1、DD CEN/TS15790:2008ICS 07.100.30; 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWDRAFT FOR DEVELOPMENTAnimal feeding stuffs PCR typing ofprobiotic strainsof Saccharomycescerevisiae (yeast)This Draft for Developmentwas published under theauthority of the StandardsPolicy a
2、nd StrategyCommittee on 31 January2009 BSI 2009ISBN 978 0 580 61806 2Amendments/corrigenda issued since publicationDate CommentsDD CEN/TS 15790:2008National forewordThis Draft for Development is the UK implementation of CEN/TS15790:2008.This publication is not to be regarded as a British Standard.It
3、 is being issued in the Draft for Development series of publications andis of a provisional nature. It should be applied on this provisional basis,so that information and experience of its practical application can beobtained.Comments arising from the use of this Draft for Development arerequested s
4、o that UK experience can be reported to the internationalorganization responsible for its conversion to an international standard.A review of this publication will be initiated not later than 3 years afterits publication by the international organization so that a decision can betaken on its status.
5、 Notification of the start of the review period will bemade in an announcement in the appropriate issue of Update Standards.According to the replies received by the end of the review period,the responsible BSI Committee will decide whether to support theconversion into an international Standard, to
6、extend the life of theTechnical Specification or to withdraw it. Comments should be sent tothe Secretary of the responsible BSI Technical Committee at BritishStandards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Anim
7、al feeding stuffs.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confe
8、r immunityfrom legal obligations.DD CEN/TS 15790:2008TECHNICAL SPECIFICATIONSPCIFICATION TECHNIQUETECHNISCHE SPEZIFIKATIONCEN/TS 15790December 2008ICS 07.100.30; 65.120English VersionAnimal feeding stuffs - PCR typing of probiotic strains ofSaccharomyces cerevisiae (yeast)Aliments des animaux - Typa
9、ge ACP des souchesprobiotiques de Saccharomyces cerevisiae (levure)Futtermittel - PCR-Typisierung der probiotischen Stmmevon Saccharomyces cerevisiae (Hefe)This Technical Specification (CEN/TS) was approved by CEN on 25 August 2008 for provisional application.The period of validity of this CEN/TS is
10、 limited initially to three years. After two years the members of CEN will be requested to submit theircomments, particularly on the question whether the CEN/TS can be converted into a European Standard.CEN members are required to announce the existence of this CEN/TS in the same way as for an EN an
11、d to make the CEN/TS availablepromptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)until the final decision about the possible conversion of the CEN/TS into an EN is reached.CEN members are the national standa
12、rds bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdo
13、m.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. CEN/TS 15790:2008: EDD CEN/T
14、S 15790:2008CEN/TS 15790:2008 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Principle .53 Reagents 53.1 PCR .53.1.1 Primers .53.1.2 dNTP mix 53.1.3 Buffer 63.1.4 Magnesium Chloride solution 63.1.5 DNA Taq polymerase 63.2 Gel Electrophoresis.63.2.1 Agarose, molecular-grade agarose free from
15、 DNase and RNase contamination. 63.2.2 Molecular weight marker 63.2.3 Tris-Borate-EDTA buffer .63.2.4 Loading dye .63.2.5 Water .63.2.6 Ethidium bromide 64 Apparatus .64.1 PCR .64.1.1 PCR Tubes .64.1.2 Pipets and sterile tips .74.1.3 Thermal cycler .74.2 Gel Electrophoreses .74.2.1 Horizontal gel el
16、ectrophoresis system .74.2.2 Microwave oven .74.2.3 Conical flask 74.2.4 Balance .74.2.5 Transilluminator 74.2.6 Image analysis system or Polaroid camera 75 Procedure .75.1 PCR reaction 75.2 Thermal Cycle 85.3 Agarose gel electrophoresis 86 Analysis of the results 9Annex A (informative) Example Gel
17、- PCR of probiotic strains of Saccharomyces cerevisiae . 10Annex B (informative) Validation data from the European Collaborative trial 45 . 11Bibliography . 12DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 3 Foreword This document (CEN/TS 15790:2008) has been prepared by Technical Committee CEN/TC 327 “A
18、nimal feeding stuffs Methods of sampling and analysis”, the secretariat of which is held by NEN. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such pate
19、nt rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association, and supports essential requirements of EU Directive(s). According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
