Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of presumptive Bacillus cereus - Colony-count technique at 30 degrees C - Amendment 1: Inclusion of optional tests (ISO 7932:2004/Amd 1:2020, Corrected version 2020-08)

Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur Zählung von präsumtivem Bacillus cereus - Koloniezählverfahren bei 30 °C - Änderung 1: Aufnahme optionaler Testmethoden (ISO 7932:2004/Amd 1:2020, korrigierte Fassung 2020-08)

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Microbiologie des aliments - Méthode horizontale pour le dénombrement de Bacillus cereus présomptifs - Technique par comptage des colonies à 30 degrés C - Amendement 1: Ajout de tests optionnels (ISO 7932:2004/Amd 1:2020, Version corrigée 2020-08)

Mikrobiologija živil in krme - Splošno navodilo za štetje domnevno prisotnih Bacillus cereus - Štetje kolonij pri 30 °C - Dopolnilo A1: Vključitev izbirnega preskusa (ISO 7932:2004/Amd 1:2020)

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Publication Date
14-Apr-2020
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6060 - Definitive text made available (DAV) - Publishing
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15-Apr-2020
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15-Apr-2020

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SLOVENSKI STANDARD
SIST EN ISO 7932:2005/A1:2020
01-junij-2020
Mikrobiologija živil in krme - Splošno navodilo za štetje domnevno prisotnih
Bacillus cereus - Štetje kolonij pri 30 °C - Dopolnilo A1: Vključitev izbirnega
preskusa (ISO 7932:2004/Amd 1:2020)

Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration

of presumptive Bacillus cereus - Colony-count technique at 30 degrees C - Amendment

1: Inclusion of optional tests (ISO 7932:2004/Amd 1:2020)

Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur Zählung

von präsumtivem Bacillus cereus - Koloniezählverfahren bei 30 °C - Änderung 1:
Aufnahme optionaler Testmethoden (ISO 7932:2004/Amd 1:2020)

Microbiologie des aliments - Méthode horizontale pour le dénombrement de Bacillus

cereus présomptifs - Technique par comptage des colonies à 30 degrés C -
Amendement 1: Ajout de tests optionnels (ISO 7932:2004/Amd 1:2020)
Ta slovenski standard je istoveten z: EN ISO 7932:2004/A1:2020
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 7932:2005/A1:2020 en

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

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SIST EN ISO 7932:2005/A1:2020
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SIST EN ISO 7932:2005/A1:2020
EN ISO 7932:2004/A1
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2020
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of food and animal feeding stuffs - Horizontal
method for the enumeration of presumptive Bacillus
cereus - Colony-count technique at 30 degrees C -
Amendment 1: Inclusion of optional tests (ISO
7932:2004/Amd 1:2020)

Microbiologie des aliments - Méthode horizontale pour Mikrobiologie von Lebensmitteln und Futtermitteln -

le dénombrement de Bacillus cereus présomptifs - Horizontales Verfahren zur Zählung von präsumtivem

Technique par comptage des colonies à 30 degrés C - Bacillus cereus - Koloniezählverfahren bei 30 °C -

Amendement 1: Ajout de tests optionnels (ISO Änderung 1: Aufnahme optionaler Testmethoden (ISO

7932:2004/Amd 1:2020) 7932:2004/Amd 1:2020)

This amendment A1 modifies the European Standard EN ISO 7932:2004; it was approved by CEN on 22 March 2020.

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, Turkey 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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 7932:2004/A1:2020 E

worldwide for CEN national Members.
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SIST EN ISO 7932:2005/A1:2020
EN ISO 7932:2004/A1:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 7932:2005/A1:2020
EN ISO 7932:2004/A1:2020 (E)
European foreword

This document (EN ISO 7932:2004/A1:2020) 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 October 2020, and conflicting national standards shall

be withdrawn at the latest by October 2020.

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.

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, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 7932:2004/Amd1:2020 has been approved by CEN as EN ISO 7932:2004/A1:2020

without any modification.
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SIST EN ISO 7932:2005/A1:2020
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SIST EN ISO 7932:2005/A1:2020
INTERNATIONAL ISO
STANDARD 7932
Third edition
2004-06-15
AMENDMENT 1
2020-03
Microbiology of food and animal
feeding stuffs — Horizontal method
for the enumeration of presumptive
Bacillus cereus — Colony-count
technique at 30 degrees C
AMENDMENT 1: Inclusion of optional
tests
Microbiologie des aliments — Méthode horizontale pour le
dénombrement de Bacillus cereus présomptifs — Technique par
comptage des colonies à 30 degrés C
AMENDEMENT 1: Ajout de tests optionnels
Reference number
ISO 7932:2004/Amd.1:2020(E)
ISO 2020
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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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Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(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 ISO Technical Committee 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).

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.
© ISO 2020 – All rights reserved iii
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SIST EN ISO 7932:2005/A1:2020
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
Microbiology of food and animal feeding stuffs —
Horizontal method for the enumeration of presumptive
Bacillus cereus — Colony-count technique at 30 degrees C
AMENDMENT 1: Inclusion of optional tests
In the Scope
Designate the existing NOTE as NOTE 1 and add the following new NOTE:
[21][22]

NOTE 2 The diversity within the Bacillus cereus group is large with 7 phylogenetic groups and a growing

number of species.
After 9.4
Add the following new subclause 9.5:
9.5 Optional tests
9.5.1 General

All the tests mentioned below are optional and intended for complementary investigations

(i.e. epidemiological) on isolated Bacillus cereus group strains obtained in 9.4.1, following the procedures

described in Annexes C to F.

In this amendment, the term “B. cereus group” is used instead of “presumptive B. cereus”, as it is

[28]

scientifically more precise, as explained in the EFSA scientific opinion published in 2016 .

9.5.2 Detection of cytK-1 or cytK-2 gene variants of the gene encoding Cytotoxin K

Some strains within the B. cereus group bacteria carry one of the two variants found for the gene

[17][22]

encoding Cytotoxin K, cytK-1 and cytK-2. The cytK-1 gene is specific to Bacillus cytotoxicus and

[20]

thus constitutes the possibility to rapidly identify B. cytotoxicus . The procedure in Annex C describes

a validated PCR method that targets both cytK gene variants and, if present, indicates which of the two

forms is present. It also allows confirmation of isolates as B. cytotoxicus.
9.5.3 Detection of Bacillus cereus group strains able to produce cereulide

Some strains within the B. cereus group bacteria are able to produce a heat-stable dodecadepsipeptide,

named cereulide. This cereulide, when produced in food, can cause an emetic food poisoning syndrome.

