SIST EN ISO 21571:2005
(Main)Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction (ISO 21571:2005)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction (ISO 21571:2005)
ISO 21571:2005 provides general requirements and specific methods for DNA extraction/purification and quantification. These methods are described in Annexes A and B.
ISO 21571:2005 has been established for food matrices, but could also be applicable to other matrices, such as grains and feed.
It has been designed as an integral part of nucleic-acid-based analytical methods, in particular ISO 21569 on qualitative analytical methods, and ISO 21570 on quantitative analytical methods.
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihren Produkten - Nukleinsäureextraktion (ISO 21571:2005)
Diese Internationale Norm legt allgemeine Anforderungen an und spezifische Verfahren für die Extraktion/Reinigung von DNA und die quantitative Bestimmung fest. Diese Verfahren sind in den Anhängen A und B beschrieben.
Diese Internationale Norm wurde für Lebensmittelmatrices erarbeitet, könnte jedoch mit einigen Anpassungen auch auf andere Matrices wie Getreide und Futtermittel angewendet werden.
Diese Norm wurde als ein integraler Bestandteil von auf Nukleinsäuren basierenden analytischen Verfahren, insbesondere ISO 21569 zu qualitativen analytischen Verfahren und ISO 21570 zu quantitativen analytischen Verfahren erarbeitet.
Produits alimentaires - Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés - Extraction des acides nucléiques (ISO 21571:2005)
L'ISO 21571:2005 indique les exigences générales et les méthodes spécifiques pour l'extraction, la purification et la quantification de l'ADN. Ces méthodes sont décrites dans les Annexes A et B..
L'ISO 21571:2005 a été élaborée pour les matrices alimentaires, mais peut également s'appliquer à d'autres matrices (par exemple grains et aliments pour animaux).
Elle a été conçue dans le cadre d'une série de méthodes d'analyse fondées sur l'utilisation de l'acide nucléique, notamment l'ISO 21569 (méthodes d'analyse qualitative) et l'ISO 21570 (méthodes d'analyse quantitative).
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in njihovih produktov - Izolacija nukleinskih kislin (ISO 21571:2005
Ta mednarodni standard določa splošne zahteve in posebne metode za izolacijo/purifikacijo in kvantifikacijo DNK. Te metode so opisane v dodatkih A in B. Ta mednarodni standard je bil pripravljen za živilske matrice, a se lahko uporablja tudi za druge matrice, npr. za žita in krmo. Zasnovan je bil kot sestavni del analitskih metod na osnovi nukleinske kisline, zlasti v okviru standarda ISO 21569 o kvalitativnih analitskih metodah in standarda ISO 21570 o kvantitativnih analitskih metodah.
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in njihovih produktov - Izolacija nukleinskih kislin (ISO 21571:2005Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihren Produkten - Nukleinsäureextraktion (ISO 21571:2005)Produits alimentaires - Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés - Extraction des acides nucléiques (ISO 21571:2005)Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction (ISO 21571:2005)67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN ISO 21571:2005SIST EN ISO 21571:2005en01-junij-2005SIST EN ISO 21571:2005SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN ISO 21571February 2005ICS 67.050English versionFoodstuffs - Methods of analysis for the detection of geneticallymodified organisms and derived products - Nucleic acidextraction (ISO 21571:2005)Produits alimentaires - Méthodes d'analyse pour ladétection des organismes génétiquement modifiés et desproduits dérivés - Extraction des acides nucléiques (ISO21571:2005)Lebensmittel - Verfahren zum Nachweis von gentechnischmodifizierten Organismen und irhen Produkten -Nukleinsäureextraktion (ISO 21571:2005)This European Standard was approved by CEN on 14 February 2005.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2005 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 21571:2005: ESIST EN ISO 21571:2005
Foreword
This document (EN ISO 21571:2005) has been prepared by Technical Committee CEN/TC 275 "Food analysis - Horizontal methods", the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC 34 "Agricultural food products".
