oSIST prEN 17711:2023
(Main)Plant biostimulants - Detection of Vibrio spp.
Plant biostimulants - Detection of Vibrio spp.
This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which causes human illness in or via the intestinal tract [1]. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the following:
- microbial plant biostimulants.
NOTE 1 The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V. vulnificus are the major contaminants of Vibrio spp. [1].
NOTE 2 For confirmation, it is possible to use PCR tests; in this case the laboratory must validate the procedure and data generated.
Pflanzen-Biostimulanzien - Nachweis von Vibrio spp.
Dieses Dokument legt ein horizontales Verfahren zum Nachweis von enteropathogenen Vibrio spp. fest, die Erkrankungen des Menschen am oder über den Darmtrakt verursachen [1]. Die Spezies, deren Nachweis die hier aufgeführten Verfahren einbeziehen, sind Vibrio parahaemolyticus, Vibrio cholerae und Vibrio vulnificus.
Das Dokument gilt für
- mikrobielle Pflanzen-Biostimulanzien.
ANMERKUNG 1 Die Weltgesundheitsorganisation (WHO; en: World Health Organization) hat V. parahaemolyticus, V. cholerae und V. vulnificus als die wichtigsten Kontaminanten von Vibrio spp. identifiziert [1].
ANMERKUNG 2 Eine Bestätigung durch PCR Tests ist möglich; in diesem Fall muss das Labor das Verfahren und die erzeugten Daten validieren.
Biostimulants des végétaux - Détection de Vibrio spp.
Le présent document spécifie une méthode horizontale pour la recherche des espèces entéropathogènes de Vibrio provoquant des maladies dans ou via le tractus intestinal chez l’homme [1]. Les espèces détectables par les méthodes spécifiées incluent Vibrio parahaemolyticus, Vibrio cholerae et Vibrio vulnificus.
Il s’applique :
- aux biostimulants microbiens des végétaux.
NOTE 1 L’Organisation Mondiale de la Santé (OMS) a identifié V. parahaemolyticus, V. cholerae et V. vulnificus comme les principales espèces de Vibrio contaminantes [1].
NOTE 2 Pour la confirmation, il est possible de recourir aux essais de PCR ; dans ce cas, le laboratoire doit valider le mode opératoire et les données générées.
Rastlinski biostimulanti - Ugotavljanje prisotnosti Vibrio spp.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17711:2023
01-maj-2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti Vibrio spp.
Plant biostimulants - Detection of Vibrio spp.
Pflanzen-Biostimulanzien - Nachweis von Vibrio spp.
Biostimulants des végétaux - Détection de Vibrio spp.
Ta slovenski standard je istoveten z: prEN 17711
ICS:
65.080 Gnojila Fertilizers
oSIST prEN 17711:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17711:2023
DRAFT
EUROPEAN STANDARD
prEN 17711
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2023
ICS 65.080 Will supersede CEN/TS 17711:2022
English Version
Plant biostimulants - Detection of Vibrio spp.
Biostimulants des végétaux - Détection de Vibrio spp. Pflanzen-Biostimulanzien - Nachweis von Vibrio spp.
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 455.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
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.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17711:2023 E
worldwide for CEN national Members.
