SIST EN ISO 10272-2:2017/A1:2023
(Amendment)Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique - Amendment 1: Inclusion of methods for molecular confirmation and identification of thermotolerant Campylobacter spp. and changes in the performance testing of culture media (ISO 10272-2:2017/Amd 1:2023)
Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique - Amendment 1: Inclusion of methods for molecular confirmation and identification of thermotolerant Campylobacter spp. and changes in the performance testing of culture media (ISO 10272-2:2017/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 2: Koloniezählverfahren - Änderung 1 (ISO 10272-2:2017/Amd 1:2023)
No scope available
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement de Campylobacter spp. - Partie 2: Technique par comptage des colonies - Amendment 1: Ajout de méthodes pour la confirmation et l’identification moléculaires de Campylobacter spp. thermotolérants, et modification des essais de performance des milieux de culture (ISO 10272-2:2017/Amd 1:2023)
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila Campylobacter spp. - 2. del: Tehnika štetja kolonij - Dopolnilo A1: Vključitev metod za molekularno potrditev in identifikacijo toplotno stabilnih bakterij Campylobacter spp. ter spremembe pri preskušanju učinkovitosti gojišč (ISO 10272-2:2017/Amd 1:2023)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 10272-2:2017/A1:2023
01-maj-2023
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Campylobacter spp. - 2. del: Tehnika štetja kolonij - Dopolnilo
A1: Vključitev metod za molekularno potrditev in identifikacijo toplotno stabilnih
bakterij Campylobacter spp. ter spremembe pri preskušanju učinkovitosti gojišč
(ISO 10272-2:2017/Amd 1:2023)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 2: Colony-count technique - Amendment 1: Inclusion of
methods for molecular confirmation and identification of thermotolerant Campylobacter
spp. and changes in the performance testing of culture media (ISO 10272-2:2017/Amd
1:2023)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter spp. - Teil 2: Koloniezählverfahren - Änderung 1 (ISO 10272
-2:2017/Amd 1:2023)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 2: Technique par comptage des colonies
- Amendment 1: Ajout de méthodes pour la confirmation et l’identification moléculaires
de Campylobacter spp. thermotolérants, et modification des essais de performance des
milieux de culture (ISO 10272-2:2017/Amd 1:2023)
Ta slovenski standard je istoveten z: EN ISO 10272-2:2017/A1:2023
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 10272-2:2017/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 10272-2:2017/A1:2023
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SIST EN ISO 10272-2:2017/A1:2023
EN ISO 10272-2:2017/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 2:
Colony-count technique - Amendment 1: Inclusion of
methods for molecular confirmation and identification of
thermotolerant Campylobacter spp. and correction of the
performance testing of the media (ISO 10272-2:2017/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 2: Technique par comptage Campylobacter spp. - Teil 2: Koloniezählverfahren -
des colonies - Amendment 1: Ajout de méthodes pour Änderung 1 (ISO 10272-2:2017/FDAM 1:2022)
la confirmation et l'identification moléculaires de
Campylobacter spp. thermotolérants, et modification
des essais de performance des milieux de culture (ISO
10272-2:2017/FDAM 1:2022)
This amendment A1 modifies the European Standard EN ISO 10272-2:2017; 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 10272-2:2017/A1:2023 E
worldwide for CEN national Members.
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SIST EN ISO 10272-2:2017/A1:2023
EN ISO 10272-2:2017/A1:2023 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 10272-2:2017/A1:2023
EN ISO 10272-2:2017/A1:2023 (E)
European foreword
This document (EN ISO 10272-2:2017/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 10272-2:2017/Amd 1:2023 has been approved by CEN as EN ISO 10272-
2:2017/A1:2023 without any modification.
