rut INTERNATIONAL ISO

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Thinh ' 01'TP'HCl,l STANDARD 2L87z--1
TEL:3829 7040'FAX :3829 1957

First edition
2077-06

Add: 8 Hoang Ouoc Viet, Cau HN

Microbiolo lh''1-
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Part 1:
Detection of potentially
enteropathogenic Vibrio
p arahaemolytictts, Vibrio cholerae and
Vibrio vulnificus
Microbiologie de la chatne alimentqire Mdthode horizontale pour
la ddtermination des Vibrio spp.-
-
Pqrtie 1: Recherche des espices de Vibrio parahaemolyticus, Vibrio
cholerae ef Vibrio vulnificus potentiell em ent entd rop ath o g dnes

Reference number
ISO 27872'7:201.7(E)

I
v o IS0 2017
rso 21872-rz20t7(E)

A COPYRIGHT PROTECTED DOCUMENT

O ISO 2017, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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the requester.
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Fax +41 22 749 09 47
copyright@iso.org
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 tSO 21872-I:20L7 (E)

Contents Page

.'."."'.......'.'....,.,.'.,..'.'.''v
Foreword,,
.............v1

1 1

2 L

3 2

4 2
4.1, 2
4.2 Primary enrichment in a liquid selective medium.."..... 2
4.3 Secondary enrichment in a Iiquid selective medium........ 3
4.4 5
4.5 3

5 3
5.1 Enrichment medium: alkaline saline peptone water (ASPW)'.'"."... 4
4
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium ITCBSJ 4
4
4
4
5.5 4
5.5.1 L-lysinedecarboxylasesalinemedium ILDC)...'....'.'.'. 4
5.5.2 Arginine dihydrolase saline medium (ADH).......'....' 4
5.5.3 Reagent for detection of B-galactosidase.'...... 5
5.5.4 Saline medium for detection of indole'. 5
5
5
5.6 5
5.6.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance
r
5.6.2 5
5.6.3 5
5.6.4 5
5.6.5 Negative extraction control 6

6 ".'......'.'.'....,6

7 Sampling.... 6

B Preparation of the test samp lo 6

9 Procedure (See Figure A.1) ,.',',,,7

7
.7

9.3 Secondaryselectiveenrichment 7
9.4 Isolationandidentification............. B
B
I
9.5.2
9.5.3
9.5.4
Selection of colonies for confirmation and preparation
Testsforpresumptiveidentification..........."......'..
Biochemical confirmation.......................
:::::::::llll : 3
10
9.5.5 PCR confirmation 12
9.5.6
9.5.7
9.5.8
13

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Itt
$A 21872-1:2O17(E)

1.L.2 Sensitivity L4
11.3 Specificity t4
1.1.4 LOD5s 74
L4
15
Annex B (normative) Composition and preparation of the culture media and reagents..............".........^""17

Annex C (informative) Conventional PCR for the detecti on of Vibrio parahoemolyticus,

thermostable direct haemolysin (fdh) and thermostable direct related haemolysin

Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus,

thermostable direct haemolysin gene (tdh) and,Vibrio vulnificus.. ...^.^..."....^.^..............".27

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 tSO 21872-1:2017 (E)

Foreword
a worldwide federation of national standards
IS0 (the International 0rganization for Standardization) is
bodies (lSO member bodies). The work of preparing International Standards is normally carried out
througtr ISO technical committees. Each member body interested in a subject for which a technical
co-*ittee 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.
IS0 collaboratei closely with the International Electrotechnical Commission (lEC) on all matters of
electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are
desciibed in the ISO/lEC 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/lEC Directives, Part2 (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
iny 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 meaning of ISO specific terms and expressions related to conformity assessment,
as well ai 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
with ISO Technical
Committee CEN/TC 275, Food analysis
- Horizontal methods, in collaboration
Committee TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the agreement
on technical cooperation between ISO and CEN (Vienna Agreement)'

This first edition cancels and replaces ISO/TS 21872-7:2002 which has been technically revised, It also
i ncorp orates I SO/ T S 2187 2-7:20 07 / Cor.l:2O 08.

The main changes are as follows:

introduction of optional molecular identification methods for major food borne Vibrio spp.
(V. parahaemolyticus, including potentially enteropathogenic strain s, V. vulnificus and V. cholerae);

performance characteristics of the method have been added in Annex E'

A list of all parts in the ISO 21872 series can be found on the ISO website.

V
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tSO 21872-Lz2Ot7(E)

Introduction
Because of the large variety of food and feed products, the horizontal method described in this
document may not be appropriate in every detail for certain products. In this case, different methods,
which are specific to these products may be used if absolutely necessary for justified technical reasons.
Nevertheless, every attempt will be made to apply this horizontal method as far as possible.

The main changes, listed in the foreword, introduced in this document compared to ISO/TS 21872-
L20A7 are considered as major (see ISO 17468).

When this document is next reviewed, account will be taken of all information then available regarding
the extent to which this horizontal method has been followed and the reasons for deviations from this
method in the case of particular products.

The harmonization of test methods cannot be immediate and, for certain groups of products,
International Standards and/or national standards may already exist that do not comply with this
horizontal method. It is hoped that when such standards are reviewed they will be changed to comply
with this document so that eventually the only remaining departures from this horizontal method will
be those necessary for well-established technical reasons.

vi @ ISO 2017 - All rights reserved

INTERNATIONAT STANDARD rSO 21872-1:ZAfi (E)

Add: 8 Hoang Quoc Viel, Cau Giay, HN

Tel: (84.4) 37564268 . Fax: (84.4)
38361556

Microbiology of the food ch Sthod fo

the determination of Vibrio s Fliffiopy fias [estr ntade [y infopmatiom
genlen lon $tandards,
lHetrolo[I and 0ualit U
Part L:
Detection of potentially enteropathogenic Vibrio
parahaemolyticus, Vibrio cholerae and Vibrio vulnificus
WARNING In order to safeguard the health of laboratory personnel, it is essential that
-
tests for detection ol Vibrio spp., and particularly toxigenic Vibrio cholerae, be conducted
only in laboratories equipped for this purpose and under the supervision of an experienced
microbiologist, and that great care is exercised in the disposal of contaminated material.

L Scope
This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which
causes human illness in or via the intestinal tract. The species detectable by the methods specified
include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.

It is applicable to the following:

products intended for human consumption and the feeding of animals;

environmental samples in the area of food production and food handling.

NOTE 1 This method may not be appropriate in every detail for certain products (see IntroductionJ'

NOTE 2 The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and Z
vulnificus are the major food-b orne Vibrio spp. However, the method in this document can also be appropriate for
the identification of other Vibrio spp. causing illness in humans.El

2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies'

ISO 6887-1:2077, Microbiotogy of the food chain Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
- Part 1: General rules for the preparation of the initial
suspension and decimal dilutions
-
ISO 6887-3, Microbiology of the food chain - Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination Part 3: Specific rules for the preparation of fish and fishery
products
-
ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for
mi c r o b i o I o g i c al exami n ati o ns
-
ISO 11133, Ivlicrobiology of food, animol feed and water Preparation, production, storage and
performance testing of culture media
-

- Polymerase chain reaction

ISO 22178, Microbiotogy of food and animal feeding stuffs (PCR) for the
detection and quantification of food-borne pathogens Performonce characteristics
-

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rSO 21872-1:20L7(E)

ISO 22LI9, Microbiology of food and animal feeding stuffs

the detection offood-barne pathogens
- Real-time polymerase chain reaction (PCR)
General requirements and definitions
for
-
$A 22174, Microbiology of food and animal feeding stuffs - Polymerase chain reaction IPCR) for the
detection offood-borne pathogens General requirements and definitions
-
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply,

ISO and IEC maintain terminological databases for use in standardization at the following addresses:
IEC Electropedia: available at http://www.electropedia.org/
ISO 0nline browsing platform: available at http://www.iso.org/obp

3.1
potentially enteropathogenic Vibrio spp,
microorganism which forms typical colonies on solid selective media and which possesses the described
biochemical or molecular characteristics when the test is performed in accordance with this document

Note 1 to entry: This document describes specific procedures for V. parahaemolyticus, V. cholerae and V. vulnificus.

3.2
detection of potentially enteropathogenic Vibrio spp.
determination of the presence or absence of potentially enteropathogenic Vibrio spp. (3Jl
[V. parahaemolyticus, V. cholerae and 14 vulnificus) in a determined quantity of product, when the test is
performed in accordance with this document

4 Principle

4.1 General
The detection of potentially enteropathogenic Vibrio spp. (V. parahaemolyticus, V. cholerae and
V. vulnificus) requires four successive phases, as shown in the procedure diagram in Annex A.

