SIST-TS CEN ISO/TS 17919:2014

SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 17919:2014
01-januar-2014
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za
ugotavljanje prisotnosti patogenih mikroorganizmov v živilih - Ugotavljanje
prisotnosti klostridijev, ki tvorijo botulinusne nevrotoksine tipov A, B, E in F
(ISO/TS 17919:2013)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of
food-borne pathogens - Detection of botulinum type A, B, E and F neurotoxin-producing
clostridia (ISO/TS 17919:2013)
Mikrobiologie von Lebensmitteln, Futtermitteln und Umgebungsproben - Polymerase-
Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln
- Nachweis von Botulinum-Neurotoxin-Typ A, B, E und F produzierenden Clostridien
(ISO/TS 17919:2013)
Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR)
pour la détection de micro-organismes pathogènes dans les aliments - Détection des
clostridies productrices de neurotoxine botulique de type A, B, E et F (ISO/TS
17919:2013)
Ta slovenski standard je istoveten z: CEN ISO/TS 17919:2013
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.020 Procesi v živilski industriji Processes in the food
industry
SIST-TS CEN ISO/TS 17919:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN ISO/TS 17919:2014

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SIST-TS CEN ISO/TS 17919:2014

TECHNICAL SPECIFICATION
CEN ISO/TS 17919

SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION
November 2013
ICS 07.100.30
English Version
Microbiology of the food chain - Polymerase chain reaction
(PCR) for the detection of food-borne pathogens - Detection of
botulinum type A, B, E and F neurotoxin-producing clostridia
(ISO/TS 17919:2013)
Microbiologie de la chaîne alimentaire - Réaction de Mikrobiologie von Lebensmitteln, Futtermitteln und
polymérisation en chaîne (PCR) pour la détection de micro- Umgebungsproben - Polymerase-Kettenreaktion (PCR)
organismes pathogènes dans les aliments - Détection des zum Nachweis von pathogenen Mikroorganismen in
clostridies productrices de neurotoxine botulique de type A, Lebensmitteln - Nachweis von Botulinum-Neurotoxin-Typ A,
B, E et F (ISO/TS 17919:2013) B, E und F produzierenden Clostridien (ISO/TS
17919:2013)
This Technical Specification (CEN/TS) was approved by CEN on 16 April 2013 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 17919:2013 E
worldwide for CEN national Members.

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SIST-TS CEN ISO/TS 17919:2014
CEN ISO/TS 17919:2013 (E)
Contents Page
Foreword .3

2

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SIST-TS CEN ISO/TS 17919:2014
CEN ISO/TS 17919:2013 (E)
Foreword
This document (CEN ISO/TS 17919:2013) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the
secretariat of which is held by DIN.
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.
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, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany,
Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO/TS 17919:2013 has been approved by CEN as CEN ISO/TS 17919:2013 without any
modification.

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SIST-TS CEN ISO/TS 17919:2014
TECHNICAL ISO/TS
SPECIFICATION 17919
First edition
2013-11-01
Microbiology of the food chain —
Polymerase chain reaction (PCR) for
the detection of food-borne pathogens
— Detection of botulinum type A, B, E
and F neurotoxin-producing clostridia
Microbiologie de la chaîne alimentaire — Réaction de polymérisation
en chaîne (PCR) pour la détection de micro-organismes pathogènes
dans les aliments — Détection des clostridies productrices de
neurotoxine botulique de type A, B, E et F
Reference number
ISO/TS 17919:2013(E)
©
ISO 2013

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SIST-TS CEN ISO/TS 17919:2014
ISO/TS 17919:2013(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2013
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved

