analyser des échantillons
WO2019143812A1 (fr) * 2018-01-18 2019-07-25 Biomeme, Inc. Procédés de dosage pour déceler la présence de micro-organismes
JP7483745B2 (ja) 2019-03-21 2024-05-15 バイオミーム インコーポレイテッド 多機能性分析デバイス
JP2023545631A (ja) 2020-09-18 2023-10-31 バイオミーム，インコーポレイテッド 試料を分析するための運搬可能デバイスおよび方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1223225A1 (fr) * 2001-01-10 2002-07-17 Stichting Nederlands Instituut Voor Zuivelonderzoek Détection de Mycobacterium avium ssp. paratuberculosis
EP1233076A3 (fr) * 2001-02-19 2002-12-04 Universite Catholique De Louvain Diagnostic différentiel d'espèces de mycobactéries et de pseudomonas avec des sondes génétiques spécifiques de la région en amont du gène p34
NZ519469A (en) * 2002-06-10 2005-01-28 Agres Ltd Nucleic acid probes for detecting the presence of Mycobacterium paratuberculosis and distinguishing between cattle and sheep strains

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006074878A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2270202A1 (fr) * 2009-07-03 2011-01-05 John Ikonomopoulos Détection mycobactérienne

Also Published As

Publication number Publication date
CA2592859A1 (fr) 2006-07-20
AU2006205914A1 (en) 2006-07-20
DE102005001788A1 (de) 2006-07-20
US20080145848A1 (en) 2008-06-19
WO2006074878A2 (fr) 2006-07-20
WO2006074878A3 (fr) 2006-11-30

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