Recovery of certain Vibrio spp. from foodstuffs may be improved by the use of different incubation
temperatures depending upon the target species or state of the food matrix. For example, recovery of
V. parahaemolyticus and V. cholerae in fresh products is enhanced by enrichment at 41,5 "C whereas
for V. vulnificut and for V. parahaemolyticus and V. cholerae in deep frozen [<-18 "C),[Z] dried or salted
products, recovery is enhanced by enrichment at 37 "C.lf detection of V. parahaemolyticus, V. cholerae
and V. vulnificus is required, all specified incubation temperatures should be used. If detection of
V. parahaemolyticus, V. cholerae and IZ vulnificus together is not required, the specific procedure(sJ may
be selected according to the species being sought. Such a selection should be clearly specified in the
test report.
NOTE V. parahaemolyticus, V. cholerae and V. vulnificus may be present in small numbers and are often
accompanied by a much larger number of other microorganisms belonging to the Vibrionaceae family or to other
families.

4.2 Primary enrichment in a liquid selective medium

Inoculation of the test portion in the primary enrichment medium alkaline saline peptone water
(ASPW) [5.1J at ambient temperature, followed by incubation at 41,5 oC for 6 h and/or 37 'C for 6 h.

The incubation conditions are determined by the target species and food product state.

For detection of all target species in deep frozen, dried or salted products, primary enrichment should
be at 37 'C.

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 tSO 21872-1=20L7(E)

For detection of Z vulnificus in all products, primary enrichment should be at 37 'C.

For detection of 7 parahaemolyticus and/or V. cholerae only, in fresh products, primary enrichment
should be at 41,5 "C.

4.3 Secondary enrichment in a liquid selective medium

Inoculation of the second enrichment medium IASPW) with the cultures obtained in 4.2.
Incubation of inoculated enrichment medium at47,5 'C for 18 h and/or 37 "C for 18 h.

For detection of V. vulnificus in all products, secondary enrichment should be at 37 'C.

For detection of V. parahaemolyticus and/or V. cholerae only, in all products, secondary enrichment
should be at 41,5 'C.

4.4 Isolation and identification

From the cultures obtained in 4.2 and in 4.3. inoculation of two solid selective media:

thiosulfate citrate bile and sucrose agar ITCBS) medium (52.1);

another appropriate solid selective medium Ieft to the choice of the laboratory), such as chromogenic
agar, complementary to the TCBS medium (5.2.2).

Incubation of the TCBS medium at37 "C,then examination after 24 h. Incubation of the second selective
medium according to the manufacturer's recommendations.

4.5 Confirmation
Presumptive colonies of V. porahaemolyticus, V. cholerae and V. vulnificus isolated in 4.4 are subcultured
and confirmed by means of appropriate biochemical and/or polymerase chain reaction (PCR) tests.

Biochemical and/or PCR confirmation of isolates may be used for species identification, however,
reliable detection of enteropathogenic V. parahaemolyticus as determined by presence of the direct
thermostable haemolysin (tdh) and/or direct related haemolysin (frh) genes can only be carried out
using PCR tests.

It has been demonstrated that by screening ASPW broths using conventional PCR, the absence of
amplification of Vp-foxR can indicate no detection of 7 porahaemolyfrcus.[3] To reduce the amount
of downstream testing and, if shown to be reliable by the user laboratory, screening of incubated
enrichment broths may be used. This approach is only recommended after secondary enrichment and
does not apply to molecular targets for other Vibrio spp.

NOTE 1 Validation data generated in the preparation of this document demonstrated that PCR based
identification can be achieved by conventional PCR for V, parahoemolyticus, V. vulnificus and V. cholerae or real-
time PCR for V. parohaemolyticus and V. vulnificus. The PCR methods used in the development of this document
are given in Annexes C and D.

NOTE 2 Validation data for screening of secondary enrichment broths for amplification of Vp-foxR were not
generated in the preparation ofthis document.

5 Culture media and reagents

For general laboratory practice, refer to ISO 7218.

For clarity of the text, details of the composition of culture media and reagents and their preparation
are described in Annex B.

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tSO 21872-tz20t7(E)

For performance testing of culture media, refer to ISO L1133.

NOTE Primers, probes and PCR running conditions used in the development of this document are given in
Annexes C and D.

5.1 Enrichment medium: alkaline saline peptone water (ASPW)

As snecified in 8.1.

5.2 Solid selective isolation media

5,2,L First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS)

As specified in 8,2. See Table 1 for performance testing data.

Table 1 Performance testing of thiosulphate, citrate, bile and sucrose agar medium ITCBS)
-
Function Incubation Control strains WDCM A Method of Criteria e Characteristic
control reactions
Productivity 37"C!loCfor Vib r i o p araha e moly ti c u s 00185 b
Qualitative Good Green colonies
24h !3h growth (21 (sucrose negative)
37"Ct1oCfor Vibrio furnissii 00186 b
Qualitative Good Yellow colonies
24hr3h growth [2J [sucrose positive)
Selectivity 37"Ct1uCfor Escherichia coli cd 00072, Qualitative Total
24h r3 h 00013 or inhibition
00090 t0l
a World Data Centre for Microorganisms (WDCMJ strain catalogue available at http://refs.wdcm.org
b Strain to be used as a minimum (see ISO 11133J.
c SomenationalrestrictionsanddirectionsmayrequiretheuseofadifferentE coliserovar.Makereferencetonational
requ i rements relating to the choice of E. coli serova rs.
d Strain free ofchoice; one ofthe strains shall be used as a minimum (see ISO 11133).
e Growth is categorized as 0: no growth, L: weak growth (partial inhibition), and 2: good growth I 13 3J.

5,2.2 Second medium

The selection of the second medium is left to the choice of the test laboratory. Preparation of the medium
should be strictly according to the manufacturer's instructions.

5.3 Saline nutrient agar (SNA)

As snecified in B.3.

5.4 Reagent for detection of oxidase

As snecified in B.4.

5.5 Biochemical tests

5.5.1 L-lysine decarboxylase saline medium (LDC)

As snecified in B.5.

5.5.2 Arginine dihydrolase saline medium (ADH)

As soecified in 8.6.

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 tSO 2L872-r=20L7 (E)

5.5.3 Reagent for detection of p'galactosidase

As specified in B.7.

5.5.4 Saline medium for detection of indole

As soecified in B.B.

5.5.5 Saline peptone waters

As specified in 8.9.

5.5.6 Sodium chloride solution

As specified in 8.10.

5.6 PCR

5.6.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the
purpose)
As snecified in B.11.

5,6.2 Mastermix
Reagents shall be added in quantities as specified by the manufacturer's instructions. See Annexes C

and D for example details of master mixes used in the development of this document.

5.6.3 Primers and probes

Primer (and hydrolysis probe) sequences if required shall be published in a peer-reviewed journal and
be verified for use against a broad range of target Vibrio spp. and non-target strains. With advances
in whole genome sequencing of bacterial strains, more appropriate species-specific markers may be
identified in the future.
For V. parahaemolyticus the target region should be toxR,

For determination of pathogenic strains of V. parahaemolyticus genes encoding the thermostable direct
(TDH) and the thermostable direct related (TRH) haemolysins should be targeted.

For V. cholerae the target region for conventional PCR should be the 165-235 rRNA intergenic spacer
region prVC.

For V. vulnificus the target region should be the V. vulnificus haemolysin region (vvho).

Target regions other than those specified above for the identification of V. parahaemolyticus, V. vulnificus
and 7 cholerae can be used if they have been shown to demonstrate equivalent performance to those
used in the development of this document and described in Annexes C or D are published in a peer-
reviewed journal and are verified against a broad range oftarget Vibrio spp. and non-target strains.

See Annex C for example details of primers used for conventional PCR and Annex D for primers and
hydrolysis probes for real-time PCR used in the development of this document.

5.6.4 Positive control material

Separate control material shall be used for each target Vibrio spp. See Annexes C and D for example
details of control strains used in the development of this document.

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rSO 2L872-L:2OL7(E)

5.6.5 Negative extraction control

Nuclease free water or sterile NaCl 0,85 7o extracted according to 9.5.6.

6 Equipment and consumables

Disposable equipment is acceptable in the same way as reusable glassware, if the specifications are
similar.

Ordinary microbiology laboratory equipment as specified in ISO 7218, and in particular the following.

6.L Refrigerator, adjustable to 5 oC + 3,0 oC.

6.2 Incubator, adjustable to 37 oC + 1,0 oC.

6.3 Incubator; adjustable to 41,5 oC + 1,0 oC.

6.4 Freezer, adjustable to <-15 'C.

6.5 Micro-centrifuge tubes, with a capacity of 1,5 ml and 2,0 ml.

6.6 Micro-centrifuge, for reaction tubes with a capacity of 1,5 ml and2,0 ml and capable of running at
10 0009.

6.7 Heating block capable of operating at 95 "C + 2,0 "C or equivalent.

6.8 Vortex.

6.9 Graduated pipettes and pipette filter tips, for volumes between 1 pl and 1 000 pl.

6.10 Associated consumables for conventional or real-time PCR, e.g. optical plates and caps, optical
plate holde6, suitable for use with the selected PCR machine.

6.11 Conventional or real-time PCR machine, gel electrophoresis and UV visualization equipment as
appropriate.

7 Sampling
It is important that the laboratory receives a truly representative sample which has not been damaged
or modified during transport and storage.

Sampling does not form part of the method specified in this document. See the International Standard
specific to the relevant product. If a specific document does not exist, it is recommended that the
relevant parties reach agreement on this subject.

