EN ISO 10272-2:2017/prA1

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)

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/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)

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)

General Information

• Status: Not Published
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463/WG 3 - Campylobacter
• Current Stage: 4599 - Dispatch of FV draft to CMC - Finalization for Vote
• Due Date: 15-Feb-2021
• Ref Project: SIST EN ISO 10272-2:2017/oprA1:2022 - 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)

RELATIONS

• Amends: EN ISO 10272-2:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp. - Part 2: Colony-count technique (ISO 10272-2:2017)
• Effective Date: 30-Jan-2019

Standards Content (sample)

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
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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

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Published in Switzerland
© ISO 2021 – All rights reserved
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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.
iii
© ISO 2021 – All rights reserved
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SIST EN ISO 10272-2:2017/oprA1:2022
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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:
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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
Hydrolysis of hippurate (9.5.3) + – – –
Indoxyl acetate (9.5.4) + + – +
Key: + = positive; – = negative.
Some hippurate-negative C. jejuni strains have been reported.
The same characteristics can appear also for other Campylobacter spp.
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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SIST EN ISO 10272-2:2017/oprA1:2022
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
Medium Function Incubation Control strains Criteria
numbers media control of target
microor-
ganism
mCCD Productivity Campylobacter 00156 or Greyish,
agar jejuni 00005 flat and
Quantita- moist,
Blood agar P ≥ 0,5
Campylobacter 00004
tive sometimes
coli
with me-
(44 ± 4) h/
tallic sheen
(41,5 ± 1) °C
Selectivity Escherichia coli 00012 Total or No
microaerobic
partial char-ac-
or 00013 — Qualitative
atmosphere
inhibition teristic
(0–1) colonies
Staphylococcus 00032 or Total
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
atmosphere coli
validated
00004 —
Media
batch
mCCDA Qualitative P ≥ 0,7
already
validated

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 .
Not applicable.

Growth is categorized as 0: no growth; 1: weak growth; 2: good growth, P = productivity ratio (see ISO 11133).

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:
© ISO 2021 – All rights reserved
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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.
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.
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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

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.
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.
© ISO 2021 – All rights reserved
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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
Taq DNA Polymerase 1 IU as required
PCR-buffer (without MgCl ) 1 x 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
Hot Start Taq DNA Polymerase was used in the validation of the method.

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
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SIST EN ISO 10272-2:2017/oprA1:2022
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.
© ISO 2021 – All rights reserved
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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.
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.
1) 10 × means 10-fold; i.e. the concentration of the PCR buffer
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SIST EN ISO 10272-2:2017/oprA1:2022
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.

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.

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
PCR-buffer (without MgCl ) 1 x 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 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
...