[10]
NOTE The method for cereulide quantification is described in ISO 18465 .
[16]

A cereulide peptide synthetase (ces) gene is involved in the non-ribosomal synthesis of cereulide .

The procedure in Annex D describes a rapid and validated PCR method that targets the ces gene.

9.5.4 Motility test for B. anthracis screening

The motility test described in Annex E allows for screening for presumptive B. anthracis among isolated

B. cereus group bacteria.

NOTE This test has nevertheless strong limitations as indicated in Annex E (see E.1 and Table E.1).

© ISO 2020 – All rights reserved 1
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)

9.5.5 Microscopic examination of the parasporal crystal from Bacillus thuringiensis

B. thuringiensis, one of the B. cereus group species, can be distinguished from the other species of this

group by the microscopic examination of the parasporal crystal formation.

The procedure for the examination of the parasporal crystal formation is described in Annex F.

After Annex B
Add the following as Annexes C, D, E and F.
2 © ISO 2020 – All rights reserved
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
Annex A
(informative)
Polymerase chain reaction for the detection of cytK-1 or cytK-2
gene variants of cytotoxin K in isolated strains of Bacillus cereus
group and identification of Bacillus cytotoxicus
A.1 General

The chromosomally located cytK-2 gene encodes cytotoxin K, an enterotoxin that is present in B. cereus

[22]
sensu stricto and B. thuringiensis strains .
[29]

Presence of cytK-2 genes are also mentioned in strains of other B. cereus group species . CytK-1 gene

is a variant of cytK-2 gene due to a marked polymorphism and encodes to a more cytotoxic form of

[18]
cytotoxin K that is present only in B. cytotoxicus .

This method is applicable to well-isolated colonies of B. cereus group strains, after appropriate

preparation of the DNA.
A.2 Principles
A.2.1 General
The method comprises the following consecutive steps:
a) nucleic acid extraction;
b) amplification of target gene and interpretation.
A.2.2 Nucleic acid extraction

Bacterial cells are harvested from well isolated colonies and the nucleic acid is extracted for use in PCR

reaction.
A.2.3 Amplification of target gene and interpretation

The extracted nucleic acid is selectively amplified using PCR. Detection of the PCR products is achieved

by electrophoresis on agarose. Interpretation is deduced from presence or absence of the expected band.

A.3 Reagents
A.3.1 General

All reagents needed for this annex are molecular grade reagents and consumables suitable for molecular

[11] [12]
biology. They shall be used as given in ISO 20837 and ISO 20838 .
A.3.2 Nucleic acid extraction

Nucleic acid extraction procedure and reagents appropriate for Gram-positive bacteria shall be used.

Commercial kits can also be used.
© ISO 2020 – All rights reserved 3
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
A.3.3 Reagents for PCR
[14] [12]
Refer to ISO 22174 and ISO 20838 .
A.3.4 Primers
The primers used for detection of cytotoxin K genes are listed in Table C.1.

Table C.1 — Sequences of oligonucleotides, characteristics and resulting amplicon

Gene Position on Amplicon
Primer Sequence (5′ – > 3′)
variant cytK gene size (bp)
CK1F F CAA TTC CAG GGG CAA GTG TC cytK-1 314–333
Accession
426
CK1R R CCT CGT GCA TCT GTT TCA TGA G 740–719
number
DQ885233.1
CK2F F CAA TCC CTG GCG CTA GTG CA cytK-2 314–333
Accession
585
CK2R R GTG IAG CCT GGA CGA AGT TGG 899–879
number
AJ318876.2
Key
F: forward
R: reverse
Make reference to the publicly available nucleotide sequences available at
http:// www .www .ncbi .nlm .nih .gov
A.4 Equipment and consumables
A.4.1 General
Appropriate equipment according to the method and, in particular, the following.
A.4.2 Equipment for nucleic acid extraction
C.4.2.1 Micro-centrifuge tubes, with capacities of 1,5 ml or 2,0 ml.

C.4.2.2 Centrifuge, for reaction tubes with a capacity of 1,5 ml or 2,0 ml and capable of achieving an

acceleration up to approximately 14 000 g.
C.4.2.3 Thermoblock, with heating capacity of up to 100 °C.

C.4.2.4 Graduated pipettes and pipette filter tips, for volumes between 1 μl to 1 000 μl.

C.4.2.5 Mixer.
A.4.3 Equipment for PCR
C.4.3.1 PCR thermal cycler.

C.4.3.2 Thin-walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR microplates or

other suitable equipment.
A.4.4 Equipment for the detection of PCR products
[12]
Refer to ISO 20838 .
4 © ISO 2020 – All rights reserved
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
A.5 Procedure
A.5.1 General
See Figure C.1.
Figure C.1 — Flow diagram for PCR detection of cytotoxin K gene

(cytK-1 or cytK-2 variants) in B. cereus group strains and identification of B. cytotoxicus

A.5.2 Nucleic acid extraction

Confirmed B. cereus group colonies according to 9.4 should be used for DNA extraction. Prior to DNA

extraction, the colonies can be optionally washed by centrifugation in 1 ml of nuclease free water. Any

nucleic acid extraction procedure appropriate for Gram-positive bacteria suitable for this purpose can

be used (e.g. Reference [15]).

A 10 µl loopfull of colony material is harvested (from MYP or non selective agars) and suspended in 1 ml

of nuclease free water, pelleted at 11 000g for 15 min. The pellet is resuspended in 500 µl extraction

buffer [1,7 g/l sodium dodecylsulfate, 200 mmol/l Tris-HCl (pH 8), 20 mmol/l EDTA, 200 mmol/l NaCl].

The suspension is incubated at 55 °C for 1 h with 25 µl of proteinase K (10 µg/µl). DNA is extracted

with one volume of phenol and subsequently with one volume of chloroform. The aqueous phase is

precipitated with 2,5 volumes of cold ethanol (100 % volume fraction) and centrifuged at 11 000g for

20 min. The supernatant is discarded and the pellet washed once with 800 µl of cold ethanol (70 %

volume fraction). After drying, the pellet is dissolved in 50 µl nuclease free water and stored at −20 °C.

DNA amount is quantified by absorbance at 260 nm in a spectrophotometer and shall be adjusted to a

concentration compatible with the sensitivity of the PCR (see C.6.3).