8 Preparation of the test sample

Prepare the test sample in accordance with ISO 6BB7-1 and ISO 6887-3 and/or the document concerning
the product to be examined. If a specific document does not exist, it is recommended that the relevant
parties reach agreement on this subject.

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9 Procedure (See Figure A.1)

9.L Test portion and initial suspension

This document has been validated for test portions of up to 25 gor 25 ml. A smaller test portion may be
used without the need for additional validation/verification provided that the same ratio between (pre-)
enrichment broth and test portion is maintained. A larger test portion than that initially validated may
be used ifa validation/verification study has shown that there are no adverse effects on the detection
of Vibrio spp.

NOTE Validation can be conducted in accordance with the appropriate document of ISO 16140 (all parts).
Verification for pooling samples can be conducted in accordance with the protocol described in ISO 6887-1:2012
Annex D.

For the preparation of the initial suspensions, use the first enrichment medium IASPW) specified in 5.]..

Take test portions [25 g or 25 ml) and homogenize in 225 ml of enrichment medium.

In the case of large quantities (greater than 25 g or 25 ml), the ASPW should be warmed to 37 "C t 1 oC
and/or 41,5 oC + L oC (4.2) before inoculation with the test portion.
In order to reduce the amount of examination work, where more than one 25 g or 25 ml test portion
stemming from a determined batch of food is to be examined, and where proof is available indicating
that a mixture (gathering together the test portions) does not modify the results concerning this
product in particular, the test portions may be mixed. For example, if 10 test portions of 25 g or 25 ml
are to be examined, it is possible to combine these 10 units in order to obtain a composite sample of
250 g or 250 ml and to add 2,251 of enrichment medium.

Cell counts of Vibrio spp. potentially decline significantly on storage at refrigeration temperatures.
Storage of samples and, to a lesser extent, of suspensions at such temperatures should be avorded where
possible and should otherwise be kept to a minimum.

9.2 Primary selective enrichment

Incubatetheinitialsuspensions(9.1)at41,5"C+LoCand/or3ToCiloCfor6htLhaccordingto
Table 2.

Table 2 Primary incubation and target species/product state

-
Target Vibrio spp. in fresh product
Vibrio Vibrio vulnificus
lncubation temperature a Vibrio cholerue
parohaemolyticus
41,5'C+1'C
37oC+1oC
Target Vibria spp. in deep frozen. dried or salted product (such as bacalhau, stockfish, boknafisk, katsuo-
bushi, obamboJ
Vibrio Vibrio vulnificus
Incubation temperature a Vibrio chalerqe
parahaemolyticus
41,5'C + 1 'C
37'C t 1 "C
a Vibrio spp. other than those listed in Reference [], such as V. alginolytlcus, may be recovered at these incubation
temperatures.

9.3 Secondaryselectiveenrichment
Transfer 1 ml of the culture obtained in 9.2 taken from the surface into a tube containing 10 ml of ASPW
(5J). It is recommended that the sample is not agitated before taking the aliquot.

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ISO 21872-7:2Afi(E)

IncubatetheASPWat4'1",5oC+LoCand/or37"Ct1oCfor18htLhaccordingtoTable3.
NOTE Cultures and/or boiled broths obtained in 9.3 can be screened using PCR

Table 3 Secondary incubation and target species/product state

-
Target Vibrio spp. in 4[ product states
Incubation temperature a Vibrio p arahaemoly ticus Vibrio cholerae Vibrio vulnificus
41,5"C11'C
37"Ct1"C
a Vibrio spp. other than those listed in Reference El, such as V. alginolyticus, may be recovered at these incubation
temperatures.

9.4 Isolation and identification

From the cultures obtained in the ASPW (92and 9.3), inoculate with a 1" pl sampling loop the surface of
a TCBS agar plate (5.2.1), so as to permit the development of well-isolated colonies.

Proceed likewise with the chosen second selective isolation medium (5.2.2) using a fresh sampling loop.

Invert the agar plates:

for TCBS agar plates, incubate at 37 'C t 1 'C for 24h !3h;
for the second isolation medium, incubate according to the manufacturer's instructions.

After incubation, examine the TCBS and second selective medium for the presence of typical colonies of
presumptive pathogeni c Vibrio spp. Mark their position on the bottom of the plates.

On TCBS agar V. parahaemolyticus, V. vulnificus, and Z cholerae exhibit different typical colony
morphologies:

typical colonies of V. parahaemolyticus and V. vulnificus are smooth, green [negative sucrose) and of
2 mm to 3 mm in diameter;

typical colonies of V. cholerqe are smooth, yellow (positive sucrose) and of l- mm to 2 mm in diameter.
For the second selective medium, examine for the presence of colonies, which, according to their
characteristics, may be considered as possible isolates of V. parahoemolyticus, V. vulnificus, andf or V.
cholerae.

NOTE 1 Vibrio spp. other than those listed in Reference [], such as V. alginolyticus, can be recovered on TCBS
and the second selective medium.

NOTE 2 Recognition of colonies of Vibrio spp. is largely a question of experience and their appearance can
sometimes vary, not only from one species to another, but also from one batch of culture medium to another.

NOTE 3 For enhanced recovery of V. vulnificus, medium containing derivatives of cellobiose-polymyxin

B-colistin and cellobiose-colistin has been shown to be effective.[4]

9.5 Confirmation

9.5.1 General

Using this document, V. parahaemolyticus, V. vulnificus, and V, cholerae can be confirmed by molecular
PCR and/or biochemical approaches. Confirmation may be carried out at the end user laboratory or by a
specialist reference laboratory. 0ther Vibrio spp. may be isolated using this document.

Not all V. parahaemolyticus possess pathogenicity traits. In order to confirm the pathogenic character
of the strains, it is preferable to detect the presence of thermostable direct haemolysin (rdh) or TDH-

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 tSO 21872-1:20L7 (E)

related haemolysin [trh) genes. This should be carried out using PCR tests. PCR based confirmation
also may reduce the subjective interpretation of biochemical identification tests and accelerate the
identification process.

If shown to be reliable, commercially available biochemical test kits may be used to identify Vibrio to
the species level provided they are inoculated with a suspension of the bacteria to be identified in a
sufficiently saline medium or dilution fluid, and as long as the database or identification table for the
product has been based on reactions obtained using similar media to those described in this document.
These kits shall be used in accordance with the manufacturer's instructions.

If shown to be reliable, commercially available molecular detection kits may be used to identify Vibrio
to the species level. These kits shall be used in accordance with the manufacturer's instructions,

9.5.2 Selection of colonies for confirmation and preparation of pure cultures

For confirmation, subculture from each selective medium (9.4) at least one well isolated colony
considered to be typical or similar to each of the potentially patho genic Vibrio spp. sought. If the result
of the first isolated colony tested is negative, a further four well isolated colonies should be tested.

In samples where it is considered important to optimize the detection of potentially pathogenic 7

parahaemolyticus based upon the presence of thermostable direct and thermostable direct related
haemolysins, it is recommended that at least five and, where possible, all colonies exhibiting typical V.
parahaemolyficus colony morphology are sub-cultured for downstream testing.

Inoculate the colonies selected onto the surface of plates of saline nutrient agar (SNA) (L3J or suitable
medium of the laboratory's choice to obtain isolated colonies.lncubate at37'C + 1
oC
for24 h t 3 h.
NOTE 1 Food, especially seafood, may contain large numbers of bacteria, including non-pathogenic /ibrio spp.
which may grow through the selective culture process. Subculture of small numbers of colonies may resuit in
potentially pathogenic species being missed. This is particularly critical with the use of TCBS on which non Vibrio
spp. or non-enteropathogenic Vibrio spp. colonies may be morphologically similar to enteropathogenic species.

NOTE 2 The proportion of pathogenic strains in environmental and food samples is usually low ffor 7
porahaemolyficus they usually represent less than 5 %o of the total Il parahaemolyticus present in a sample) and
thus the likelihood of detecting pathogenic strains by sub-culturing a small number of colonies for identification
and confirmation is very low. Confirmation of as many colonies as possible and preferably all colonies increases
the likelihood of detecting pathogenic strains.

9.5.3 Tests for presumptive identification

9.5.3.1 Oxidase test

Using a sampling loop, platinum iridium straight wire or glass rod, take a portion of the pure culture
from the saline nutrient agar (9.5.2) and streak onto the filter paper moistened with oxidase reagent
(5,4J or use a commercially available test, following the manufacturer's instructions. Neither a nickel-
chromium sampling loop nor a metallic wire shall be used. Nickel or chrome wire sampling loops may
give false-positive results, and should be avoided.

9,5.3,2 Microscopic examination (optional)

For each pure culture obtained in 9.4. test according to a) and b)

aJ Prepare a see ISO 7218. After staining, examine for morphology and the
film for Gram staining;
Gram reaction using a microscope and record the results.

bl Inoculate a tube of ASPW (5J).Incubateat3T "C t 1'C for t h to 6 h. Deposit a drop of the culture
onto a clean slide, cover with a cover slip and examine for the motility under the microscope. Note
the cultures showing a positive result for motility.

