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1、DD CEN/TS 15790:2008 ICS 07.100.30; 65.120 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW DRAFT FOR DEVELOPMENT Animal feeding stuffs PCR typing of probiotic strains of Saccharomyces cerevisiae (yeast) Licensed Copy: London South Bank University, South Bank University, 02/02/
2、2009 03:26, Uncontrolled Copy, (c) BSI This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 31 January 2009 BSI 2009 ISBN 978 0 580 61806 2 Amendments/corrigenda issued since publication DateComments DD CEN/TS 15790:2008 National foreword Thi
3、s Draft for Development is the UK implementation of CEN/TS 15790:2008. This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on this provisional basis, so that informat
4、ion and experience of its practical application can be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can be reported to the international organization responsible for its conversion to an international standard. A review of this publication
5、 will be initiated not later than 3 years after its publication by the international organization so that a decision can be taken on its status. Notification of the start of the review period will be made in an announcement in the appropriate issue of Update Standards. According to the replies recei
6、ved by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into an international Standard, to extend the life of the Technical Specification or to withdraw it. Comments should be sent to the Secretary of the responsible BSI Technical Committee at
7、 British Standards House, 389 Chiswick High Road, London W4 4AL. The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not pur
8、port to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immunity from legal obligations. Licensed Copy: London South Bank University, South Bank University, 02/02/2009 03:26, Uncontrolled Copy, (c
9、) BSI DD CEN/TS 15790:2008 TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 15790 December 2008 ICS 07.100.30; 65.120 English Version Animal feeding stuffs - PCR typing of probiotic strains of Saccharomyces cerevisiae (yeast) Aliments des animaux - Typage ACP des souche
10、s probiotiques de Saccharomyces cerevisiae (levure) Futtermittel - PCR-Typisierung der probiotischen Stmme von 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 limited ini
11、tially to three years. After two years the members of CEN will be requested to submit their comments, 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 and to make
12、the CEN/TS available promptly 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 standards bod
13、ies 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 Kingdom. EU
14、ROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2008 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 15790:2008: E License
15、d Copy: London South Bank University, South Bank University, 02/02/2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008 CEN/TS 15790:2008 (E) 2 Contents Page Foreword 3? Introduction .4? 1?Scope 5? 2?Principle .5? 3?Reagents 5? 3.1?PCR .5? 3.1.1?Primers .5? 3.1.2?dNTP mix 5? 3.1.3?Buffer 6? 3
16、.1.4?Magnesium Chloride solution 6? 3.1.5?DNA Taq polymerase 6? 3.2?Gel Electrophoresis.6? 3.2.1?Agarose, molecular-grade agarose free from DNase and RNase contamination. 6? 3.2.2?Molecular weight marker 6? 3.2.3?Tris-Borate-EDTA buffer .6? 3.2.4?Loading dye .6? 3.2.5?Water .6? 3.2.6?Ethidium bromid
17、e 6? 4?Apparatus .6? 4.1?PCR .6? 4.1.1?PCR Tubes .6? 4.1.2?Pipets and sterile tips .7? 4.1.3?Thermal cycler .7? 4.2?Gel Electrophoreses .7? 4.2.1?Horizontal gel electrophoresis system .7? 4.2.2?Microwave oven .7? 4.2.3?Conical flask 7? 4.2.4?Balance .7? 4.2.5?Transilluminator 7? 4.2.6?Image analysis
18、 system or Polaroid camera 7? 5?Procedure .7? 5.1?PCR reaction 7? 5.2?Thermal Cycle 8? 5.3?Agarose gel electrophoresis 8? 6?Analysis of the results 9? Annex A (informative) Example Gel - PCR of probiotic strains of Saccharomyces cerevisiae . 10? Annex B (informative) Validation data from the Europea
19、n Collaborative trial 45 . 11? Bibliography . 12? Licensed Copy: London South Bank University, South Bank University, 02/02/2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008 CEN/TS 15790:2008 (E) 3 Foreword This document (CEN/TS 15790:2008) has been prepared by Technical Committee CEN/TC 3
20、27 “Animal 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
21、 patent 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 foll
22、owing 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
