WO2007019945A2 - Procede et kit de detection de fractions de tissus bovins dans des produits d'origine animale fabriques dans des conditions de decomposition - Google Patents

Procede et kit de detection de fractions de tissus bovins dans des produits d'origine animale fabriques dans des conditions de decomposition Download PDF

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Publication number
WO2007019945A2
WO2007019945A2 PCT/EP2006/007305 EP2006007305W WO2007019945A2 WO 2007019945 A2 WO2007019945 A2 WO 2007019945A2 EP 2006007305 W EP2006007305 W EP 2006007305W WO 2007019945 A2 WO2007019945 A2 WO 2007019945A2
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WO
WIPO (PCT)
Prior art keywords
dna
bovine
pcr
probes
control
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Application number
PCT/EP2006/007305
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German (de)
English (en)
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WO2007019945A3 (fr
Inventor
Roger Stephan
Taurai Tasara
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Universität Zürich
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Publication date
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Publication of WO2007019945A2 publication Critical patent/WO2007019945A2/fr
Publication of WO2007019945A3 publication Critical patent/WO2007019945A3/fr

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes

Definitions

  • the invention relates to a method and a kit for the detection of bovine tissue fractions in products of animal origin produced under decomposing conditions.
  • An essential application of the invention is the examination or certification of products for which it is to be ensured that they contain no bovine tissue components.
  • the invention relates to the study of gelatin.
  • the desire for a reliable certification of gelatin for the absence of bovine components is due to the BSE crisis.
  • the EU has issued a directive regarding the production of edible gelatine, which requires documentary evidence that the starting materials used to produce the gelatine are derived from animals suitable for human consumption.
  • This is relatively costly in the case of BSE, as bones and slaughterhouse waste from many different cattle are pooled during manufacture, which means that a corresponding number of BSE tests would need to be documented for each batch.
  • the object of the invention is to provide a detection method which also functions reliably in such products.
  • the object is achieved by a method according to claim 1 and a kit according to claim 23.
  • a DNA extract is obtained from a sample of the product.
  • the DNA extract is then processed by means of PCR, wherein the primers used in the PCR are designed so that they recognize as a target sequence a bovine-specific sequence with a length of less than 150 nucleotides. Finally, it is then checked whether an amplification has taken place during the PCR with the primers used.
  • a DNA extract is obtained from the sample to be investigated, which can be further processed by means of PCR.
  • the sample can be homogenized, for example, then incubated in the presence of a lysis buffer and / or a proteinase, in particular proteinase K, and then the DNA contained can be extracted and purified.
  • a lysis buffer and / or a proteinase in particular proteinase K
  • the DNA contained can be extracted and purified.
  • the DNA is then further extracted and purified by means of, in particular, alcoholic precipitation, if appropriate after prior precipitation of the proteins.
  • the lysate can also be applied to a DNA-binding column and the column subsequently eluted as prescribed in the kit "High Pure Template Preparation Kit Cat No. 1796 828" from Roche Diagnostics. The eluate is then further processed as a DNA extract in the PCR.
  • DNA extracts of e.g. preferably examined gelatin still contain species-specific by PCR detectable target sequences.
  • An essential aspect is that the invention uses very short target sequences, since it has been found that such short sequences are still intact after treatment under extreme conditions and are present in an amount sufficient for analysis.
  • the target sequence used is particularly preferably a boin-specific sequence which occurs several times in the genome or the mitochondrial DNA. Basically, it can be said that with the co- The number of statistical proofs increases, which can be an important argument, especially for the desired certification.
  • the work-up takes place in the presence of primers which specifically recognize the ATPase 8 subunit.
  • the ATPase 8 subunit is encoded by region 7766-7966 (SEQ ID NO 1) of bovine mitochondrial DNA.
  • SEQ ID NO 1 The entire mitochondrial genome is published under No. AF492351 in the database of EMBL-EBI.
  • primer pair having the following sequences:
  • the primer Fpc F2 corresponds to the section 7799-7823 in the mitochondrial DNA (34-58 in SEQ ID NO 1) and the region 4-24 of the primer Fpc R (SEQ ID NO 3) corresponds to Section 7898- 7918 in the mitochondrial DNA or in the section 133-154 in the ATPase 8 sequence (SEQ ID NO 1).
  • the first 3 nucleotides at the 5 'end are not complementary and have been inserted for reasons of melting behavior of the primer.
  • Target sequence primer sequence (5 ⁇ -30 position amplicon (oligonucleotides) (bp)
  • DNase 1 is added to the master mix in a particularly preferred embodiment in order to avoid potential Exclude contamination by bovine components not derived from the product. It could e.g. demonstrated that 0.2 UDNase I on 10 microliter of master mix was sufficient to exclude contaminating DNA without adversely affecting the PCR reaction.
  • a fundamental problem with PCR approaches is that, due to PCR inhibitors possibly contained in the samples examined, the reaction does not proceed or does not take place sufficiently.
  • the invention therefore provides that the PCR is carried out in the presence of an internal amplification control.
  • Suitable amplification controls may be any desired target DNA sequences (control DNA) that are appropriately designed with primers standardized and reproducible under similar conditions as the ATPase 8 subunit amplify sequences.