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Contents Page
European foreword . 6
Introduction . 7
1 Scope . 8
2 Normative references . 8
3 Terms and definitions . 8
4 Principle . 9
4.1 General . 9
4.2 Primary enrichment in a liquid selective medium . 9
4.3 Secondary enrichment in a liquid selective medium . 9
4.4 Isolation and identification . 10
4.5 Confirmation . 10
5 Culture media and reagents . 10
5.1 Enrichment medium: alkaline saline peptone water (ASPW) . 10
5.2 Solid selective isolation media . 10
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS) . 10
5.2.2 Second medium . 11
5.3 Saline nutrient agar (SNA) . 11
5.4 Reagent for detection of oxidase . 11
5.5 Reagent for Biochemical tests . 11
5.5.1 L-lysine decarboxylase saline medium (LDC) . 11
5.5.2 Arginine dihydrolase saline medium (ADH) . 11
5.5.3 Reagent for detection of β-galactosidase . 11
5.5.4 Saline medium for detection of indole . 11
5.5.5 Saline peptone water . 12
5.5.6 Sodium chloride solution . 12
5.5.7 Toluene, p.a. . 12
6 Equipment and consumables . 12
7 Sampling . 12
8 Preparation of the test sample . 12
9 Procedure (see Figure A.1) . 13
9.1 Test portion and initial suspension . 13
9.2 Primary selective enrichment . 13
9.3 Secondary selective enrichment . 14
9.4 Isolation and identification . 14
9.5 Confirmation . 15
9.5.1 General . 15
9.5.2 Selection of colonies for confirmation and preparation of pure cultures . 15
9.5.3 Tests for presumptive identification . 16
9.5.4 Biochemical confirmation . 16
10 Expression of results . 18
11 Performance characteristics of the method . 18
11.1 Sensitivity . 18
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11.2 Specificity . 18
11.3 LOD50 . 18
11.4 Precision . 18
12 Test report . 18
Annex A (normative) Diagram of procedure . 20
Annex B (normative) Composition and preparation of the culture media and reagents . 22
B.1 Introduction . 22
B.2 Water . 22
B.3 Alkaline saline peptone water (ASPW) . 22
B.3.1 Composition . 22
B.3.2 Preparation . 22
B.4 Thiosulfate citrate bile and sucrose agar (TCBS) . 23
B.4.1 Composition . 23
B.4.2 Preparation . 23
B.4.3 Preparation of the agar dishes . 23
B.5 Saline nutrient agar (SNA) . 23
B.5.1 Composition . 23
B.5.2 Preparation . 24
B.5.3 Preparation of the agar dishes . 24
B.5.4 Preparation of slants of saline nutrient agar . 24
B.6 Reagent for detection of oxidase . 24
B.6.1 Composition . 24
B.6.2 Preparation . 24
B.7 L-lysine decarboxylase saline medium (LDC). 24
B.7.1 Composition . 24
B.7.2 Preparation . 24
B.8 Arginine dihydrolase saline medium (ADH) . 25
B.8.1 Composition . 25
B.8.2 Preparation . 25
B.9 Detection of β-galactosidase . 25
B.9.1 ONPG solution . 25
B.9.2 Buffer solution . 25
B.9.3 Complete reagent . 26
B.10 Saline medium for detection of indole . 26
B.10.1 Tryptophan saline medium . 26
B.10.2 Kovacs reagent . 26
B.11 Saline peptone water . 27
B.11.1 Composition . 27
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B.11.2 Preparation . 27
B.12 Sodium chloride solution . 27
B.12.1 Composition . 27
B.12.2 Preparation . 27
B.13 Tris acetate EDTA (TAE) buffer . 27
B.13.1 Composition . 27
B.13.2 Preparation . 27
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 . 28
C.1 General . 28
C.2 Equipment . 28
C.2.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the
purpose) . 28
C.2.2 Mastermix . 28
C.3 DNA extraction . 29
C.4 Procedure Conventional PCR . 29
C.5 Primers and probes . 30
C.5.1 General . 30
C.5.2 Vibrio parahaemolyticus primers (conventional PCR) . 30
C.5.3 Thermostable direct haemolysin (tdh) and thermostable direct related haemolysin
(trh) genes Vibrio parahaemolyticus primers (conventional PCR) . 31
C.5.4 Vibrio cholerae primers (conventional PCR) . 31
C.5.5 Vibrio vulnificus primers (conventional PCR) . 31
C.5.6 Cycling parameters — VptoxR and VVH . 31
C.5.7 Cycling parameters — prVC . 31
C.5.8 Cycling parameters — tdh and trh . 32
C.6 Control material — conventional PCR . 32
Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin gene (tdh) and Vibrio vulnificus . 34
D.1 General . 34
D.2 Equipment . 34
D.2.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the
purpose) . 34
D.2.2 Mastermix . 34
D.2.3 Composition of mastermix . 34
D.3 DNA extraction . 34
D.4 Real-time PCR . 35
D.5 Vibrio parahaemolyticus — primers and hydrolysis probes . 35
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D.6 Thermostable direct haemolysin (tdh) gene Vibrio parahaemolyticus — primers and
hydrolysis probes . 35
D.7 Vibrio vulnificus — primers and hydrolysis probes . 36
D.8 Cycling parameters . 36
D.9 Control material – real-time PCR . 36
Annex E (informative) Repeatability and Reproducibility data . 37
E.1 Materials used in the interlaboratory study . 37
E.2 Samples . 37
E.3 Procedure . 37
E.4 Replicate determination. 37
E.5 Results . 38
Annex ZA (informative) Relationship of this European Standard and the essential
requirements of Regulation (EU) 2019/1009 making available on the market of EU
fertilising products aimed to be covered . 39
Bibliography . 40
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European foreword
This document (prEN 17711:2023) has been prepared by Technical Committee CEN/TC 455
“Plant Biostimulants”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
This document will supersede CEN/TS 17711:2022.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of EU
Directive(s) / Regulation(s).