Other methods or commercial ready-to-use purification kits can be used if controls (see C.5.3.2) are

scrupulously used.
© ISO 2020 – All rights reserved 5
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
A.5.3 PCR amplification
A.5.3.1 General

The total PCR volume is 15 μl per reaction. The reagents are listed in Table C.2. The final concentrations

of reagents as outlined in the table have proven to be suitable.
Table C.2 — PCR reaction reagents
Reagent (concentration) Final concentration Volume per reaction (μl)
DNA polymerase buffer (10X) 1x 1,5
dNTPs mix (5 mmol/l each) 0,2 mmol/l each 0,6
CK1F (10 μmol/l) 0,25 µmol/l 0,375
CK1R (10 μmol/l) 0,25 µmol/l 0,375
CK2F (10 μmol/l) 0,25 µmol/l 0,375
CK2R (10 μmol/l) 0,25 µmol/l 0,375
MgCl (25 mmol/l) 2,5 mmol/l 1,5
DNA polymerase 0,75 U 0,15
Template DNA (Genomic - 25 ng/µl) 2,5
Adjust the volume to 15 μl using nuclease free water
a 1)

This protocol has been validated using commercially available AmpliTaq®10x Buffer and AmpliTaq® polymerase

and Master Mix containing the four dNTPs.

AmpliTaq®10x Buffer and AmpliTaq® polymerase are products supplied by Applied Biosystems, Forster City, CA, USA.

Master Mix is a product supplied by Eurogentec. This information is given for the convenience of users of this document and

does not constitute an endorsement by ISO of the products named. Equivalent products can be used if they can be shown to

lead to the same results.

Different protocols for PCR amplification can be used, depending on the DNA polymerase and DNA

preparation that is used. However, the PCR reaction shall be stringent, using the primers described

in Table C.1 with appropriate hybridization temperature (see Table C.4) and appropriate controls (see

C.5.3.2), with the reliability of primers being validated with a specific hybridization temperature. The

control strains are listed in Table C.3.
Table C.3 — Control strains to be included in PCR assays
WDCM number (species) cytK-1 cytK-2
WDCM 00218 Negative control Positive control
(Bacillus cereus)
WDCM 00220 Positive control Negative control
(Bacillus cytotoxicus)
WDCM 00222 (Bacillus Negative control Negative control
weihenstephanensis)

Refer to the reference strain catalogue available on http:// www .wfcc .info for information on

culture collection strain numbers and contact details.
A.5.3.2 PCR controls
[14]

All appropriate controls as given in ISO 22174 shall be performed. At least a positive and a negative

control, represented for each gene variant by a known positive and a known negative bacterial strain

DNA respectively, shall be included in the PCR assay to check the conditions of amplification.

[14]

DNA positive controls (including process controls as given in ISO 22174 ) should be obtained by the

same DNA extraction protocol as used for test isolates.
6 © ISO 2020 – All rights reserved
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
A.5.3.3 Temperature-time programme

The temperature-time programme, as outlined in Table C.4, has been used in the evaluation study.

Table C.4 — Temperature-time programme
Initial denaturation 94 °C for 5 min
Denaturation 94 °C for 15 s
Amplification Hybridization 57 °C for 30 s
Elongation 72 °C for 30 s
Number of cycles 30
Final extension 72 °C for 7 min
A.5.3.4 Detection of the PCR products

The PCR products are detected after electrophoresis on agarose gel (1,5 %) with an appropriate

[12]
molecular weight marker (refer to ISO 20838 ).
A.5.4 Interpretation of the PCR result

The result obtained, including the controls specified above (see C.5.3.2), should be as follows. Otherwise,

the PCR shall be repeated.
The PCR result will be one of the following:

a) positive for cytK-1 variant, if a specific PCR product of 426 bp has been detected and all the controls

give expected results, or

b) positive for cytK-2 variant, if a specific PCR product of 585 bp has been detected and all the controls

give expected results, or

c) negative for cytotoxin K genes, if a specific PCR product has not been detected, and all controls give

expected results.
A.5.5 Confirmation of the PCR product
[14]
Refer to ISO 22174 .
A.6 Performance characteristics
A.6.1 General

This method was evaluated in a single-laboratory validation study. It was tested on a total of 160 B.

[23]

cereus group strains and 10 outgroup species, including Southern blotting or PCR product sequencing ,

[22]

and then applied on 391 strains . The assay turned out to be highly reliable with 0 % false-negative

[25]

reactions and 0 % false-positive reactions. BLASTN analysis also showed that there were no targets

in the bacterial organisms other than cytK gene for the four primers included in the PCR reaction. The

[23]

specificity for cytK-1 and cytK-2 variant was 100 % under the PCR conditions described and with the

recommended controls (see C.5.3.2).
A.6.2 Selectivity
A.6.2.1 General

Selectivity was performed in duplex PCR using the primers CK1F, CK1R, CK2F, CK2R, working by couple

(CK1F/CK1R and CK2F/CK2R). It was checked that the respective primer pairs run exclusively on cytK-1

variant or cytK-2 variant and target low conserved regions between the two variants.

© ISO 2020 – All rights reserved 7
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
A.6.2.2 Inclusivity

Inclusivity of the PCR assay for cytK-2 fragment was tested on 66 target strains. A 100 % inclusivity

was obtained. Inclusivity of the PCR assay for cytK-1 fragment was tested on 5 target strains. A

100 % inclusivity was obtained. The tested strains for cytK-2 variant were B. cereus sensu stricto and

B. thuringiensis strains. Target strains for cytK-1 correspond to all 5 B. cytotoxicus strains that were

available at the time of evaluation. They were enough distant from the remaining species of the group

to exhibit a particular polymorphism resulting in cytK-1 variant for cytK gene.
A.6.2.3 Exclusivity

Exclusivity of the assay for cytK-1 fragment was tested on 157 non-target strains. A 100 % exclusivity

was obtained. Exclusivity of the assay for cytK-2 fragment was tested on 94 non-target strains. A 100 %

exclusivity was obtained.

Strains tested were from all species of the B. cereus group and eight diverse food-related species.

A.6.3 Sensitivity

The limits for PCR amplification ranged from 15 ng to 75 ng of genomic DNA per 15 µl of final reaction.