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Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 1
4.1 Symbols . 1
4.2 Abbreviated terms . 2
5 Principle . 2
5.1 General . 2
5.2 Microbial enrichment . 2
5.3 Nucleic acid extraction . 2
5.4 Amplification by PCR . 2
5.5 Detection of PCR products . 2
5.6 Confirmation . 2
6 Reagents . 3
6.1 General . 3
6.2 Culture media . 3
6.3 Nucleic acid extraction . 4
6.4 Reagents for PCR . 5
7 Apparatus and equipment . 5
7.1 General . 5
7.2 Equipment for sample preparation prior to enrichment . 5
7.3 Equipment for microbial enrichment . . 5
7.4 Equipment used for nucleic acid extraction . 6
7.5 Equipment used for PCR . 6
7.6 Equipment used for the detection of the PCR product . 6
8 Sampling . 7
9 Procedure. 7
9.1 Sample preparation prior to enrichment . 7
9.2 Microbial enrichment . 7
9.3 Nucleic acid preparation. 8
9.4 PCR amplification . 9
9.5 Confirmation of a positive PCR result . 9
Annex A (normative) Flow-diagram of the procedure .10
Annex B (informative) Multiplex PCR assays for detection of genes encoding botulinum
neurotoxin types A, B, E, and F using agarose gel electrophoresis .11
Annex C (informative) Assays for detection of genes encoding botulinum neurotoxin types A, B, E,
and F using real-time PCR .29
Annex D (informative) Preparation of C. botulinum spores.40
Bibliography .46
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SIST-TS CEN ISO/TS 17919:2014
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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.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of document:
— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical
experts in an ISO working group and is accepted for publication if it is approved by more than 50 %
of the members of the parent committee casting a vote;
— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a
technical committee and is accepted for publication if it is approved by 2/3 of the members of the
committee casting a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for
a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or
ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be
transformed into an International Standard or be withdrawn.
ISO/TS 17919 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, Subcommittee SC 9, Microbiology, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
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Introduction
Botulinum neurotoxin-producing clostridia are ubiquitous in the environment. Botulism is a severe
neuroparalytic disease resulting from the action of botulinum neurotoxins (BoNTs). Seven different
serotypes of BoNTs (type A to G) and a number of subtypes have been identified to date.
BoNT type A (BoNT/A), type B (BoNT/B), type E (BoNT/E) and type F (BoNT/F) are mainly responsible
for botulism in humans and the genes encoding these toxins are the targets of this Technical Specification.
BoNT type A, B, E, and F-producing clostridia exist in four physiologically distinct groups (Group I
Clostridium botulinum, Group II C. botulinum, C. baratii, C. butyricum).
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 patents.
ISO take no position concerning the evidence, validity and scope of this patent right.
The holder of this patent right has assured ISO that they are 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:
Applied Biosystems, LLC
Scott Miller, Legal Department
5781 Van Allen Way
CARLSBAD
CA 92008
USA
Tel: +1 760 476 4387
Fax: +1 760 476 6048
e-mail: Scott.miller@lifetechnologies.com
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.
ISO (www.iso.org/patents) maintains an on-line database of patents relevant to its documents. Users
are encouraged to consult the databases for the most up-to-date information concerning patents.
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SIST-TS CEN ISO/TS 17919:2014
TECHNICAL SPECIFICATION ISO/TS 17919:2013(E)
Microbiology of the food chain — Polymerase chain
reaction (PCR) for the detection of food-borne pathogens
— Detection of botulinum type A, B, E and F neurotoxin-
producing clostridia
1 Scope
This Technical Specification specifies a horizontal method for the molecular detection of clostridia