For relationship with EU Directive(s) / Regulation(s), see informative Annex ZA, which is an
integral part of this document.
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prEN 17711:2023 (E)
Introduction
This document was prepared by the experts of CEN/TC 455 “Plant Biostimulants”. The European
Committee for Standardization (CEN) was requested by the European Commission (EC) to draft
European standards or European standardization deliverables to support the implementation of
Regulation (EU) 2019/1009 of 5 June 2019 laying down rules on the making available on the
market of EU fertilizing products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as SR M/564 and M/564 Amd1, also contributes to the
Communication on “Innovating for Sustainable Growth: A Bio economy for Europe”. The Working
Group 5 “Labelling and denominations”, was created to develop a work program as part of this
Request. The technical committee CEN/TC 455 “Plant Biostimulants” was established to carry out
the work program that will prepare a series of standards. The interest in biostimulants has
increased significantly in Europe as a valuable tool to use in agriculture. Standardization was
identified as having an important role in order to promote the use of biostimulants. The work of
CEN/TC 455 seeks to improve the reliability of the supply chain, thereby improving the confidence
of farmers, industry, and consumers in biostimulants, and will promote and support
commercialisation of the European biostimulant industry.
Because of the large variety of Plant Biostimulant 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 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.
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.
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1 Scope
This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp.,
which causes human illness in or via the intestinal tract [1]. The species detectable by the methods
specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the following:
— microbial plant biostimulants.
NOTE 1 The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and
V. vulnificus are the major contaminants of Vibrio spp. [1].
NOTE 2 For confirmation, it is possible to use PCR tests; in this case the laboratory must validate the
procedure and data generated.
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.
1
prEN 17702-1:— , Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
2
prEN 17724:—, Plant biostimulants — Terminology
3
EN ISO 7218:2007, Microbiology of food and animal feeding stuffs – General requirements and
guidance for microbiological examinations (ISO 7218:2007)
4
EN ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production,
storage and performance testing of culture media (ISO 11133:2014)
EN ISO 3696:1995, Water for analytical laboratory use — Specification and test methods
(ISO 3696:1987)
3 Terms and definitions
5
For the purposes of this document, the terms and definitions given in prEN 17724:— and the
following apply.
ISO and IEC maintain terminological databases for use in standardization at the following
addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
1
Under preparation.
2
Under preparation.
3
As amended by EN ISO 7218:2007/A1:2013.
4
As amended by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020.
5
Under preparation.
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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. (3.1) (V.
parahaemolyticus, V. cholerae and V. 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. can be improved by the use of different incubation temperatures
depending upon the target species or state of the matrix. In liquid products, recovery of V.
parahaemolyticus and V. cholerae is enhanced by enrichment at 41,5 °C and the recovery of V.
vulnificus is enhanced by enrichment at 37 °C. Whereas in solid products, for V. vulnificus, V.
parahaemolyticus and V. cholerae recovery is enhanced by enrichment at 37 °C.
If 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 V. vulnificus
together is not required, the specific procedure(s) may be selected according to the species being
sought. Such a selection should be clearly specified in the test report.
V. parahaemolyticus, V. cholerae and V. vulnificus can 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) (5.1) at ambient temperature, followed by
...
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