This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by August 2005, and conflicting national standards shall be withdrawn at the latest by August 2005.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
Reference numberISO 21571:2005(E)© ISO 2005
INTERNATIONAL STANDARD ISO21571First edition2005-02-15Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Nucleic acid extraction Produits alimentaires — Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés — Extraction des acides nucléiques
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ii © ISO 2005 – All rights reserved
ISO 21571:2005(E) © ISO 2005 – All rights reserved iii Contents Page Foreword.iv Introduction.v 1 Scope.1 2 Normative references.1 3 Principle.1 3.1 General.1 3.2 DNA extraction.2 3.3 DNA quantitation.2 4 General laboratory requirements.2 5 Procedure.2 5.1 Preparation of the test portion.2 5.2 DNA extraction/purification.4 5.3 Quantitation of the extracted DNA.5 5.4 Stability of extracted DNA.6 6 Interpretation.6 7 Test report.6 Annex A (informative)
Methods for DNA extraction.7 Annex B (informative)
Methods for the quantitation of the extracted DNA.34 Bibliography.41
ISO 21571:2005(E) iv © ISO 2005 – All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. ISO 21571 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with Technical Committee ISO/TC 34, Food products, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). SIST EN ISO 21571:2005
ISO 21571:2005(E) © ISO 2005 – All rights reserved v Introduction The search for genetically modified origin of ingredients is performed by means of the following successive (or simultaneous) steps. After sample collection, nucleic acids are extracted from the test portion. Extracted nucleic acids can be further purified, simultaneously or after the extraction process. Afterwards, they are quantified (if necessary), diluted (if necessary) and subjected to analytical procedures (such as PCR). These steps are detailed in this and the following International Standards: ISO 21568, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Sampling. ISO 21569, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Qualitative nucleic acid based methods. ISO 21570, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Quantitative nucleic acid based methods. Further information about definitions and general items involving the steps cited above are collected in: ISO 24276, Foodstuffs — Nucleic acid based methods of analysis for the detection of genetically modified organisms and derived products — General requirements and definitions. The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that compliance with this document may involve the use of a patent concerning the silica-based extraction method (No. EP 0389063/USP 5,234,809) given in Clause A.4. ISO takes no position concerning the evidence, validity and scope of this patent right. The holder of this patent right has assured the ISO that he/she is willing to negotiate licences under reasonable and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the statement of the holder of this patent right is registered with ISO. Information may be obtained from: Jean Deleforge, BioMérieux Chemin de l'Orme,
69280 Marcy-l'Étoile, France. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights.
INTERNATIONAL STANDARD ISO 21571:2005(E) © ISO 2005 – All rights reserved 1 Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Nucleic acid extraction 1 Scope This International Standard provides general requirements and specific methods for DNA extraction/purification and quantitation. These methods are described in Annexes A and B. This International Standard has been established for food matrices, but could also be applicable to other matrices, such as grains and feed. It has been designed as an integral part of nucleic-acid-based analytical methods, in particular ISO 21569 on qualitative analytical methods, and ISO 21570 on quantitative analytical methods. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 24276:—1), Foodstuffs — Nucleic acid based methods of analysis for the detection of genetically modified organisms and derived products — General requirements and definitions ISO 21568, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Sampling 3 Principle 3.1 General The objective of nucleic acid extraction methods is to provide nucleic acids suitable for subsequent analysis (see ISO 24276). NOTE The “quality” of DNA depends on the average length of the extracted DNA molecules, the chemical purity and the structural integrity of the DNA sequence and of the double helix (e.g. intra-, inter-strand linking between DNA bases, single-strand gaps, cross-linking with polyols, haemin, etc). Moreover, such alterations are often sequence-specific and consequently not randomly distributed all over the genome (see References [1], [2], [3] and [4]). Users of this International Standard should note that some methods (e.g. all silica-based methods), might be covered by patent rights.
1) To be published. SIST EN ISO 21571:2005
