SIST-TS CEN ISO/TS 17919:2014

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

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.

Contents Page
Foreword .3

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.
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
ISO/TS 17919:2013(E)
© 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
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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

ISO/TS 17919:2013(E)
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
ISO/TS 17919:2013(E)
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).
iv © ISO 2013 – All rights reserved

ISO/TS 17919:2013(E)
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.
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
ISO/TS 17919:2013(E)
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.
2 © ISO 2013 – All rights reserved

ISO/TS 17919:2013(E)
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
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
ISO/TS 17919:2013(E)
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 .
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) =
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.
4 © ISO 2013 – All rights reserved

ISO/TS 17919:2013(E)
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.
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.
ISO/TS 17919:2013(E)
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
other suitable equipment.
7.5.4 Thermal cycler.
7.6 Equipment used for the detection of the PCR product
Appropriate equipment according to the method and, in particular, the following.
7.6.1 Gel-based PCR
7.6.1.1 Horizontal gel system.
7.6.1.2 Power supply.
7.6.1.3 Ultraviolet (UV) transilluminator or UV light box.
7.6.1.4 Gel documentation system.
7.6.2 Real-time PCR
7.6.2.1 Real-time PCR thermal cycler.
7.6.2.2 Appropriate detection and analysis software.
6 © ISO 2013 – All rights reserved

ISO/TS 17919:2013(E)
8 Sampling
Sampling is not part of the method specified in this Technical Specification. If there is no specific
International Standard dealing with the sampling of the product concerned, it is recommended that the
parties concerned come to an agreement on the subject.
It is important the laboratory receive a truly representative sample which has not been damaged or
changed during transport or storage.
9 Procedure
9.1 Sample preparation prior to enrichment
9.1.1 General
See Figure A.1.
It is recommended that at least 25 g be analysed, particularly for honey samples; however, if supply is
limited, smaller sample sizes may be used.
9.1.2 Preparation of the sample
[9]–[13]
Prepare and homogenize the sample according to ISO 6887-1 and the relevant parts of ISO 6887
concerning the relevant matrix.
9.1.3 Preparation of honey samples
Place the vessel of honey in a water bath (7.2.1) at 50 °C ± 1 °C for 30 min to melt the honey. Invert the
vessel several times to mix the sample.
Weigh 25 g ± 2 g of honey into a sterile centrifuge tube with a capacity of 100 ml (7.2.4) and add at least
50 ml of sterile distilled or deionized water containing 1 % volume fraction polysorbate 80, preheated
to 50 °C ± 1 °C. Mix until the solution is homogeneous. Centrifuge the mixture at 12 000 × g (7.2.2) for
30 min. Remove the supernatant carefully and pass it through a 0,45 µm membrane filter (7.2.3). In
case of blockage, pass any remaining supernatant through a fresh filter. Use all filters in the subsequent
steps. Store the sediment temporarily at 5 ± 3 °C.
9.2 Microbial enrichment
9.2.1 Inoculation
9.2.1.1 General
Remove dissolved oxygen from the enrichment medium (6.2) by boiling for 10 min to 15 min in a water
bath (7.3.1).
If the sample is acidic or acidifying, the enrichment broth shall be prepared according to 6.2.4.
9.2.1.2 Inoculation of test portion
9.2.1.2.1 Recovery of vegetative cells and spores
Adjust the temperature of the enrichment medium to 30 °C ± 1 °C in a water bath (7.3.1). Transfer the
−1
test portion into the degassed enrichment medium to give a final dilution of 10 .
ISO/TS 17919:2013(E)
9.2.1.2.2 Recovery of spores
Adjust the temperature of the enrichment medium to 65 °C ± 1 °C in a water bath (7.3.1). Transfer the
−1
test portion into the preheated enrichment medium to give a final dilution of 10 at 65 °C ± 1 °C. After
inoculation, maintain the flask or bottle at 65 °C ± 1 °C for further 10 min, and then quickly cool to
30 °C ± 1 °C in a water bath (7.3.1).
9.2.1.3 Inoculation of honey test portions
Adjust the temperature of the enrichment broth to 65 °C ± 1 °C (7.3.1). Transfer the sediment (9.1.3)
to one flask or bottle (7.3.4) and the filter or filters (9.1.3) into a second flask or bottle (7.3.4) each
containing at least 10 ml of the heated enrichment broth, but in all cases ensure sufficient liquid covers
the filters. Incubate both flasks or bottles at 65 °C ± 1 °C for 10 min in a water bath (7.3.1).
NOTE Honey samples are only analysed for spores.
9.2.2 Incubation
Incubate under anaerobic conditions (7.3.2) at 30 °C ± 1 °C. After 24 h ± 2 h of incubation, remove 1 ml of
the enrichment for the PCR analysis and return immediately to anaerobic conditions.
If the result of the first PCR is negative, continue incubation under the same conditions for a further
48 h ± 2 h, then transfer 1 ml of the enrichment into a flask or bottle (7.3.4) containing 9 ml of fresh
enrichment broth (6.2.3). Incubate anaerobically (7.3.2) at 30 °C ± 1 °C for 18 h ± 2 h and perform a
second PCR run.
9.2.3 Process controls
Positive and negative process controls shall be included according to ISO 22174.
An example of a method for the preparation of spores is given in Annex D.
9.3 Nucleic acid preparation
9.3.1 General
An appropriate nucleic acid extraction procedure for Gram-positive bacteria shall be used.
An example of a procedure is given in 9.3.2 to 9.3.4. This procedure consists of a lysis step — thermal
lysis in the presence of CTAB — followed by several extraction steps in order to remove inhibitors, such
as polysaccharides and proteins.
Once the matrix test portion has been prepared, apply the DNA extraction and purification protocol
given in 9.3.2 to 9.3.4.
Scale adaptation of masses and buffer volumes is required as a function of the selected size of the test portion.
9.3.2 Sample extraction
Transfer 1 000 µl of the enrichment culture (9.2.2) into a microcentrifuge tube (7.4.1). Centrifuge (7.4.4)
for 5 min at approximately 12 000 × g. Discard the supernatant (aqueous).
Add 500 µl prewarmed (65 °C) CTAB extraction-buffer (6.3.11) to the pellet and mix gently until the
pellet is lysed. Incubate for 30 min at 65 °C, under agitation (7.4.2). Add 20 µl of proteinase-K solution
(6.3.7), gently mix the tube and incubate for 30 min at 65 °C, under agitation (7.4.2). Centrifuge for
10 min at approximately 12 000 × g. Transfer the supernatant to a new tube, add 0,7 to 1 volume of
chloroform (6.3.1) and mix thoroughly.
Centrifuge for 15 min at approximately 12 000 × g. Transfer the supernatant (aqueous) to a new tube.
8 © ISO 2013 – All rights reserved