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tSO 21872-1:2O17(E)

9.5.3.3 Selection of the cultures

For confirmation, retain the oxidase-positive colonies.

For confirmation, also retain Gram-negative colonies which give a positive result in the motility test
(if examinedJ.

9.5.4 Biochemicalconfirmation

9.5.4.1 General

Using an inoculation loop, inoculate the media indicated in9.5.4.2 to 9.5.4.6 with each of the cultures
obtained from the colonies retained in 9.5.2.

9.5.4,2 L-lysine decarboxylase saline medium (5,5J)

lnoculate the liquid medium just below the surface. Add about 1 ml of sterile mineral oil on the top of
the medium.

Incubate at37 "C t 1'C for 24ht3h.

Turbidity and a violet colour after incubation indicate a positive reaction [bacterial growth and
decarboxylation of the L-lysineJ. A yellow colour indicates a negative reaction.

9.5.4.3 Arginine dihydrolase saline medium (5.5.2)

Inoculate the liquid medium just below the surface. Add about 1 ml of sterile mineral oil on the top of
the medium.

Incubate at37 "C t 1'C for 24h!3h.

Turbidity and a violet colour after incubation indicate a positive reaction (bacterial growth and
dihydrolation of arginine). A yellow colour indicates a negative reaction.

9.5.4.4 Detection of B-galactosidase (5.5'3)

Inoculate the presumptive colony into a tube containing 0,25 ml of sodium chloride solution (5.5.6).

Add one drop oftoluene and shake the tube.

oC + oC
Place the tube in the incubator (6.2) set at 37 1 and leave to stand for approximately 5 min.

Add 0,25 mi of the reagent for the detection of B-galactosidase and mix'

Replace the tube in the water bath set at 37 "C + 1

oC, leave to stand for 24 h t 3 h, examining it from
time to time.
A yellow colour indicates a positive reaction (presence of B-galactosidase). The reaction is often visible
after 20 min. Absence of colouring after 24 h indicates a negative reaction.

If ready-to-use paper disks (5.5.3J are used, follow the manufacturer's instructions.

9.5.4.5 Detection of indole (5.5.4)

Inoculate a tube containing 5 ml of the tryptophan saline medium with the presumptive colony.

Incubate at 37 'C t 1 oC for 24h t 3 h. After incubation, add 1 ml of Kovacs reagent (554).
The formation of a red ring indicates a positive reaction (formation of indole). A yellow-brown ring
indicates a negative reaction.

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9.5.4.6 Halotolerancetest(5.5.5)
Produce a series of saline peptone waters with increasing salt (NaCl) concentration; 0 0/0, 6 0/o and 10 o/o
(s.s.s).

Inoculate each of the tubes [5.5.5) with the presumptive colony.

Incubate at37 "Cl 1 oC for 24h t 3 h.

Observation ofturbidity indicates that the bacterium can grow at the concentration ofsodium chloride
present in the tube of saline peptone water.

9.5.4.7 Interpretation of biochemical tests

V. parahaemolyticus, V. cholerae and V. vulnificus generally give the reactions indicated in Table 4.

Table 4 Interpretation of biochemical tests

-
Vibrio Vibrio Vibrio Vibrio Vihrio
Test a parahaemolyticus a vulnificus a mimicusab alginolyticus ab
cholerae
0xidase + + + + +

LDC + + + + +

ADFI
ONPG hydrolysis + + +

Production of indole + + + + +

Growth in peptone
water with
0 % NaCl + +

6 % NaCl + + +

10 o/o NaCl +

The sign + denotes 76o/oto 89 % positive reactions

b Provided for reference purposes.

N0TE 1 The reactions given in Table 4 are a guide to the identification of the listed species. Additional
phenotypic tests can be required to fully distinguish these species from each other and from non-pathogenic
Vibrio spp.

N0TE 2 It is preferable to conduct serology for V. cholerae (at least to determine whether they are serogroups
O1 or 0139) or in appropriate PCR-based test to determine toxigenic strains, such as those that carry the cholera
toxin gene cfx. This can be carried out by the end user laboratory or a specialist reference laboratory'

NSTE3 There are some rare instances where V. parahaemolyticus strains are positive for ONPG hydrolysis'
Additional phenotypic and/or molecular tests may be required to fully distinguish these strains from each other
and from non-pathogenic Vibrio spp.

9.5.4.8 Step by step confirmation (optional)

Both 7 cholerae and. V. parahaemolyticus have different salinity tolerances. With the cultures that
are selected in 9.5.3.3. undertake tests for growth in 10 % saline peptone water (5'5'5) and arginine
dihydrolase (5.5.2). Continue with the other confirmation tests on any colonies that do not show growth
in 10 o/o saline peptone water and which give a negative arginine dihydrolase reaction.
It is preferable to subculture onto saline nutrient agar (5.3) at the same time in order to make sure that
the "no growth" in the 10 % saline peptone water is not due to a dead culture.

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rSO 2L872-l:2017(E)

9.5.5 PCR confirmation

The minimum requirements for the amplification and detection of nucleic acid sequences by PCR are
laid outin ISO 221.\8,15O22719 and ISO 22174.
PCR may be carried out by conventional or real-time PCR formats. See 9.5.7 and Annex C for example
details of conventional PCR, and 9.5.8 and Annex D for real time PCR used in the development of this
document.

For PCR, use colonies retained in 9.5.3.3.

For transport to a specialist laboratory, inoculate colonies onto saline nutrient agar slants [5.3] or
other suitable media.

9.5.6 DNA extraction

Prepare a bacterial suspension using an inoculating loop. Take one well isolated colony from the saline
nutrient agar (53) and inoculate into 500 pl of sterile 0,85 % NaCl (5.5.6) or nuclease free water in a
1,5 ml micro-centrifuge tube.

Heat the micro-centrifuge tubes in a heating block (-6,7J set at 95 "C + 2

oC
for 5 min t 1 min.
Centrifuge the micro-centrifuge tubes at 10 0009 for 1 min. Retain the supernatant for PCR testing.

If shown to be reliable, commercially available DNA extraction kits may be used to extract bacterial
DNA. These kits shall be used in accordance with the manufacturer's instructions.
For each batch of samples tested a negative extraction control shall be included. DNA extraction shall
be carried out in parallel on 500 pl of nuclease free water or sterile 0,85 % NaCl.
oC
For long term storage a temperature of <-15 is recommended. Short-term storage [<1 month) samples
can be refrigerated at 5'C 3 t
oC.

9,5,7 Conventional PCR

Prepare master mix using appropriate primers, adjusting volumes depending on the required number
of reactions.

Place x pl of extracted DNA to a clearly labelled tube and addy pl of master mix.

Load PCR tubes into the thermocycler and set the appropriate running conditions depending on the
primer set used.
Thermocycler running conditions should be carried out as single reactions and run for 20 to 30 cycles
depending upon the target (see Annex C). The duration and temperature of each stage (denaturation,
annealing and extension) depend on the reagents used. See Annex C for details of the PCR primers and
running conditions used in the development of this document.
Prepare a 2 o/o agarose gel by mixing 2 g of agarose with 100 ml of 1X TAE buffer (or equivalent buffer)
(5.6.1). Allow to cool before adding a few drops of ethidium bromide or alternative dye enabling
visualization of the product.
Place an appropriate sized gel comb to the mould before pouring the gel and allow to solidify before
removing the comb.

Load the gel with L0 pl of 100 bp DNA ladder and the following wells with 20 pl of PCR product prepared
previously, using an appropriate loading dye as necessary, Run the gel at 130 V for 25 min to 30 min.
Alternative volumes of PCR product and DNA ladder may be used according to the individual laboratory
procedures and manufacturers' recommendations.

Following electrophoresis, visualize the gel using an ultraviolet transilluminator. See Annex C for
details of expected product sizes for primers used in the development of this document.

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 rSO 2t$72a:2Afi (E)

PCR products should be confirmed by an appropriate method, following ISO 22174,

PCR may be used to screen for the presence of target bacterium in primary (9.1) and secondary
enrichment broths (9.2) following incubation. This may enable downstream targeting of identification,
It is recommended that for samples considered of importance, e.g. official controls and,for outbreak
investigations, colony isolation and confirmation is carried out according to this document.

9.5.8 Real-time PCR

Add 20 pl of the relevant mastermix to each well. Alternative volumes of PCR reagents may be modified
according the individual laboratory procedures or from published protocols'

For every batch of samples in a cycler run, prepare an optical plate with the following:
at least L well with 5 pl of undiluted sample DNA;

at least 1 well with 5 pl of negative extraction control;

at least 1 well with 5 pl of control DNA for each target assay;

at least 1 well with 5 pl of nuclease free water.

Subject the plate to at least 45 cycles. The duration and temperature of each stage (denaturation,
annealing and extension) depend on the reagents used; they should be based upon the manufacturer's
recommendations.

For real-time PCR machines where the user can set the point of fluorescence measurement, this shall be
set at the end ofthe extension stage.

Analyse the amplification plots using the approach recommended by the manufacturer of the real-time
PCR machine. The threshold should ideally be set so that it crosses the area where the amplification
plots (logarithmic view) are parallel (the exponential phase).
Negative controls [nuclease-free water and negative extraction controls) shall always be negative;
if positive results occur in these controls then any samples giving positive results shall be retested.
See Annex D for example details of an amplification method used in the development of this document.