ISO 21571:2005(E) 2 © ISO 2005 – All rights reserved 3.2 DNA extraction The basic principle of DNA extraction consists of releasing the DNA present in the matrix and further, concurrently or subsequently, purifying the DNA from polymerase chain reaction (PCR) inhibitors. DNA extraction/purification methods are described in Annex A. Method-selection is an experience-based choice of the user, taking into account the scope and examples of matrices as given in each method. Alternative protocols are suitable provided that the method has been validated on the respective matrix under investigation. 3.3 DNA quantitation Quantitation of extracted DNA could be useful for subsequent PCR analysis. It may be performed by either physical (e.g. measure of absorbance at a specific wavelength), chemical-physical (e.g. use of intercalating or binding agents able to emit fluorescence), enzymatic (e.g. bioluminescence detection) methods or by quantitative PCR. The latter method is especially suitable for composite matrices or for samples with a low DNA content or whose DNA is degraded. There are several methods available to quantify the DNA present in a solution, as described in Annex B. It is for the user to choose the most appropriate one to be applied, depending on the amount and quality of DNA to be quantified and, consequently, on the matrix from which the DNA has been extracted. Alternative protocols are suitable, provided that the method has been validated on the respective matrix under investigation. 4 General laboratory requirements Accidental contamination of DNA can originate from dust and spreading aerosols. As a consequence, the organization of the work area in the laboratory is logically based on
=the systematic containment of the methodological steps involved in the production of the results, and = a “forward flow” principle for sample handling. The latter ensures that the DNA to be analysed and the amplified DNA generated by PCR remain physically segregated. Further details can be found in ISO 24276. 5 Procedure 5.1 Preparation of the test portion 5.1.1 General Commodity-specific variables (e.g. humidity) and processing can impact the amount and quality of DNA extracted from the material under investigation. Therefore the performance characteristics of a given DNA extraction method depend on the matrix. Take appropriate measures to ensure that the test portion is representative of the laboratory sample. The test portion shall be of sufficient size and shall contain a sufficient number of particles to be representative of the laboratory sample (e.g. 3 000 particles at an LOD of 0,1 %) to allow a statistically valid conclusion to be made (see ISO 21568). SIST EN ISO 21571:2005
ISO 21571:2005(E) © ISO 2005 – All rights reserved 3 For practical/technical reasons, it is not recommended to exceed a size of 2 g. Any restrictions that arise from the size of the test portion which prevent it from being representative shall be reported and taken into consideration in the interpretation of the analytical results. The methods for DNA extraction in Annex A describe test portions from 200 mg to 500 mg, which are usually adequate for DNA-rich raw materials (e.g. ground grains, flour). However, for certain matrices containing very low amounts or degraded DNA, insufficient DNA suitable for analysis can be extracted. In these cases, the test portion may be increased. DNA extractions shall be carried out at least on two test portions. Storage of standards, samples and test portions shall comply with ISO 24276 and shall be organized in such a way as to preserve the biochemical parameters to be analysed (for details, see ISO/IEC 17025). 5.1.2 Samples All operations for the preparation of test samples (e.g. grinding, homogenization, division, drying) shall be carried out in accordance with the procedures described in ISO 24276, taking care to prevent all contamination of the sample or modification of its composition. Laboratory samples shall be sufficiently homogeneous before reducing the laboratory sample and taking the test portion. For liquid samples, shake the vessel containing the sample to improve the homogenization of the product. In the case of non-homogenous products like raw oils, check that the sediments have been completely removed from the walls of the vessel. For solid matrices that cannot easily be suspended, the matrix shall be ground to reduce the particle size and/or facilitate the extractability of DNA. In such a case, attention shall be paid to the particle size. The test portion subjected to extraction shall contain a minimum number of particles as specified in ISO 21568. Milling/grinding devices should be capable of being thoroughly cleaned and shall be selected in order to achieve the expected particle number and particle size distribution within the test portion as defined in ISO 21568. If components of the laboratory sample have been removed prior to extraction, then such procedures shall be reported. Final food products that are solid or paste and have high lipid contents are often not easy to grind to the desired particle size in a single step. Several procedures may therefore be added, such as lipid removal using hexane after intermediate grinding, freezing or freeze-drying before grinding. In order to facilitate the grinding of paste or viscous products, it is possible to apply one of the following treatments to certain matrices: =heating to a maximum temperature of 40 °C; =dissolving in an appropriate liquid such as water; =freezing at a temperature below or equal to −20 °C. Homogenize the whole laboratory sample. Sample the two test portions, taking into account possible dilutions or concentrations. During milling/grinding, precautions should be taken to ensure that the heating of the sample is kept to a minimum since heating can have a negative impact on the quality of the extracted DNA. Milling/grinding techniques with a high risk of cross-contamination (such as the combined use of liquid nitrogen and mortar) shall be avoided as far as possible. As a rule of good practice, any dust-producing methodological step should be contained from all other analytical steps. SIST EN ISO 21571:2005
