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: Aufnahme von Verfahren zur molekularen Bestätigung und Identifizierung von thermotoleranten Campylobacter spp., Verwendung von Wachstumszusatz zur Preston-Bouillon und Änderungen der Leistungsprüfung von Nährmedien (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
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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.
Contents Page
European foreword . 3
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.
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 10272-1:2017/Amd.1:2023(E)
© 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
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Published in Switzerland
ii
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 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.
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].
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.
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.
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:
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.
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.
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.
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
MgCl solution 2,5 mM as required
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.
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 laboratories and in an interlaboratory study in accordance with
[25]
ISO 16140-6 , see Reference [26]. The data of the interlaboratory study are summarized 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/EMBL/
® 3)
DDBJ database (NCBI Blast search , EMBL database, 22 September 2015). The result of the search
confirmed a 100 % similarity only with the expected target sequences.
NOTE A 100 % similarity only with the expected target sequences does not exclude the presence of false-
positive and/or false-negative results. These are addressed in the original publications and in Table D.4.
D.7.3 Inclusivity and exclusivity
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 strains showed
the expected results in comparison with the reference method (see also Table D.4).
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 with the reference method (see also Table D.4).
Table D.4 — Inclusivity and exclusivity
Inclusivity/ Number of strains Inclusivity Inclusivity Exclusivity Exclusivity
exclusivity agreement deviation agreement deviation
Inclusivity 265 265 0 Not applicable Not applicable
Exclusivity 142 Not applicable Not applicable 142 0 ®
3) NCBI Blast search is an example of a suitable product freely available under https:// blast .ncbi .nlm .nih
.gov/ Blast .cgi. This information is given for the convenience of users of this document and does not constitute an
endorsement by ISO of this product. Equivalent products may be used if they can be shown to give the same results.
ISO 10272-1:2017/Amd.1:2023(E)
NOTE Table D.4 shows a comparison of the results of the reference method with the results of the PCR method
described in Annex D. Considering the real identity of the strains, false-positive results were obtained with both
the reference method and the Annex D PCR method for 2 C. upsaliensis, 1 C. peloridis and 1 C. insulaenigrae strains,
but the latter did not grow on the selective media at 41,5 °C. The reference method was not able to distinguish
between the target organisms of the Annex D PCR method (C. jejuni, C. coli and C. lari), and other Campylobacter
spp. able to grow on the selective media at 41,5 °C.
ISO 10272-1:2017/Amd.1:2023(E)
Annex E
(informative)
PCR methods for molecular confirmation and identification of
thermotolerant Campylobacter spp.
E.1 General
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.
E.2 Gel-based multiplex PCR assay for confirmation and identification of
thermotolerant Campylobacter spp.
E.2.1 General
This clause 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
[20]
For quality of reagents used, see ISO 22174 . Ready-to-use reagents may be commercially available.
The manufacturer’s instructions for use should be considered.
E.2.3.1 Reagents for nucleic acid extraction
E.2.3.1.1 NaCl, 0,9 % (mass fraction).
E.2.3.1.2 TE-buffer.
E.2.3.2 Reagents for PCR
E.2.3.2.1 PCR grade water.
E.2.3.2.2 PCR buffer solution, 10×.
The PCR buffer solution is usually delivered with the DNA polymerase, which may include MgCl in
a concentration specified by the manufacturer. The final MgCl concentration is method specific and
therefore listed in Table E.2 (see E.2.5.2).
E.2.3.2.3 MgCl solution.
E.2.3.2.4 Thermostable Taq DNA polymerase.
E.2.3.2.5 dNTP solution.
E.2.3.2.6 Oligonucleotides.
ISO 10272-1:2017/Amd.1:2023(E)
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
[27]
C. jejuni (hipO) CJF (forward) ACT TCT TTA TTG CTT GCT GC 323
CJR (reverse) GCC ACA ACA AGT AAA GAA GC
[27]
C. coli (glyA) CCF (forward) GTA AAA CCA AAG CTT ATC GTG 126
CCR (reverse) TCC AGC AAT GTG TGC AAT G
[28]
C. lari (cpn60) JH0015 (forward) TCT GCA AAT TCA GAT GAG AAA A 180
JH0016 (reverse) TTT TTC AGT ATT TGT AAT GAA ATA TGG
C. upsaliensis (glyA) CUF (forward) AAT TGA AAC TCT TGC TAT CC 204
[27]
CUR (reverse) TCA TAC ATT TTA CCC GAG CT
Campylobacter spp. 23SF (forward) TAT ACC GGT AAG GAG TGC TGG AG 650
[28]
(23S rRNA)
23SR (reverse) ATC AAT TAA CCT TCG AGC ACC G
NOTE The system detecting Campylobacter spp. (23S rRNA) can also be used as an internal amplification
control (IAC) for Campylobacter. The method comparison study showed that this system (23S rRNA) also targets
Arcobacter and Helicobacter spp.
