SIST EN ISO 10272-1:2017/A1:2023
(Amendment)Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method - Amendment 1: Inclusion of methods for molecular confirmation and identification of thermotolerant Campylobacter spp., the use of growth supplement in Preston broth and changes in the performance testing of culture media (ISO 10272-1:2017/Amd 1:2023)
Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 1: Detection method - Amendment 1: Inclusion of methods for molecular confirmation and identification of thermotolerant Campylobacter spp., the use of growth supplement in Preston broth and changes in the performance testing of culture media (ISO 10272-1:2017/Amd 1:2023)
Inclusion of methods for molecular confirmation and identification of thermotolerant Campylobacter spp. and change of the performance testing of culture media
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur Zählung von Campylobacter spp. - Teil 1: Nachweisverfahren - Änderung 1 (ISO 10272-1:2017/Amd 1:2023)
No scope available
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement de Campylobacter spp. - Partie 1: Méthode de recherche - Amendment 1: Ajout de méthodes pour la confirmation et l’identification moléculaires de Campylobacter spp. thermotolérants, de l’utilisation d’un supplément de croissance dans le bouillon de Preston, et modification des essais de performance des milieux de culture (ISO 10272-1:2017/Amd 1:2023)
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila Campylobacter spp. - 1. del: Metoda za ugotavljanje prisotnosti - Dopolnilo A1: Vključitev metod za molekularno potrditev in identifikacijo toplotno stabilnih bakterij Campylobacter spp. z uporabo rastnega dodatka v prestonskem bujonu ter spremembe v preskušanju učinkovitosti gojišč (ISO 10272-1:2017/Amd 1:2023)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 10272-1:2017/A1:2023
01-maj-2023
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Campylobacter spp. - 1. del: Metoda za ugotavljanje
prisotnosti - Dopolnilo A1: Vključitev metod za molekularno potrditev in
identifikacijo toplotno stabilnih bakterij Campylobacter spp. z uporabo rastnega
dodatka v prestonskem bujonu ter spremembe v preskušanju učinkovitosti gojišč
(ISO 10272-1:2017/Amd 1:2023)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 1: Detection method - Amendment 1: Inclusion of methods for
molecular confirmation and identification of thermotolerant Campylobacter spp., the use
of growth supplement in Preston broth and changes in the performance testing of culture
media (ISO 10272-1:2017/Amd 1:2023)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter spp. - Teil 1: Nachweisverfahren - Änderung 1 (ISO 10272-
1:2017/Amd 1:2023)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 1: Méthode de recherche - Amendment
1: Ajout de méthodes pour la confirmation et l’identification moléculaires de
Campylobacter spp. thermotolérants, de l’utilisation d’un supplément de croissance dans
le bouillon de Preston, et modification des essais de performance des milieux de culture
(ISO 10272-1:2017/Amd 1:2023)
Ta slovenski standard je istoveten z: EN ISO 10272-1:2017/A1:2023
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 10272-1:2017/A1:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10272-1:2017/A1:2023
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SIST EN ISO 10272-1:2017/A1:2023
EN ISO 10272-1:2017/A1
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2023
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for
detection and enumeration of Campylobacter spp. - Part 1:
Detection method - Amendment 1: Inclusion of methods
for molecular confirmation and identification of
thermotolerant Campylobacter spp., the use of growth
supplement in Preston broth and changes in the
performance testing of culture media (ISO 10272-
1:2017/Amd 1:2023)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche et le dénombrement de Verfahren zum Nachweis und zur Zählung von
Campylobacter spp. - Partie 1: Méthode de recherche - Campylobacter spp. - Teil 1: Nachweisverfahren -
Amendment 1: Ajout de méthodes pour la confirmation Änderung 1 (ISO 10272-1:2017/Amd 1:2023)
et l'identification moléculaires de Campylobacter spp.
thermotolérants, de l'utilisation d'un supplément de
croissance dans le bouillon de Preston, et modification
des essais de performance des milieux de culture (ISO
10272-1:2017/Amd 1:2023)
This amendment A1 modifies the European Standard EN ISO 10272-1:2017; it was approved by CEN on 29 November 2022.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for inclusion of
this amendment into the relevant national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This amendment exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the
same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10272-1:2017/A1:2023 E
worldwide for CEN national Members.
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SIST EN ISO 10272-1:2017/A1:2023
EN ISO 10272-1:2017/A1:2023 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 10272-1:2017/A1:2023
EN ISO 10272-1:2017/A1:2023 (E)
European foreword
This document (EN ISO 10272-1:2017/A1:2023) has been prepared by Technical Committee ISO/TC 34
"Food products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food
chain” the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by August 2023, and conflicting national standards shall
be withdrawn at the latest by August 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 10272-1:2017/Amd 1:2023 has been approved by CEN as EN ISO 10272-
1:2017/A1:2023 without any modification.
3
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SIST EN ISO 10272-1:2017/A1:2023
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SIST EN ISO 10272-1:2017/A1:2023
INTERNATIONAL ISO
STANDARD 10272-1
Second edition
2017-06
AMENDMENT 1
2023-01
Microbiology of the food chain —
Horizontal method for detection and
enumeration of Campylobacter spp. —
Part 1:
Detection method
AMENDMENT 1: Inclusion of methods
for molecular confirmation and
identification of thermotolerant
Campylobacter spp., the use of growth
supplement in Preston broth and changes
in the performance testing of culture
media
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Campylobacter spp. —
Partie 1: Méthode de recherche
AMENDEMENT 1: Ajout de méthodes pour la confirmation et
l’identification moléculaires de Campylobacter spp. thermotolérants,
de l’utilisation d’un supplément de croissance dans le bouillon de
Preston, et modification des essais de performance des milieux de
culture
Reference number
ISO 10272-1:2017/Amd.1:2023(E)
© ISO 2023
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
A list of all the parts in the ISO 10272 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iii
© ISO 2023 – All rights reserved
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SIST EN ISO 10272-1:2017/A1:2023
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
Microbiology of the food chain — Horizontal method for
detection and enumeration of Campylobacter spp. —
Part 1:
Detection method
AMENDMENT 1: Inclusion of methods for molecular
confirmation and identification of thermotolerant
Campylobacter spp., the use of growth supplement in Preston
broth and changes in the performance testing of culture media
3.1
Replace the text with the following:
3.1
Campylobacter
genus of microorganisms of the family Campylobacteraceae, forming characteristic colonies on solid
selective media, such as modified Charcoal Cefoperazone Deoxycholate (mCCD) agar, when incubated
in a microaerobic atmosphere at 41,5 °C and displaying certain characteristics with biochemical
confirmation tests and by microscopy
Note 1 to entry: Microscopy, the biochemical confirmation tests and the characteristics of Campylobacter are
described in 9.5.
