Animal feeding stuffs - PCR typing of probiotic strains of Saccharomyces cerevisiae (yeast)

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.

Futtermittel - PCR-Typisierung der probiotischen Stämme von Saccharomyces cerevisiae (Hefe)

Diese Technische Spezifikation legt eine Polymerase-Kettenreaktion-(PCR)-Methodik für die Identifizierung
von probiotischen Saccharomyces cerevisiae-Hefestämmen fest. Zusätzlich wird ein Verfahren für die
Extraktion hochreiner DNA der Hefe vorgeschlagen.

Aliments des animaux - Typage par réaction de polymérisation en chaîne (PCR) des souches probiotiques de Saccharomyces cerevisiae (levure)

La présente Spécification technique définit une méthode d’amplification en chaîne par polymérase (ACP)
destinée à l’identification des souches de levure probiotiques de Saccharomyces cerevisiae. Elle présente
également une méthode d’extraction d’un ADN de bonne qualité à partir de la levure.

Krma - PCR-tipizacija probiotičnih sevov Saccharomyces cerevisiae (kvasovk)

General Information

Status
Published
Publication Date
09-Dec-2008
Current Stage
9093 - Decision to confirm - Review Enquiry
Due Date
26-May-2011
Completion Date
26-May-2011

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SLOVENSKI STANDARD
SIST-TS CEN/TS 15790:2009
01-april-2009
.UPD3&5WLSL]DFLMDSURELRWLþQLKVHYRY6DFFKDURP\FHVFHUHYLVLDH NYDVRYN

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
SIST-TS CEN/TS 15790:2009 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 15790:2009
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SIST-TS CEN/TS 15790:2009
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.
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SIST-TS CEN/TS 15790:2009
CEN/TS 15790:2008 (E)
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

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SIST-TS CEN/TS 15790:2009
CEN/TS 15790:2008 (E)
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.
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SIST-TS CEN/TS 15790:2009
CEN/TS 15790:2008 (E)
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.
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SIST-TS CEN/TS 15790:2009
CEN/TS 15790:2008 (E)
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 aga
...

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