E.2.3.3 Reagents for gel electrophoresis
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.2.3.3.1 Agarose, suitable for DNA electrophoresis and for the intended size separation of the DNA
fragments.
E.2.3.3.2 Boric acid (H BO ), for electrophoresis with TBE buffer system only.
3 3
E.2.3.3.3 Bromophenol blue (C H Br O SNa) and/or xylene cyanole FF (C H N O S Na).
19 9 4 5 25 27 2 6 2
E.2.3.3.4 DNA molecular mass standard, e.g. a commercial preparation containing DNA fragments
from very high to very low molecular mass (but to be at least 100 bp).
E.2.3.3.5 Glacial acetic acid (CH COOH), for electrophoresis with the TAE buffer system only.
E.2.3.3.6 Ethylenediaminetetraacetic acid disodium salt (Na -EDTA) (C H N O Na ).
2 10 14 2 8 2
E.2.3.3.7 Ethidium bromide (EtBr) (C H N Br) or other appropriate DNA intercalating dyes.
21 20 3
Take care when using ethidium bromide solution as it is mutagenic/teratogenic. Other intercalating
dyes can be used, but refer to the manufacturer's material safety data sheet.
E.2.3.3.8 Glycerol (C H O ).
3 8 3
E.2.3.3.9 Sodium acetate (C H O Na), for electrophoresis with TAE buffer only.
2 3 2
E.2.3.3.10 Hydrochloric acid, w (HCl) = 37 % (volume fraction).
E.2.3.3.11 Sodium hydroxide (NaOH).
E.2.3.3.12 Tris(hydroxymethyl)-aminomethane (Tris) (C H NO ).
4 11 3
E.2.3.3.13 TAE buffer solution (1×), c (Tris) = 0,050 mol/l, c (C H O Na) = 20 mmol/l, c (Na -
2 3 2 2
EDTA) = 0,001 mol/l.
ISO 10272-1:2017/Amd.1:2023(E)
Adjust the pH to 8,0 with glacial acetic acid or NaOH at 25 °C. It is advisable to prepare the TAE buffer
solution as a concentrated stock solution (maximum 50-fold concentrated). Discard it if a precipitate is
visible. Dilution of the concentrated electrophoresis buffer can be carried out, immediately before its
use, with non-sterile, (mono)-distilled or deionized water.
E.2.3.3.14 TBE buffer solution (0,5×), c (Tris) = 0,055 mol/l, c (boric acid) = 0,055 mol/l,
c (Na EDTA) = 0,001 mol/l.
Adjust the pH to 8,0 with HCl or NaOH at 25 °C. It is advisable to prepare the TBE buffer solution as
a concentrated stock solution (maximum 10-fold concentrated). Discard it if precipitation is visible.
Dilution of the concentrated electrophoresis buffer can be carried out, immediately before its use, with
non-sterile, (mono)-distilled or deionized water.
When using TBE take care that it is toxic to reproduction and teratogenic. The TAE buffer is preferable.
E.2.3.3.15 Sample loading buffer solution (5×), c (glycerol) = 50 % (volume fraction), ρ
(bromophenol blue) = 2,5 g/l and/or c (xylene cyanol) = 2,5 g/l, dissolved in electrophoresis
buffer solution.
NOTE Other concentrations of loading buffer solution can also be used.
E.2.4 Apparatus
Appropriate equipment according to the method and, in particular, the following shall be used.
E.2.4.1 Equipment used for thermal lysis
E.2.4.1.1 Microcentrifuge tubes, of capacities of 1,5 ml and 2,0 ml.
E.2.4.1.2 Thermo block, with a temperature capacity up to +95 °C.
E.2.4.1.3 Graduated pipettes and pipette filter tips, for volumes between 1 µl and 1 000 µl.
E.2.4.1.4 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.
E.2.4.1.5 Mixer, e.g. type vortex.
E.2.4.2 Equipment used for PCR
E.2.4.2.1 Pipettes and pipette filter tips, having a capacity between 1 µl and 1 000 µl.
E.2.4.2.2 Microcentrifuge tubes, having a capacity of 1,5 ml and 2,0 ml.
E.2.4.2.3 Thin-walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR microplates
or other suitable equipment.
E.2.4.2.4 Thermal cycler.
E.2.4.3 Equipment used for detection of the PCR product
E.2.4.3.1 Microwave oven or boiling water bath.