ISO 21571:2005(E) 4 © ISO 2005 – All rights reserved If salts, spices, powdered sugars and/or other substances that could potentially interfere with the extraction or analytical method are present, appropriate purification steps should be considered according to the selected method (see Annex A). For example, in samples from composite matrices, the target matrix (e.g. the breading layer of fish sticks) can be isolated for DNA extraction. 5.2 DNA extraction/purification 5.2.1 General The following considerations apply for the design of extraction methods. The quality and yield of nucleic acid extracted using a given method on a given matrix should be both repeatable and reproducible in terms of analysis, provided sufficient nucleic acid is present in the matrix from which it has been extracted. In order to obtain a good quality DNA, it is advisable, where relevant, to remove the following: =polysaccharides (pectin, cellulose, hemi-cellulose, starch, thickeners, etc.) using appropriate enzyme treatments (e.g. pectinase, cellulase, hemi-cellulase, α-amylase) or organic extraction (e.g. CTAB/chloroform); =RNA and/or proteins using an appropriate treatment, such as enzymatic treatment by RNase and proteinase, respectively; =the lipid fractions using for example enzyme treatments, or solvents (e.g. n-hexane); =salts (e.g. from the extraction/lysis buffer, from the precipitation step) able to interfere with the subsequent analysis. In particular for solid or dried samples, the volume of lysis/extraction buffer should be adapted to guarantee the DNA is dissolved. NOTE 1 DNA purification can be performed by different means such as fractionated precipitation, using solvents like phenol, chloroform, ethanol, isopropanol, and/or by adsorption on solid matrices (anion exchange resin, silica or glass gel, diatomaceous earth, membranes, etc.). Several DNA purification principles may be combined. If appropriate, extraction and purification can be performed within the same step. Should a DNA co-precipitant such as glycogen, PEG or t-RNA be used to improve the DNA recovery during the precipitation steps, it should neither contain any detectable level of nuclease activity or PCR inhibitors/competitors, nor bear any sequence similarity with the potential PCR target under study. For genetically modified plants, a carrier DNA may be used (e.g. salmon or herring sperm DNA). When using vacuum freeze dryers to dry the DNA pellets obtained after a precipitation step, the risk of cross contamination should be taken into account. Re-suspend the DNA in water or in a buffer solution that prevents DNA from degradation. When setting up a new type of DNA extraction, or when applying one of the methods described in Annex A to a new matrix, the potential quality and integrity of the extracted DNA using the chosen protocol should be estimated by the following approach. A known quantity of a tracer DNA is added to the lysis buffer plus sample used for DNA extraction. When the chosen tracer is a predetermined amount of DNA or represents a predetermined number of copies of a particular DNA-sequence mixed to a matrix at start of DNA extraction, attention shall be paid to ascertain the lack of DNA sequence similarity between the tracer DNA and the target DNA sequence under study. NOTE 2 The use of a tracer DNA is a good approximation to a real situation where DNA of a given matrix, complexed to other components (e.g. proteins) is expected. Such a method may also be used to estimate the presence of soluble and trans-acting PCR inhibitors in the extracted DNA (see ISO 24276, ISO 21569 and ISO 21570). SIST EN ISO 21571:2005
ISO 21571:2005(E) © ISO 2005 – All rights reserved 5 However, tracer DNA may give a misleading impression of recovery, since tracer DNA may be much easier to separate from matrix than the target DNA. 5.2.2 Controls The controls to be included are described in Table 1 of ISO 24276:—. These should as a minimum include an extraction blank control and a positive extraction control, but may also include an environment control. 5.2.3 Control of DNA purity: Internal PCR control When setting up a new type of extraction, the presence of PCR inhibitors in the extracted DNA may be estimated using DNA spikes (see ISO 24276, ISO 21569