ISO/TS 17919:2013(E)
9.3.3 CTAB precipitation
Add 2 volumes of the CTAB precipitation buffer (6.3.12). Incubate for 60 min at room temperature without
agitation. Centrifuge for 15 min at 12 000 × g. Discard the supernatant. Dissolve the precipitated DNA by
adding 350 µl of NaCl solution (6.3.13). Add 350 µl of chloroform (6.3.1) and mix thoroughly. Centrifuge
for 10 min at 12 000 × g. Transfer the aqueous phase into a new tube.
NOTE CTAB-precipitation is not necessary for all matrices, only for protein- and polysaccharide-rich
matrices. Alternatively, a solid-phase purification of the DNA (e.g. by the use of spin columns) is possible assuming
the results are equivalent.
9.3.4 DNA precipitation
Add 0,6 volume of isopropanol (6.3.6), mix smoothly by inverting the tube and keep the tube at room
temperature for 20 min. Centrifuge for 15 min at 12 000 × g. Discard the supernatant. Add 500 µl of
ethanol solution (6.3.14) to the tube and invert several times. This is the critical step ensuring the
complete removal of CTAB. Centrifuge for 10 min at 12 000 × g. Discard the supernatant. Dry the DNA
pellet and redissolve it into 100 µl of an appropriate buffer, e.g. TE buffer (6.3.16). This is the DNA master
stock. The DNA can be stored at −20 °C until use.
9.4 PCR amplification
Different procedures for PCR amplification can be used. The detection of the PCR product can be either
gel based or by detection of the fluorescence signal.
All requirements for the PCR amplification are specified in ISO 20838.
Examples of gel-based PCR methods are described in Annex B and of real-time PCR methods in Annex C.
9.4.1 PCR controls
PCR controls shall be in accordance with ISO 22174.
9.4.2 Detection of PCR products
Different procedures for detection of PCR products can be used. Examples of gel-based PCR methods are
described in Annex B and of real-time PCR methods in Annex C.
9.5 Confirmation of a positive PCR result
Follow the procedure specified in ISO 20838.
9.5.1 Interpretation of the results
The results obtained, including the controls specified in ISO 22174, should be unambiguous and the
controls should yield expected results, otherwise the procedure shall be repeated.
The PCR result is:
a) positive, if a specific PCR product has been detected and confirmed and all the controls give
expected results;
b) negative within the limits of detection, if a specific PCR product has not been detected, and all
controls give expected results.
A positive PCR result does not automatically indicate the presence of botulinum neurotoxins. To confirm
the presence of the neurotoxins suitable methods targeting the toxins directly shall be applied.
ISO/TS 17919:2013(E)
Annex A
(normative)
Flow-diagram of the procedure
Prepare sample (9.1.1)
Test portion
x g or x ml
9x ml enrichment-broth (6.2.3)
Incubate under anaerobic conditions at 30 °C ± 1 °C for 24 h ± 2 h (7.3.2)
Remove 1 ml of the enrichment for PCR analysis and return to anaerobic conditions
Positive PCR result Negative PCR result:
Incubate for a further 48 h ± 2 h
under anaerobic conditions (7.3.2)
Transfer 1 ml of enrichment broth into
9 ml of fresh enrichment broth (6.2.3)
Incubate under anaerobic conditions
at 30 °C ± 1 °C for 18 h ± 2 h (7.3.2)
Second PCR run
Figure A.1
10 © ISO 2013 – All rights reserved