A.7 Limitations of the PCR assay

This PCR assay allows to a) detect cytotoxin K genes (and particularly one of its two variants), and b)

identify B. cytotoxicus strains, two characteristics that are both associated with food poisoning. Even if

that indicates a potential risk, the sole presence of the gene or species identification does not allow to

confirm cytK gene expression.
8 © ISO 2020 – All rights reserved
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
Annex B
(informative)
Polymerase chain reaction for the detection of ces gene encoding
cereulide peptide synthetase in strains of Bacillus cereus group
B.1 Gel-based PCR assay for detection of ces gene encoding cereulide peptide
synthetase in strains of Bacillus cereus group
B.1.1 General

This annex describes a method for the amplification and detection of the ces gene specific for the

production of cereulide in Bacillus cereus group strains using agarose gel electrophoresis.

B.1.2 Principle

Specific DNA fragment of the ces gene is amplified by PCR using two primers. The detection of the PCR

product is done using agarose gel-electrophoresis.
B.1.3 Reagents
B.1.3.1 General

All reagents needed for this annex are molecular grade reagents and consumables suitable for molecular

[11] [12]
biology. They shall be used as given in ISO 20837 and ISO 20838 .
B.1.3.2 Reagents for nucleic acid extraction

Nucleic acid extraction procedure and reagents appropriate for Gram-positive bacteria shall be used.

Commercial kits can also be used.
B.1.3.3 Reagents for PCR
D.1.3.3.1 PCR buffer solution.

The 10x PCR buffer solution is usually delivered with the DNA polymerase, which may or may not

include MgCl in a concentration specified by the manufacturer. The final MgCl concentrations are

2 2

method specific and are therefore listed in Table D.2. Commercially available ready-to-use reagents

may be used. The manufacturer’s instructions for use should be considered.
D.1.3.3.2 MgCl solution, c (MgCl ) = 25 mmol/l.
2 2
D.1.3.3.3 Thermostable DNA polymerase (for hot-start PCR), 5 IU/µl.
D.1.3.3.4 dNTP solution, c (dNTP) = 10 mmol/l, for the mix.
D.1.3.3.5 Oligonucleotides.
Sequences of the oligonucleotides are listed in Table D.1.
© ISO 2020 – All rights reserved 9
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SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
Table D.1 — Sequences of oligonucleotides
Amplicon
Gene Primer Sequence (5′ – 3′)
size (bp)
ces EM1F GAC AAG AGA AAT TTC TAC GAG CAA GTA CAA T 635
EM1R GCA GCC TTC CAA TTAC TCC TTC TGC CAC AGT
B.1.3.4 Reagents for gel electrophoresis
[12]
Refer to ISO 20838 .
B.1.4 Equipment and consumables
B.1.4.1 General
Appropriate equipment according to the method and, in particular, the following.
B.1.4.2 Equipment used for nucleic acid extraction
D.1.4.2.1 Micro-centrifuge tubes, with capacities of 1,5 ml or 2,0 ml.

D.1.4.2.2 Centrifuge, for reaction tubes with a capacity of 1,5 ml or 2,0 ml and capable of achieving

an acceleration up to approximately 14 000g.
D.1.4.2.3 Thermoblock, with heating capacity of up to 100 °C.

D.1.4.2.4 Graduated pipettes and pipette filter tips, for volumes between 1 μl to 1 000 μl.

D.1.4.2.5 Mixer.
B.1.4.3 Equipment used for PCR

D.1.4.3.1 Pipettes and pipette filter tips, having a capacity between 1 µl and 1 000 µl.

D.1.4.3.2 Microcentrifuge tubes, having a capacity of 1,5 ml or 2,0 ml.

D.1.4.3.3 Thin walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR microplates

or other suitable equipment.
D.1.4.3.4 Thermal cycler.
B.1.4.4 Equipment used for the detection of the PCR product
[12]
Refer to ISO 20838 .
B.1.5 Procedure
B.1.5.1 Nucleic acid extraction

Confirmed B. cereus group colonies according to 9.4 should be used for DNA extraction. Prior to DNA

extraction, the colonies can be optionally washed by centrifugation in 1 ml of nuclease free water. The

protocol described in C.5.2 can be used as well as any nucleic acid extraction procedure appropriate for

Gram-positive bacteria and suitable for this purpose (e.g. Reference [15]).
B.1.5.2 PCR set-up

The method is described for a total PCR volume of 50 µl per reaction with the reagents as listed in

Table D.2. The PCR can also be carried out in a smaller volume if the solutions are adjusted accordingly.

The final concentrations of reagents as outlined in Table D.2 have proven to be suitable.

10 © ISO 2020 – All rights reserved
---------------------- Page: 20 ----------------------
SIST EN ISO 7932:2005/A1:2020
ISO 7932:2004/Amd.1:2020(E)
Table D.2 — PCR reaction reagents
Reagent (concentration) Final concentration Volume per reaction (µl)
DNA polymerase buffer without MgCl (10X) 5
MgCl (25 mmol/l) 150 µmol/l 0,3
dNTP mix (10 mmol/l each) 800 µmol/l 4
EM1F (5 µmol/l) 0,5 µmol/l 2,5
EM1R (5 μmol/l) 0,5 µmol/l 2,5
Template DNA / 1
Thermostable DNA polymerase, 5 IU/µl 0,5 IU/µl 5
Adjust the volume to 50 µl using nuclease-free water

If the DNA polymerase buffer already contains MgCl , the final concentration of MgCl in the reaction mixture is

2 2
adjusted to 0,15 mmol/l.
B.1.5.3 PCR controls
[14]
All appropriate controls as given in ISO 22174 shall be performed.
The following strain can be used as positive controls.
Bacillus cereus, emetic strain WDCM 00219.
B.1.5.4 Temperature-time-programme
The temperature-time pro
...

SLOVENSKI STANDARD
SIST EN ISO 7932:2005/oprA1:2018
01-december-2018
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Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of

presumptive Bacillus cereus - Colony-count technique at 30 degrees C - Amendment 1:

Inclusion of optional tests (ISO 7932:2004/DAM 1:2018)

Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur Zählung

von präsumtivem Bacillus cereus - Koloniezählverfahren bei 30 °C - Änderung 1:
Aufnahme optionaler Testmethoden (ISO 7932:2004/DAM 1:2018)

Microbiologie des aliments - Méthode horizontale pour le dénombrement de Bacillus

cereus présomptifs - Technique par comptage des colonies à 30 degrés C -
Amendement 1: Ajout de tests optionnels (ISO 7932:2004/DAM 1:2018)
Ta slovenski standard je istoveten z: EN ISO 7932:2004/prA1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 7932:2005/oprA1:2018 en