and ISO 21570). The amount of added DNA shall not exceed the maximum level supported by PCR and shall contain a definite number of target sequence copies. This number should be determined individually for each target sequence and indicated as a multiple of the existing lower limit of detection. Ideally, the target concentration of the positive control PCR should correspond to the sensitivity needed in the analysis. Care shall be taken when using highly concentrated cloned target DNA. As far as possible, the positive controls shall conform to the conditions of the test material with regard to the nucleic acids they contain. 5.3 Quantitation of the extracted DNA 5.3.1 General The quality, integrity and amount of the nucleic acid template influences the performance of the analytical method, and hence the analytical results obtained. The limit of detection of a specific method may therefore depend on the amount of nucleic acids used. Quantitation of DNA is helpful =to compare the efficiency of different DNA extraction protocols for a given matrix (repeatability), and =to measure the concentration of nucleic acids prior to analysis. 5.3.2 Range of application Each method of quantitation shall be applied within its dynamic range, also considering its level of precision. 5.3.3 Quantity standards The accuracy of the quantitation methods depends on the nucleic acid standards used to calibrate the method. If using a method that is sensitive to the size and/or quality of the nucleic acid fragments, then the nucleic acid standards that match the size and/or quality of the expected nucleic acid as extracted from the sample shall be used. The reference material used should ensure traceability to stated references, usually national or international Standards, through an unbroken chain of comparison [see ISO Guide 30]. When a method using intercalating agents is employed, high molecular mass DNA standard should be used when high molecular mass DNA is to be quantified. Low molecular mass DNA should be used when low molecular mass DNA is to be quantified. High molecular mass nucleic acid usually also contains a certain amount of lower molecular mass fragments. This means that many methods for DNA quantitation suffer from a certain degree of inaccuracy, which should be taken into account. NOTE Additionally, depending on the matrix and type of extraction method, a certain portion of the extracted DNA may be recovered as single-stranded DNA (with much poorer intercalation capacity), leading to an underestimation of the overall DNA content. In contrast, single-stranded DNA is equally well detected by physical measurements. SIST EN ISO 21571:2005
ISO 21571:2005(E) 6 © ISO 2005 – All rights reserved At least three points (preferably replicated) are required for the construction of a good calibration curve. The amount of standard DNA used for each calibration point depends on the sensitivity of the method and on the dynamic range under consideration. 5.4 Stability of extracted DNA The DNA extracted shall be stored under such conditions that the stability is ensured to perform the subsequent analyses. Repeated freezing and thawing of DNA solutions should be avoided. 6 Interpretation The DNA extraction method employed shall be appropriate to obtain the quality and quantity of nucleic acid required for the subsequent analysis. The quality of the extracted nucleic acid should be assessed using an analytical method that is influenced by the same quality parameters as the analysis to be performed on the extracted nucleic acid (e.g. if the analysis to be performed is PCR, then an additional PCR should be used for the assessment of the quality of the extracted DNA). Further parameters for method compatibility can be found in ISO 21569, ISO 21570 and ISO 24276. 7 Test report When issuing the final test report in accordance with ISO 24276, the following additional information to document the activity of the laboratory shall be included: =a statement describing the derivation of the test portions, and any preliminary processing of the sample before nucleic acid extraction; =the size of the test portions used for the nucleic acid extraction; =the nucleic acid extraction method used; =any special observations made during testing; =any operation not specified in the method or considered to be optional but that can have an effect on the results; =the interpretation of the results; =the experimentor's name. Handling and storage of raw data are described in ISO/IEC 17025 and related quality assurance schemes. Consistency should be achieved. SIST EN ISO 21571:2005
ISO 21571:2005(E) © ISO 2005 – All rights reserved 7 Annex A (informative)
Methods for DNA extraction A.1 Preparation of PCR-quality DNA using phenol/chloroform-based DNA extraction methods A.1.1 Basic phenol/chloroform method A.1.1.1 General This routine method (see Reference [5]) is suitable for the extraction of DNA from a wide variety of matrices (see A.1.1.8). Phenol is usually very suitable for nuclease destruction and protein denaturation. When foliar or green material (e.g. chicory leaves, dried alfalfa) is investigated, many PCR inhibitors may also be co-precipitated together with DNA. For this reason, difficulties may be encountered in obtaining PCR-amplifiable DNA reproducibly. The corrosive hazardous properties of phenol must be taken into consideration, thus the use of DNA extraction methods based on CTAB and/or PVP and/or silica adsorption are favoured as primary alternatives. A.1.1.2 Validation status The method has been widely applied in all areas of biology, agronomy and medicine, over the past 40 years, but has never been evaluated through interlaboratory studies for GMO detection in foodstuffs. A.1.1.3 Principle The method consists of a lysis step (thermal lysis in presence of sodium dodecyl sulfate and a high EDTA content) followed by the removal of contaminants (such as lipophylic molecules, polysaccharides and proteins) and nucleases from the DNA-containing aqueous phase using phenol and chloroform. A final DNA precipitation with ethanol concentrates the DNA and eliminates salts and residual chloroform. The critical step of the method is the lysis step [5]. A.1.1.4 Safety precautions A fume hood is necessary for handling organic chemicals. A.1.1.5 Reagents A.1.1.5.1 Ethanol, volume fraction φ (C2H5OH) = 96 % Store and use at −20 °C. SIST EN ISO 21571:2005