E.2.4.3.2 Horizontal gel system.
E.2.4.3.3 Power supply.
E.2.4.3.4 UV transilluminator or UV light box.
E.2.4.3.5 Gel documentation system.
ISO 10272-1:2017/Amd.1:2023(E)
E.2.5 Procedure
E.2.5.1 Nucleic acid (DNA) 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 2,5 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.
E.2.5.2 PCR set-up
The method is described for a total PCR volume of 25 µl per reaction, containing 2,5 µl of template
DNA, with the reagents as listed in Table E.2. The PCR can also be carried out in a larger volume if the
solutions are adjusted accordingly. The final concentrations of reagents as outlined in Table E.2 have
proven to be suitable.
Table E.2 — Reagents
Reagent Final concentration Volume per sample
µl
Template DNA maximum 250 ng 2,5
Taq DNA polymerase 1,25 IU as required
a
PCR-buffer (without MgCl ) 1× as required
MgCl solution 2 mM as required
dNTP solution 0,2 mM of each as required
PCR primers C. jejuni and C. lari (according 0,5 µM each as required
to Table E.1)
PCR primers C. coli (according to Table E.1) 1 µM each as required
PCR primers C. upsaliensis (according to Table E.1) 2 µM each as required
PCR primers 23S rRNA (according to Table E.1) 0,2 µM each as required
PCR grade water — as required
Total volume — 25
a
If the PCR buffer solution already contains MgCl , the final concentration of MgCl in the reaction mixture is adjusted
2 2
to 2 mM.
E.2.5.3 PCR controls
[20]
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 Campylobacter spp., positive for all target sequences (C. jejuni, C.
coli, C. lari, and C. upsaliensis) is used as positive control.
c) Amplification control: The primer-system for detection of 23S rRNA of Campylobacter genus is used
as internal amplification control.
E.2.5.4 Temperature-time programme
The temperature-time programme as outlined in Table E.3 has been used in the validation of the ®
method using thermal cyclers Eppendorf Mastercycler Gradient, Bio-Rad S1000 and T100, Analytic
ISO 10272-1:2017/Amd.1:2023(E)
4)
Jena Biometra, Applied Biosystems Veriti and Simpli Amp . The use of other thermal cyclers can make
an adaptation necessary. The time for activation/initial denaturation depends on the polymerase used.
Table E.3 — Temperature-time programme
Steps Temperature-time combination
Activation/initial denaturation 3 min/95 °C
30 s/95 °C
Amplification 30 s/59 °C
30 s/72 °C
Number of cycles (amplification) 30
Final extension 7 min/72 °C
E.2.5.5 Detection of PCR products (gel electrophoresis)
E.2.5.5.1 General
The agarose gel electrophoresis may be carried out with TAE buffer or with TBE buffer. Use the same
buffer to dissolve the agarose and to fill the electrophoresis tank.
E.2.5.5.2 Agarose gel preparation
The amplified PCR products should be detected using a 1,5 % (mass fraction) agarose gel. Weigh an
appropriate amount of agarose (E.2.3.3.1) and add it to the electrophoresis buffer solution (E.2.3.3.13
or E.2.3.3.14). Allow the solution to boil in a microwave oven or in a water bath (E.2.4.3.1) until the
agarose is completely dissolved, cool down the solution to about 60 °C and keep it at this temperature
until use. Prepare a gel support (gel tray) with a suitable sample comb placed in the right position. Pour
the agarose solution onto the gel tray and allow the gel to solidify at room temperature (1 h is usually
recommended).
E.2.5.5.3 Agarose gel electrophoresis
Following the amplification step add the loading buffer to 10 µl PCR product in the ratio 1:4 (e.g. add
2,5 µl of loading buffer to 10 µl of PCR product) and mix.
NOTE Other concentrations of loading buffer solution can also be used.
Carefully remove the sample comb from the gel. Transfer the gel (with its gel tray) to the electrophoresis
cell, so that the wells are close to the cathode (negative electrode). Fill the cell with the electrophoresis
buffer (E.2.3.3.13 or E.2.3.3.14). Overlay the gel with approximately 2 mm of the same buffer and load
the mixture to the sample slots (wells) using a micropipette. If the unknown samples are suspected to
be too concentrated, also prepare some dilutions and load them onto the gel.
To determine the size of the PCR products, add the loading buffer (E.2.3.3.15) and DNA molecular mass
standard (E.2.3.3.4) in the proportion of 1:4. The DNA molecular mass standard is loaded on the gel at
least before the first and after the last sample well.
Carry out the electrophoresis at room temperature at the appropriate voltage and power intensity
(gen
...
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