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

---------------------- Page: 1 ----------------------
SIST EN ISO 7932:2005/oprA1:2018
---------------------- Page: 2 ----------------------
SIST EN ISO 7932:2005/oprA1:2018
DRAFT AMENDMENT
ISO 7932:2004/DAM 1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2018-10-26 2019-01-18
Microbiology of food and animal feeding stuffs —
Horizontal method for the enumeration of presumptive
Bacillus cereus — Colony-count technique at 30 degrees C
AMENDMENT 1: Inclusion of optional tests

Microbiologie des aliments — Méthode horizontale pour le dénombrement de Bacillus cereus

présomptifs — Technique par comptage des colonies à 30 degrés C
AMENDEMENT 1: Ajout de tests optionnels
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
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 7932:2004/DAM 1:2018(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 2018
---------------------- Page: 3 ----------------------
SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
ISO 7932:2004/DAM 1:2018(E)
Microbiology of food and animal feeding stuffs -- Horizontal
method for the enumeration of presumptive Bacillus cereus --
Colony-count technique at 30 degrees C
AMENDMENT 1: Inclusion of optional tests
Page 6 after Subclause 9.4
Add Subclause 9.5
9.5 Optional tests

All the tests mentioned below are optional and intended for complementary investigations (i.e.

epidemiological) on isolated B. cereus group strains obtained in 9.4.1, following the procedures

described in annexes C to F.

9.5.1 Detection of cytK-1 or cytK-2 gene variants of the gene encoding Cytotoxin K

Some strains within the B. cereus group bacteria carry one of the two variants found for the gene

encoding Cytotoxin K, cytK-1 and cytK-2. The cytK-1 gene is specific to B. cytotoxicus [17, 22] and

thus constitutes a way to rapidly identify B. cytotoxicus [20]. The procedure in annex C describes

a rapid and validated PCR method that targets both cytK gene variants and, if present, indicates

which of the two forms is present. It also allows to identify isolates to B. cytotoxicus.

9.5.2 Detection of [cereulide-producing] Bacillus cereus group strains [able to produce

cereulide]

Some strains within the B. cereus group bacteria are able to produce a heat-stable

dodecadepsipeptide, named cereulide. This cereulide, when produced in food, may cause an

emetic food poisoning syndrome.

A cereulide peptide synthetase (ces) gene is involved in the non-ribosomal production of cereulide

[16]. The procedure in annex D describes a rapid and validated PCR method that targets the ces

gene.
9.5.3 Motility and hemolysis tests

These tests allows to screen presumptive B. anthracis among isolated B. cereus group bacteria.

Note these tests have strong limitations that are presented in annex E (E.1 and Table E.1).

9.5.4 Microscopic examination of the parasporal crystal from B. thuringiensis

The B. thuringiensis species, one of the B. cereus group species, can be distinguished of the other

COPYRIGHT PROTECTED DOCUMENT

species of this group by the microscopic examination of the parasporal crystal that its isolated

strains are able to produce under the sporulation conditions described in annex F.

© ISO 2018

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.
Page 12 after Annex B
ISO copyright office
Add the following as Annexes C, D, E and F:
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
© ISO 2018 – All rights reserved 1
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 4 ----------------------
SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
Microbiology of food and animal feeding stuffs -- Horizontal
method for the enumeration of presumptive Bacillus cereus --
Colony-count technique at 30 degrees C
AMENDMENT 1: Inclusion of optional tests
Page 6 after Subclause 9.4
Add Subclause 9.5
9.5 Optional tests

All the tests mentioned below are optional and intended for complementary investigations (i.e.

epidemiological) on isolated B. cereus group strains obtained in 9.4.1, following the procedures

described in annexes C to F.

9.5.1 Detection of cytK-1 or cytK-2 gene variants of the gene encoding Cytotoxin K

Some strains within the B. cereus group bacteria carry one of the two variants found for the gene

encoding Cytotoxin K, cytK-1 and cytK-2. The cytK-1 gene is specific to B. cytotoxicus [17, 22] and

thus constitutes a way to rapidly identify B. cytotoxicus [20]. The procedure in annex C describes

a rapid and validated PCR method that targets both cytK gene variants and, if present, indicates

which of the two forms is present. It also allows to identify isolates to B. cytotoxicus.

9.5.2 Detection of [cereulide-producing] Bacillus cereus group strains [able to produce

cereulide]

Some strains within the B. cereus group bacteria are able to produce a heat-stable

dodecadepsipeptide, named cereulide. This cereulide, when produced in food, may cause an

emetic food poisoning syndrome.

A cereulide peptide synthetase (ces) gene is involved in the non-ribosomal production of cereulide

[16]. The procedure in annex D describes a rapid and validated PCR method that targets the ces

gene.
9.5.3 Motility and hemolysis tests

These tests allows to screen presumptive B. anthracis among isolated B. cereus group bacteria.

Note these tests have strong limitations that are presented in annex E (E.1 and Table E.1).

9.5.4 Microscopic examination of the parasporal crystal from B. thuringiensis

The B. thuringiensis species, one of the B. cereus group species, can be distinguished of the other

species of this group by the microscopic examination of the parasporal crystal that its isolated

strains are able to produce under the sporulation conditions described in annex F.

Page 12 after Annex B
Add the following as Annexes C, D, E and F:
© ISO 2018 – All rights reserved 1
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
Annex C
(informative)
Polymerase chain reaction for the detection of cytK-1 or cytK-2 gene
variants of cytotoxin K in isolated strains of Bacillus cereus group and
identification of B. cytotoxicus
C.1 Introduction

The chromosomally located cytK-2 gene encodes cytotoxin K, an enterotoxin that is present only

in B. cereus sensu stricto and B. thuringiensis [22], although all strains do not carry cytK-2 genes.

CytK-1 gene is a variant of cytK-2 gene due to a marked polymorphism and encodes to a more

cytotoxic form of cytotoxin K that is present only in B. cytotoxicus [18].

This method is applicable on well isolated colonies of B. cereus group strains, after appropriate

preparation of the DNA.
C.2 Principles
C.2.1 General
The method comprises the following consecutive steps:
a) Nucleic acid extraction
b) Amplification of target gene and interpretation
C.2.2 Nucleic acid extraction

Bacterial cells are harvested from well isolated colonies and the nucleic acid is extracted for use

in PCR reaction.
C.2.3 Amplification of target gene and interpretation

The extracted nucleic acid is selectively amplified using PCR. Detection of the PCR products is

achieved by electrophoresis on agarose. Interpretation is deduced from presence or absence of

the expected band.
C.3 Reagents
C.3.1 General

All reagents needed for this annex are molecular grade reagents and consumables suitable for

molecular biology. They shall be used as given in ISO 20837 [10] and ISO 20838 [11].