ISO 21571:2005(E) 8 © ISO 2005 – All rights reserved A.1.1.5.2 Glacial acetic acid (CH3COOH). A.1.1.5.3 Potassium acetate (C2H3O2K). A.1.1.5.4 Hydrochloric acid, φ (HCl) = 37 %. A.1.1.5.5 Isoamyl alcohol [(CH3)2CHCH2CH2OH]. A.1.1.5.6 Phenol (C6H5OH). A.1.1.5.7 Chloroform (CHCl3). A.1.1.5.8 Tris(hydroxymethyl)-aminomethane (Tris) (C4H11NO3). A.1.1.5.9 Ethylenediaminetetraacetic acid dipotassium salt (K2EDTA) (C10H14N2O8K2). A.1.1.5.10 Potassium hydroxide (KOH). A.1.1.5.11 Potassium chloride (KCl). A.1.1.5.12 Sodium dodecyl sulfate (SDS) (C12H25O4SNa). A.1.1.5.13 Proteinase K, approximately 20 Units/mg lyophilisate. A.1.1.5.14 RNase-A, DNase-free, from bovine pancreas, approximately 50 Kunitz Units/mg of lyophilisate. A.1.1.5.15 Equilibrated phenol, pH [ 7,8. Use phenol equilibrated against extraction buffer (A.1.1.5.18) without SDS, or prepared according to Reference [5], or according to the manufacturers recommendations. A.1.1.5.16 Chloroform-isoamyl alcohol Mix 24 volume parts of chloroform (A.1.1.5.7) with 1 volume part of isoamyl alcohol (A.1.1.5.5). A.1.1.5.17 Phenol-chloroform-isoamyl alcohol Mix 1 volume part of equilibrated phenol (A.1.1.5.15) with 1 volume part of the chloroform-isoamyl alcohol solution (A.1.1.5.16). A.1.1.5.18 Extraction/lysis buffer, substance concentration c(Tris) = 0,050 mol/l, c(K2EDTA) = 0,050 mol/l, mass concentration ρ(SDS) = 30 g/l. Adjust the pH to 8,0 with HCl or KOH. A.1.1.5.19 TE buffer, c(Tris) = 0,010 mol/l, c(K2EDTA) = 0,001 mol/l. Adjust the pH to 8,0 with HCl or KOH. A.1.1.5.20 Proteinase-K solution, ρ = 20 mg/ml, dissolved in sterile water. Do not autoclave. Store at −20 °C, but avoid repeated freezing and thawing. SIST EN ISO 21571:2005
ISO 21571:2005(E) © ISO 2005 – All rights reserved 9 A.1.1.5.21 RNase-A solution, ρ = 10 mg/ml lyophylisate. Store at −20 °C, but avoid repeated freezing and thawing. A.1.1.5.22 Ethanol solution, φ (C2H5OH) = 70 %. Store and use at −20 °C. A.1.1.5.23 Potassium acetate solution, c(C2H3O2K) = 3 mol/l. Adjust the pH to 5,2 with glacial acetic acid. Do not autoclave. If necessary, filter through a 0,22 µm filter. A.1.1.6 Apparatus and equipment Usual laboratory equipment and, in particular, the following. A.1.1.6.1 Centrifuge, capable of achieving a minimum acceleration of 10 000 g. In some steps a refrigerated centrifuge is required. A.1.1.6.2 Water bath or incubator, working in a temperature range from 60 °C to 70 °C. A.1.1.6.3 Vacuum dryer (optional). A.1.1.6.4 Freeze dryer (optional). A.1.1.6.5 Mixer, e.g. Vortex2) A.1.1.6.6 Reaction vessels, resistant to freezing in liquid nitrogen. A.1.1.7 Procedure A.1.1.7.1 General Once the matrix test portion has been prepared, apply the following DNA extraction/purification protocol. Scale-adaptation of masses and buffer volumes is required as a function of the selected size of the test portion. A.1.1.7.2 Extraction procedure Weigh 0,25 g of the test sample into a microtube. Add 1,6 ml of extraction buffer (A.1.1.5.18) and, when necessary (e.g. in protein-rich matrices), 50 µl of proteinase K solution (A.1.1.5.20.) Incubate at 60 °C to 70 °C, usually for between 30 min to 2 h (overnight incubation is also possible). Add RNase A (A.1.1.5.21) up to a final concentration of 0,1 µg/ml. Centrifuge at 5 000 g for 30 min and recover the supernatant in a fresh tube. Add 1 volume of equilibrated phenol (A.1.1.5.15) to the supernatant, then mix gently and thoroughly. Centrifuge at 5 000 g for 15 min and recover the upper aqueous phase in a fresh tube. Add 1 volume of phenol-chloroform isoamyl alcohol (A.1.1.5.17) to the supernatant, then mix gently and thoroughly. Centrifuge at 5 000 g for 15 min and recover the aqueous phase in a fresh tube. Repeat this step once or more times (depending on the matrix) until the interface between the phases is clean.
2) Vortex is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN ISO 21571:2005
ISO 21571:2005(E) 10 © ISO 2005 – All rights reserved Add 1 volume of chloroform/isoamyl alcohol (A.1.1.5.16) to the supernatant, then mix gently and thoroughly. Centrifuge at 5 000 g for 10 min and recover the upper aqueous phase in a fresh tube. Repeat, if necessary, until the interface between the phases is clear. Mix the supernatant with 0,1 volume of potassium acetate solution (A.1.1.5.23.) and 2,5 volumes of 96 % ethanol (A.1.1.5.1), then mix thoroughly by inversion. Incubate for at least 5 min in liquid nitrogen, or 1 h at −80 °C, or overnight at −20 °C. Centrifuge at 10 000 g (or up to 13 000 g) at 4 °C for at least 15 min, then carefully discard the supernatant.