3
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SIST EN ISO 10272-2:2017/A1:2023
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SIST EN ISO 10272-2:2017/A1:2023
INTERNATIONAL ISO
STANDARD 10272-2
First edition
2017-06
AMENDMENT 1
2023-01
Microbiology of the food chain —
Horizontal method for detection and
enumeration of Campylobacter spp. —
Part 2:
Colony-count technique
AMENDMENT 1: Inclusion of methods
for molecular confirmation and
identification of thermotolerant
Campylobacter spp. 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 2: Technique par comptage des colonies
AMENDEMENT 1: Ajout de méthodes pour la confirmation et
l’identification moléculaires de Campylobacter spp. thermotolérants,
et modification des essais de performance des milieux de culture
Reference number
ISO 10272-2:2017/Amd.1:2023(E)
© ISO 2023
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/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 10272-2:2017/A1:2023
ISO 10272-2:2017/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.
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SIST EN ISO 10272-2:2017/A1:2023
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/Amd.1:2023(E)
Microbiology of the food chain — Horizontal method for
detection and enumeration of Campylobacter spp. —
Part 2:
Colony-count technique
AMENDMENT 1: Inclusion of methods for molecular
confirmation and identification of thermotolerant
Campylobacter spp. 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 9.4.
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.
9.4.1
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.
9.5.1, 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.
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/Amd.1:2023(E)
9.5.1
Add the following text as the second paragraph:
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 9.5.2 to 9.5.5.
9.5.4, 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.
9.5.5, Table 2
Replace the table with the following:
b b
Characteristic C. jejuni C. coli C. lari C. upsaliensis
Catalase (9.5.2) + + + – or weak
a
Hydrolysis of hippurate (9.5.3) + – – –
c
Indoxyl acetate (9.5.4) + + – +
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 [12].
Clause B.2
Replace the text with the following:
See the ISO 6887 series.
Clause B.9, Table B.1
Replace the table with the following:
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/Amd.1:2023(E)
Table B.1 — Performance testing of culture media for Campylobacter
Characteristic
Refer- Method
Control WDCM reactions of
b
Medium Function Incubation ence of Criteria
a
strains numbers target
media control
microorganism
Campylobacter 00156 or
Greyish, flat and
c
jejuni 00005
Blood Quantita- moist, sometimes
Productivity P ≥ 0,5
R
agar tive with metallic
Campylobacter 00004 or
sheen
c
coli 00072
(44 ± 4) h/
(41,5 ± 1) °C
Total or
mCCD
Escherichia 00012 or Qualita- partial No characteristic
agar
microaerobic —
c
coli 00013 tive inhibition colonies
atmosphere
(0 to 1)
Selectivity
Total
Staphylococcus 00032 or Qualita-
— inhibition —
c
aureus 00034 tive
(0)
Campylobacter 00156 or
24 h to 48 h/
d
Columbia jejuni 00005
(41,5 ± 1) °C
Confirma- Qualita- Good
blood or or — —
tion tive growth (2)
microaerobic
agar Campylobacter 00004 or
d
atmosphere
coli 00072
a
WDCM: World Data Centre for Microorganisms. Refer to the reference strain catalogue available at www .wfcc .info for information on
[10]
culture strain numbers and contact details.
b
Growth is categorized as 0: no growth; 1: weak growth; 2: good growth, P = productivity ratio (see ISO 11133).
R
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.
After Annex C
Add the following as Annexes D, E and F:
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/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 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
[13]
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 10272-2:2017/A1:2023
ISO 10272-2:2017/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
[14]
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.
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/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
[13]
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 10272-2:2017/A1:2023
ISO 10272-2:2017/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
[15]
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 [16] and [17].
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
[18]
ISO 16140-6 , see Reference [19]. 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.
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/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.
8
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/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
[13]
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
2
a concentration specified by the manufacturer. The final MgCl concentration is method specific and
2
therefore listed in Table E.2 (see E.2.5.2).
E.2.3.2.3 MgCl solution.
2
E.2.3.2.4 Thermostable Taq DNA polymerase.
E.2.3.2.5 dNTP solution.