C.3.2 Nucleic acid extraction

Nucleic acid extraction procedure and reagents appropriate for Gram-positive bacteria shall be

used.
Commercial kits can also be used.
C.3.3 Reagents for PCR
Refer to ISO 22174 [13] and ISO 20838 [11].
C.3.4 Primers
The primers used for detection of cytotoxin K genes are listed below.
© ISO 2018 – All rights reserved 2
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
Table C.1 Sequences of oligonucleotides, characteristics and resulting amplicon
Position
Gene Amplicon
Primer Sequence (5' —> 3') on cytK
variant size (bp)
gene
cytK-1
CAA TTC CAG GGG CAA GTG TC
CK1F F 314-333
Accession
426
CCT CGT GCA TCT GTT TCA TGA G number
CK1R R 740-719
DQ885233.1
cytK-2
CAA TCC CTG GCG CTA GTG CA
CK2F F 314-333
Accession
585
number
GTG IAG CCT GGA CGA AGT TGG
CK2R R 899-879
AJ318876.2
F : Forward ;
R : Reverse

Make reference to the publicly available nucleotide sequences available on http://www.

www.ncbi.nlm.nih.gov
C.4 Equipment and consumables
C.4.1 Equipment for nucleic acid extraction
C.4.1.1 Micro-centrifuge tubes, with capacities of 1,5 ml and 2,0 ml.

C.4.1.2 Centrifuge, for reaction tubes with a capacity of 1,5 ml and 2,0 ml and capable of

achieving an acceleration up to approximately 14 000 x g.
C.4.1.3 Thermoblock, with heating capacity of up to 100 °C.

C.4.1.4 Graduated pipettes and pipette filter tips, for volumes between 1 μl to 1000

μl.
C.4.1.5 Mixer
C.4.2 Equipment for PCR
C.4.2.1 PCR thermal cycler

C.4.2.2 Thin-walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR

microplates or other suitable equipment.
C.4.3 Equipment for the detection of PCR products
Refer to ISO 20838 [11]
C.5 Procedure
C.5.1 General
See Figure C.1
© ISO 2018 – All rights reserved 3
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
B. cereus group colony
(well isolated – see
9.4.1)
DNA preparation
PCR Analysis
Detection of cytK-1
Detection of cytK-2
cyt-K genes not
variant variant
detected
B. cereus group
B. cereus group (other
B. cytotoxicus
(other than B.
than B. cytotoxicus)
cytotoxicus) with
without cyt-K genes
cytK-2 variant

Figure 1: Flow diagram for PCR detection of cytotoxin K gene (cytK-1 or cytK-2 variants) in

B. cereus group strains and identification of B. cytotoxicus
C.5.2 Nucleic acid extraction

Confirmed B. cereus group colonies according to subclause 9.4 should be used for DNA extraction.

Prior to DNA extraction, the colonies can be optionally washed by centrifugation in 1 ml of

nuclease free water. Any nucleic acid extraction procedure appropriate for Gram-positive

bacteria suitable for this purpose can be used (e.g. reference [23]).

A 10 µl loopfull of colony material is harvested (from MYP or non selective agars) and suspended

in 1 ml of nuclease free water, pelleted at 11 000 x g for 15 min. The pellet is resuspended in 500

µl extraction buffer (1,7 % sodium dodecylsulfate, 200 mmol/l Tris-HCl (pH 8), 20 mmol/l EDTA,

200 mmol/l NaCl). The suspension is incubated at 55°C for 1 h with 25 µl of proteinase K (10 µg

/µl). DNA is extracted with one volume of phenol and subsequently with one volume of

chloroform. The aqueous phase was precipitated with 2,5 volumes of cold 100 % ethanol and

centrifuged at 11 000 x g for 20 min. The supernatant was discarded and the pellet washed once

with 800 µl of cold 70 % ethanol. After drying, the pellet was dissolved in 50 µl nuclease free water

and stored at -20 °C. DNA amount is quantified by absorbance at 260 nm in a spectrophotometer

and must be adjusted to a concentration compatible with the sensitivity of the PCR (see C.6.3).

Also other methods or commercial ready-to-use purification kits can be used if controls (see

C.5.3.2) are scrupulously used.
C.5.3 PCR amplification
C.5.3.1 General

The total PCR volume is 15 μl per reaction. The reagents are listed in Table C.2. The final

concentrations of reagents as outlined in the table have proven to be suitable.
© ISO 2018 – All rights reserved 4
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
Table C.2 – PCR reaction reagents
Reagent (Stock conc.) Final concentration Volume per sample (μl)
10x DNA Polymerase Buffer 1x 1,5
dNTPs mix (5 mmol/l each) 0,2 mmol/l each 0,6
CK1F (10 μmol/l) 0,25 µmol/l 0,375
CK1R (10 μmol/l) 0,25 µmol/l 0,375
CK2F (10 μmol/l) 0,25 µmol/l 0,375
CK2R (10 μmol/l) 0,25 µmol/l 0,375
MgCl (25 mmol/l) 2,5 mmol/l 1,5
DNA polymerase 0,75 U 0,15
Genomic DNA (25 ng/µl) 2,5
Adjust the volume to 15 μl using nuclease free water

This protocol has been validated using commercial available AmpliTaq®10x Buffer and AmpliTaq®

polymerase (Applied Biosystems, Forster City, CA, USA) and Master Mix containing the four dNTPs

(Eurogentec)

Different protocols for PCR amplification can be used, depending on used DNA polymerase and

DNA preparation that is used. However, the PCR reaction must be stringent, using the primers

described in Table C.1 with appropriate hybridization temperature (Table C.3) and appropriate

controls (C.5.3.2), the reliability of primers being validated with a specific hybridization

temperature.
C.5.3.2 PCR controls

All appropriate controls as given in ISO 22174 [13] must be performed. At least a positive and a

negative control, represented for each gene variant by a known positive and a known negative

bacterial strain DNA respectively, need to be included in the PCR assay to check the conditions of

amplification.

DNA positive controls (including process controls as given in ISO 22174) should be obtained by

the same DNA extraction protocol as used for test isolates.
Table C.3 – Control strains to be included in PCR assays
WDCM number cytK-1 cytK-2
WDCM xxxx (ATCC14579 ) Negative control Positive control
WDCM xxxx DSM 22905 Positive control Positive control
WDCM xxxx DSM 11821 Negative control Negative control

Make reference to the reference strain catalogue available on http://www.wfcc.info

for information on culture collection strain numbers and contact details.
C.5.3.3 Temperature-time program

The temperature-time program as outlined below has been used in the evaluation study.