  • the internal amplification control is carried out in the same approach, in which the detection of the Linderspezifischen sequence is carried out.
  • the control DNA used in the invention preferably PuC 19 plasmid DNA. Details of this plasmid, in particular the sequence, are e.g. from “Yanish-Perron et al.” in “Gene 33 (1), 103-119 (1985)". PuC 19 can be obtained under No. ATCC 37254 / L09137 or from New England Biolabs (Cat. No. N3041S).
  • Suitable control primers which recognize PuC 19 may be e.g. have the following sequences:
  • Target sequence oligonucleotides sequence (5i-3 ⁇ ) position amplicon (bp)
  • PuC19 plasmid DNA PuC 19fw (SEQ ID CGG AGA CGG TCA CAG CT 49-65 400
  • the method according to the invention can use a conventional PCR by means of subsequent evaluation in the gel.
  • the work-up of the DNA extract takes place by means of real-time PCR.
  • Real-time PCR methods have been known for a long time and differ essentially from conventional PCR methods in that the PCR approach additionally contains probes which generate a changing signal correlated with the increase of amplified DNA. Furthermore, the PCR apparatus used (cycler) has a measuring device which detects the signals generated during the PCR in the approaches, 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.
  • specific probes which specifically generate a signal only when amplifying bovine sequences and / or the internal control DNA sequence are particularly preferably used for detecting the amplicons generated during the PCR. It is understood that the signals generated while working up the amplification control must be differentiable in a common approach from the signals generated in amplification of the bovine sequence, which is not a problem by appropriate marker selection.
  • Specific probes typically have a portion of DNA chosen to specifically hybridize to a portion of the target DNA. The DNA segment is linked to a marker or a marker system which generates a different signal upon hybridization than in the non-hybridized state. Suitable probes have become known in the art, for example, under the term "TAQMAN probes".
  • This system uses 2 probes per target, both of which hybridize to adjacent regions of the target sequence.
  • One probe is labeled with fluorescein, the other with a fluorescent dye, e.g. LC-Red-705 or LC-Red-640 (Roche Diagnostics).
  • the fluorescein can activate the fluorescent dyes, which then generate a signal with the respective specified wavelengths 705 nm or 640 nm.
  • bovine probes with the following sequences have been found to be particularly suitable:
  • SEQ ID NO 7 (BovSP-5'LC Red 640): 5'TAT CAC AAT CCA GAA CTG ACA CCA AC 3 '
  • control DNA which can be worked up with the same primers as the ATPase 8 subunit.
  • a suitable control DNA in this context may, for example, have the following sequence:
  • SEQ ID NO 8 AT GATCTTATCA ATATTCTTGA CCCTTTTAT CATCTTTCAA CTAAAAGTTT CAAAACACAA CTTT CAGGGT
  • SEQ ID NO 8 corresponds to the region 34-153 of the bovine ATPase sequence (SEQ ID NO 1), wherein the section 67-90 in SEQ ID NO 8 was replaced by a 21 bp fragment, that of the region 355 -375 of the puC19 plasmid.
  • the main advantage of using the described control DNA according to SEQ ID NO 8 is that an amplification can be carried out with the same primers as for the bovine sequence, whereby not only a basic PCR control but also a primer control is given ,
  • control DNA according to SEQ ID 8 is used as the internal amplification control, then the further advantage is that only one control probe which binds to the PuC19 insert is required, which in turn reacts with the bovine probe according to SEQ ID NO. BovSP-3'Fluo).
  • this control probe has a sequence according to:
  • Target sequence probes oligonucleotide sequence (5-30 position de
  • the invention further relates to a kit in which the reagents required for carrying out the method are contained.
  • the kit according to the invention contains at least reagents for obtaining a DNA extract from sample material, and reagents for carrying out a PCR, in particular a real-time PCR, containing a specific for a bovine sequence primer pair, an internal amplification control and hybridizing with the bovine sequence and the internal amplification control probes each having different detectable markers.
  • Gelatin samples each weighing 500 mg, were homogenized in a Ryboly- ser (Hybaid, Ashford) in the presence of 1 ml of the lysis buffer contained in the kit and 60 ⁇ l proteinase K ( ⁇ .Smsec 1 for 45 s) and then for 30 min 70 ° C and incubated at 90 ° C for 15 minutes.
  • the genomic DNA was then treated with the appropriate column as prescribed in the kit protocol. nigt.
  • the DNA templates were then washed out with 100 ⁇ l of the elution buffer (Kit). 5 ⁇ l aliquots were then used in the PCR.
  • Real-time PCR was performed in 20 ⁇ l glass capillaries using Light Cycler 2.0 instrument (Roche Diagnostics).
  • the reaction mixture consists of QuantiTect PCR Master Mix (Qiagen), 0.5 ⁇ M of each primer (Fpc F2 / SEQ ID ID ID 1, Fpc R / SEQ NO ID 2), 0.2 ⁇ M of each probe (BovSP-3'Fluo, BovSP - 5'LC Red 640, ⁇ uC19-5'LC Red 705) and 0.008 atomol of the internal amplification control (SEQ ID NO 5).
  • Amplification started with an initial preincubation at 95 ° C for 20 minutes, followed by 55 cycles (95 ° C for 0s, 55 ° C for 30s, and 72 ° C for 30s).
  • the fluorescence was measured during the annealing phase at 55 ° C at 640 nm (channel 4) for the bovine sequence and at 705 nm (channel 6) for the amplification control.
  • the DNA was extracted and purified from mixtures (varying amounts of bovine gelatin in porcine gelatin) and the respective DNA extracts were processed by real-time PCR.
  • the results are shown in Table 4.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
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  • Physics & Mathematics (AREA)
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  • 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

