CEN/TS 15790:2008

SLOVENSKI STANDARD
01-april-2009
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Animal feeding stuffs - PCR typing of probiotic strains of Saccharomyces cerevisiae
(yeast)
Futtermittel - PCR-Typisierung der probiotischen Stämme von Saccharomyces
cerevisiae (Hefe)
Aliments des animaux - Typage ACP des souches probiotiques de Saccharomyces
cerevisiae (levure)
Ta slovenski standard je istoveten z: CEN/TS 15790:2008
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN/TS 15790
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
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 souches Futtermittel - PCR-Typisierung der probiotischen Stämme
probiotiques de Saccharomyces cerevisiae (levure) 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 initially 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 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 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 Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15790:2008: E
worldwide for CEN national Members.

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
3.1.4 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 bromide .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 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 European Collaborative trial [4][5] . 11
Bibliography . 12

Foreword
This document (CEN/TS 15790:2008) has been prepared by Technical Committee CEN/TC 327 “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 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 following
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, Slovenia, Spain,
Sweden, Switzerland and United Kingdom.
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 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 characterise the yeast strains require high quality high
molecular weight genomic DNA. The method of DNA extraction from the yeast must facilitate these
requirements.
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 upon the amplification of δ 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
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 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 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 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
3.1.1 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 µM
and stored at least - 20 °C.
3.1.2 dNTP 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.
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/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 (MgCl ).
3.1.5 DNA Taq polymerase
DNA Taq Polymerase with a concentration of 5 U/µl 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 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 % 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 µm filtered.
3.2.6 Ethidium bromide
A commercially produced 10 mg/ml 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.
4.1.2 Pipets and sterile tips
Capable of dispensing between 0,5 µl and 200 µl.
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 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
M
...