Algae and algae products - Identification of the biomass of microalgae, macroalgae, cyanobacteria and Labyrithulomycetes - Detection and identification with morphological and/or molecular methods

This document specifies a method for the detection and identification of microalgae, macroalgae (seaweed), cyanobacteria and Labyrinthulomycetes by using morphological methods and/or molecular methods.
The morphological methods in this document are applicable to harvested wet biomass and to harvested dried unground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have been grown and/or harvested for further processing and/or use.
The molecular methods in this document are applicable to harvested wet biomass and to harvested dried and/or ground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have been grown and/or harvested for further processing and/or use.
This document describes a toolbox, consisting of several identification methods that can be chosen according to the applicability and purpose of the identification:
—   morphological methods based on observation and referring to scientific literature on taxonomy:
—   macroscopic observation;
—   light microscopic observation;
—   molecular methods of sequencing and blasting of sequences:
—   16S-rDNA sequencing;
—   18S-rDNA sequencing;
—   rbcL DNA sequencing;
—   ITS sequencing;
—   COX 1 gene sequencing;
—   tufA gene sequencing.
This document does not deal with genetic purity of the biomass or quantification of the identified taxa.

Algen und Algenprodukte - Identifizierung der Biomasse von Mikroalgen, Makroalgen, Cyanobakterien und/oder Labyrinthulomycetes - Erkennung und Identifizierung mit morphologischen und/oder molekularen Methoden

Dieses Dokument legt ein Verfahren für den Nachweis und die Identifizierung von Mikroalgen, Makroalgen (Seetang), Cyanobakterien und Labyrinthulomycetes (Netzschleimpilze) mittels morphologischer und/oder molekularer Verfahren fest.
Die in diesem Dokument dargelegten morphologischen Verfahren gelten für geerntete feuchte Biomasse und geerntete trockene, ungemahlene Biomasse von Mikroalgen, Makroalgen, Cyanobakterien und Labyrinthulo¬mycetes, die für die Weiterverarbeitung und/oder Verwendung kultiviert und/oder geerntet wurden.
Die in diesem Dokument dargelegten molekularen Verfahren gelten für geerntete feuchte Biomasse und geerntete trockene und/oder gemahlene Biomasse von Mikroalgen, Makroalgen, Cyanobakterien und Labyrinthulomycetes, die für die Weiterverarbeitung und/oder Verwendung kultiviert und/oder geerntet wurden.
In diesem Dokument wird eine Sammlung von Verfahren beschrieben, die aus mehreren Identifizierungs-verfahren besteht, welche je nach Anwendbarkeit und Zweck der Identifizierung gewählt werden können:
   morphologische Verfahren, die auf Beobachtung unter Bezug auf die wissenschaftliche Literatur zur Taxonomie basieren:
   makroskopische Beobachtung;
   lichtmikroskopische Beobachtung;
   molekulare Verfahren der Sequenzierung und des Sequenzvergleichs („Blasting“):
   16S rDNS Sequenzierung;
   18S rDNS Sequenzierung;
   rbcL DNS Sequenzierung;
   ITS Sequenzierung;
   COX1 Gensequenzierung;
   tufA Gensequenzierung.
Dieses Dokument behandelt weder die genetische Reinheit der untersuchten Biomasse noch die Quantifizierung der identifizierten Taxa.

Algues et produits d'algues - Identification de la biomasse de microalgues, macroalgues, cyanobactéries et/ou Labyrinthulomycètes - Détection et identification à l'aide de méthodes morphologiques et/ou moléculaires