Note 2 to entry: This document targets the thermotolerant Campylobacter species relevant to human health.
The most frequently encountered and relevant to human health are Campylobacter jejuni and Campylobacter coli.
However, other species have been described (Campylobacter lari, Campylobacter upsaliensis and others).
Note 3 to entry: Campylobacter is usually capable of growth in the selective enrichment media Bolton broth and
Preston broth.
4.1
Add the following note after the last paragraph:
NOTE The enrichment broth used in Detection procedure B (Preston broth) can be too selective to allow
[18]
the recovery of strains of Campylobacter species (particularly C. coli), see ISO 17995 . The addition of growth
supplement to Preston broth enhances the recovery of Campylobacter spp. and some strains will not grow
without it. This issue arose after the publication of ISO 10272-1:2017 and is due to properties of the antibiotic
solution. Supporting data are available at: https://st andards. iso. org/i so/10 272/- 1/ed -1 /e n/a md/1 /.
9.5.1
Add the following text after the last paragraph:
NOTE PCR tests for confirmation and species identification are described in Annexes D and E. The results
for the ILS study are described in Annex F.
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
9.6.1, second sentence
Replace the text with the following:
However, other species have been described (Campylobacter lari, Campylobacter upsaliensis and
others); the characteristics given in Table 2 permit their differentiation from Campylobacter jejuni and
Campylobacter coli.
9.6.1
Add the following text as the third sentence:
Additionally, Annex E describes molecular methods for identification of thermotolerant Campylobacter
species, which can be used as an alternative to the biochemical identification described in 9.6.2 to 9.6.5.
9.6.4, second paragraph
Replace the text with the following:
If the indoxyl acetate is hydrolysed, a colour change to blue occurs within 5 min to 10 min. If there is an
unclear result after 10 min, a better result can be obtained after waiting for another 20 min. No colour
change indicates hydrolysis has not taken place.
9.6.5, Table 2
Replace the table with the following:
b b
Characteristic C. jejuni C. coli C. lari C. upsaliensis
Catalase (9.6.2) + + + – or weak
a
Hydrolysis of hippurate (9.6.3) + – – –
c
Indoxyl acetate (9.6.4) + + – +
Key
+ = positive
– = negative
a
Some hippurate-negative C. jejuni strains have been reported.
b
The same characteristics can appear also for other Campylobacter spp.
c
Indoxyl acetate negative C. upsaliensis strains have been reported.
11.1
Add the following text after the first sentence:
The results have been published, see Reference [19].
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
Clause B.3
Replace the clause with the following:
B.3 Preston broth
B.3.1 Basic medium
B.3.1.1 Composition
Enzymatic digest of animal tissues 10,0 g
Peptone 10,0 g
®a
Sodium chloride (CAS Registry Number 7647-14-5) 5,0 g
Water 940 ml
a ®
CAS Registry Number is a trademark of CAS corporation. This information is given for the conven-
ience of users of this document and does not constitute an endorsement by ISO of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
B.3.1.2 Preparation
Dissolve the basic components or the dehydrated complete basic medium in the water, by heating if
necessary.
Adjust the pH, if necessary, so that after sterilization, the pH of the complete medium is 7,4 ± 0,2 at
25 °C. Dispense the basic medium into containers of suitable capacity. Sterilize in the autoclave set at
121 °C for 15 min.
B.3.2 Sterile lysed horse blood
Use horse blood saponin-lysed or lysed by freezing then thawing.
B.3.3 Antibiotic solution
B.3.3.1 Composition
Polymyxin B sulfate (CAS No. 1405-20-5) 5 000 IU
Rifampicin (CAS No. 13292-46-1) 0,01 g
Trimethoprim lactate salt (CAS No. 23256-42-0) 0,01 g
Amphotericin B (CAS No. 1397-89-3) 0,01 g
Ethanol, 95 % (volume fraction) 5 ml
B.3.3.2 Preparation
Dissolve the components in the ethanol.
B.3.4 Growth supplement
B.3.4.1 Composition
Sodium pyruvate (CAS No. 113-24-6) 0,25 g
Sodium metabisulfite (CAS No. 7681-57-4) 0,25 g
Iron(II) sulfate hydrate (CAS No. 13463-43-9) 0,25 g
Water 5 ml
B.3.4.2 Preparation
Dissolve the components in the water and sterilize by filtration. Aliquots of 5 ml should be stored at
(−20 ± 5) °C for not more than 12 months.
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
B.3.5 Complete medium
B.3.5.1 Composition
Basic medium (B.3.1) 940 ml
Sterile lysed horse blood (B.3.2) 50 ml
Antibiotic solution (B.3.3) 5 ml
Growth supplement (B.3.4) 5 ml
B.3.5.2 Preparation
To the basic medium, cooled down to below 47 °C, add the antibiotic solution and then the growth
supplement and finally the lysed blood aseptically, and mix. Dispense the medium aseptically into tubes,
bottles or flasks of suitable capacity to obtain the portions necessary for the test. If the enrichment
medium has been prepared in advance, store it in the dark at 5 °C (6.7) for up to seven days.