ISO/TS 17919:2013(E)
Annex B
(informative)
Multiplex PCR assays for detection of genes encoding botulinum
neurotoxin types A, B, E, and F using agarose gel electrophoresis
B.1 Method 1
B.1.1 Introduction
This clause describes a method for the specific amplification and detection of the genes encoding
botulinum neurotoxin types A, B, E, and F using agarose gel electrophoresis.
For limitations see B.1.7.6.
B.1.2 Performance characteristics
B.1.2.1 General
The method has been validated for DNA extracted from various C. botulinum type A, B, E, and F reference
strains and from naturally contaminated samples.
The method appears in References [1][2][5].
B.1.2.2 Theoretical evaluation of the method
Theoretical evaluation was done by performing a sequence similarity search against the GenBank/EMBL/
DDBJ database, where EMBL is the European Molecular Biology Laboratory and DDBJ is the DNA Database
of Japan (Reference [16], 2009-09-20). The result of the search confirmed a complete identity only with
the expected target sequences.
B.1.2.3 Selectivity
B.1.2.3.1 Inclusivity test
The inclusivity of the method was tested with 86 C. botulinum type A strains, 70 C. botulinum type B,
15 C. botulinum/butyricum type E and six C. botulinum type F strains, see Table B.1.
ISO/TS 17919:2013(E)
Table B.1 — Inclusivity of the multiplex PCR using target strains
Type of botulinum neurotoxin gene detected
Number
Strain, type and subtype
of strains
Type A Type B Type E Type F
a
C. botulinum type A subtype A1 3 3 0 0 0
b
C. botulinum type A subtype A2 9 9 0 0 0
c
C. botulinum type A subtype A3 1 1 0 0 0
d
C. botulinum type A subtype A5 4 4 0 0 0
e
C. botulinum type A undetermined subtype 69 69 0 0 0
f
C. botulinum type Ba subtype A4 1 1 1 0 0
g
C. botulinum type Ab subtype A2 4 4 4 0 0
h
C. botulinum type B 70 0 70 0 0
i
C. botulinum type E 4 0 0 4 0
j
C. butyricum type E 11 0 0 11 0
k
C. botulinum type F 6 0 0 0 6
a
C. botulinum National Collection of Type Cultures (NCTC 4587), C. botulinum strain 62A, C. botulinum NCTC 7272.
b
Strains isolated from Italian National Reference Centre for Botulism (NRCB).
c
C. botulinum NCTC 2012 Loch Maree.
d
C. botulinum strains from Institute of Food Research (IFR), UK.
e
A total of 64 strains from NRCB collection and five strains from the Consultant Laboratory for Anaerobic Bacteria
(CLAB) of the University of Leipzig, Germany.
f
C. botulinum strain 657Ba from IFR, UK.
g
Strains isolated by NRCB, Italy.
h
C. botulinum NCTC 7273 and 69 strains from NRCB collection.
i
Three strains from the CLAB and one strain from NRCB.
j
Seven strains isolated in Italy by NRCB, four strains provided by Reference [17].
k
C. botulinum NCTC 10281: three strains isolated in Italy by NRCB; two strains isolated in Germany by CLAB.
B.1.2.3.2 Exclusivity test
The exclusivity of the method was tested with 34 non-target organisms, see Table B.2. No cross-reactivity
was observed with the non-target bacteria.
12 © ISO 2013 – All rights reserved