Carefully wash the DNA pellet with 2 volumes of 70 % ethanol solution (A.1.1.5.22). Centrifuge at 10 000 g to 13 000 g at 4 °C for 15 min, then discard carefully the supernatant. This step is essential for the removal of the precipitating salts that could interfere with the subsequent analysis (e.g. PCR). Dry the pellet and re-dissolve it in 100 µl of water or appropriate buffer, e.g. TE buffer (A.1.1.5.19). This is the DNA master stock. Add RNase-A (A.1.1.5.21) up to a final concentration of 0,1 µg/ml. A.1.1.8 List of examples The method has been successfully applied to extract DNA3) from the following matrices: acidified soya beans3), dehydrated alfalfa, baby biscuits3), baby milk3), bacteria and spores thereof, barley seeds, beef/pork paté3), beer3), blue cheese, brownies3), canned corn, carrot seeds, cereal bars3), cheese, chicken nuggets, chicory leaves, chicory roots, chocolate cookies3), chocolate paste3), cinnamon cookies3), compotes, cornflakes3), cracked rice, dessert cream3), dried pea seeds, maize biscuits3), maize feeding oil cakes, maize flour, maize gluten feed, maize seeds, manioc hard pellets for feed, manioc tapioca meat, fresh and cooked3) meats (beef, pork, chicken and turkey), melon fruit pulp, melon seeds, minced meat, muesli ingredients3), muesli3), mung bean sprouts3), oat seeds, potato tuber, rapeseed feeding oil cake, glupacolza, rapeseed seeds, sausages (slicing3) and cocktail3) (see A.1.2 for an improved extraction method), schnitzel, sooie hoi sin3), soup balls, soya protein in meat preparations3), soya lecithin (raw brown and yellow refined3)), soya sprouts3), soya drinks, soya beans, soya bean cream, soya bean feeding oil cake, soya bean tofu, spaghetti sauce3), spelt seeds, sugar beet (dried pulp), sugar beet (fresh root), sugar beet seeds, sunflower seeds, tofu, tomato fresh fruit, tomato purée3), tomato seeds, vegetarian hamburger, waffles (with3) and without3) chocolate), wheat bran, wheat flour, wheat gluten feed, wheat seeds, wheat semolina, yoghurt3) (see A.1.3 for an improved extraction method). A.1.2 Phenol/chloroform method: Protocol for starter cultures of fermented sausages A.1.2.1 General This method is designed to isolate the total DNA, including bacterial genomic DNA, from sausages. The applicability of the method to obtain DNA of high quality suitable for the specific detection of recombinant DNA by PCR has been demonstrated for fermented sausages[6] as well as for thermally treated fermented sausages, so-called summer sausages[7]. In addition, the extraction method was shown to be suitable to isolate total DNA from cream to detect specifically Staphylococcus aureus in this food matrix[8]. (For a summary of the matrices for which the method is suitable, see A.1.2.8.) A.1.2.2 Validation status This method has been validated by an interlaboratory study on a test portion of 0,4 g (see A.1.2.9).
3) Repeatability may depend on the batch of the matrix and/or to its production technology. In some cases, DNA could not be found or was degraded in such a way that PCR results were below the limit of detection of the method, irrespective of the PCR primers/protocols used. This may be a source of low reproducibility between laboratories. SIST EN ISO 21571:2005
ISO 21571:2005(E) © ISO 2005 – All rights reserved 11 A.1.2.3 Principle The method consists of the recovery of bacterial cells from the food matrix by homogenization of the sausage samples, followed by a centrifugation step. The sediment contains not only bacterial cells but also meat particles. For specific lysis of the bacterial cells, the cell walls are degraded by addition of lysozyme. To improve the degradation of cell walls of meat lactobacilli, mutanolysin can be added. Complete lysis of the cells is performed by addition of the detergent SDS (sodium dodecyl sulfate) and proteinase-K, followed by several extractions of the aqueous phase with phenol and/or chloroform. The phenol/chloroform extraction step is important to eliminate any nuclease activities and PCR-inhibiting substances including those arising from the food matrix (e.g. haematin). A final precipitation of DNA by ethanol is performed. A.1.2.4 Safety precautions A fume hood is necessary for handling organic chemicals. A.1.2.5 Reagents A.1.2.5.1 Isopropanol [CH3CH(OH)CH3]. A.1.2.5.2 Ethanol, ϕ(C2H5OH) = 96 %. Store and use at −20 °C. A.1.2.5.3 Glacial acetic acid (CH3COOH). A.1.2.5.4 Hydrochloric acid, =φ(HCl) = 37 %. A.1.2.5.5 Sodium hydroxide (NaOH). A.1.2.5.6 Isoamyl alcohol [(CH3)2CHCH2CH2OH)]. A.1.2.5.7 Phenol (C6H5OH). A.1.2.5.8 Chloroform (CHCl3). A.1.2.5.9 Tris(hydroxymethyl)-aminomethane (Tris) (C4H11NO3). A.1.2.5.10 Ethylenediaminetetraacetic acid disodium salt (Na2EDTA) (C10H14N2O8Na2). A.1.2.5.11 Sodium dodecyl sulfate (SDS) (C12H25O4SNa). A.1.2.5.12 Lysozyme 50 000 U/mg protein (1 U will produce a ∆A450 of 0,001 per minute at pH 6,24 and 25 °C, using a suspension of Micrococcus lysodeikticus as substrate, in a 2,6 ml reaction mixture using a 1 cm light path). A.1.2.5.13 Sucrose (C12H22O11). A.1.2.5.14 Proteinase-K, approximately 20 Units/mg lyophilisate. A.1.2.5.15 Sodium acetate (C2H3O2Na). A.1.2.5.16 Equilibrated phenol, saturated with Tris/HCl (pH [ 7,8) buffer, or prepared according to Reference [5], or according to the manufacturer's recommendations. SIST EN ISO 21571:2005
ISO 21571:2005(E) 12 © ISO 2005 – All rights reserved A.1.2.5.17 Chloroform-isoamyl alcohol Mix 24 volume parts of chloroform (A.1.2.5.8) with 1 volume part of isoamyl alcohol (A.1.2.5.6). A.1.2.5.18 Phenol-chloroform-isoamyl alcohol Prepare by mixing 25 volume parts of equilibrated phenol (A.1.2.5.16) with 24 volume parts of chloroform (A.1.2.5.8) and 1 volume part of isoamyl alcohol (A.1.2.5.6). A.1.2.5.19 Mutanolysin-solution, containing 500 U/ml or 5 000 U/ml mutanolysin, dissolved in sterile water. Do not autoclave. Store at –20 °C, but avoid repeated freezing and thawing.
