FprCEN/TS 17711

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 validates the procedure and data generated.

Biostimulanzien für die pflanzliche Anwendung - 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 Biostimulanzien für die pflanzliche Anwendung.
ANMERKUNG 1   Die Weltgesundheitsorganisation (WHO; en: World Health Organization) 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 validiert das Labor das Verfahren und die erzeugten Daten.

[Not translated]

General Information

• Status: Not Published
• ICS: 65.080 - Fertilizers
• Technical Committee: CEN/TC 455 - Plant Biostimulants and Agricultural Micro-Organisms
• Drafting Committee: CEN/TC 455/WG 3 - Pathogenic and non-pathogenic microorganisms
• Current Stage: 5060 - Closure of Vote - Formal Approval
• Due Date: 02-Dec-2021
• Completion Date: 02-Dec-2021
• Directive: 2019/1009 - Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC) No 1107/2009 and repealing Regulation (EC) No 2003/2003
• Mandate: M/564 - Fertilising products in support of Regulation (EU) 2019/1009
• Ref Project: kSIST-TS FprCEN/TS 17711:2021 - Plant biostimulants - Detection of Vibrio spp

Standards Content (sample)

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17711:2021
01-november-2021
[Not translated]
Plant biostimulants - Detection of Vibrio spp
Biostimulanzien für die pflanzliche Anwendung - Nachweis von Vibrio spp
Ta slovenski standard je istoveten z: FprCEN/TS 17711
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17711:2021 en,fr,de

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

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kSIST-TS FprCEN/TS 17711:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17711
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Detection of Vibrio spp
Biostimulanzien für die pflanzliche Anwendung -
Nachweis von Vibrio spp

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 455.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

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 Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17711:2021 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 ...................................................................................................... 9

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 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 ................................................................................ 12

5.5.5 Saline peptone waters .............................................................................................................. 12

5.5.6 Sodium chloride solution ........................................................................................................ 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 ................................................................................... 15

9.5.4 Biochemical confirmation ....................................................................................................... 16

10 Expression of results ................................................................................................................. 18

11 Performance characteristics of the method ..................................................................... 18

11.1 Sensitivity ...................................................................................................................................... 18

11.2 Specificity ...................................................................................................................................... 18

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11.3 LOD50 ............................................................................................................................................. 18

12 Test report .................................................................................................................................... 18

Annex A (normative) Diagram of procedure ................................................................................... 20

Annex B (normative) Composition and preparation of the culture media and reagents 21

B.1 Introduction ................................................................................................................................. 21

B.2 Water .............................................................................................................................................. 21

B.3 Alkaline saline peptone water (ASPW) .............................................................................. 21

B.3.1 Composition ................................................................................................................................. 21

B.3.2 Preparation .................................................................................................................................. 21

B.4 Thiosulfate citrate bile and sucrose agar (TCBS) ........................................................... 22

B.4.1 Composition ................................................................................................................................. 22

B.4.2 Preparation .................................................................................................................................. 22

B.4.3 Preparation of the agar dishes .............................................................................................. 22

B.5 Saline nutrient agar (SNA) ...................................................................................................... 22

B.5.1 Composition ................................................................................................................................. 22

B.5.2 Preparation .................................................................................................................................. 22

B.5.3 Preparation of the agar dishes .............................................................................................. 23

B.5.4 Preparation of slants of saline nutrient agar ................................................................... 23

B.6 Reagent for detection of oxidase .......................................................................................... 23

B.6.1 Composition ................................................................................................................................. 23

B.6.2 Preparation .................................................................................................................................. 23

B.7 L-lysine decarboxylase saline medium (LDC) .................................................................. 23

B.7.1 Composition ................................................................................................................................. 23

B.7.2 Preparation .................................................................................................................................. 23

B.8 Arginine dihydrolase saline medium (ADH) .................................................................... 23

B.8.1 Composition ................................................................................................................................. 23

B.8.2 Preparation .................................................................................................................................. 24

B.9 Detection of β-galactosidase .................................................................................................. 24

B.9.1 ONPG solution .............................................................................................................................. 24

B.9.2 Buffer solution ............................................................................................................................. 24

B.9.3 Complete reagent ....................................................................................................................... 24

B.10 Saline medium for detection of indole ............................................................................... 25

B.10.1 Tryptophan saline medium .................................................................................................... 25

B.10.2 Kovacs reagent ............................................................................................................................ 25

B.11 Saline peptone water ................................................................................................................ 25

B.11.1 Composition ................................................................................................................................. 25

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B.11.2 Preparation................................................................................................................................... 25

B.12 Sodium chloride solution ........................................................................................................ 26

B.12.1 Composition ................................................................................................................................. 26

B.12.2 Preparation................................................................................................................................... 26

B.13 Tris acetate EDTA (TAE) buffer ............................................................................................. 26

B.13.1 Composition ................................................................................................................................. 26

B.13.2 Preparation................................................................................................................................... 26

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 ................................. 27

C.1 General ........................................................................................................................................... 27

C.2 Equipment ..................................................................................................................................... 27

C.2.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the

purpose) ........................................................................................................................................ 27

C.2.2 Mastermix ..................................................................................................................................... 27

C.3 DNA extraction ............................................................................................................................ 28