ISO/TS 17919:2013(E)
Table B.2 — Exclusivity of the multiplex PCR using non-target strains
Type of botulinum neurotoxin gene detected
Number
a
Strains
of strains
Type A Type B Type E Type F
C. sporogenes WDCM 00008 1 0 0 0 0
C. perfringens (WDCM 00007 and field strains) 3 0 0 0 0
C. carnis NCTC 13036 1 0 0 0 0
C. histolyticum NCTC 503 1 0 0 0 0
C. butyricum NCTC 7423 1 0 0 0 0
C. barati NCTC 10986 1 0 0 0 0
Bacillus subtilis WDCM 00003 1 0 0 0 0
B. cereus (NCTC 11143 and field strains) 3 0 0 0 0
Thermophilic Campylobacter spp. (field strains) 2 0 0 0 0
Escherichia coli (WDCM 00013 and field strains) 3 0 0 0 0
Salmonella spp. (WDCM 00030 and field strain) 2 0 0 0 0
Listeria spp. (WDCM 00017 and WDCM 00109) 2 0 0 0 0
Brochotrix thermospacta WDCM 00071 1 0 0 0 0
Enterococcus faecalis WDCM 00087 1 0 0 0 0
Citrobacter freundii WDCM 00078 1 0 0 0 0
Pseudomonas spp. (field strains) 3 0 0 0 0
Yersinia enterocolitica (field strains) 3 0 0 0 0
Lactobacillus fermentum (field strain) 1 0 0 0 0
Aspergillus spp. (field strains) 2 0 0 0 0
Saccharomyces cervisiae (field strain) 1 0 0 0 0
a WDCM ≡ World Data Centre for Microorganisms.
B.1.2.4 Sensitivity
B.1.2.4.1 Sensitivity tests using artificially contaminated samples (Reference [2])
The limit of detection was assessed by measuring artificially contaminated samples with different
inoculation levels (0,1 cfu to 10 cfu and 10 cfu to 100 cfu before enrichment) in 10 g of various food matrices
(canned fish, canned sausages, and honey) under investigation using the culture method (Reference [1]).
According to the results, the false-positive rate for the method using artificially contaminated samples
is 0 % and the false-negative rate is 0 %. The limit of detection for the method described is 1 cfu to 10 cfu
or spores per 10 g before enrichment.
B.1.2.4.2 Sensitivity tests using naturally contaminated samples (Reference [2])
The limit of detection was assessed by measuring 382 naturally contaminated samples (e.g. honey,
vegetable matter, and canned meats) using the mouse bioassay as reference method. According to the
results the false-positive rate for the method using naturally contaminated samples is 0 % and the false-
negative rate is 0 %.
NOTE The experi
...