Clause B.11, Table B.1
Replace Table B.1 with the following:
Table B.1 — Performance testing of culture media for Campylobacter
Characteristic
Control WDCM Method of reactions
b
Medium Function Incubation Criteria
a
strains numbers control of target
microorganism
(5 ± 1) h /
Campylobacter 00156 or
(37 ± 1) °C
c
jejuni 00005
then
Bolton + Escherichia 00012 or
Productivity (44 ± 4) h /
c
broth coli 00013
(41,5 ± 1) °C
+ Staphylococcus 00032 or
> 10 Greyish, flat and
microaerobic
c
aureus 00034
characteristic moist, sometimes
atmosphere
Qualitative
colonies on with metallic
Campylobacter 00004 or
mCCD agar sheen
c
coli 00072
+ Escherichia 00012 or
c
coli 00013
+ Staphylococcus 00032 or
c
aureus 00034
Escherichia 00012 or
c < 10
coli 00013
Selectivity Qualitative colonies on —
Staphylococcus 00032 or
TSA
c
aureus 00034
a
WDCM: World Data Centre for Microorganisms. Refer to the reference strain catalogue available at www .wfcc .info for information on
[17]
culture strain numbers and contact details.
b
Growth/turbidity is categorized as 0: no growth/turbidity; 1: weak growth/turbidity; 2: good growth/turbidity (see ISO 11133).
c
Strain free of choice, one of the strains has to be used as a minimum.
d
Strain free of choice, one of the Campylobacter strains has to be used as a minimum.
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
TTabablele B B.11 ((ccoonnttiinnueuedd))
Characteristic
Control WDCM Method of reactions
b
Medium Function Incubation Criteria
a
strains numbers control of target
microorganism
Campylobacter 00156 or
c
jejuni 00005
(24 ± 2) h /
(41,5 ± 1) °C
Preston + Escherichia 00012 or
Productivity
c
broth coli 00013
microaerobic
atmosphere
+ Staphylococcus 00032 or
> 10 Greyish, flat and
c
aureus 00034
characteristic moist, sometimes
Qualitative
colonies on with metallic
Campylobacter 00004 or
c mCCD agar sheen
coli 00072
+ Escherichia 00012 or
c
coli 00013
+ Staphylococcus 00032 or
c
aureus 00034
Escherichia 00012 or
c Total
coli 00013
Selectivity Qualitative inhibition (0) —
Staphylococcus 00032 or
on TSA
c
aureus 00034
(44 ± 4) h / Campylobacter 00156 or
Greyish, flat and
c
(41,5 ± 1) °C jejuni 00005
Good moist colonies,
mCCD agar Productivity Qualitative
growth (2) sometimes with
microaerobic Campylobacter 00004 or
c metallic sheen
atmosphere coli 00072
Total or
Escherichia 00012 or partial No characteristic
Selectivity Qualitative
c
coli 00013 inhibition colonies
(0–1)
Staphylococcus 00032 or Total
Qualitative —
c
aureus 00034 inhibition (0)
Campylobacter 00156 or
24 h to 48 h /
d
jejuni 00005
(41,5 ± 1) °C
Columbia Good
Confirmation or or Qualitative —
blood agar growth (2)
microaerobic
Campylobacter 00004 or
atmosphere d
coli 00072
a
WDCM: World Data Centre for Microorganisms. Refer to the reference strain catalogue available at www .wfcc .info for information on
[17]
culture strain numbers and contact details.
b
Growth/turbidity is categorized as 0: no growth/turbidity; 1: weak growth/turbidity; 2: good growth/turbidity (see ISO 11133).
c
Strain free of choice, one of the strains has to be used as a minimum.
d
Strain free of choice, one of the Campylobacter strains has to be used as a minimum.
Annex C
Add the following text after the fourth sentence:
NOTE Validation data were collected from participants using Preston broth without growth supplement as
well as from participants using Preston broth with growth supplement (see 4.1). Therefore, the performance
[19]
characteristics are considered to be still valid and thus re-verification is not required.
After Annex C
Add the following as Annexes D, E and F:
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
Annex D
(informative)
Multiplex real-time PCR assay for confirmation of thermotolerant
Campylobacter spp.
D.1 General
This annex describes a probe-based multiplex real-time PCR method for confirmation of thermotolerant
Campylobacter spp. (C. jejuni, C. coli, C. lari).
D.2 Principle
A specific fragment of the 16S rRNA of thermotolerant Campylobacter spp. C. jejuni, C. coli, C. lari is
amplified by multiplex real-time PCR. The PCR product is detected by measuring fluorescence of the
hydrolysed probe.
D.3 Reagents
[20]
For quality of reagents used, see ISO 22174 . Ready-to-use reagents can be commercially available.
The manufacturer’s instructions for use should be considered.
D.3.1 Reagents for nucleic acid extraction
D.3.1.1 NaCl, 0,9 % (mass fraction).
D.3.1.2 PCR grade water.
D.3.1.3 TE-buffer.
D.3.2 Reagents for real-time PCR
D.3.2.1 PCR grade water.
D.3.2.2 PCR buffer solution, 10×.
NOTE 10× means 10-fold, i.e. the concentration of the PCR buffer.
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 concentration is method
2 2
specific and therefore listed in Table D.2 (see D.5.2).
D.3.2.3 MgCl solution.
2
D.3.2.4 Thermostable Taq DNA polymerase (for hot-start PCR).
D.3.2.5 dNTP solution.
D.3.2.6 Oligonucleotides.
Sequences of the oligonucleotides are listed in Table D.1.
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
D.3.2.7 IPC-ntb2 plasmid.
A vector plasmid carrying a 125-bp sequence of the gene rbcMT-T encoding Ribulose-1,5-bisphosphate
[21]
carboxylase/oxygenase N-methyltransferase from Nicotiana tabacum. The plasmid is used as an
1)
internal amplification control.