20、 countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slo
21、venia, Spain, Sweden, Switzerland and United Kingdom. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 4 Introduction This methodology is based on specific polymerase chain reaction (PCR) amplification of a genetic sequence for the detection of Saccharomyces cerevisiae isolated from animal feed or animal f
22、eed probiotic supplement. The aim of this method is to identify authorised probiotic yeast strains. Molecular typing methods and especially PCR amplification based methods used to characterise the yeast strains require high quality high molecular weight genomic DNA. The method of DNA extraction from
23、 the yeast must facilitate these requirements. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 5 1 Scope This Technical Specification defines a polymerase chain reaction (PCR) methodology for the identification of S. cerevisiae probiotic yeast strains. Additionally, a method for the extraction of high qua
24、lity DNA from yeast is suggested. 2 Principle This method is based upon the amplification of elements which are present in the yeast genome. Two primers are used for the PCR reaction, which are a modification of Ness et al. 1. Distinct patterns are produced for probiotic S. cerevisiae strains when s
25、eparated in agarose gels by electrophoresis. Patterns are visualised under UV light after electrophoresis and ethidium bromide staining of the agarose gel. The PCR analysis of individual yeast colonies isolated from agar plates involved the following steps: 1. DNA extraction and purification; 2. PCR
26、 reaction; 3. Gel electrophoresis; 4. Analysis of results. Individual and typical colonies can be obtained following growth on appropriate agar media whereby the standard enumeration procedure is recommended that uses yeast extract dextrose chloramphenicol agar (CGYE) 1. Typical colonies are picked
27、from agar plates to inoculate 10 ml malt extract broth which is cultured overnight at 30 C in a shaking incubator e.g. an orbital incubator revolving at 100 rpm, or equivalent. The cells are subsequently harvested and DNA is extracted following the instructions from manufacturers when using kits or
28、other appropriate procedures. The DNA extraction procedure is a sequential process of outer cell wall removal, lysis of nuclei, protein precipitation and removal, followed by precipitation of the nucleic acid. An extraction procedure is described e.g. by Hoffman and Winston 2. 3 Reagents 3.1 PCR 3.1
29、.1 Primers The following primer sequences are used. Delta 1 modified primer: 5 CAA ATT CAC CTA TTT CTC A 3 Delta 2 Primer 5 GTG GAT TTT TAT TCC AAC A 3 Stock solutions of each primer are made by diluting in sterile water (3.2.5) to a final concentration of 50 M and stored at least - 20 C. 3.1.2 dNTP
30、 mix A 2 mM equimolar stock solution of dATP, dTTP, dGTP, dCTP is made from a dNTP mix set and stored at least - 20 C. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 6 3.1.3 Buffer A commercial 10x stock solution of reaction buffer is used. The composition is 100 mM Tris-HCl (pH 9,0), 500 mM KCl, 1 % (v/
31、v) Triton X-100. The buffer is stored at least - 20 C. 3.1.4 Magnesium Chloride solution A commercial 10x stock solution is used and stored at least - 20 C. The composition is 25 mM magnesium chloride (MgCl2). 3.1.5 DNA Taq polymerase DNA Taq Polymerase with a concentration of 5 U/l is used and stor
32、ed at least - 20 C. Alternative appropriate enzyme preparation may be applicable. 3.2 Gel Electrophoresis 3.2.1 Agarose, molecular-grade agarose free from DNase and RNase contamination. 3.2.2 Molecular weight marker A 100 bp ladder is recommended. 3.2.3 Tris-Borate-EDTA buffer Commercially produced
33、10x TBE diluted with distilled water to 1x (e.g. from Sigma). TBE (1x) is composed of 89 mM Tris Borate-EDTA-buffer, pH 8,3, containing 2 mM EDTA. 3.2.4 Loading dye A commercially produced 6x loading dye is used. This is composed of 0,4 % orange G, 0,03 % bromophenol blue, 0,03 % xylene cyanol FF, 1
34、5 % Ficoll 400, 10 mM tris-HCl (pH 7,5) and 50 mM EDTA (pH 8,0). Appropriate alternative standard loading dyes may be equally applicable. 3.2.5 Water DNase and RNase free, 18 Ohms, 0,2 m filtered. 3.2.6 Ethidium bromide A commercially produced 10 mg/ml solution is used. 4 Apparatus Usual equipment a
35、ppropriate for a molecular laboratory and, in particular the following. 4.1 PCR 4.1.1 PCR Tubes Thin walled DNAse free microtubes appropriate for the thermal cycler that is used. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 7 4.1.2 Pipets and sterile tips Capable of dispensing between 0,5 l and 200 l.