It is recommended that for samples considered of importance, e.g. official controls and/or outbreak
investigations, colony isolation and confirmation is carried out according to this document.

10 Expression of results
Depending on the interpretation of results, indicate that potentially enteropathogenic Vibrio spp. is
detected or not detected in a test portion of x grams or x ml of product (see ISO 72IB), specifying the
name ofthe relevant species and any pathogenicity characteristics ifthey have been tested.

11 Performance characteristics of the method

1 1.1 Interlaboratory study

The performance characteristics of the method were determined in an interlaboratory study to
determine the specificity, sensitivity and, when possible, the LODso of the method. The data are
summarized in Annex E. The values derived from the interlaboratory study may not be applicable to
food types other than those given in Annex E.

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$A 2L872-1:2017(E)

11.2 Sensitivity
The sensitivity is defined as the number of samples found positive divided by the number of samples
tested at a given level of contamination. The results are thus dependent on the level of contamination of
the sample.

11.3 Specificity
The specificity is defined as the number of samples found negative divided by the number of blank
samples tested.

11.4 LODso
The LODso is the concentration [cfu/sampleJ for which the probability of detection is 50 %0.

12 Testreport
The test report shall specify:

the reference of this document, i.e.lSO 21872-L;

all information required for the complete identification of the sample;

the sampling method used, if known;

any deviation with respect to the enrichment medium or the incubation conditions used;

all operating details not specified in this document, or regarded as optional, together with details of
any incidents which may have influenced the results;

the results obtained, and in particular if the pathogenicity markers of the isolated strains were
confirmed;

whether a positive result has been obtained when using an isolation medium (5.2.2) not specified in
this document.

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 tSO 21872-l=20L7 (E)

Annex A
[normative)

Diagram of procedure

Identification of trh positive V. parahoemolyticus is by conventional PCR only (refer to Annex C),
identification of 7 cholerae is by conventional PCR and/or biochemical tests (refer to Annexes B and C)'

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tSO 2L872-l:2O17(E)

ASPW at arnbient temperature

25 gor 25 ml of test portion in 225 ml of ASPW (9,1)
F
Z
LI
a,

d
zlr.t (Temperature dependent upon target ylbrro spp. md state ofproduct e.g deep frozen, fresh, etcJ

d
oC
tI At 37 "C * 1 "C (9.2)
E At 41,5 "C (9.2)
V, poruhaemollticus and. V. cholerae in deep frozen, dried or
d porohaemoljtlcus and y. cioleroe in fresh products
r V.
salted products, and y. vulntlclr all product states

1 ml of culture obtained above in 10 ml ASPW

1 ml of culture obtained above in 10 ml ASPW
>2 incubation at 41,5 'C t oC
for 18 h t t h (9.31
<= L Incubation at
j Incubation at
2,!. 41,5 "C L'C for
18h11h(9.3) 37'Ci1"Cfor
a)4 V. po rohae moly tl cus and V. 18h11h[9.3)
a& cio,€roe itr deep frozen, dried ,a ,ulrrlcus ail product states
or slted products

PCR Saening of 18 h i t h brcths followint incubation (optional) (trote 1 in 9.3)

PCR ScEenirg of 18 h 1 t h broths folloMng incubaton (optional) (note 1 in 9,3)

Plating on TCBS medium, incubation at 37 "C t l"Cfor 24 h I 3 h,

and plating onto a second medium of the laboratories own choice [9.4J

Q<
z)
<o
>2 Sub-culture from each medium at least one colony considered typical of the target
;) onto SNA, incubate at 37 oC ! 1 'C for 24 h t 3 h
Ei (for tdft and trh detection in V. parahoemolyticus take at least 5 or all colonies) (9.5.2)
;2
-o
tII
0 Choose at least one ofthe following confirmation steps

Confirmation by biochemical tests Confirmation by PCR Confirmation by real-time PCR

(9.5.4) (Annex B) (9.5.7J (Annex CJ [9.5.8) (Annex D)
z
ti

&
lt
z
o
Q

Further confi rmation of pathogenicity

markers

Expression ofresults {Clause 10)

Figure A.1 Diagram of procedure for the detection of enteropathogenic V. parahaemolyticus,

- V. cholerae and Z vulnificus

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 ISO 2L872-1:2017(E)

Annex
Add: 8 Hoang Ouoc Viet, Cau Giay, HN
[normati Tel: (844) 9z56426g"Far (84.4)
98961556
Website: WWW ,0r9, vn

Composition and preparation of th

Tlris crpI fias [een made [y inlonmalion
{ienter Isn $tandapds, Melnolo[y and
0uality

8.1 Alkaline saline peptone water (ASPW)

B.1.1 Composition
Peptone 20,0 g

Sodium chloride 20,0 g

Water 1 000 ml

8.L.2 Preparation
Dissolve the components in the water, by heating if necessary.

If necessary, adjust the pH so that, after sterilization, it is 8,6 ! 0,2 at25 "C.

Dispense the medium, in quantities required for the examination.

oC
Sterilize in an autoclave set att?l for 15 min.

8,2 Thiosulfate citrate bile and sucrose agar (TCBS)

8.2.1 Composition
Peptone 1.0,0 g

Yeast extract 5,0 g

Sodium citrate 10,0 g

Sodium thiosulfate 10,0 g

Iron [ll) citrate 1,0 g

Sodium chloride 10,0 g

Dried bovine bile 8,0 g

Sucrose 20,0 g

Bromothymol blue 0,04 g

Thymol blue 0,04 g

Agar-agar 8,0 g to 18,0 g a

Water 1 000 ml

, Depending ofthe gel strength ofthe agar-agar.

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ISO 21872-t:20L7(E)

8.2.2 Preparation
Dissolve the components or the complete dehydrated medium in the water, by bringing to the boil.
oC.
Adjust the pH, if necessary, so that it is 8,6 + 0,2 at25

Do not autoclave.

8.2.3 Preparation of the agar dishes

oC,
Dispense L5 ml to 20 ml of the medium, cooled down to approximately 50 into sterile Petri dishes
and leave to solidify.

8.3 Saline nutrient agar [SNA)

8.3.1 Composition
Meat extract 5,0 g

Peptone 3,0 g

Sodium chloride 10,0 g

Agar-agar Bgtol8ga
Water 1 000 ml

a Depending on the gel strength ofthe agar-agar.

8.3.2 Preparation
Dissolve the dehydrated components or the complete dehydrated medium in the water, by heating if
necessary.

Adjust the pH so that, after sterilization, itis 7,2 + 0,2 at 25 "C.

Transfer the medium into containers of appropriate capacity.

Sterilize in an autoclave set atl2l "C for l-5 min.

8.3.3 Preparation of the agar dishes

oC,
Dispense 15 ml to 20 ml of the medium, cooled down to approximately 50 into sterile Petri dishes
and leave to solidify.

Just prior to use, carefully dry the dishes of agar medium (preferably after having removed the lids and
inverted the dishes), in an incubator (6.2) until the agar surface is dry.

8.3.4 Preparation of slants of saline nutrient agar

ml of the medium, cooled down to approximately 50 oC, into tubes of
Dispense approximately 10
appropriate capacity,
Leave to settle and solidify in an inclined position.

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 ISO 21872-1,.2Afi(E)

8.4 Reagent for detection of oxidase

8.4.L Composition
N,N, N', N'-Tetramethyl-p-phenylenediamine 1,0 g
dihydrochloride

Water 100 ml

8.4.2 Preparation
Dissolve the components in the cold water immediately before use.

8.5 L-lysine decarboxylase saline medium (LDC)

8.5.1 Composition
monohydrochloride
L-lysine 5,0 g

Yeast extract 3,0 g

Glucose 1,0 g

Bromocresol purple 0,015 g

Sodium chloride 10,0 g

Water 1 000 ml

8.5.2 Preparation
Dissolve the components in the watel by heating if necessary.
If necessary, adjust the pH to 6,8 t 0,2 at 25 "C after sterilization.

Dispense the medium, in volumes of 2 ml to 5 ml into narrow tubes.

Sterilize in an autoclave set atl2l 'C for 15 min.

8.6 Arginine dihydrolase saline medium (ADH)

8.6.1 Composition
Argininemonohydrochloride 5,0 g

Yeast extract 3,0 g

Glucose 1,0 g

Bromocresol purple 0,015 g

Sodium chloride 10,0 g

Water 1 000 ml

8.6.2 Preparation
Dissolve the components in the water, by heating if necessary'

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ISO 21872-1:2O17(E)

If necessary, adjust the pH to 6,8 t 0,2 at25 oC

after sterilization.

Dispense the medium in volume of 2 ml to 5 ml, into narrow tubes

Sterilize in an autoclave set at12'1. 'C for 15 min.

8.7 Detection of B-galactosidase

8.7.1 ONPG solution

B.7.l.L Composition

2-orthonitrophenyl- B-D-galactosipyranoside 0,08 g

(oNPGJ

water 15 ml

8.7,L.2 Preparation
oC t 1
Dissolve the ONPG in the water at 50 'C.