Table D.1 — Sequences of oligonucleotides
Gene Primer/probe Sequence (5′ — 3′)
Jos-F1 (forward) CCT GCT TAA CAC AAG TTG AGT AGG
16S rRNA Jos-R1 (reverse) TTC CTT AGG TAC CGT CAG AAT TC
a b
Jos-P (probe) FAM - TGT CAT CCT CCA CGC GGC GTT GCT GC-NFQ
IPC-ntb2-fw (forward) ACC ACA ATG CCA GAG TGA CAA C
Internal amplification
IPC-ntb2-re (reverse) TAC CTG GTC TCC AGC TTT CAG TT
control (IAC)
a b
IPC-ntb2-probe (probe) ROX -CAC GCG CAT GAA GTT AGG GGA CCA-NFQ
a
Equivalent reporter dyes and/or quencher dyes may be used for the probes if they can be shown to yield similar
or better results. The alternative combinations FAM-HEX, FAM-TAMRA, FAM-JOE and FAM-Cy5 have been used with
equivalent result in the validation of the method.
b
NFQ: Non-fluorescence quencher (dark quencher).
D.4 Apparatus
Appropriate equipment according to the method and, in particular, the following shall be used.
D.4.1 Equipment used for nucleic acid extraction
D.4.1.1 Microcentrifuge tubes, of capacities of 1,5 ml and 2,0 ml.
D.4.1.2 Thermo block, obtaining a temperature of 95 °C.
D.4.1.3 Pipettes and pipette filter tips, for volumes between 1 µl and 1 000 µl.
D.4.1.4 Centrifuge, for microcentrifuge tubes having a capacity of 1,5 ml and 2,0 ml, e.g.
microcentrifuge, capable of achieving an acceleration of up to 12 000g. In some steps a refrigerated
centrifuge is required.
D.4.2 Equipment used for real-time PCR
D.4.2.1 Pipettes and pipette filter tips, having a capacity between 1 µl and 1 000 µl.
D.4.2.2 Microcentrifuge tubes, having a capacity of 1,5 ml and 2,0 ml.
D.4.2.3 Thin-walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR microplates or
other suitable consumables.
D.4.2.4 Real-time PCR instrument.
1) The plasmid IPC-ntb2 was used as an internal amplification control in the validation study of the PCR system.
This information is given for convenience of users of this document and does not constitute an endorsement by ISO
of the product named. Alternative internal amplification control systems may be used if they can be shown to give
equivalent or better results. If necessary, adapt the amounts of the reagents and the temperature-time programme.
7
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
D.5 Procedure
D.5.1 Nucleic acid extraction
One 1 µl-loop of suspected colonies (see 9.5.2) is suspended in 1 ml of 0,9 % NaCl solution and DNA is
extracted with a thermal lysis step (15 min at 95 °C). After an additional centrifugation step for 3 min
at 10 000g, 5 µl of the supernatant is used as DNA template. If the DNA will be stored, TE-buffer should
be used instead of 0,9 % NaCl. Other methods for DNA extraction can be used if they have been shown
to be suitable. Before addition to the PCR mastermix, the template should be 100-fold diluted in sterile
water.
D.5.2 PCR set-up
The method is described for a total PCR volume of 25 µl per reaction with the reagents as listed in
Table D.2. The PCR can also be carried out in a larger volume if the solutions are adjusted appropriately.
The final concentrations of reagents as outlined in Table D.2 have proven to be suitable.
Table D.2 — Reagents
Reagent Final concentration Volume per sample
µl
Template DNA (1:100 dilution) maximum 250 ng 2,5 µl
a
Taq DNA Polymerase 1 IU as required
b
PCR-buffer (without MgCl ) 1× as required
2
MgCl solution 2,5 mM as required
2
dNTP solution 0,2 mM of each dNTP as required
PCR primers (according to Table D.1) 500 nM each primer as required
PCR probes (according to Table D.1) 100 nM each probe as required
PCR grade water — as required
IPC-ntb2 plasmid 25 copies per reaction as required
Total volume — 25
a
Hot Start Taq DNA Polymerase was used in the validation of the method.
b
If the PCR buffer solution already contains MgCl , the final concentration of MgCl in the reaction mixture is adjusted
2 2
to 2,5 mM.
D.5.3 PCR controls
[22]
In accordance with ISO 22174 the following controls are necessary:
a) Negative PCR control: PCR grade water is used as negative control.
b) Positive PCR control: DNA from C. jejuni, C. coli or C. lari is used as positive control.
c) Amplification control: The system contains an internal amplification control (see D.3.2.7).
D.5.4 Temperature-time programme
The temperature-time programme as outlined in Table D.3 has been used in the validation of the method
using thermal cyclers Applied Biosystem 7500 Fast, Stratagene MX3000P, Biorad CFX 96 and iCycler
2)
iQ5 . The use of other thermal cyclers can make an adaptation necessary. The time for activation/
initial denaturation depends on the polymerase used.
2) Applied Biosystem 7500 Fast, Stratagene MX3000P, Biorad CFX 96 and iCycler iQ5 are examples of suitable
products available commercially from ThermoFisher Scientific, Agilent Technologies and Bio-Rad. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO of these
products. Equivalent products may be used if they can be shown to give the same results.
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SIST EN ISO 10272-1:2017/A1:2023
ISO 10272-1:2017/Amd.1:2023(E)
Table D.3 — Temperature-time programme
Steps Temperature-time combination
Activation/initial denaturation 3 min/95 °C
Number of cycles (amplification) 45
15 s/95 °C
Amplification 60 s/60 °C
30 s/72 °C
D.6 Interpretation of the results
The threshold value to determine the cycle of threshold (Cq) shall be defined by the analyst or by the
cycler-specific software. A positive sample generates an amplification plot with at least the exponential
[22]
phase of a typical amplification curve, see ISO 22119 . The amplification curve of these samples
crosses the defined threshold setting after a certain number of cycles. A sample with a fluorescence
signal above the threshold is considered positive. In the validation of the method, all true positive
samples generated Cq values below 38.
D.7 Performance characteristics of the method
D.7.1 General
The method (including inhouse validation data) has been published, see References [23] and [24].
Additionally, the performance characteristics of the method were determined in a method comparison
study conducted in two different labo
...