Le présent document spécifie une méthode de détection et d’identification des microalgues, macroalgues (algues marines), cyanobactéries et Labyrinthulomycètes à l’aide de méthodes morphologiques et/ou moléculaires.
Les méthodes morphologiques utilisées dans le présent document sont applicables à la biomasse humide récoltée et à la biomasse sèche non broyée récoltée provenant des microalgues, macroalgues, cyanobactéries et Labyrinthulomycètes qui ont été cultivées et/ou récoltées en vue d’un traitement et/ou d’une utilisation ultérieur(e).
Les méthodes moléculaires utilisées dans le présent document sont applicables à la biomasse humide récoltée et à la biomasse sèche et/ou broyée récoltée provenant des microalgues, macroalgues, cyanobactéries et Labyrinthulomycètes qui ont été cultivées et/ou récoltées en vue d’un traitement et/ou d’une utilisation ultérieur(e).
Le présent document décrit une boîte à outils, comprenant plusieurs méthodes d’identification qui peuvent être choisies en fonction de l’applicabilité et de l’objectif de l’identification :
—   méthodes morphologiques reposant sur l’observation et faisant référence aux ouvrages scientifiques sur la taxonomie :
—   observation macroscopique ;
—   observation au microscope optique ;
—   méthodes moléculaires de séquençage et d’alignement de séquences :
—   séquençage de l’ADNr 16S ;
—   séquençage de l’ADNr 18S ;
—   séquençage de l’ADN rbcL ;
—   séquençage de l’ITS ;
—   séquençage du gène COX1 ;
—   séquençage du gène tufA.
Le présent document ne traite ni de la pureté génétique de la biomasse ni de la quantification des taxa identifiés.

Alge in izdelki iz alg - Ugotavljanje biomase pri mikroalgah, makroalgah, cianobakterijah in labirintulomicetah - Odkrivanje in prepoznavanje z morfološkimi in/ali molekularnimi metodami

General Information

Status
Published
Public Enquiry End Date
31-Mar-2020
Publication Date
24-Aug-2021
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Aug-2021
Due Date
24-Oct-2021
Completion Date
25-Aug-2021

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SLOVENSKI STANDARD
SIST EN 17477:2021
01-oktober-2021
Alge in izdelki iz alg - Ugotavljanje biomase pri mikroalgah, makroalgah,

cianobakterijah in labirintulomicetah - Odkrivanje in prepoznavanje z morfološkimi

in/ali molekularnimi metodami

Algae and algae products - Identification of the biomass of microalgae, macroalgae,

cyanobacteria and Labyrithulomycetes - Detection and identification with morphological

and/or molecular methods

Algen und Algenprodukte - Identifizierung der Biomasse von Mikroalgen, Makroalgen,

Cyanobakterien und/oder Labyrinthulomycetes - Erkennung und Identifizierung mit
morphologischen und/oder molekularen Methoden

Algues et produits d'algues - Identification de la biomasse de microalgues, macroalgues,

cyanobactéries et/ou Labyrinthulomycètes - Détection et identification à l'aide de

méthodes morphologiques et/ou moléculaires
Ta slovenski standard je istoveten z: EN 17477:2021
ICS:
13.020.55 Biološki izdelki Biobased products
SIST EN 17477:2021 en,fr,de

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

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SIST EN 17477:2021
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SIST EN 17477:2021
EN 17477
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2021
EUROPÄISCHE NORM
ICS 13.020.55
English Version
Algae and algae products - Identification of the biomass of
microalgae, macroalgae, cyanobacteria and
Labyrinthulomycetes - Detection and identification with
morphological and/or molecular methods

Algues et produits d'algues - Identification de la Algen und Algenprodukte - Identifizierung der

biomasse de microalgues, macroalgues, cyanobactéries Biomasse von Mikroalgen, Makroalgen,

et Labyrinthulomycètes - Détection et identification à Cyanobakterien und Labyrinthulomycetes - Erkennung

l'aide de méthodes morphologiques et/ou moléculaires und Identifizierung mit morphologischen und/oder

molekularen Verfahren
This European Standard was approved by CEN on 7 June 2021.