E.2.3.2.6 Oligonucleotides.
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SIST EN ISO 10272-2:2017/A1:2023
ISO 10272-2:2017/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
[20]
C. jejuni (hipO) CJF (forward) ACT TCT TTA TTG CTT GCT GC 323
CJR (reverse) GCC ACA ACA AGT AAA GAA GC
[20]
C. coli (glyA) CCF (forward) GTA AAA CCA AAG CTT ATC GTG 126
CCR (reverse) TCC AGC AAT GTG TGC AAT G
[21]
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
[20]
CUR (reverse) TCA TAC ATT TTA CCC GAG CT
Campylobacter spp. 23SF (forward) TAT ACC GGT AAG GAG TGC TGG AG 650
[20]
(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 ge
...
SLOVENSKI STANDARD
SIST EN ISO 10272-2:2017/oprA1:2022
01-februar-2022
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti in števila Campylobacter spp. - 2. del: Tehnika štetja kolonij - Dopolnilo
A1: Vključitev metod za molekularno potrditev in identifikacijo termotolerantnih
bakterij Campylobacter spp. ter popravek preskušanja učinkovitosti gojišč (ISO
10272-2:2017/DAM 1:2021)
Microbiology of the food chain - Horizontal method for detection and enumeration of
Campylobacter spp. - Part 2: Colony-count technique - Amendment 1: Inclusion of
methods for molecular confirmation and identification of thermotolerant Campylobacter
spp. and correction of the performance testing of the media (ISO 10272-2:2017/DAM
1:2021)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis und zur
Zählung von Campylobacter spp. - Teil 2: Koloniezählverfahren - Änderung 1 (ISO
10272 2:2017/DAM 1:2021)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le
dénombrement de Campylobacter spp. - Partie 2: Technique par comptage des colonies
- Amendement 1: Ajout de méthodes de confirmation et d’identification moléculaires de
Campylobacter spp. thermotolérants, et correction des essais de performance des
milieux (ISO 10272-2:2017/DAM 1:2021)
Ta slovenski standard je istoveten z: EN ISO 10272-2:2017/prA1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 10272-2:2017/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 10272-2:2017/oprA1:2022
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SIST EN ISO 10272-2:2017/oprA1:2022
DRAFT AMENDMENT
ISO 10272-2:2017/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 2:
Colony-count technique
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 2: Technique par comptage des colonies
AMENDEMENT 1
ICS: 07.100.30
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 10272-2:2017/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 10272-2:2017/oprA1:2022
ISO 10272-2:2017/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
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Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/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 on 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 10272-2:2017/oprA1:2022
ISO 10272-2:2017/DAM 1:2021(E)
Microbiology of the food chain — Horizontal method for
detection and enumeration of Campylobacter spp. —
Part 2:
Colony-count technique
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, forming characteristic colonies on
solid selective media, like modified Charcoal Cefoperozone Deoxycholate agar (mCCD agar) 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 9.4.
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).
9.4.1
Add the following text after the last paragraph:
PCR tests for confirmation and species identification are described in Annex D and E.
9.5.1, 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.
9.5.1
Add the following text as the third paragraph:
1
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/DAM 1:2021(E)
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 9.5.2 to 9.5.5.
9.5.4, second sentence
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.
9.5.5
Replace the table with the following table:
b b
Characteristic C. jejuni C. coli C. lari C. upsaliensis
Catalase (9.5.2) + + + – or weak
a
Hydrolysis of hippurate (9.5.3) + – – –
c
Indoxyl acetate (9.5.4) + + – +
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 [18].
B.2
Replace the text with the following:
See ISO 6887.