SLOVENSKI STANDARD
SIST EN ISO 10272-1:2017/oprA1:2022
01-februar-2022
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Campylobacter spp. - 1. del: Metoda za ugotavljanje
prisotnosti - Dopolnilo A1: Vključitev metod za molekularno potrditev in
identifikacijo termotolerantnih bakterij Campylobacter spp. ter popravek
preskušanja učinkovitosti gojišč (ISO 10272-1:2017/DAM 1:2021)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 1: Detection method - Amendment 1: Inclusion of methods for
molecular confirmation and identification of thermotolerant Campylobacter spp., and
correction of the performance testing of the media (ISO 10272-1:2017/DAM 1:2021)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter spp. - Teil 1: Nachweisverfahren - Änderung 1 (ISO 10272
1:2017/DAM 1:2021)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 1: Méthode de recherche - Amendement
1: Ajout de méthodes pour la confirmation et l’identification moléculaires de
Campylobacter spp. thermotolérants, et correction des essais de performance des
milieux (ISO 10272-1:2017/DAM 1:2021)
Ta slovenski standard je istoveten z: EN ISO 10272-1:2017/prA1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 10272-1:2017/oprA1:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10272-1:2017/oprA1:2022
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SIST EN ISO 10272-1:2017/oprA1:2022
DRAFT AMENDMENT
ISO 10272-1:2017/DAM 1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2021-11-23 2022-02-15
Microbiology of the food chain — Horizontal method for
detection and enumeration of Campylobacter spp. —
Part 1:
Detection method
AMENDMENT 1: Inclusion of methods for molecular
confirmation and identification of thermotolerant
Campylobacter spp., and correction of the performance testing
of the media
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche et le dénombrement de
Campylobacter spp. —
Partie 1: Méthode de recherche
AMENDEMENT 1
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO 10272-1:2017/DAM 1:2021(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2021
---------------------- Page: 3 ----------------------
SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www.iso.org/iso/foreword.html.
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in collaboration with ISO 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).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
A list of all the parts in the ISO 10272 series can be found on the ISO website.
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SIST EN ISO 10272-1:2017/oprA1:2022
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
Microbiology of the food chain — Horizontal method for
detection and enumeration of Campylobacter spp. —
Part 1:
Detection method
AMENDMENT 1: Inclusion of methods for molecular
confirmation and identification of thermotolerant
Campylobacter spp., and correction of the performance testing
of the media
3.1
Replace the text with the following:
Campylobacter
genus of microorganisms of the family Campylobacteraceae, usually capable of growth, in the selective
enrichment media Bolton broth and Preston broth, forming characteristic colonies on solid selective
media, like modified Charcoal Cefoperozone Deoxycholate agar (mCCD agar), when incubated in
a microaerobic atmosphere at 41,5 °C and displaying certain characteristics with biochemical
confirmation tests and by microscopy
Note 1 to entry Microscopy, the biochemical confirmation tests and the characteristics of Campylobacter are
described in 9.5.
Note 2 to entry This document targets the thermotolerant Campylobacter species relevant to human health.
The most frequently encountered and relevant to human health are Campylobacter jejuni and Campylobacter coli.
However, other species have been described (Campylobacter lari, Campylobacter upsaliensis and others).
4.1
Add the following note after the last paragraph:
Note 1 to entry The enrichment broth used in Detection procedure B (Preston broth) may be too selective to
allow the recovery of some strains of C. coli, see ISO 17995 [18]. The addition of growth supplement to Preston
broth enhances the recovery of Campylobacter spp. and some strains will not grow without it. This issue arose
after the publication of ISO 10272-1:2017 and is due to properties of the antibiotic solution. Supporting data are
available at: https:// standards .iso .org/ iso/ 10272/ -1/ ed -1/ en/ amd/ 1/ .
9.5.1
Add the following text after the last paragraph:
PCR tests for confirmation and species identification are described in Annex D and E. The results for
the ILS study are described in Annex F.
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
9.6.1, second sentence
Replace the text with the following:
However, other species have been described (Campylobacter lari, Campylobacter upsaliensis and
others); the characteristics given in Table 2 permit their differentiation from Campylobacter jejuni and
Campylobacter coli.
9.6.1
Add the following text as third sentence:
Additionally, Annex D and E describe molecular methods for confirmation and identification of
thermotolerant Campylobacter species, which can be used as an alternative to the biochemical
identification described in 9.6.2 to 9.6.5.
9.6.4, second paragraph
Replace the text with the following:
If the indoxyl acetate is hydrolysed, a colour change to blue occurs within 5 min to 10 min. If there is an
unclear result after 10 min., a better result can be obtained after waiting for another 20 min. No colour
change indicates hydrolysis has not taken place.
9.6.5, Table 2
Replace the table with the following:
b b
Characteristic C. jejuni C. coli C. lari C. upsaliensis
Catalase (9.6.2) + + + – or weak
a
Hydrolysis of hippurate (9.6.3) + – – –
c
Indoxyl acetate (9.6.4) + + – +
Key: + = positive; – = negative.
a
Some hippurate-negative C. jejuni strains have been reported.
b
The same characteristics can appear also for other Campylobacter spp.
c
Indoxyl acetate negative C. upsaliensis strains have been reported.
11.1
Add after the first sentence the following:
The results have been published [19].
B.3
Replace the clause with the following:
B.3 Preston broth
B.3.1 Basic medium
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
B.3.1.1 Composition
Enzymatic digest of animal tissues 10,0 g
Peptone 10,0 g
Sodium chloride (CAS No. 7647-14-5) 5,0 g
Water 940 ml
B.3.1.2 Preparation
Dissolve the basic components or the dehydrated complete basic medium in the water, by heating if
necessary.
Adjust the pH, if necessary, so that after sterilization, the pH of the complete medium is 7,4 ± 0,2 at
25 °C. Dispense the basic medium into containers of suitable capacity. Sterilize in the autoclave set at
121 °C for 15 min.
B.3.2 Sterile lysed horse blood
Use horse blood saponin-lysed or lysed by freezing then thawing.