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. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

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

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

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

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17477:2021 E

worldwide for CEN national Members.
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SIST EN 17477:2021
EN 17477:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 7

3 Terms and definitions ................................................................................................................................... 7

4 Abbreviations ................................................................................................................................................ 11

5 Reagents .......................................................................................................................................................... 12

5.1 Reagents for morphological methods ................................................................................................... 12

5.1.1 Isotonic solution ........................................................................................................................................... 12

5.2 Reagents for molecular methods ............................................................................................................ 12

5.2.1 Primer .............................................................................................................................................................. 12

5.2.2 Deoxynucleotide triphosphate mix (dNTPs) ...................................................................................... 12

5.2.3 Thermostable DNA polymerase .............................................................................................................. 12

5.2.4 PCR reaction buffer ..................................................................................................................................... 12

5.2.5 Agarose gel ..................................................................................................................................................... 12

5.2.6 Gel electrophoresis buffer ........................................................................................................................ 13

5.2.7 Loading buffer ............................................................................................................................................... 13

5.2.8 DNA Ladder .................................................................................................................................................... 13

6 Apparatus ........................................................................................................................................................ 13

6.1 General ............................................................................................................................................................. 13

6.2 Apparatus for morphological identification methods .................................................................... 13

6.2.1 Low-magnifying optical system .............................................................................................................. 13

6.2.2 Light microscope .......................................................................................................................................... 13

6.2.3 Scientific literature on taxonomy ........................................................................................................... 13

6.2.4 Microscope slide ........................................................................................................................................... 14

6.2.5 Microscope cover glass .............................................................................................................................. 14

6.3 Apparatus for molecular identification methods ............................................................................. 14

6.3.1 Thermocycler ................................................................................................................................................ 14

6.3.2 Gel electrophoresis device ........................................................................................................................ 14

6.3.3 DNA sequencer .............................................................................................................................................. 14

6.3.4 Plastic consumables, DNA free, disposable ........................................................................................ 14

7 Principle .......................................................................................................................................................... 14

7.1 General ............................................................................................................................................................. 14

7.2 Morphological methods ............................................................................................................................. 15

7.3 Molecular methods ...................................................................................................................................... 15

8 Procedure........................................................................................................................................................ 15

8.1 General laboratory requirements .......................................................................................................... 15

8.2 Choice of methods ........................................................................................................................................ 15

9 Morphological identification methods ................................................................................................. 17

9.1 General ............................................................................................................................................................. 17

9.2 Macroscopic identification with the naked eye or a magnifying glass ..................................... 17

9.3 Light microscopy .......................................................................................................................................... 17

9.3.1 General ............................................................................................................................................................. 17

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SIST EN 17477:2021
EN 17477:2021 (E)

9.3.2 Staining ............................................................................................................................................................. 17

9.3.3 Preparation of microscope slides ........................................................................................................... 17

9.3.4 Microscopic identification ......................................................................................................................... 18

9.3.5 Use of identification keys ........................................................................................................................... 18

10 Molecular identification methods .......................................................................................................... 18

10.1 General ............................................................................................................................................................. 18

10.2 DNA extraction and purification ............................................................................................................. 19

10.3 DNA Amplification ........................................................................................................................................ 19

10.3.1 Principle of DNA Amplification ................................................................................................................ 19

10.3.2 Method .............................................................................................................................................................. 19

10.4 Selection of primers ..................................................................................................................................... 20

10.5 Control reactions .......................................................................................................................................... 20

10.6 Evaluation of PCR products ....................................................................................................................... 21

10.7 PCR product cloning .................................................................................................................................... 21

10.8 PCR product sequencing ............................................................................................................................ 21

10.9 Evaluation of sequence data ..................................................................................................................... 21

10.10 Sequence analysis/comparison with reference sequences in public databases ................... 22

11 Test report ...................................................................................................................................................... 23

Annex A (informative) Examples of applicable primers ............................................................................ 24

Annex B (informative) Scientific literature that may be used for identification ................................ 26

Bibliography ................................................................................................................................................................. 28

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SIST EN 17477:2021
EN 17477:2021 (E)
European foreword

This document (EN 17477:2021) has been prepared by Technical Committee CEN/TC 454 “Algae and

algae products”, the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of

an identical text or by endorsement, at the latest by February 2022 and conflicting national standards

shall be withdrawn at the latest by February 2022.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

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).

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,

Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,

Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North

Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United

Kingdom.
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SIST EN 17477:2021
EN 17477:2021 (E)
Introduction

This document has been prepared by the experts of CEN/TC 454 'Algae and algae products'.

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

Article 3 of Directive 2009/28/EC for algae and algae-based products or intermediates.