carrying botulinum neurotoxin A, B, E, and F genes by a PCR method. This method detects the genes
and not the toxins, therefore a positive result does not necessarily mean the presence of these toxins in
the sample investigated. This Technical Specification is applicable to products for human consumption,
animal feed, and environmental samples.
The PCR assays for detection of genetic sequences encoding specific toxin types are described
in Annexes B and C.
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 6887-1, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the
initial suspension and decimal dilutions
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance
testing of culture media
ISO 20837, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — Requirements for sample preparation for qualitative detection
ISO 20838:2006, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — Requirements for amplification and detection for qualitative methods
ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — General requirements and definitions
3 Terms and definitions
For the purpose of this document, the terms and definitions given in ISO 22174 apply.
4 Symbols and abbreviated terms
4.1 Symbols
c substance concentration
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ρ mass concentration
φ volume fraction
w mass fraction
4.2 Abbreviated terms
BoNT botulinum neurotoxin
5 Principle
5.1 General
The method comprises the following consecutive steps:
a) microbial enrichment (see 5.2);
b) nucleic acid extraction (see 5.3);
c) amplification (see 5.4);
d) detection of PCR products (see 5.5);
e) confirmation (see 5.6).
NOTE Real-time-PCR combines steps c) to e).
5.2 Microbial enrichment
The number of BoNT-producing clostridia (spores or vegetative cells) to be detected is increased by
encouraging their germination and growth in non-selective liquid nutrient medium tryptone–peptose–
glucose–yeast extract broth under anaerobic conditions.
5.3 Nucleic acid extraction
Bacterial cells are separated from the nutrient medium, lysed and the nucleic acids are extracted for use
in the PCR reaction.
5.4 Amplification by PCR
The extracted nucleic acid is transferred to the PCR mix and the amplification is carried out in a
thermal cycler.
5.5 Detection of PCR products
PCR products are detected by gel electrophoresis or an appropriate alternative.
5.6 Confirmation
The identity of the PCR products shall be confirmed by any appropriate method, e.g. sequencing,
hybridization or restriction analysis.
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6 Reagents
6.1 General
For all stages 5.1 b) to e), use only reagents of recognized analytical grade and consumables suitable for
molecular biology applications as specified in ISO 20837 and ISO 20838.
Reagent requirements specified in ISO 20838:2006, Clause 5, apply.
6.2 Culture media
6.2.1 General
Follow ISO 11133 for the preparation, production and performance testing of culture media.
6.2.2 Diluent
[9]–[13]
Follow ISO 6887-1 and the relevant part of ISO 6887 dealing with the product to be examined.
6.2.3 Non–selective enrichment culture medium, tryptone–peptone–glucose–yeast extract
broth (TPGY broth) (Reference [7])
6.2.3.1 General
Other approved non-selective enrichment culture media can be used provided equivalent
performance is shown.
6.2.3.2 Composition and pH
Tryptone 50 g
Peptone 5 g
Yeast extract 20 g
d-Glucose 4 g
Sodium thioglycolate, HSCH COONa 1 g
2
Water to 1 000 ml
pH 7,0 ± 0,2
6.2.3.3 Preparation
Dissolve the components in the water by boiling. After sterilization, adjust to pH 7,0 ± 0,2 at 25 °C.
Dispense the base into flasks or bottles of appropriate capacity. Sterilize for 15 min at 121 °C. Store in a
refrigerator at 5 °C ± 3 °C. Discard unused medium 4 weeks after preparation.
6.2.4 TPGY broth buffered — for acidic and acidifying foodstuffs only
6.2.4.1 Stock solution (phosphate buffer)
6.2.4.1.1 Solution 1
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Sodium dihydrogenphosphate monohydrate [NaH PO ·H O] 138 g
2 4 2
Water to 1 000 ml
6.2.4.1.2 Solution 2
Disodium hydrogenphosphate [Na HPO ] 142 g
2 4
Water to 1 000 ml
6.2.4.1.3 Preparation
Dissolve the components in the water by boiling. To 250 ml solution 1, add solution 2 until the pH reaches