Procédé et kit de détection de fractions de tissus bovins dans des produits d'origine animale fabriqués dans des conditions de décomposition. Selon ledit procédé, un extrait d'ADN est prélevé à partir d'un échantillon du produit, l'extrait d'ADN est traité par PCR, les amorces utilisées dans la PCR étant ainsi conçues qu'elles reconnaissent une séquence spécifique des bovins ayant une longueur inférieure à 150 nucléotides, et on vérifie ensuite si une amplification s'est produite pendant la PCR à l'aide des amorces utilisées.
PCT/EP2006/007305 2005-08-12 2006-07-25 Procede et kit de detection de fractions de tissus bovins dans des produits d'origine animale fabriques dans des conditions de decomposition WO2007019945A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200510038214 DE102005038214A1 (de) 2005-08-12 2005-08-12 Verfahren und Kit zur Detektion von Anteilen bovinen Gewebes in unter zersetzenden Bedingungen hergestellten Produkten tierischen Ursprungs
DE102005038214.2 2005-08-12

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WO2007019945A2 true WO2007019945A2 (fr) 2007-02-22
WO2007019945A3 WO2007019945A3 (fr) 2007-07-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106987647A (zh) * 2017-05-25 2017-07-28 上海瑞丰农业科技有限公司 一种检测猪源性成分的rpa引物、试剂盒及检测方法