This request, presented as Mandate M/547, also contributes to the Communication on “Innovating for

Sustainable Growth: A Bio economy for Europe”.

The former working group CEN Technical Board Working Group 218 “Algae”, was created in 2016 to

develop a work programme as part of this Mandate. The technical committee CEN/TC 454 'Algae and

algae products' was established to carry out the work programme that will prepare a series of standards.

The interest in algae and algae-based products or intermediates has increased significantly in Europe as

a valuable source including but not limited to, carbohydrates, proteins, lipids, and several pigments.

These materials are suitable for use in a wide range of applications from food and feed purposes to other

sectors, such as textile, cosmetics, biopolymers, biofuel and fertilizer/biostimulants. Standardization was

identified as having an important role in order to promote the use of algae and algae products.

The work of CEN/TC 454 should improve the reliability of the supply chain, thereby improving the

confidence of industry and consumers in algae, which include macroalgae, microalgae, cyanobacteria,

Labyrinthulomycetes, algae-based products or intermediates and will promote and support

commercialisation of the European algae industry.

This document has been developed with the aim to cover the horizontal definitions for algae and algae

based products or intermediates. Hence, other terms and definitions are given in the other standards

developed by CEN/TC 454 “Algae and algae products”.

For food, feed and non-food, non-feed applications additional definitions may exist in other product

specific standards.
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SIST EN 17477:2021
EN 17477:2021 (E)
1 Scope

This document specifies a method for the detection and identification of microalgae, macroalgae,

cyanobacteria and Labyrinthulomycetes by using morphological methods and/or molecular methods.

The morphological methods in this document are applicable to harvested wet biomass and to harvested

dried unground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have

been grown and/or harvested for further processing and/or use.

The molecular methods in this document are applicable to harvested wet biomass and to harvested dried

and/or ground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have

been grown and/or harvested for further processing and/or use.

This document describes a toolbox, consisting of several identification methods that can be chosen

according to the applicability and purpose of the identification:

— morphological methods based on observation and referring to scientific literature on taxonomy:

— macroscopic identification;
— light microscopic identification.
— molecular methods for sequencing and blasting of sequences:
— 16S rDNA sequencing;
— 18S rDNA sequencing;
— rbcL DNA sequencing;
— ITS sequencing;
— COX1 gene sequencing;
— tufA gene sequencing.

This document does not deal with genetic purity of the biomass or quantification of the identified taxa.

This document is not suitable for the analysis of highly processed biomass with highly degraded DNA

where the fragments’ length are not sufficient for amplification of the targets and the morphological

characteristics cannot be assessed.
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SIST EN 17477:2021
EN 17477:2021 (E)
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 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — General requirements and definitions
EN 17399:2020, Algae and algae products — Terms and definitions
3 Terms and definitions

For the purposes of this document, the terms and definitions in EN 17399:2020, ISO 24276:2006 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
16S rDNA sequencing

process of determining the sequence of nucleotides in a complete or partial gene coding for the 16S

ribosomal ribonucleic acid

Note 1 to entry: The largest amount of 16S rDNA gene sequencing work concerns prokaryotes.

3.2
18S rDNA sequencing

process of determining the sequence of nucleotides in a complete or partial gene coding for the 18S

ribosomal ribonucleic acid
Note 1 to entry: The 18S rDNA gene sequencing work concerns eukaryotes.
3.3
alignment

process or result of matching up the nucleotide residues of two or more biological sequences to achieve

maximal levels of identity
3.4
basic local alignment search tool
BLAST

sequence comparison algorithm optimized for speed that is used to search sequence databases for

optimal local alignments to a query

Note 1 to entry: This algorithm directly approximates alignments that optimize a measure of local similarity, the

maximum signal pair (MST) score or high-scoring segment pair (HSP) score.