B.3.3 Antibiotic solution
B.3.3.1 Composition
Polymyxin B sulfate (CAS No. 1405-20-5) 5 000 IU
Rifampicin (CAS No. 13292-46-1) 0,01 g
Trimethoprim lactate salt (CAS No. 23256-42-0) 0,01 g
Amphotericin B (CAS No. 1397-89-3) 0,01 g
Ethanol, 95 % (volume fraction) 5 ml
B.3.3.2 Preparation
Dissolve the components in the ethanol.
B.3.4 Growth supplement
B.3.4.1 Composition
Sodium pyruvate (CAS No. 113-24-6) 0,25 g
Sodium metabisulfite (CAS No. 7681-57-4) 0,25 g
Iron(II) sulfate hydrate (CAS No. 13463-43-9) 0,25 g
Water 5 ml
B.3.4.2 Preparation
Dissolve the components in the water and sterilize by filtration. Aliquots of 5 ml should be stored at
(−20 ± 5) °C for not more than twelve months.
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
B.3.5 Complete medium
B.3.5.1 Composition
Basic medium (B.3.2) 940 ml
Sterile lysed horse blood (B.3.2) 50 ml
Antibiotic solution (B.3.3) 5 ml
Growth supplement (B.3.4) 5 ml
B.3.5.2 Preparation
To the basic medium, cooled down to below 47 °C, add the antibiotic solution and then the growth
supplement and finally the lysed blood aseptically, and mix. Dispense the medium aseptically into tubes,
bottles or flasks of suitable capacity to obtain the portions necessary for the test. If the enrichment
medium has been prepared in advance, store it in the dark at 5 °C (6.7) for up to seven days.
B.11, Table B.1
Replace Table B.1 with the following table:
Table B.1 — Performance testing of culture media for Campylobacter
Characteristic
reactions
Control WDCM Method of
c
Medium Function Incubation Criteria of target
a
strains numbers control
microorgan-
ism
Bolton Productiv- (5 ± 1) h /
Campylobacter 00156 or
broth ity (37 ± 1) °C
d
jejuni 00005
then
+ Escherichia 00012 or
(44 ± 4) h /
d
coli 00013
(41,5 ± 1) °C
>10
+ Staphylococ- 00032 or
Greyish, flat
microaerobic
d characteris-
cus aureus 00034
and moist,
atmosphere
Qualitative tic colonies
sometimes with
on mCCD
Campylobacter
metallic sheen
00004
agar
coli
00012 or
+ Escherichia
00013
d
coli
00032 or
+ Staphylococ-
00034
d
cus aureus
Selectivity Escherichia 00012 or
d < 10
coli 00013
Qualitative colonies on —
Staphylococcus 00032 or
TSA
d
aureus 00034
a
WDCM: World Data Centre for Microorganisms. Refer to the reference strain catalogue available at www .wfcc .info for
[17]
information on culture strain numbers and contact details .
b
Not applicable.
c
Growth/turbidity is categorized as 0: no growth/turbidity; 1: weak growth/turbidity; 2: good growth/turbidity (see
ISO 11133).
d
Strain free of choice, one of the strains has to be used as a minimum.
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
Table B.1 (continued)
Characteristic
reactions
Control WDCM Method of
c
Medium Function Incubation Criteria of target
a
strains numbers control
microorgan-
ism
Preston Productiv- (24 ± 2) h / Campylobacter 00156 or
d
broth ity (41,5 ± 1) °C jejuni 00005
microaerobic + Escherichia 00012 or
d
atmosphere coli 00013
>10
+ Staphylococ- 00032 or
Greyish, flat
characteris-
d
cus aureus 00034
and moist,
Qualitative tic colonies
sometimes with
Campylobacter
on mCCD
00004
metallic sheen
coli
agar
00012 or
+ Escherichia
00013
d
coli
00032 or
+ Staphylococ-
00034
d
cus aureus
Selectivity Escherichia 00012 or
d Total
coli 00013
Qualitative inhibition —
Staphylococcus 00032 or
(0) on TSA
d
aureus 00034
mCCD Productiv- (44 ± 4) h / Campylobacter
Greyish, flat and
00156 or
d
agar ity (41,5 ± 1) °C jejuni
Good moist colonies,
00005
Qualitative
growth (2) sometimes with
microaerobic Campylobacter
00004
metallic sheen
atmosphere coli
Selectivity Total or
Escherichia 00012 or partial No characteris-
Qualitative
d
coli 00013 inhibition tic colonies
(0–1)
Total
Staphylococcus 00032 or
Qualitative inhibition —
d
aureus 00034
(0)
Columbia Productiv- 24 h to 48 h / 00156
blood ity (41,5 ± 1) °C
Campylobacter
or
agar
d
jejuni
microaerobic Good
00005 Qualitative
atmosphere growth (2)
Campylobacter
d or
coli
00004
a
WDCM: World Data Centre for Microorganisms. Refer to the reference strain catalogue available at www .wfcc .info for
[17]
information on culture strain numbers and contact details .
b
Not applicable.
c
Growth/turbidity is categorized as 0: no growth/turbidity; 1: weak growth/turbidity; 2: good growth/turbidity (see
ISO 11133).
d
Strain free of choice, one of the strains has to be used as a minimum.
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
Annex C
Add after the fourth sentence the following:
NOTE The introduction of growth supplement to the composition of Preston broth (see 4.1) does not have an
impact on the results of the validation study. The results are still valid and does not need to be verified [19].
After Annex C
Add the following as Annexes D, E and F:
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
Annex D
(informative)
Multiplex real-time-PCR assay for confirmation of thermotolerant
Campylobacter spp.
D.1 Introduction
This annex describes a probe-based multiplex real-time PCR method based on 5’ exonuclease activity
for the detection of a fragment of the 16S rRNA of thermotolerant Campylobacter spp. This assay can be
used for confirmation of thermotolerant Campylobacter spp. [20].
D.2 Principle
A specific fragment of the 16S rRNA of thermotolerant Campylobacter spp. is amplified by multiplex-
real-time-PCR. The PCR product is detected by measuring fluorescence of the hydrolysed probe.
D.3 Reagents
D.3.1 General
For quality of reagents used, see ISO 22174 [22]. Ready-to-use reagents may be commercially available.