Cool the solution.

8.7.2 Buffer solution

8.7.2,L Composition

Sodiumdihydrogen-orthophosphate(NaH2POa) 6,99
Sodium hydroxide [0,1 mol/l solution) approx.3 ml

Water, quantity sufficient for a final volume of 50 ml

8,7.2.2 Preparation
Dissolve the sodium dihydrogen-orthophosphate in about 45 ml of water, in a volumetric flask.

Adjust the pH to 7,0 t 0,2 at25'C with a 0,1- mol/l solution of sodium hydroxide.

Make up to 50 ml with water.

8.7.3 Complete reagent

8.7.3.1 Composition
(&1-02)
Buffer solution 5 ml

ONPG solution (8.10.1) L5 ml

8.7.3,2 Preparation
Add the buffer solution to the ONPG solution.

store at 3 "c t 2,0 "c.

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 tSO 21872-l:20L7(E)

B.B Saline medium for detection of indole

8.8.1 Tryptophan saline medium

8.8.1.1 Composition
Enzymatic digest of casein L0,0 g

Dl-trytophan 1,0 g

Sodium chloride L0,0 g

Water 1000 ml

8.8.1.2 Preparation
Dissolve the components in the water, by heating if necessary, and filter.

If necessary, adjust the pH, so that, after sterilization, it is Z0 t 0,2 at25 "C.

Dispense the medium in quantities of 5 ml into tubes of appropriate capacity.

Sterilize in an autoclave set atl2l 'C for 15 min.

8.8.2 Kovacs reagent

B.B.2.t Composition
Dimethylamino-4 benzaldehyde 5g

Hydrochloric acid, p = 7,18 g/ml to 1,,1i g/ml 25 ml

Methyl-Z butan-2-ol 75 ml

8.8.2.2 Preparation
Mix the components.

8.9 Saline peptone water

8.9.1 Composition
Peptone 1og

NaCl 0gor60gorL00g
Water 1 000 ml

8.9.2 Preparation
Dissolve the component in the water, by heating if necessary.
oC
If necessary, adjust the pH, so that, after sterilization, it is Z5 + 0,2 at25

Dispense into tubes of appropriate capacity.

Sterilize in an autoclave set at\27 'C for L5 min.

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tSO 21872-t:2$t7(E)

B.10 Sodium chloride solution

8.10.1 Composition

chioride
Sodium 8,5 g

Water 1 000 ml

B.lO.2 Preparation
Dissolve the component in the water, by heating if necessary.

If necessary, adjust the pH, so that, after sterilization, it is Z5 + 0,2 at25 "C.

Dispense into tubes ofappropriate capacity.

oC
Sterilize in an autoclave set atl21' for 15 min.

B.11Tris acetate EDTA (TAE) buffer

8.11.1 Composition

buffer
50X TAE 20 ml

Water L 000 ml

8.1L.2 Preparation
Add the buffer solution to the water.

Store at 3 oC + 2,0 oC.

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 rSO 2t872-lz2OL7(E)

Annex C
(informative)

Conventional PCR for the detection of Vibrio parahaemolyticus,

thermostable direct haemolysin (tdh) and thermostable direct
related haemolysin (trh) genes, Vibrio cholerae and Vibrio
vulnificus

C.1 Composition of mastermix

See Table C.1.

Table C.1- Composition of mastermix

Reagent Volume per reaction
pl
Reaction buffer (5x) a 10
MgCI2 (25 mM) 5

dNTPs (20 mMJ 0,625

Forward primer (nMJ [100 pM) 0,5
Reverse primer [nM) (100 pMJ 0,5
Water 30,625
Taq polymerase 0,25
Total volume 47,5
a Dependent upon the initial concentration ofthe reaction buffer

C.2 Vibrio parahaemolyticus primers (conventional PCR)

VpToxR (FW): GTC TTC TGA CGC AAT CGT Tctsl

VpToxR (REV): ATA CGA GTG GTT GCT GTC ATGtsl

The target region used for detection of toxR is based on previously published and validated data.[S]
The toxR region of all publicly available and sequen ced V. parahaemolyficus strains, as well as publicly
deposited foxR sequences was used to ensure specificity. Sequence alignment using all sequenc€s
available in GenBank of the assay target region demonstrates that this primer/probe set is adequate for
the quantification of all foxR sequences. The primer sequences do not align with any other sequences
available in GenBank.

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ISO 21872-l:2OL7(E)

C.3 Thermostable direct haemolysin (fdh) and thermostable direct related

haemolysin (trh) genes Vibrio parohaemolyticus primers (conventional PCR)

Thermosrable direct haemolysin L-rdh [FW): GTA AAG GTC TCT GAC TTT TGG ACt6l

Thermostable direct haemolysin R-rdh (REV): TGG AAT AGA ACC TTC ATC TTC ACCt6l

Thermostable direct related haemolysin L-frh (FW): TTG GCT TCG ATA TTT TCA GTA TCTt6l

Thermostable direct related haemolysin R-frh (REV):CAT AAC AAA CAT ATG CCC ATT TCC Gt6I

The target region used for detection of tdh and trh is based on previously published and validated data
[6] The primer sequences do not align with any other sequences available in GenBank.

C.4 Vibrio cholerae primers (conventional PCR)

pTVC {FW): TTA AGC STT TTC RCT GAG AAT GtZ]

prrC (REVJ: AGT CAC TTA ACC ATA CAA CCC GtzI

The target region used for detection of prVC is based on previously published and validated data.[Z]
The primer sequences do not align with any other sequences available in GenBank.

C.5 Vibrio vulnificus primers (conventional PCR)

VVH {FW): CCG GCG GTA CAG GTT GGC GCts]

77H IREVJ: CGC CAC CCA CTT TCG GGC Ct8]

The target region used for detection of vvH is based on previously published and validated data.[8]
The primer sequences do not align with any other sequences available in GenBank.

C.6 Cycling parameters - VptoxR and WH

See Table C.2.

Table C,2 Cycling parameters VptoxR andVVH

- -
Step description Temperature and time Number of cycles
Pre-heating 96'C for 5 min 1

Denaturation 94'C for 1 min

Amplification Annealing 63'C for 1,5 min 30
Extension 7 2 "C for 1,5 min
Post amplification 72"Cfor7 min 1

C.7 Cycling parameters - prVC

See Table C.3.

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Table C.3 Cycling parameters prVC

- -
Step description Temperature and time Number of cycles
Pre-heating 94 "C for 2 min 1

Denaturation 94 "C for 1 min

Amplification Annealing 50'C for 1 min 30
Extension 72 "C for 1,5 min
Post amplification 72'C for 10 min 1

C.B Cycling parameters tdh and trh

-
See Table C.4.

Table C.4 Cycling parameters tdh and, trh

- -
Step description Temperature and time Number of cycles
Pre-heating 94'C for 3 min I
Denaturation 94'C for 1 min
Amplification Annealing 58'C for 1 min 30
Extension 72 oC for 1 min
Post amplification 72 'C for 5 min 1.

C.9 Control material conventional PCR

-
For each target assay DNA was extracted from characterized reference strains (see Table C.5).

A bacterial suspension was prepared by taking one to three colonies from a saline nutrient agar (5,3)
and inoculating 500 pl of sterile NaCl0,85 % (5.5.6) in a 1,5 ml micro-centrifuge tube.

Each micro-centrifuge tube was placed in a heating block (6.7) set at 95

oC +
1 "C for 5 min t 1 min.
The micro-centrifuge tubes were centrifuged at 10 0009 for 1 min and the supernatant removed for
PCR testing.

For long term storage a temperature of <-15 'C is recommended.

Table C.5 Reference strains

-
P roduct
Function Target WDCM a control strains Identification size
bp
foxR Vibrio p or ahoemolyticu s WDCM 00185 Vibrio p arahaemoly ticu sb 368
Vibrio p arah a emoly ticu s NCTC 10884
tdh thermostable direct haemolysin c 269
IWDCM number claimed)
Specificity thermostable direct related 500
trh Vibrio parahaemolyticus WDCM 00037
haemolysin c

prVC Vibrio choleroe WDCM 00136 Vibrio choleraeb 295 to 3L0

VVH Vibrio vulnificus WDCM 00139 Vibrio vulnificusb 519
a World Data Centre for Microorganisms (WDCM) strain catalogue available at http://refs.wdcm.org
b Denotes species identification.
c Indicates pathogenic strain of Vibrio parohaemolyticus.