[SOURCE: ISO 20813:2019, 3.1, modified – Note 2 to entry and Note 3 to entry have been deleted]

3.5
blasting of sequences

sequence comparison against commonly used gene sequence databases using the BLAST algorithm

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SIST EN 17477:2021
EN 17477:2021 (E)
3.6
COX1 gene sequencing

process of determining the sequence of the COX1 gene that codes for the cytochrome C oxidase subunit 1

Note 1 to entry: In literature, COX1 gene can also be called CO1 or COI.
3.7
detection
discovery of the target organism using a suitable method
3.8
DNA extraction

sample treatment for the release and separation of DNA from other cellular components

[SOURCE: ISO 16577:2016, 3.44, modified – the word 'liberation' has been replaced by 'release']

3.9
DNA purification
method resulting in a more purified DNA

Note 1 to entry: In this context, purity refers to the reduction of observable and measurable effects of PCR

inhibitors.
[SOURCE: ISO 24276:2006, 3.2.2]
3.10
DNA sequence
order of nucleotides within a deoxyribonucleic acid molecule
3.11
external amplification control
spiked amplification control

DNA added to an aliquot of the extracted nucleic acid in a defined amount or copy number serving as a

control for amplification in nucleic acid-based reactions
[SOURCE: ISO 16577:2016, 3.60]
3.12
FASTA

GIR (genomic information representation) that includes a name and a nucleotide sequence for each

sequencing read

[SOURCE: ISO/IEC 23092-2:2020, 3.7, modified – Note 1 to entry has been left out]

3.13
GenBank
comprehensive public database of genetic reference sequences

Note 1 to entry: GenBank at National Center for Biotechnology Information (NCBI) is part of the International

Nucleotide Sequence Database Collaboration, which comprises the DNA DataBank of Japan (DDBJ), the European

Nucleotide Archive (ENA). These three organizations exchange data on a daily basis.

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SIST EN 17477:2021
EN 17477:2021 (E)
3.14
internal amplification control

gene sequence naturally present in template DNA that is amplified to serve as a control for amplification

in nucleic acid-based reactions

Note 1 to entry: A housekeeping gene with known copy numbers/genome can be used as an internal

amplification control.
3.15
Internal Transcribed Spacer
ITS
piece of non-coding DNA located between structural ribosomal rDNA subunits
3.16
ITS sequencing

process of determining the partial or complete sequence of the internal transcribed spacer (ITS)

Note 1 to entry: The largest amount of ITS sequencing work concerns eukaryotes and prokaryotes.

3.17
macroscopic identification
identification with the naked eye, based on taxonomic identification keys
3.18
microscopic identification

identification with magnification by using magnifying glasses, binoculars or microscopes, based on

taxonomic identification keys
3.19
molecular identification method

set of tools that rely on the comparison of the nucleic acid sequences of DNA obtained from an organism

using a PCR-based method with public/documented data of known organisms

Note 1 to entry: The data obtained using the respective follow-up tools like gene sequencing, can be compared

with sequences of known species accessible via public databases (see 3.4).

Note 2 to entry: These methods allow detection of low concentrations of DNA in non-viable organisms.

3.20
negative DNA target control

well-characterized DNA preparation material that does not contain target nucleic acid sequences

[SOURCE: ISO 16577:2016, 3.118]
3.21
negative extraction control

negative control reaction generated by performing all required steps in an extraction procedure except

for the addition of the test portion
Note 1 to entry: For example, by substitution of water for the test portion.

Note 2 to entry: This control is used to demonstrate the absence of contamination during extraction steps.

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SIST EN 17477:2021
EN 17477:2021 (E)
3.22
negative process control

well-characterized reference sample lacking target analyte and that should be put through the exact same

process steps as the test samples

[SOURCE: ISO 16577:2016, 3.119, modified – the word 'recognized' has been replaced by 'well-

characterized']
3.23
morphological identification method
identification method based on morphological characteristics
3.24
polymerase chain reaction
PCR

in vitro enzymatic technique to increase the number of copies of a specific DNA fragment by several

orders of magnitude
Note 1 to entry: PCR is used to selectively amplify DNA target.
3.25
PCR product
DNA molecule / fragment amplified by PCR

Note 1 to entry: If necessary the PCR product can be purified by using commercial kits.