23、, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Licensed Copy: London South Bank University, South Bank University, 02/02/2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008 CEN/TS 15790:2008 (E) 4 Introduction This methodology is based on specific polymerase chain reaction (PCR)
24、amplification of a genetic sequence for the detection of Saccharomyces cerevisiae isolated from animal feed or animal feed 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 c
25、haracterise the yeast strains require high quality high molecular weight genomic DNA. The method of DNA extraction from the yeast must facilitate these requirements. Licensed Copy: London South Bank University, South Bank University, 02/02/2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008
26、CEN/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 quality DNA from yeast is suggested. 2 Principle This method is based
27、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 separated in agarose gels by electrophoresis. Patterns are visualise
28、d 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 reaction; 3. Gel electrophoresis; 4. Analysis of results. Individu
29、al 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 from agar plates to inoculate 10 ml malt extract broth which is cul
30、tured 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 other appropriate procedures. The DNA extraction procedure is a seq
31、uential 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 3.1.1 Primers The following primer sequences are used. Delta 1 modifie
32、d 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 mix A 2 mM equimolar stock solution of dATP, dTTP, dGTP, dCTP is m
33、ade from a dNTP mix set and stored at least - 20 C. Licensed Copy: London South Bank University, South Bank University, 02/02/2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008 CEN/TS 15790:2008 (E) 6 3.1.3 Buffer A commercial 10x stock solution of reaction buffer is used. The composition i
34、s 100 mM Tris-HCl (pH 9,0), 500 mM KCl, 1 % (v/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
35、 with a concentration of 5 U/l is used and stored 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
36、 Tris-Borate-EDTA buffer Commercially produced 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
37、 % bromophenol blue, 0,03 % xylene cyanol FF, 15 % 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
38、solution is used. 4 Apparatus Usual equipment appropriate 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. Licensed Copy: London South Bank University, South Bank University, 02/02/
39、2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008 CEN/TS 15790:2008 (E) 7 4.1.2 Pipets and sterile tips Capable of dispensing between 0,5 l and 200 l. 4.1.3 Thermal cycler An appropriate and reliable thermal cycler which is technically maintained and calibrated. 4.2 Gel Electrophoreses 4.2
40、.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 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 a
41、ppropriate apparatus to visualise the banding patterns in the agarose gel following ethidium bromide staining. 4.2.6 Image analysis system or Polaroid camera Appropriate system to record and analyse results. 5 Procedure 5.1 PCR reaction This method has been slightly altered to incorporate modificati
42、on 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 premixture containing all the reagents except template DNA is prepared according to Table 1. Licensed Copy: London South Bank University, South Bank
43、 University, 02/02/2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008 CEN/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 MgCl2 25 mM 1,5 mM 3 Delta 1 modified primer 50 M 1 M 1 Delta 2 primer 50 M 1 M 1 Taq polymerase 5
44、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 (3.2.5) for negative control)
45、. 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 ensure that this is the ca
46、se by appropriate standardisation procedures applicable for the instrument. 95 C - 2 min cycles4 min 2 - C 72 s 30 - C 45 s 30 - C 95 cycles30 min 2- C 72 s 30- C 45 s 30- C 95 72 C - 10 min 4 C 8 min 5.3 Agarose gel electrophoresis A 2 % (w/v) agarose gel is made with Molecular Biology Grade agaros
47、e 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. Licensed Copy: London South Bank University, South Bank University, 02/02/2009 03:26, Uncontrolled Copy, (c) BSI DD CEN/TS 15790:2008 CE
48、N/TS 15790:2008 (E) 9 10 l of loading dye is added to the reaction and mixed. 20 l of the reaction 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
49、 visualised under UV light. 6 Analysis of the results A reference photo of an agarose gel (Annex A) 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 in base pairs (bp) of individual bands using the corresponding molecular weight ladders. It i