A.1.2.5.20 Lysozyme solution, containing 10 mg/ml, dissolved in sterile water. Do not autoclave. Store at –20 °C, but avoid freezing and thawing.
A.1.2.5.21 Sucrose solution, ρ(C12H22O11) = 400 g/l. A.1.2.5.22 Buffer solution A, c(Tris) = 0,020 mol/l, c(Na2EDTA) = 0,020 mol/l, c(NaCl) = 0,1 mol/l. Adjust the pH to 8,0 with HCl or NaOH. A.1.2.5.23 Extraction/lysis buffer, containing one volume part of buffer solution A (A.1.2.5.22) and one volume part of sucrose solution (A.1.2.5.21). A.1.2.5.24 SDS solution, ρ(SDS) = 250 g/l. A.1.2.5.25 Proteinase-K solution, containing 20 mg/ml, stored at −20 °C, dissolved in sterile water. Do not autoclave. Store at – 20 °C, but avoid repeated freezing and thawing.
A.1.2.5.26 Ethanol solution, φ(C2H5OH) = 70 %. Store and use at −20 °C. A.1.2.5.27 Sodium acetate solution, c(C2H3O2Na) = 3 mol/l. Adjust the pH to 5,2 with glacial acetic acid. A.1.2.5.28 TE buffer, c(Tris) = 0,010 mol/l, c(Na2EDTA) = 0,001 mol/l. Adjust the pH to 8,0 with HCl or NaOH. A.1.2.6 Apparatus and equipment Usual laboratory equipment and, in particular, the following. A.1.2.6.1 Instrument and/or material for chopping tissue (e.g. scalpel). A.1.2.6.2 Centrifuge, capable of achieving an acceleration of 12 000 g. In some steps a refrigerated centrifuge is required. SIST EN ISO 21571:2005
ISO 21571:2005(E) © ISO 2005 – All rights reserved 13 A.1.2.6.3 Water bath or incubator. A.1.2.6.4 Vacuum dryer (optional). A.1.2.6.5 Mixer, e.g. Vortex2). A.1.2.7 Procedure A.1.2.7.1 General Once the matrix test portion has been prepared, apply the following DNA extraction/purification protocol. Scale-adaptation of masses and buffer volumes is required as a function of the selected size of the test portion. A.1.2.7.2 Sample preparation Chop the meat sausage, homogenize it and add 200 mg to 500 mg to 3 volumes of water (up to 1,5 ml). Keep at room temperature for about 10 min. A.1.2.7.3 Extraction procedure Carefully transfer 500 µl of the aqueous phase (suspension) to a new reaction vessel. Centrifuge for 10 min at 12 000 g. Discard the supernatant and re-suspend the pellet in 500 µl of extraction/lysis buffer (A.1.2.5.23). Add 50 µl of lysozyme solution (A.1.2.5.20). Incubate at 37 °C for 1 h. If the results are not satisfactory, lysozyme may be combined with 10 U of mutanolysin (A.1.2.5.19), but the matrix-specific effect of this should be tested prior to routine application. Add 25 µl of SDS solution (A.1.2.5.24) and 25 µl of proteinase-K solution (A.1.2.5.25), then incubate for 10 min at 60 °C. Add 1 volume of phenol/chloroform/isoamyl alcohol (A.1.2.5.18) and mix. Centrifuge the mixture for 3 min at about 12 000 g. Transfer the u
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