7,2. Store in a refrigerator at 5 °C ± 3 °C.
6.2.4.2 Preparation of the complete medium
Dissolve the components given for the base (6.2.3.2) in 500 ml water by boiling. Add 100 ml phosphate
buffer (6.2.4.1) The final phosphate concentration of the complete medium is 0,1 mol/l. Add water up
to 1 000 ml. Dispense the complete medium into flasks or bottles of appropriate capacity. Sterilize for
15 min at 121 °C. Store in a refrigerator at 5 °C ± 3 °C. Discard unused medium 4 weeks after preparation.
6.3 Nucleic acid extraction
6.3.1 Chloroform, CHCl .
3
6.3.2 Ethanol, φ(C H OH) = 96 %.
2 5
6.3.3 Ethylenediaminetetraacetic acid disodium salt (Na EDTA), C H N O Na .
2 10 14 2 8 2
6.3.4 Hexadecyl(trimethyl)ammonium bromide [(cetyl(trimethyl)ammonium bromide, CTAB],
C H BrN.
19 42
6.3.5 Hydrochloric acid, φ(HCl) = 37 %.
6.3.6 Isopropanol, CH CH(OH)CH .
3 3
6.3.7 Proteinase-K, approximately 20 units/mg of lyophilizate.
6.3.8 Sodium chloride, NaCl.
6.3.9 Sodium hydroxide, NaOH.
6.3.10 Tris(hydroxymethyl)aminomethane (tris), C H NO .
4 11 3
6.3.11 CTAB extraction buffer, ρ(CTAB) = 20 g/l, c(NaCl) = 1,4 mol/l, c(tris) = 0,1 mol/l, c(Na EDTA) =
2
0,02 mol/l.
Adjust to pH 8,0 with HCl or NaOH.
6.3.12 CTAB-precipitation buffer, ρ(CTAB) = 5 g/l, c(NaCl) = 0,04 mol/l.
6.3.13 Sodium chloride solution, c(NaCl) = 1,2 mol/l.
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6.3.14 Ethanol solution, φ(C H OH) = 70 %.
2 5
6.3.15 Proteinase-K solution, ρ = 20 mg/ml, dissolved in sterile water.
Do not autoclave. Store at −20 °C, but avoid repeated freezing and thawing.
6.3.16 Tris–EDTA (TE) buffer, c(tris) = 0,01 mol/l, c(Na EDTA) = 0,001 mol/l.
2
Adjust to pH 8,0 with HCl or NaOH.
6.4 Reagents for PCR
6.4.1 Thermostable DNA polymerase, as specified in ISO 20838 and ISO 22174.
6.4.2 Deoxyribonucleoside triphosphates (dNTPs) containing dATP, dCTP, dGTP and dTTP or dUTP,
as specified in ISO 20838 and ISO 22174.
6.4.3 PCR buffer solution, as specified in ISO 20838 and ISO 22174.
The PCR buffer solution is usually delivered with the DNA polymerase, which may or may not include
MgCl in a concentration specified by the manufacturer. The final MgCl concentrations are method
2 2
specific and are therefore listed in the annexes. It is possible that ready-to-use reagents are commercially
available. If so, follow the manufacturer’s instructions for use.
6.4.4 Primers and probes
Primers and probes for specific detection of the neurotoxin gene sequences are listed in Annexes B and C.
7 Apparatus and equipment
7.1 General
Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.
7.2 Equipment for sample preparation prior to enrichment
7.2.1 Water bath, capable of being maintained at 50 °C ± 1 °C.
7.2.2 Centrifuge, for 50 ml and 100 ml tubes and with an adjustable acceleration of up to 12 000 × g.
7.2.3 Membrane filter, nitrocellulose-filter, pore size 0,45 µm.
7.2.4 Centrifuge tubes, of capacities of 50 ml and 100 ml.
7.3 Equipment for microbial enrichment
7.3.1 Water baths, capable of being maintained at 30 °C ± 1 °C, 65 °C ± 1 °C and 100 °C ± 1 °C.
7.3.2 Anaerobic jar or anaerobic cabinet, capable of being maintained at 30 °C ± 1 °C, according to
ISO 7218.
7.3.3 Incubator, capable of operating at 30 °C ± 1 °C.
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7.3.4 Flasks or bottles, of appropriate capacity.
7.4 Equipment used for nucleic acid extraction
Appropriate equipment according to ISO 20837 and, in particular, the following.
7.4.1 Microcentrifuge tubes, of capacities of 1,5 ml and 2,0 ml.
7.4.2 Thermo block, with a mixing frequency between 300 r/min and 1 400 r/min.
7.4.3 Graduated pipettes and pipette filter tips, for volumes between 1 µl and 1 000 µl.
7.4.4 Centrifuge, for reaction tubes having a capacity of 1,5 ml and 2,0 ml, e.g. microcentrifuge, capable
of achieving an acceleration of up to 12 000 × g.
7.4.5 Mixer, e.g. vortex type.
7.5 Equipment used for PCR
Appropriate equipment according to the method and, in particular, the following.
7.5.1 Pipettes and pipette filter tips, having a capacity between 1 µl and 1 000 µl.
7.5.2 Microcentrifuge tubes, having a capacity of 1,5 ml and 2,0 ml.
7.5.3 Thin-walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR microplates or
ot
...