The manufacturer’s instructions for use should be considered. All information concerning commercially
available products in the document is given for convenience of users of this document and does not
constitute an endorsement by ISO of the product names. Equivalent products from other manufacturers
may be used if they can be shown to give equivalent or better results. If necessary, adapt the amounts of
the reagents and the temperature-time programme.
D.3.2 Reagents for nucleic acid extraction
D.3.2.1 NaCl, 0,9%.
D.3.2.2 PCR grade Water.
D.3.2.3 TE-buffer.
D.3.3 Reagents for real-time-PCR
D.3.3.1 PCR grade Water.
D.3.3.2 PCR buffer solution, 10 x.
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 concentration is method
2 2
specific and therefore listed in Table D.2.
D.3.3.3 MgCl solution.
2
D.3.3.4 Thermostable Taq DNA polymerase (for hot-start PCR).
D.3.3.5 dNTP solution.
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
D.3.3.6 Oligonucleotides
Sequences of the oligonucleotides are listed in Table D.1.
D.3.3.7 ntb2-plasmid
A vector plasmid carrying a 125-bp sequence of the gene rbcMT-T encoding Ribulose-1,5-bisphosphate
carboxylase/oxygenase N-methyltransferase from Nicotiana tabacum [27].
Table D.1 — Sequences of oligonucleotides
gene Primer/probe Sequence (5´ — 3´)
Jos-F1 CCT GCT TAA CAC AAG TTG AGT AGG
16S rRNA [20] Jos-R1 TTC CTT AGG TAC CGT CAG AAT TC
a b
Jos-P FAM - TGT CAT CCT CCA CGC GGC GTT GCT GC-NFQ
IPC-ntb2-fw ACC ACA ATG CCA GAG TGA CAA C
Internal Amplification
IPC-ntb2-re TAC CTG GTC TCC AGC TTT CAG TT
Control (IAC)
a b
IPC-ntb2-probe ROX -CAC GCG CAT GAA GTT AGG GGA CCA-NFQ
a
Equivalent reporter dyes and/or quencher dyes may be used for the probes if they can be shown to yield similar
or better results. The alternative combinations FAM-HEX, FAM-TAMRA, FAM-JOE and FAM-Cy5 have been used with
equivalent result in the validation of the method.
b
NFQ: Non-fluorescence quencher (dark quencher)
D.4 Apparatus
D.4.1 General
Appropriate equipment according to the method and, in particular, the following.
D.4.2 Equipment used for nucleic acid extraction
D.4.2.1 Microcentrifuge tubes, of capacities of 1,5 ml and 2,0 ml.
D.4.2.2 Thermo block, with a mixing frequency between 300 rpm and 1 400 rpm.
D.4.2.3 Pipettes and pipette filter tips, for volumes between 1 µl and 1 000 µl.
D.4.2.4 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 x g. In some steps a refrigerated centrifuge is
required.
D.4.3 Equipment used for real-time-PCR
D.4.3.1 Pipettes and pipette filter tips, having a capacity between 1 µl and 1 000 µl.
D.4.3.2 Microcentrifuge tubes, having a capacity of 1,5 ml and 2,0 ml.
D.4.3.3 Thin-walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR microplates or
other suitable consumables.
D.4.3.4 Real-time PCR instrument.
8
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
D.5 Procedure
D.5.1 Nucleic acid extraction
One 1 µl-loop of suspected colonies is (see 9.5.2.2) suspended in 1 ml of 0,9 % NaCl solution and DNA is
extracted with a thermal lysis step (15 min at 95° C). After an additional centrifugation step for 3 min
at 10 000 x g 5 µl of the supernatant is used as DNA template. If the DNA will be stored, TE-buffer should
be used instead of 0,9 % NaCl. Other methods for DNA extraction can be used if they have been shown
to be suitable. Before addition to the PCR mastermix, the template should be 100-fold diluted in sterile
water.
D.5.2 PCR-Setup
The method is described for a total PCR volume of 25 µl per reaction with the reagents as listed in
Table D.2. The PCR can also be carried out in a larger volume if the solutions are adjusted appropriately.
The final concentrations of reagents as outlined in Table D.2 have proven to be suitable.
Table D.2 — Reagents
Reagent Final concentration Volume per sample (µl)
Template DNA (1:100 dilution) maximum 250 ng 2,5 µl
a
Taq DNA Polymerase 1 IU as required
b
PCR-buffer (without MgCl ) 1 x as required
2
MgCl solution 2,5 mM as required
2
dNTP solution 0,2 mM of each dNTP as required
PCR primers (according to Table D.1) 500 nM each primer as required
PCR probes (according to Table D.1) 100 nM each probe as required
PCR grade water / as required
IPC-ntb2-plasmid 25 copies per reaction as required
Total volume / 25
a
Hot Start Taq DNA Polymerase was used in the validation of the method.
b
If the PCR buffer solution already contains MgCl , the final concentration of MgCl in the reaction mixture is adjusted
2 2
to 2,5 mM.
D.5.3 PCR controls
In accordance with ISO 22174 [22] the following controls are necessary.
D.5.3.1 Negative PCR control
PCR grade water is used as negative control.
D.5.3.2 Positive PCR control
DNA from C. jejuni, C. coli or C. lari is used as positive control.
D.5.3.3 Amplification control
The system contains an internal amplification control (see D.3.3.7).
D.5.4 Temperature-time programme
The temperature-time programme as outlined in Table D.3 has been used in the validation of the method
using thermal cyclers Applied Biosystem 7500 Fast, Stratagene MX3000P, Biorad CFX 96 and iCycler
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iQ5. The use of other thermal cyclers might make an adaptation necessary. The time for activation/
initial denaturation depends on the polymerase used.