2(
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C.10 Interlaboratory study

The PCR methods were validated in an interlaboratory study organized by the European Union Reference
Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs in 2011. Twenty-
three laboratories were recruited according to the requirements of ISO 5725-1 and ISO 5725-2; all
followed a single protocol. Test samples were anonymized and distributed as semi-solid marine agar
(MA) swabs inoculated with reference strains of target and non-targetVibrio spp. Swabs were inoculated
into225 mlt 5 ml ASPW and subjectto primary enrichment at41,,5 + 1oC and 37 I 1oC for 6 h t t h.
oC oC

One millilitre aliquots of each enrichment were subject to secondary enrichment in 10 ml t 0,5 ml fresh
ASPWat4l,5ocr1"Cand37oC+l"CforL8ht3h.Followingprimaryandsecondaryenrichment,lpl
of each enrichment broth was sffeaked onto the surface of TCBS and a second plating medium, TCBS
plates were incubated at 37 "C ! 1 'C for 24 h ! 3 h; second plating media were incubated according to
the manufacturers' instructions. A minimum of two colonies showing typical phenotypic characteristics
of Vibria spp. were sub-cultured onto SNA and incubated at 37 "C t 1 "C for 24 h t 3 h. Subsequent
cultures were checked visually for purity and subject to oxidase tests and microscopic examination
(motility and Gram stainJ. Oxidase positive, Gram negative, motile isolates were subject to tests for
glucose utilization, lactose and sucrose fermentation, L-lysine decarboxylation, B-galactosidase activity,
presence of arginine dihydrolase, indole production, ornithine decarboxylase production and growth
in 0 o/0,2 o/o, 6 o/s, B o/o, and 10 % NaCl. ln parallel, DNA was extracted from a single colony suspended in
500 pl of nuclease free water, the bacterial suspension was heated at 95
oC I
1 'C for 5 min t 1 min and
oC
centrifuged at 10 0009, The resultant supernatant was stored at <-15 for PCR analysis. Aliquots of
2,5 prl of extracted DNA were added to a mastermix containing 10 pl reaction buffer, 5 pl MgCl2, 0,625 1:.1
dNTPs (20 mMJ, 0,5 pl primer [forward and reverse),0,25 pl Taq polymerase and 30,625 pl nuclease
free water. All samples were subjected to PCR according to the cycling parameters described in C.6,
C.7 and C.B for foxR[S], tdhl6),trhl6),prVC[Z] and VVHtgl. Products were visualized on 2 %o agarose gels
following electrophoresis at 130 V for 25 min to 30 min.
A summary of the results from the interlaboratory study is given in Table C.6.

Table C.6 Interlaboratory study data

-
Parameters Results
Year 2077
Maximum number of laboratories reporting PCR and/or biochemical results for
ZJ
both primary enrichment temperatures
Number of samples per laboratory 5

Maximum number of results per laboratory 20

Number of accepted results 370
37 "C 4L,5 "C
PCR Bio PCR Bio
Laboratories assigning correct species identification (V. vulnificus) {o/o) 93,3 86,4 100,0 95,2
Laboratories assigning correct species identification ( V. p a r ah a e m o ly ti c u s) (o/o) 100,0 82,6 100,0 85,7
Laboratories assigning correct species identification (V. cholerae) (0/o) 87,5 69,6 73,3 61.,9

Laboratories assigning correct species identification (V. mimicus) {o/o) 100,0 73,9 100,0 71,4
Laboratories assigning correct species identification (V. fluvialis) (o/o) 100,0 77,3 100,0 76,2
PCR Bio
Total correct, all samples [%) 94,8 78,3
PCR tests for tdh and trh
tdh trh
Laboratories correctly assigning presence of tdh and trh, all samplest6J (%] 94,6 88,04
PCR = polymerase chain reaction
Bio = biochemical tests

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Annex D
(informative)

Real-time PCR for the detection of Vibrio parahqemolyticus,

thermostable direct haemolysin gene (tdh) and Vibrio vulnificus

D.1 Composition of mastermix

See Table D.1.

Table D.1 Composition of mastermix

-
Reagent Volume per reaction
pl
2 x TaqMan universal mastermix (1xJ a 72,5
Forward primer (100 nM) a 0,45
Reverse primer (100 nM) a 0,45
Probe (500 nM) a 1.

Water 5,6
DNA sample 5

Total volume 25
a Final concentration

D.2 Vibrio parahaemolyticus - primers and hydrolysis probes

VpToxR (FW): GAA CCA GAA GCG CCA GTA GTIeI

VpToxR (REV): AAA CAG CAG TAC GCA AAT CGI9I

VpToxR [PROBE): TCA CAG CAG AAG CCA CAG GTG Cte]

Probes used in the interlaboratory study were labelled 5' 6-carboxyfluorescein (FAM), 3' 6-carboxy-
tetramethylrhodamine (TAMRA).
The target region used for detection of toxR is based on previously published and validated data'[9]
The primer sequences do not align with any other sequences available in GenBank. The toxR region
of alipublicly available and sequencedV. parahaemolyficus strains, as well as publicly deposited toxR
s"quences wis used to ensure specificity. Sequence alignment using all sequences available in GenBank
of fhe assay target region demonstrates that this primer/probe set is adequate for the quantification of
all roxR sequences. Tlie primer sequences do not align with any other sequences available in GenBank.

D.3 Thermostable direct haemolysin [tdh) gene Vibrio parqhaemolyticus -

primers and hydrolysis probes
rdh (FWl: TCC CTT TTC CTG CCC Cctlol

rdh (REV): CGC TGC CAT TGT ATA GTC TTT ATCtlol

rdh (PROBEJ: TGA CAT CCT ACA TGA CTG Tctl0l

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Probes used in the interlaboratory study were labelled 5' 6-carboxyfluorescein (FAM), 3' 6-carboxy-
tetramethylrhodamine (TAM RA).
The target region used for detection of tdh is based on previously published and validated data.[10]

D.4 Vibrio vulnificus * primers and hydrolysis probes

VVHA [FWJ: TGT TTA TGG TGA GAA CGG TGA CAITL]

VVHA [REVJ: TTC TTT ATC TAG GCC CCAAAC TTGIll]

VVHA (PROBEJ: CCG TTA ACC GAA CCA CCC GCA Atl1]

Probes used in the interlaboratory study were labelled 5' 6-carboxyfluorescein (FAM), 3' 6-carboxy-
tetramethylrhodamine ITAM RA).
The target region used for detection of vvhA is based on previously published and validated data.[.Lt]
Laboratories involved in the interlaboratory study used a reverse primer of the vvHA with a guanine
base missing. The following verification study was carried out by the European Union Reference
Laboratory (EURL) to ascertain impact on performance.[20]
NOTE Real-time primers and probes for V. cholerae and trh genes are excluded from Annex D as data
generated in the interlaboratory study was not reliable.

D.5 Cycling parameters

See Table D.2.

Table D.2 Cycling parameters

-
Step description PCR step Temperature and time Number of cycles
Preheating Initial denaturation 95'C for 10 min 1

Denaturation 95 'C for 15 s

Amplification 45
Annealing - extension 60 "C for 1 min

D.6 Control material - real-time PCR

See Table D.3.

Table D.3 Reference strains

-
Function Target WDCM a control strains Identification
toxR Vibrio p arahaemo ly ticu s WDCM 00185 Vibrio parahaemolyticu s b
Specificity tdh Vibrio parohaemolyticus NCTC 10884 thermostable direct haemolysin c

VVH Vibrio vulnificus WDCM 00139 Vibrio vulinificusb

World Data Centre for Microorganisms (WDCM) strain catalogue available at http://refs.wdcm.org
b Denotes species identification.
c Indicates pathogenic strain of Il parahaemolyticus.

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Annex E
Iinformative)

Results of an interlaboratory study

An international interlaboratory study involving 13 collaborators in 9 countries was carried out for
detection of V. porahaemolyticus (including thermostable direct and direct related strains), V. vulnificus
and I4 cholerae in raw and cooked seafood, (oysters and prawns). The food samples were each tested
for each determinant at two different levels of contamination, plus a negative control. The study was
organized in 2013 by the European Union Reference Laboratory for monitoring bacteriological and
viral contamination of bivalve molluscs.
The method specified in this document was used to carry out the interlaboratory study' The values
of the performance characteristics derived from this interlaboratory study are shown per bacterial
determinant and type of sample in TableS-E-L to E.6. Identification of target Vibrio spp. were by
biochemicai, conveniional and/or real-time PCR. Real-time PCR for identification of V. cholerae was
tested in this international interlaboratory study but data were not reliable. Details are excluded from
Annex D. Real-time PCR identification of trh genes of V. parahaemolyticuswas tested in this international
interlaboratory study but data were not reliable. Details are excluded from Annex D.

Table E.1- Results of data analysis obtained withVibrio parahaemolyticus in raw oysters
Low level contamination High level contamination
Parameters Blank
(2 x 1,0 cfu/25 g) (2x706 cfu/25 g)a
Number of participating laboratories 13 13 13

Number of retained participating 10 10 10

laboratories after evaluation of
the data
Number of samples 104 104 1.04

Number of samples retained after 80 BO BO

evaluation ofthe data
25 25
Sample size (g / ml / cm2/item) 25
Sensitivity, 7o b 90 72,5 100

Specificity, % b 1.11.

LODso, cfu/gc 0,43 (0,32 to 0,57)

a For spiked samples inocula levels prior to inoculation were estimated using optical density measurements at 600 nm
with refeience to pieviously prepared growth curves for each strain under test, confirmation ofinoculum was by a 100 pl
spread plate method on marine agar in triplicate.
b Sensitivity and specificity were calculated according to 772 and I[J, specificity exceeds ]"00 % where positive
identifications were reported in'blank' samples.
c LODso was calculated using the methods described in Reference U,21. Currently estimated as per g, 95 7o confidence
intervals in parentheses.