Citations (2)

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JP2003164287A (ja) * 2001-11-30 2003-06-10 Fertilizer & Feed Inspection Station プライマー配列
US6756495B1 (en) * 1997-05-05 2004-06-29 Centre National De La Recherche Scientifique Method for detecting the presence of biological matters of bovine origin, and oligonucleotides for its implementation

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US8158772B2 (en) * 2004-04-16 2012-04-17 National Institute Of Agrobiological Sciences Oligonucleotide sequences that identify species of animal

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US6756495B1 (en) * 1997-05-05 2004-06-29 Centre National De La Recherche Scientifique Method for detecting the presence of biological matters of bovine origin, and oligonucleotides for its implementation
JP2003164287A (ja) * 2001-11-30 2003-06-10 Fertilizer & Feed Inspection Station プライマー配列

Non-Patent Citations (7)

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AU - Tasara, T. and Stephan, R. PGF - (Poster NO: 54 of conference / meeting) PD - 28.09. - 01.10.2004 PDY - 2004 PDM - 01.10.2004 CONF- 45. AT des Arbeitsgebietes "Lebensmittelhygiene", Teil 2 - Poster ISBN- 3-938026-37-5 PUBP- Garmisch-Partenkirchen XP008077333 *
CASTELLO ANNA ET AL: "Identification of bovine material in porcine spray-dried blood derivatives using the Polymerase Chain Reaction technique" BIOTECHNOLOGIE AGRONOMIE SOCIETE ET ENVIRONNEMENT, Bd. 8, Nr. 4, 2004, Seiten 267-273, XP008077375 ISSN: 1370-6233 *
KRCMAR P ET AL: "IDENTIFICATION OF BOVINE-SPECIFIC DNA IN FEEDSTUFFS" JOURNAL OF FOOD PROTECTION, DES MOINES, IO, US, Bd. 64, Nr. 1, Januar 2001 (2001-01), Seiten 117-119, XP009014319 ISSN: 0362-028X *
LAHIFF S ET AL: "Real-time polymerase chain reaction detection of bovine DNA in meat and bone meal samples." JOURNAL OF FOOD PROTECTION JUL 2002, Bd. 65, Nr. 7, Juli 2002 (2002-07), Seiten 1158-1165, XP008077372 ISSN: 0362-028X *
STEFANO BELLORINI ET AL: "Discriminating animal fats and their origins: assessing the potentials of Fourier transform infrared spectroscopy, gas chromatography, immunoassay and polymerase chain reaction techniques" ANALYTICAL AND BIOANALYTICAL CHEMISTRY, SPRINGER-VERLAG, BE, Bd. 382, Nr. 4, 1. Juni 2005 (2005-06-01), Seiten 1073-1083, XP019327417 ISSN: 1618-2650 & GIZZI G ET AL: "An overview of tests for animal tissues in feeds applied in response to public health concerns regarding bovine spongiform encephalopathy" REVUE SCIENTIFIQUE ET TECHNIQUE - OFFICE INTERNATIONAL DES PIZOOTIES / SCIENTIFIC AND TECHNICAL REVIEW - INTERNATIONAL OFFICE OF EPIZOOTICS, OFFICE INTERNATIONAL DES EPIZOOTIES, PARIS, FR, Bd. 22, Nr. 1, April 2003 (2003-04), Seiten 311-331, XP002358770 ISSN: 0253-1933 *
TARTAGLIA MARCO ET AL: "Detection of bovine mitochondrial DNA in ruminant feeds: A molecular approach to test for the presence of bovine-derived materials" JOURNAL OF FOOD PROTECTION, Bd. 61, Nr. 5, Mai 1998 (1998-05), Seiten 513-518, XP001153765 ISSN: 0362-028X *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106987647A (zh) * 2017-05-25 2017-07-28 上海瑞丰农业科技有限公司 一种检测猪源性成分的rpa引物、试剂盒及检测方法

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