Table D.3 — Temperature-time program
Activation/initial denaturation 3 min/95 °C
Number of cycles (amplification) 45
15 s/95 °C
Amplification 60 s/60 °C
30 s/72 °C
D.6 Interpretation of the results
The threshold value to determine the cycle of threshold (Cq) shall be defined by the analyst or by the
cycler-specific software. A positive sample generates an amplification plot with at least the exponential
phase of a typical amplification curve, see ISO 22119 [23]. The amplification curve of these samples
crosses the defined threshold setting after a certain number of cycles. A sample with a fluorescence
signal above the threshold is considered positive. In the validation of the method, all true positive
samples generated Cq values below 38.
D.7 Performance characteristics of the method
D.7.1 General
The method (including inhouse validation data) has been published [20, 21]. Additionally, the
performance characteristics of the method were determined in a method comparison study conducted
in two different laboratories and in an interlaboratory study following ISO 16140-6 [3]. The data of the
interlaboratory study are summarised in Annex F.
D.7.2 Theoretical evaluation of the method
In silico evaluation was done by performing a sequence similarity search against the GenBank/
® th
EMBL/DDBJ database (NCBI Blast search, EMBL database, September 22 , 2015 [will be updated
before publication]). The result of the search confirmed a 100 % identity only with the expected target
sequences.
D.7.3 Inclusivity
The inclusivity of the method was tested in the method comparison study with 104 C. jejuni, 105 C. coli
and 56 C. lari strains (in total 265 strains of thermotolerant Campylobacter spp.). The method showed
the expected results in comparison to the reference method (see also Table D.4).
D.7.4 Exclusivity
The exclusivity of the method was tested in the method comparison study with 66 non-target
Campylobacter spp., and 76 strains other than Campylobacter spp. (in total 142 strains). The strains
showed the expected results in comparison to the reference method (see also Table D.4).
Table D.4 — Inclusivity and exclusivity
Number of strains Inclusivity Inclusivity Exclusivity Exclusivity
agreement deviation agreement deviation
Inclusivity 265 265 0 Not applicable Not applicable
Exclusivity 142 Not applicable Not applicable 142 0
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NOTE Both inclusivity and exclusivity of the method showed better results compared to the reference
method. False-positive results were obtained for 2 C. upsaliensis, 1 C. peloridis and 1 C. insulaenigrae strain, but
the latter did not grow on the selective media at 41,5 °C. The reference method could not distinguish between the
target organisms (C. jejuni, C. coli and C. lari), and other Campylobacter spp. able to grow on the selective media at
41,5 °C.
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SIST EN ISO 10272-1:2017/oprA1:2022
ISO 10272-1:2017/DAM 1:2021(E)
Annex E
(informative)
PCR methods for molecular confirmation and identification of
thermotolerant Campylobacter spp.
E.1 Introduction
This annex describes both a gel-based multiplex PCR assay and a probe-based multiplex real-time PCR
assay for confirmation and identification of thermotolerant Campylobacter spp. [24].
E.2 Gel-based multiplex PCR assay for confirmation and identification of
thermotolerant Campylobacter spp.
E.2.1 Introduction
This annex describes a method for the amplification and detection of genes specific for different
species of thermotolerant Campylobacter (C. jejuni, C. coli, C. lari, and C. upsaliensis) using agarose gel
electrophoresis.
E.2.2 Principle
Specific DNA fragments of the genes specific for the different Campylobacter spp. are amplified by
multiplex-PCR using five primer pairs. The detection of the PCR products is done using agarose gel-
electrophoresis.
E.2.3 Reagents
E.2.3.1 General
For quality of reagents used, see ISO 22174 [22]. Ready-to-use reagents may be commercially available.
The manufacturer’s instructions for use should be considered. All information concerning commercially
available products in the document is given for convenience of users of this document and does not
constitute an endorsement by ISO of the product names. Equivalent products from other manufacturers
may be used if they can be shown to give equivalent or better results. If necessary, adapt the amounts of
the reagents and the temperature-time programme.
E.2.3.2 Reagents for nucleic acid extraction
E.2.3.2.1 NaCl, 0,9%.
E.2.3.2.2 TE-buffer.
E.2.3.3 Reagents for PCR
E.2.3.3.1 PCR grade Water.
1)
E.2.3.3.2 PCR buffer solution, 10 x .
1) 10 × means 10-fold; i.e. the concentration of the PCR buffer.
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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 concentration is method
2 2
specific and therefore listed in Table E.2.
E.2.3.3.3 MgCl solution.
2
E.2.3.3.4 Thermostable Taq DNA polymerase.
E.2.3.3.5 dNTP solution.
E.2.3.3.6 Oligonucleotides.
Sequences of the oligonucleotides are listed in Table E.1.
Table E.1 — Sequences of oligonucleotides
Species (gene) Primer Sequence (5´ — 3´) Amplicon size (bp)
C. jejuni (hipO) CJF ACT TCT TTA TTG CTT GCT GC 323
[24]
CJR GCC ACA ACA AGT AAA GAA GC
C. coli (glyA) [24] CCF GTA AAA CCA AAG CTT ATC GTG 126
CCR TCC AGC AAT GTG TGC AAT G
C. lari (cpn60) JH0015 TCT GCA AAT TCA GAT GAG AAA A 180
[25]
JH0016 TTT TTC AGT ATT TGT AAT GAA ATA TGG
C. upsaliensis CUF AAT TGA AAC TCT TGC TAT CC 204
(glyA) [24]
CUR TCA TAC ATT TTA CCC GAG CT
Campylobacter 23SF TAT ACC GGT AAG GAG TGC TGG AG 650
spp. (23S rRNA)
23SR ATC AAT TAA CCT TCG AGC ACC G
[24]
NOTE the system detecting Campylobacter spp. (23S rRNA) can also be used as an Internal Campylobacter
Amplification control (IAC). The method comparison study showed that this system (23S rRNA) also targets
Arcobacter and Helicobacter spp.
E.2.3.4 Reagents for gel electrophoresis
E.2.3.4.1 General.
The agarose gel electrophoresis may be carried out with TAE buffer or TBE buffer. Solutions as
described in this method do not usually need to be autoclaved.
E.2.3.4.2 Agarose, suitable for DNA electrophoresis and for the intended size separation of the DNA
molecules.
E.2.3
...
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