B.9, Table B.1
Replace by the following Table:
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ISO 10272-2:2017/DAM 1:2021(E)
Table B.1 — Performance testing of culture media for Campylobacter
Charac-ter-
istic
WDCM Reference Method of reactions
c
Medium Function Incubation Control strains Criteria
a
numbers media control of target
microor-
ganism
mCCD Productivity Campylobacter 00156 or Greyish,
d
agar jejuni 00005 flat and
Quantita- moist,
Blood agar P ≥ 0,5
Campylobacter 00004
R
tive sometimes
coli
with me-
(44 ± 4) h/
tallic sheen
(41,5 ± 1) °C
d
Selectivity Escherichia coli 00012 Total or No
microaerobic
partial char-ac-
or 00013 — Qualitative
atmosphere
inhibition teristic
(0–1) colonies
Staphylococcus 00032 or Total
d
aureus 00034 — Qualitative inhibition —
(0)
Columbia Productivity 24 h to 48 h/ Campylobacter 00156 Media
, d
blood agar (41,5 ± 1) °C jejuni batch
Good
or 00005
blood agar Qualitative
growth
microaerobic Campylobacter
already
or
d
atmosphere coli
validated
00004 —
Media
batch
mCCDA Qualitative P ≥ 0,7
R
already
validated
a
WDCM: World Data Centre for Microorganisms. Refer to the reference strain catalogue available at www .wfcc .info for
[10]
information on culture strain numbers and contact details .
b
Not applicable.
c
Growth is categorized as 0: no growth; 1: weak growth; 2: good growth, P = productivity ratio (see ISO 11133).
R
d
Strain free of choice, one of the strains has to be used as a minimum.
After Annex C
Add the following as Annexes D, E and F:
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/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. [19].
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 [21]. 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 T E-bu f fer.
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 Mg C l solution.
2
D.3.3.4 Thermost able Taq DNA polymerase (for hot-start PCR).
D. 3. 3.5 dN TP solut ion .
D.3.3.6 Oligonucleot ides.
Sequences of the oligonucleotides are listed in Table D.1.
4
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/DAM 1:2021(E)
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 [26].
Table A.1 — Sequences of oligonucleotides
gene Primer/probe Sequence (5´ — 3´)
Jos-F1 CCT GCT TAA CAC AAG TTG AGT AGG
16S rRNA 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 Ther mo 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 × 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 Microcentrif uge t ubes, 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.
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 × 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.
5
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/DAM 1:2021(E)
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 [21] 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.3.2.7).
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
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
6
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ISO 10272-2:2017/DAM 1:2021(E)
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 [22]. 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 [19, 20]. 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 to ISO 16140-6 [3]. 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/DDBJ database (NCBI Blast search, EMBL database, September 22th, 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
I nc lu s i v i t y Inclusivity devi- Exclusivity agree- Exclusivity de-
Number of strains
agreement ation ment viation
Inclusivity 265 265 0 Not applicable Not applicable
Exclusivity 142 Not applicable Not applicable 142 0
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.
7
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/DAM 1:2021(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 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 [21]. 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 .
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
1) 10 × means 10-fold; i.e. the concentration of the PCR buffer
8
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ISO 10272-2:2017/DAM 1:2021(E)
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) [23] CJF ACT TCT TTA TTG CTT GCT GC 323
CJR GCC ACA ACA AGT AAA GAA GC
C. coli (glyA) [23] CCF GTA AAA CCA AAG CTT ATC GTG 126
CCR TCC AGC AAT GTG TGC AAT G
C. lari (cpn60) [24] JH0015 TCT GCA AAT TCA GAT GAG AAA A 180
JH0016 TTT TTC AGT ATT TGT AAT GAA ATA TGG
C. upsaliensis (glyA) CUF AAT TGA AAC TCT TGC TAT CC 204
[23]
CUR TCA TAC ATT TTA CCC GAG CT
23SF TAT ACC GGT AAG GAG TGC TGG AG
Campylobacter spp.
650
(23S rRNA) [23]
23SR ATC AAT TAA CCT TCG AGC ACC G
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.2.3.4.3 Boric acid (H BO ), for the TBE buffer system only.