Table C.4 – Temperature-time program
Initial denaturation 94°C for 5 min
Denaturation 94°C for 15 s
Amplification
Hybridisation 57°C for 30 s

This information is given for the convenience of the user of this standard and does not constitute an endorsement

of this product. Equivalent products can be used if they can be shown to give the same results.

© ISO 2018 – All rights reserved 5
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
Elongation 72°C for 30 s
Number of cycles 30
Final extension 72°C for 7 min
C.5.3.4 Detection of the PCR product

The PCR product is detected after electrophoresis on agarose gel (1,5 %) with an appropriate

molecular weight marker (refer to ISO 20838).
C.5.4 Interpretation of the PCR result

The result obtained, including the controls specified above (see C.5.3.2), should be as follows,

otherwise the PCR shall be repeated.
The PCR result will be either :

a) positive for cytK-1 variant, if a specific PCR product of 426 bp has been detected and all

the controls give expected results, or

b) positive for cytK-2 variant, if a specific PCR product of 585 bp has been detected and all

the controls give expected results, or

c) negative for cytotoxin K genes, if a specific PCR product has not been detected, and all

controls give expected results.
C.5.5 Confirmation of the PCR product

The specificity of this PCR method for cytK-1 and cytK-2 variant was 100 % under the PCR

conditions described (see C.6) and with the recommended controls (see C.5.3.2). Nevertheless, as

prescribed in ISO 22174 [13], the presence of the PCR product and its specificity shall be

demonstrated by a suitable confirmation reaction.
C.6 Performance characteristics
C.6.1 General

This method was evaluated on a total of 160 B. cereus group strains and 10 outgroup species

including Southern blotting or PCR product sequencing [23], and then applied on 391 strains [22].

The assay turned out to be highly reliable with 0 % false-negative reactions and 0 % false-positive

reactions. BLASTN [25] analysis also showed that there were no targets in the bacterial organisms

other than cytK gene for the four primers included in the PCR reaction. The specificity for cytK-1

and cytK-2 variant was 100 % under the PCR conditions described [23] and with the

recommended controls (see C.5.3.2).
C.6.2 Selectivity

Selectivity was performed in duplex PCR using the primers CK1F, CK1R, CK2F, CK2R, working by

couple (CK1F/CK1R and CK2F/CK2R). It was checked that the respective primer pairs run

exclusively on cytK-1 variant or cytK-2 variant and target low conserved regions between the two

variants.
C.6.2.1 Inclusivity test

Inclusivity of the PCR assay for cytK-2 fragment was tested on 66 target strains. A 100 %

inclusivity was obtained. Inclusivity of the PCR assay for cytK-1 fragment was tested on 5 target

strains. A 100 % inclusivity was obtained. The tested strains for cytK-2 variant were B. cereus

sensu stricto and B. thuringiensis strains (the two species that may carry cytK-2gene [22]). Target

strains for cytK-1 correspond to all 5 B. cytotoxicus strains that were available at the time of

evaluation. They were enough distant from the remaining species of the group to exhibit a

particular polymorphism resulting in cytK-1 variant for cytK gene.
© ISO 2018 – All rights reserved 6
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
C.6.2.2 Exclusivity test

Exclusivity of the assay for cytK-1 fragment was tested on 157 non-target strains. A 100 %

exclusivity was obtained. Exclusivity of the assay for cytK-2 fragment was tested on 94 non-target

strains. A 100 % exclusivity was obtained.

Strains tested were from all species of the B. cereus group and 8 diverse food-related species.

C.6.3 Sensitivity

The limits for PCR amplification ranged from 15 ng to 75 ng of genomic DNA per 15 µl of final

reaction.
C.7 Limitations of the PCR assay

This PCR assay allows to i) detect cytotoxin K genes (and particularly one of its two variants) and

ii) identify B. cytotoxicus strains, two characteristics that are both associated with food poisoning.

Even if that indicates a potential risk, the sole presence of the gene or species identification does

not allow to confirm cytK gene expression.
© ISO 2018 – All rights reserved 7
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
Annex D
(informative)
Method for molecular detection of cereulide-producing Bacillus cereus
group strains
D.1 Gel-based PCR assay for detection of ces-gene of cereulide producing
Bacillus cereus group strains
D.1.1 Introduction

This annex describes a method for the amplification and detection of the ces gene specific for the

production of cereulide in Bacillus cereus group strains using agarose gel electrophoresis.

D.1.2 Principle

Specific DNA fragments of the ces gene are amplified by PCR using two primers. The detection of

the PCR product is done using agarose gel-electrophoresis.
D.1.3 Reagents
D.1.3.1 General

All reagents needed for this annex are molecular grade reagents and consumables suitable for

molecular biology. They shall be used as given in ISO 20837 and 20838.
D.1.3.2 Reagents for Nucleic Acid Extraction

Nucleic acid extraction procedure and reagents appropriate for Gram-positive bacteria shall be

used. Commercial kits can also be used.
D.1.3.3 Reagents for PCR
D.1.3.3.1 PCR buffer solution

The 10x PCR buffer solution is usually delivered with the DNA polymerase, which may

or may not include MgCl in a concentration specified by the manufacturer. The final

MgCl concentrations are method specific and are therefore listed in table D.2. Ready-

to-use reagents may be commercially available. The manufacturer’s instructions for use

should be considered.
D.1.3.3.2 MgCl solution, c (MgCl )  25 mmol/l.
D.1.3.3.3 Thermostable DNA polymerase (for hot-start PCR), 5 IU/µl.
D.1.3.3.4 dNTP solution, c (dNTP)  10 mmol/l, for the mix.
D.1.3.3.5 Oligonucleotides
Sequences of the oligonucleotides are listed in Table D.1.
© ISO 2018 – All rights reserved 8
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SIST EN ISO 7932:2005/oprA1:2018
ISO/DIS 7932:2004/AMD1 (E)
Table D.1 — Sequences of oligonucleotides
Amplicon size
gene Primer Sequence (5´ — 3´)
(bp)
GAC AAG AGA AAT TTC TAC GAG CAA GTA CAA T
ces EM1F 635
EM1R GCA GCC TTC CAA TTAC TCC TTC TGC CAC AGT
D.1.3.4 Reagents for gel electrophoresis
Refer to ISO 20838 [11]
D.1.4 Equipment and consumables
D.1.4.1 General
Appropriate equipment according to the method and, in particular, the following.
D.1.4.2 Equipment used for Nucleic Acid Extraction
D.1.4.2.1 Micro-centrifuge tubes, with capacities of 1,5 ml and 2,0 ml.