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29
ISO 21872-l:2O17(E)

Table E.Z Results of data analysis obtained for tdh and trh for Vibrio parahaemolyticus in
- raw oysters
Confirmation tests a
Parameters Conventional PCR Real-time PCR
(Annex C) tAnnexj!)
tdh trh tdh
Number of participating laboratories 6 7 9
Number of retained participating laboratories after 6 7 9
evaluation ofthe data
Sensitivity, % b B6 94 /5

Specificity, o/o b 89 9L 94
a PCR methods (real time or conventional PCR applied to isolated presumptive/confirmed
V. parahaemolyficu.s colonies (n s 5) only from up to 16 samples per laboratory of V. parahaemolyticus) not all laboratories
used both conventional and real-time methods for assignment of tdh.
b Calculated as number ofisolates reported positive for fdh gene divided by number ofisolates tested.

Table E.3 Results of data analysis obtained withVibrio vulnificus in raw oysters
-
Low level contamination
Parameters Blank
(2 x 1.0 cfu/25 g) a

Number of participating laboratories 13 13

Number of retained participating 10 10

laboratories after evaluation ofthe data
Number of samples 104 1.04

Number of samples retained after 104 80

evaluation ofthe data
Sample size (g / ml / cmz 1 item) 25 25
Sensitivity o/o b 90 37,50
Specificity, % b 77,50
LOD56, cfu/gc 12,37 {8,46 to L7,91)
a Inocula levels prior to inoculation were estimated using optical density measurements at 600 nm with reference to
previously prepared growth curves for each strain under test, confirmation ofinocula was by a L00 pl spread plate method
on marine agar in triplicate.
b Sensitivity and specificity were calculated according to 1 1.2 and 11.3.
c LODso was calculated using the methods described in Reference [/]. Currently estimated as per g, 95 o/o confidence
intervals in parentheses.

Table 8.4 Results of data analysis obtained with Vibrio cholerae in cooked prawns
-
Low level contamination High level contamination
Parameters Blanl<
(1 x 10: cfu/25 g) a (1 x 10s cfu/25 g) a

Number of participating 11 11 11
collaborators
Number of collaborators retained 10 10 10
after evaluation ofthe data
Number of samples BB BB BB
a Bacterial inocula prepared as freeze-dried ampoules supplied by FEPTU, PHE, UK according to organizing laboratories
specification.
b Sensitivity and specificity were calculated according to 11.2 and 11.3.
c LODso was calculated using the methods described in Reference E2]. Currently estimated as per g, 95 0/o confidence
intervals in parentheses.

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 tSO 2L872-1:20L7(E)

Table 8.4 (continued)

Low level contamination High level contamination
Parameters BIank
[1 x 103 cfulZS g) a (L x 10s cfulT\ g) a
Number of samples retained after BO BO BO
evaluation ofthe data
Sensitivity, %o b 100 92,5 92,5
Specificity, 7o b 84
LOD5s, cfu/gc 1]-,34 (6,26 to 20,56)
a Bacterial inocula prepared as freeze-dried ampoules supplied by FEPTU, PHE, UK according to organizing laboratories
specification.
b Sensitivity and specificity were calculated according to 11.2 and 11.3.
c LODso was calculated using the methods described in Reference U,2]. Currently estimated as per g, 95 0/o confidence
intervals in parentheses

Table E.5 Results of data analysis obtained withVibriovulnificus in cooked prawns

-
Low level contamination High level contamination
Parameters Blank
(1 x 1S2 cfu/25 g) a (1 x 10+ cfu/25 g) a

Number of participating L\ 11 11
collaborators
Number of collaborators retained 10 10 10
after evaluation ofthe data
Number of samples 88 88 B8

Number of samples retained after 80 80 80

evaluation ofthe data
Sensitivity, %o b 95 61,25 71,5
Specificity, o/o b 60
LODso, cfu/gc 81.,2 (63,39 to 101,2)
a Bacterial inocula prepared as freeze-dried ampoules supplied by FEPTU, PHE, UK according to organizing laboratories
specification.
b Sensitivity and specificity were calculated according to 11.2 and 1L.3.
c LOD59 was calculated using the methods described in Reference E2]. Currently estirnated as per g, 95 %o confidence
intervals in parentheses.

Table E.6 Results of data analysis obtained withVibrio parahaemalyticus in cooked prawns
-
Level contamination
Parameters Blank
[1 x 10s cfu/25 g) a

Number of participating collaborators 71 11

Number of collaborators retained after 10 10

evaluation ofthe data
Number of samples 176 B8

Number of samples retained after evaluation 1.28 64

ofthe data
Sensitivity, %o b 100 100
Specificity, o/o b 99
a Bacterial inocula prepared as freeze-dried ampoules supplied by FEPTU, PHE, UK according to organizing laboratories
specification. All V. parihaemolyticus tested by conventional PCR (Annex C) and real-time PCR (Annex D) for tdh and trh
genes gave negative results, inoculum intended result tdh and trh negative.

b Sensitivity and specificity were calculated according to l[2 and 1.1.3.

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Bibliography

t1l FAO/WHO. 2001, Hazard identification, exposure assessment and hazard characterization
of Campylobacter spp. in broiler chickens and Vibrio spp. in seafood, a joint FAO/WHO expert
consultation, Geneva, Switzerland, 23-27 July 200 1

t21 Codex Alimentarius - Code of Practice for fish and fisheries products, 2012, Edition 2, FAO/WHO,
Rome, Italy

t3l Rosec |-P., Causse V., Cnuz B., ReuztrR |., CanNer L. The international standard IS0/TS 21872-
1 to study the occurrence of total and pathogenic Vibrio parahaemolyllcus and Vibrio cholerae in
seafood: its improvement by the use of a chromogenic medium and PCR. 1nt. J. Food Microbiol.
201.2, 157, pp. 189-194

L4) WanrurR E., & OlrvrR J.D. Refined medium for the isolation of Vibrio vulnificus from oyster
tissue and seawater. Appl. Environ. Microbiol. 2007, 73 (9J pp. 3098-3100

tsl Krrur Y.B.,Oxuna J,, Marsuuoro C.,Taxesassr N,, Hassn'roro S., NrsurBUCHr M. Identification
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Microbiol. 1999, 37 pp.1773-1177

t6l Brl A.K., ParrERsoN D.P., BRassER C.W., VIcxEnv M.C.L., Joxrs D.D., KavsNen C.A. Detection
of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR
amplification of tl, tdh and, trh. f . Microbiol. Methods. 1999, 36 pp. 215-225

t7) CHUN J., HUQ A., COLWELL R.R. Analysis of 165-235 rRNA intergenic spacer regions of Vibrio
cholerae and Vibrio mimicus. Appl. Environ. Microbiol. L999, 65 pp. 2202-2208

tBl Hnl WE., Krasr,rR S.P., TRucxsrss M.W., Fgr.rc P., KavsNsn C.A., Laupel K.A. Polymerase
chain reaction identification of Vibrio vulnificus in artificially contaminated oysters. Appl.
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tel Tarwo M., Baxrn-AusrrN C., Powell A., Hopcsoti E., NarAs O.B., Walxen D.l, Comparison of
foxR and rlh based PCR assays for Vibrio parahaemolyticus. Food Control. 2017, 77 pp.1L6-120

[10] NoRnsrRou J.L., VrcxrRv M.C.L., Blacxsronr G.M., MuRRav S.L., DsPaole A. Development of
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[1 1] CaNlpse Lr M.S., & WRIcH'r A.C. Real-time PCR analysis of Vibrio vulnificus from oysters. Appl.
Environ. Microbiol. 2003, 69 pp.7137-7144

[12) IS0 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 7:
General principles and definitions
-
ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic
[13]
method
-
for the determination of repeotability and reproducibility of a standard measurement method
u4l IS0 16140 (all parts), Microbiology of the food chain Method validation
-
[1s] Recommended protocol for the determination of limit of detection of a qualitative microbiological
method and estimate of its confidence limits (Prof. B Jarvis). Available at: http://www.wiwiss.fu
-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich/POnLOD verB.vls

ISO/TS 20836, Microbiology of food and animal feeding stuffs chain reaction (PCR)
[16]
the detection offood-borne pathogens Performance
- Polymerase
testing thermal cyclers
for - for

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 rSO 2L872-1:2oL7(E)

LL7) ISO 20832 Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR)
the detection of pathogens Requirements
-
for sample preparation for qualitative
for food-borne -
detection

ll8l ISO 20838, Microbiotogy of food and animql feeding stuffs Polymerase chain reaction (PCR)
-
for the detection of food-borne pathogens
- Requirements for amplification and detection for
qualitative methods

t19] ISO 1746B, Microbiology of the food chain

- Technical requirements and guidance on establishment
or revision of a standardized reference method

[20] https://eurlcefas.org/media/1 3971/eurl-vvha-method-comparison-pdf-pdf

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rcs 07.100.30
Price based on 33 pages

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