3 3
E.2.3.4.4 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.2.3.4.5 DNA molecular mass standard, for example a commercial preparation containing DNA
fragments from very high to very low molecular mass.
E.2.3.4.6 Glacial acetic acid (CH COOH), for the TAE buffer system only.
3
E.2.3.4.7 Ethylenediaminetetraacetic acid disodium salt (Na -EDTA) (C H N O Na ).
2 10 14 2 8 2
E.2.3.4.8 Ethidium bromide (EtBr) (C H N Br) or other appropriate DNA intercalating dyes.
21 20 3
NOTE In case of using Ethidium bromide solution take care that it is mutagenic/teratogenic. Other
intercalating dyes can be used referring to the Manufacturer's Material Safety Data Sheet.
E.2.3.4.9 Glycerol (C H O ).
3 8 3
E.2.3.4.10 Sodium acetate (C H O Na), for the TAE buffer system only.
2 3 2
E.2.3.4.11 Hydrochloric acid, c (HCl) = 37 %.
E.2.3.4.12 Sodium hydroxide (NaOH).
E.2.3.4.13 Tris(hydroxymethyl)-aminomethane (Tris) (C H NO ).
4 11 3
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/DAM 1:2021(E)
E.2.3.4.14 TAE buffer solution (1x), 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.
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 buffers can be carried out, immediately before its
use, with non-sterile, (mono)-distilled or deionised water.
E.2.4.4.15 Tris/borate (TBE) buffer solution (0,5x), c (Tris) = 0,055 mol/l, c (boric
acid) = 0,055 mol/l, c (Na EDTA) = 0,001 mol/l.
2
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 buffers can be carried out, immediately before its use,
with non-sterile, (mono)-distilled or deionised water.
NOTE In case of using TBE take care that it is toxic to reproduction and teratogenic. The TAE buffer is
preferable.
E.2.4.4.16 Sample loading buffer solution (5x), c (glycerol) = 50 %, 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.
E.2.4 Apparatus
E.2.4.1 General
Appropriate equipment according to the method and, in particular, the following.
E.2.4.2 Equipment used for thermal lysis
E.2.4.2.1 Microcentrifuge tubes, of capacities of 1,5 ml and 2,0 ml.
E.2.4.2.2 Thermo block, with a mixing frequency between 300 rpm and 1 400 rpm.
E.2.4.2.3 Graduated pipettes and pipette filter tips, for volumes between 1 µl and 1 000 µl.
E.2.4.2.4 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 g. In some steps a refrigerated
centrifuge is required.
E.2.4.2.5 Mixer, e.g. type Vortex.
E.2.4.3 Equipment used for PCR
E.2.4.3.1 Pipettes and pipette filter tips, having a capacity between 1 µl and 1 000 µl.
E.2.4.3.2 Microcentrifuge tubes, having a capacity of 1,5 ml and 2,0 ml.
E.2.4.3.3 Thin-walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR
microplates or other suitable equipment.
E.2.4.3.4 Thermal cycler.
E.2.4.4 Equipment used for detection of the PCR product
E.2.4.4.1 Microwave oven or boiling water bath.
E.2.4.4.2 Horizontal gel system.
E.2.4.4.3 Power supply.
E.2.4.4.4 UV transilluminator or UV light box.
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SIST EN ISO 10272-2:2017/oprA1:2022
ISO 10272-2:2017/DAM 1:2021(E)
E.2.4.4.5 Gel documentation system.
E.2.5 Procedure
E.2.5.1 Nucleic acid (DNA) extraction
One 1 µl-loop of suspected colonies (see 9.5.2.2) 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 10 000 × g 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 x As required
2
MgCl solution 2 mM As required
2
dNTP solution 0,2 mM of each As required
PCR primers C. jejuni and C. lari (according to Table E.1) 0,5 µM each As required
PCR primers C. coli (according to Table E.1) 1 µM each As required
PCR prime
...
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