D.1.4.2.2 Centrifuge, for reaction tubes with a capacity of 1,5 ml and 2,0 ml and capable

of achieving an acceleration up to approximately 14 000 x g.
D.1.4.2.3 Thermoblock, with heating capacity of up to 100°C.

D.1.4.2.4 Graduated pipettes and pipette filter tips, for volumes between 1 μl to 1000

μl.
D.1.4.2.5 Mixer
D.1.4.3 Equipment used for PCR

D.1.4.3.1 Pipettes and pipette filter tips, having a capacity between 1 µl and 1 000 µl.

D.1.4.3.2 Microcentrifuge tubes, having a capacity of 1,5 ml and 2,0 ml.

D.1.4.3.3 Thin walled PCR microtubes, 0,2 ml or 0,5 ml reaction tubes, multi-well PCR

microplates or other suitable equipment.
D.1.4.3.4 Thermal cycler.
D.1.4.4 Equipment used for the detection of the PCR product.
D.1.4.4.1 Microwave oven or boiling water bath.
D.1.4.4.2 Horizontal gel system.
D.1.4.4.3 Power supply.
D.1.4.4.4 UV transilluminator or UV light box.
D.1.4.4.5 Gel documentation system.
D.1.5 Procedure
D.1.5.1 Nucleic acid extraction

Confirmed B. cereus group colonies according to subclause: 9.4 should be used for DNA extraction.

Prior to DNA extraction, the colonies can be optionally washed by centrifugation in 1 ml of

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nuclease free water. Any nucleic acid extraction procedure appropriate for Gram-positive

bacteria and suitable for this purpose can be used. (e. g. reference [15])
D.1.5.2 PCR set-up

The method is described for a total PCR volume of 50 µl per reaction with the reagents as listed

in Table D.2. The PCR can also be carried out in a smaller volume if the solutions are adjusted

accordingly. The final concentrations of reagents as outlined in Table D.2 have proven to be

suitable.
Table D. 2 — Addition of reagents
Reagent Final concentration Volume per sample (µl)
Template DNA 1
Nuclease-free water 24,7
PCR-buffer 10 x (without MgCl )
MgCl solution, c (MgCl )  25 mmol/l
150 µmol/l 0,3
2 2
dNTP solution, c (dNTP)  10 mmol/l 800 µmol/l 4
PCR primers (EM1F and EM1R, according to 0,5 µmol/l each 5
table D.1), 5 µmol/l
Thermostable DNA polymerase, 5 IU/µl 0,5 IU 5

If the PCR buffer solution already contains MgCl , the final concentration of MgCl in the reaction mixture is adjusted to 4,8 mmol/l.

2 2
D.1.5.3 PCR controls
All appropriate controls as given in ISO 22174 [13] must be performed.
The following strain can be used as positive controls.
Bacillus cereus, emetic strain WDCM xxxx DSM 4312
D.1.5.4 Temperature-time-programme

The temperature-time program as outlined in Table D.3 and the MasterMix according to table D.2.

were used in the validation study [16]. The use of other thermal cyclers might make an adaptation

necessary. The time for activation/initial denaturation depends on the polymerase used. The

recommendation of the DNA polymerase manufacturer is to be followed.
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Table D.3 — Temperature-time programme
Activation/initial denaturation 95 °C for 15 min
95 °C for 30 s
Amplification 59 °C for 30 s
72 °C for 60 s
Number of cycles (amplification) 30
Final extension 72 °C for 5 min
D.1.5.5 Detection of PCR products
D.1.5.5.1 Gel electrophoresis
Refer to ISO 20838 [11]
D.1.5.5.2 Gel recording

Transfer the gel to the transilluminator surface, switch on the UV light and record the DNA

fluorescence by photography or video-documentation.
Table D.4 — Size of amplification products
gene Primer Product size (bp)
ces EM1F/EM1R 635

The target sequences are considered to be detected if the size of the PCR product corresponds to

the expected length of the target DNA sequences. For the interpretation of the results see ISO

22174 [13].
D.1.5.6 Confirmation of a positive PCR result

A positive PCR result shall be confirmed with e.g. sequencing of the PCR products. Other

appropriate methods for confirmation can also be used.
D.1.6 Performance characteristics
D.1.6.1 General

The method has been validated for DNA extracted from different emetic Bacillus cereus group

strains (Refer to [9]).
D.1.6.2 Theoretical evaluation of the method

Theoretical evaluation was done by performing a sequence similarity search against the

® th

GenBank/EMBL/DDBJ database (NCBI Blast search, EMBL database, September 22 , 2015). The

result of the search confirmed a complete identity of the primers only with the expected target

sequence.
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D.1.6.3 Selectivity
D.1.6.4 Inclusivity test

The inclusivity of the method was tested with 30 emetic Bacillus cereus group strains, see Table

D.5.
Table D.5 — Inclusivity of the PCR using target strains
Source Number of strains ces gene detected
food strains (without connection to a 5 5
foodborne illness/outbreak)
Food strains connected to foodborne 13 13
illness
human samples (vomit, faeces) 12 12
D.1.6.5 Exclusivity test

The exclusivity of the method was tested with 178 non-target organisms, see Table D.6. No cross-

reactivity was observed with the non-target bacteria.
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Table B.6 — Exclusivity of the PCR using non-target strains
Species Number of strains ces gene detected
Bacillus cereus non-emetic strains 100 0
Bacillus anthracis 7 0
Bacillus mycoides 6 0
Bacillus thuringiensis 6 0
Bacillus pseudomycoides 3 0
Bacillus weihenstephanensis 7 0
other Bacillus spp. 8 0
Staphylococcus aureus 10 0
Staphylococcus equorum 1 0
Clostridium perfringens 3 0
Listeria monocytogenes 6 0
Campylobacter spp. 3 0
Escherichia coli 4 0
Salmonella spp. 6 0
Yersinia enterocolitica 8 0
D.2 PCR assay for detection of ces-gene of cereulide producing Bacillus
cereus group strains using real-time-PCR
D.2.1 Introduction

This annex describes a probe-based real-time PCR method based on TaqMan technology for the

detection of the ces gene specific for emetic Bacillus cereus group strains.
D.2.2 Principle

Specific DNA fragment of the ces gene specific for emetic Bacillus cereus group st

...

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