[ISO 16577:2016, 3.138 – modified – note added]
3.26
positive DNA target control

well-characterized DNA preparation material containing intact target nucleic acid sequences for PCR

Note 1 to entry: Reference DNA or DNA extracted from a certified reference material is generally used to

demonstrate that PCR reagents are working as intended.
[SOURCE: ISO 16577:2016, 3.150, modified – partly rewritten]
3.27
positive PCR control

known positive (identified) sample representing the DNA-sequence of the organism under study

Note 1 to entry: This control is used to demonstrate that the PCR reagents are working as intended.

3.28
positive process control

well-characterized reference sample containing a detectable amount of a target analyte that should be

put through the exact same process steps as the test samples

Note 1 to entry: The positive process control goes through exactly the same process steps as the test samples.

[SOURCE: ISO 16577:2016, 3.151, modified – partly rewritten and added 'that should be put through the

exact same process steps as the test samples']
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EN 17477:2021 (E)
3.29
rbcL DNA sequencing

process of determining the sequence of the rbcL in a complete or partial gene coding for the large subunit

of the protein ribulose-1,5-bisphosphate carboxylase/oxygenase

Note 1 to entry: The largest amount of rbcL DNA gene sequencing work concerns eukaryotes.

3.30
sanger sequencing
cycle sequencing method using fluorescent-labelled dideoxynucleotides
3.31
sample taxon identification
determination of the genus and preferably also of the species name of an isolate
3.32
tufA gene sequencing

process of determining the sequence of the tufA in a complete or partial gene coding for the elongation

factor Tu

Note 1 to entry: Some tufA gene sequencing work concerns prokaryotes and eukaryotes.

Note 2 to entry: The plastid tufA gene is encoding the transcription factor Tu and is evaluated as a suitable

barcode marker gene for green algae.
4 Abbreviations
A Adenine
BLAST Basic Local Alignment Search Tool
C Cytosine
COX1 Cytochrome c oxidase subunit 1
DDBJ DNA Data Bank of Japan
DIC Differential Interference Contrast
DNA Deoxyribonucleic acid
dATP Deoxyadenosine 5'-triphosphate
dCTP Deoxycytidine 5'-triphosphate
dGTP Deoxyguanine 5'-triphosphate
dNTPs Deoxyribonucleotide triphosphate mix
dTTP Deoxythymidine 5'-triphosphate
ENA European Nucleotide Archive
G Guanine
GenBank GenBank at National Centre for Biotechnology Information (NCBI)
ITS Internal Transcribed Spacer
NCBI National Centre for Biotechnology Information
PBS Phosphate-Buffered Saline
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EN 17477:2021 (E)
PCR Polymerase Chain Reaction
rbcL Ribulose-1,5-bisphosphate carboxylase/oxygenase
rDNA Ribosomal DNA
T Thymine
tufA Elongation factor Tu
5 Reagents
5.1 Reagents for morphological methods
5.1.1 Isotonic solution

An isotonic solution should be used when examining biomass for morphological identification. The

isotonic solution should contain a sufficient amount of salts, so that when examining the sample, bursting

or shrinking of cells is prevented. It can be used to prepare a sample for microscopic identification.

Distilled water shall not be used for this purpose.

NOTE Examples of suitable isotonic solutions could be tap water, culture medium, phosphate-buffered saline

(PBS) or water from the local environment.
5.2 Reagents for molecular methods
5.2.1 Primer

A primer for sequencing is a synthetic DNA oligonucleotide of approximately 15–30 bases. They are

designed to bind (via
...

SLOVENSKI STANDARD
oSIST prEN 17477:2020
01-marec-2020
Alge in izdelki iz alg - Ugotavljanje biomase pri mikroalgah, makroalgah,

cianobakterijah in labirintulomicetah - Odkrivanje in prepoznavanje z morfološkimi

in/ali molekulskimi metodami

Algae and algae products - Identification of the biomass of microalgae, macroalgae,

cyanobacteria and Labyrithulomycetes - Detection and identification with morphological

and/or molecular methods

Algen und Algenprodukte - Identifizierung der Biomasse von Mikroalgen, Makroalgen,

Cyanobakterien und/oder Labyrinthulomycetes - Erkennung und Identifizierung mit
morphologischen und/oder molekularen Methoden