36、4.1.3 Thermal cycler An appropriate and reliable thermal cycler which is technically maintained and calibrated. 4.2 Gel Electrophoreses 4.2.1 Horizontal gel electrophoresis system Including cell and power supply capable of operating at a constant voltage. 4.2.2 Microwave oven 4.2.3 Conical flask 250
37、 ml size for 80 ml to 100 ml of agarose preparation. 4.2.4 Balance Capable of weighing to the nearest 0,01 g. 4.2.5 Transilluminator Most appropriate apparatus to visualise the banding patterns in the agarose gel following ethidium bromide staining. 4.2.6 Image analysis system or Polaroid camera App
38、ropriate system to record and analyse results. 5 Procedure 5.1 PCR reaction This method has been slightly altered to incorporate modification of the Delta 1 primer 1. Delta 1 modified primer: 5 CAA ATT CAC CTA TTT CTC A 3 Delta 2 Primer 5 GTG GAT TTT TAT TCC AAC A 3 For each PCR analysis a premixtur
39、e containing all the reagents except template DNA is prepared according to Table 1. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 8 Table 1 PCR reaction mix Stock Reaction Volume l/tube required Buffer 10x 1x 5 dNTPs 2 mM 200 M 5 MgCl225 mM 1,5 mM 3 Delta 1 modified primer 50 M 1 M 1 Delta 2 primer 50 M
40、 1 M 1 Taq polymerase 5 U/l 2,5 U/l 0,5 H2O (to 48 l) 32,5 The reaction mix is dispensed in 48 l aliquots into the PCR tubes and kept on ice until the template has been added and the tubes are ready to load onto the thermal cycler. Add 2 l of DNA template to final reaction mix (or 2 l of water (3.2.
41、5) for negative control). 5.2 Thermal Cycle This step of the procedure is crucial and therefore particular care needs to be taken to ensure that the thermal cycler that is used is reliable with regards to its ability to reproducibly meet the described time temperature cycles. It is recommended to en
42、sure that this is the case by appropriate standardisation procedures applicable for the instrument. 95 C - 2 min cycles4min 2 - C 72s 30 - C 45s 30 - C 95cycles30min 2- C 72s 30- C 45s 30- C 9572 C - 10 min 4 C 8 min 5.3 Agarose gel electrophoresis A 2 % (w/v) agarose gel is made with Molecular Biol
43、ogy Grade agarose and 1x TBE. Ethidium bromide is added to a final concentration of 0,1 g/ml. The running buffer is 1x TBE with a final concentration of 0,5 g/ml ethidium bromide. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 9 10 l of loading dye is added to the reaction and mixed. 20 l of the reaction
44、 and dye is loaded into a well for each reaction. The molecular weight marker used is a 100 bp ladder. The product is run at a Voltage of 150 V with a running time between 45 min. and 60 min. The gel is visualised under UV light. 6 Analysis of the results A reference photo of an agarose gel (Annex A
45、) is provided with distinct patterns from authorised probiotic S. cerevisiae strains and a commercially available reference strain (S. cerevisiae NCYC 81) for the analysis of the results. The banding patterns as obtained are compared to those in the reference gel on the photo by measuring the size i
46、n base pairs (bp) of individual bands using the corresponding molecular weight ladders. It is suggested to record the results by an appropriate camera or image analysis system to facilitate the analysis. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 10 Annex A (informative) Example Gel - PCR of probioti
47、c strains of Saccharomyces cerevisiae Key Lane 1 and lane 8 Promega 100 bp ladder Lane 2 negative control Lane 3 APYS1CBS 493.94 Lane 4 APYS1CNCM 1-1079 Lane 5 APYS1CNCM 1-1077 Lane 6 reference strain, NCYC 81 Llane 7 APYS1NCYC SC47 Figure A.1 Example Gel PCR of probiotic strains of Saccharomyces ce
48、revisiae 1 APYS = Authorised probiotic yeast strain. DD CEN/TS 15790:2008CEN/TS 15790:2008 (E) 11 Annex B (informative) Validation data from the European Collaborative trial 45 In the validation study four different samples of animal feeding stuffs containing yeast (Saccharomyces cerevisiae) at leve
49、ls between 105and 107CFU/g by Polymerase Chain Reaction (PCR) were examined. Feeds supplemented with one of four authorised probiotic strains were analysed by seven of nine invited laboratories. The laboratories received blind samples meaning that they did not know before the analysis which yeast strain was in which sample. The laboratories were provided with