C.4 Procedure Conventional PCR ................................................................................................. 28

C.5 Primers and probes ................................................................................................................... 29

C.5.1 Vibrio parahaemolyticus primers (conventional PCR) ................................................ 29

C.5.2 Thermostable direct haemolysin (tdh) and thermostable direct related
haemolysin (trh) genes Vibrio parahaemolyticus primers (conventional PCR) .. 29

C.5.3 Vibrio cholerae primers (conventional PCR) .................................................................... 30

C.5.4 Vibrio vulnificus primers (conventional PCR) .................................................................. 30

C.5.5 Cycling parameters — VptoxR and VVH ............................................................................. 30

C.5.6 Cycling parameters — prVC .................................................................................................... 30

C.5.7 Cycling parameters — tdh and trh........................................................................................ 30

C.6 Control material — conventional PCR ................................................................................ 31

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

thermostable direct haemolysin gene (tdh) and Vibrio vulnificus .......................... 32

D.1 General ........................................................................................................................................... 32

D.2 Equipment ..................................................................................................................................... 32

D.2.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance for the

purpose) ........................................................................................................................................ 32

D.2.2 Mastermix ..................................................................................................................................... 32

D.2.3 Composition of mastermix ...................................................................................................... 32

D.3 DNA extraction ............................................................................................................................ 32

D.4 Real-time PCR .............................................................................................................................. 32

D.5 Vibrio parahaemolyticus — primers and hydrolysis probes ...................................... 33

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D.6 Thermostable direct haemolysin (tdh) gene Vibrio parahaemolyticus — primers

and hydrolysis probes .............................................................................................................. 33

D.7 Vibrio vulnificus — primers and hydrolysis probes ...................................................... 34

D.8 Cycling parameters .................................................................................................................... 34

D.9 Control material – real-time PCR .......................................................................................... 34

Bibliography ............................................................................................................................................... 35

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European foreword

This document (FprCEN/TS 17711:2021) 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 Vote on TS.

This document has been prepared under a mandate M/564 given to CEN by the European

Commission and the European Free Trade Association.
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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 fertilising products (“FPR” or “Fertilising Products Regulation”). This request,

presented as SR M/564, 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 validates 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.

FprCEN/TS 17702-1, Plant biostimulants — Sampling and sample preparation — Part 1: Sampling

FprCEN/TS 17724, Plant biostimulants — Terminology

EN ISO 7218:2007, Microbiology of food and animal feeding stuffs – General requirements and

guidance for microbiological examinations ()

EN ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production,

storage and performance testing of culture media ()

EN ISO 3696:1995, Water for analytical laboratory use — Specification and test methods (ISO

3696:1987)
3 Terms and definitions

For the purposes of this document, the terms and definitions given in FprCEN/TS 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
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.
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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. may 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 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) (5.1) at ambient temperature, followed by incubation at 41,5 °C for 6 h and/or 37 °C for

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

For detection of all target species in solid products, primary enrichment should be at 37 °C.

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

For detection of V. parahaemolyticus and/or V. cholerae only, in liquid products, primary

enrichment should be at 41,5 °C.
4.3 Secondary enrichment in a liquid selective medium

Inoculation of the second enrichment medium (ASPW) with the cultures obtained in 4.2.

Incubation of inoculated enrichment medium at 41,5 °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 (TCBS) medium (5.2.1);

— another appropriate solid selective medium (left to the choice of the laboratory), such as

chromogenic agar, complementary to the TCBS medium (5.2.2).
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Incubation of the TCBS medium at 37 °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. parahaemolyticus, V. cholerae and V. vulnificus isolated in 4.4 are

subcultured and confirmed by means of appropriate biochemical test. The PCR test is also possible

to use for confirmation; the PCR methods are suggested in Annexes C and D, but the laboratory

must validate the procedure and data generated.
5 Culture media and reagents
For general laboratory practice, refer to EN ISO 7218:2007.

For clarity of the text, details of the composition of culture media and reagents and their

preparation are described in Annex B.
For performance testing of culture media, refer to EN ISO 11133:2014.
5.1 Enrichment medium: alkaline saline peptone water (ASPW)
As specified in B.1.
5.2 Solid selective isolation media
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS)
As specified in B.2. See Table 1 for performance testing data.
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Table 1 — Performance testing of thiosulphate, citrate, bile and sucrose agar medium

(TCBS)
Function Incubation Control strains a Method of e Characteristi
WDCM Criteria
control c reactions
37 °C ± 1 °C Vibrio b Qualitative Good Green
00185
for 24 h ± 3 h parahaemolyticus growth colonies
(2) (sucrose
negative)
Productivity
37 °C ± 1 °C Vibrio furnissii b Qualitative Good Yellow
00186
for 24 h ± 3 h growth colonies
(2) (sucrose
positive)
00012,
Total
37 °C ± 1 °C
c d
Selectivity Qualitative —
Escherichia coli inhibition
for 24 h ± 3 h
00013 or
(0)
00090

World Data Centre for Microorganisms (WDCM) strain catalogue available at http://refs.wdcm.org

Strain to be used as a minimum (see EN ISO 11133:2014).
Some national restrictions and directions may require the use of a
...