Algues et produits d'algues - Identification de la biomasse de microalgues, macroalgues,

cyanobactéries et/ou Labyrinthulomycètes - Détection et identification à l'aide de

méthodes morphologiques et/ou moléculaires
Ta slovenski standard je istoveten z: prEN 17477
ICS:
13.020.55 Biološki izdelki Biobased products
oSIST prEN 17477:2020 en,fr,de

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

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oSIST prEN 17477:2020
DRAFT
EUROPEAN STANDARD
prEN 17477
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2020
ICS 13.020.55
English Version
Algae and algae products - Identification of the biomass of
micro-algae, macro-algae, cyanobacteria and/or
Labyrithulomycetes - Detection and identification with
morphological and/or molecular methods
Algen und Algenprodukte - Identifizierung der
Biomasse von Mikroalgen, Makroalgen,
Cyanobakterien und/oder Labyrithulomycetes -
Erkennung und Identifizierung mit morphologischen
und/oder molekularen Methoden

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 454.

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, 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 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 NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17477:2020 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 6

4 Abbreviations ................................................................................................................................................... 8

5 Reagents ............................................................................................................................................................. 9

5.1 Reagents for morphological methods ...................................................................................................... 9

5.1.1 General ................................................................................................................................................................ 9

5.1.2 Tap water ........................................................................................................................................................... 9

5.1.3 Culture medium ............................................................................................................................................... 9

5.1.4 Phosphate-buffered saline (PBS) .............................................................................................................. 9

5.2 Reagents for molecular methods ............................................................................................................... 9

5.2.1 Thermostable DNA polymerase ................................................................................................................. 9

5.2.2 PCR reaction buffer ........................................................................................................................................ 9

5.2.3 Deoxynucleoside triphosphate mix (dNTPs) ........................................................................................ 9

5.2.4 Primer ................................................................................................................................................................. 9

5.2.5 Agarose gel ..................................................................................................................................................... 10

5.2.6 DNA Ladder .................................................................................................................................................... 10

6 Apparatus ........................................................................................................................................................ 10

6.1 General ............................................................................................................................................................. 10

6.2 Apparatus for morphological identification methods .................................................................... 10

6.2.1 Low-magnifying optical system, such as a magnifying glass or a dissecting

microscope ..................................................................................................................................................... 10

6.2.2 Light microscope .......................................................................................................................................... 10

6.2.3 Scientific literature, on taxonomy .......................................................................................................... 10

6.2.4 Microscopic slide .......................................................................................................................................... 10

6.2.5 Microscopic cover glass ............................................................................................................................. 11

6.3 Apparatus for molecular identification methods ............................................................................. 11

6.3.1 Thermocycler ................................................................................................................................................ 11

6.3.2 Gel electrophoresis device ........................................................................................................................ 11

6.3.3 DNA sequencer .............................................................................................................................................. 11

6.3.4 PCR tube .......................................................................................................................................................... 11

6.3.5 DNA free pipette ........................................................................................................................................... 11

6.3.6 Plugged sterile tip ........................................................................................................................................ 11

7 Principle .......................................................................................................................................................... 11

7.1 General ............................................................................................................................................................. 11

7.2 Morphological methods ............................................................................................................................. 11

7.3 Molecular methods ...................................................................................................................................... 12

8 Procedure........................................................................................................................................................ 12

8.1 General laboratory requirements .......................................................................................................... 12

8.2 Choice of the methods ................................................................................................................................ 12

9 Morphological identification methods ................................................................................................. 14

9.1 General ............................................................................................................................................................. 14

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9.2 Macroscopic observation with the naked eye or a magnifying glass ......................................... 14

9.3 Light microscopy ........................................................................................................................................... 14

9.3.1 General ............................................................................................................................................................. 14

9.3.2 Staining ............................................................................................................................................................. 14

9.3.3 Preparation of microscopic slides .......................................................................................................... 14

9.3.4 Microscopic observation ............................................................................................................................ 15

9.3.5 Identification using keys ............................................................................................................................ 15

10 Molecular identification methods .......................................................................................................... 15

10.1 DNA extraction ............................................................................................................................................... 15

10.2 PCR Amplification ......................................................................................................................................... 15

10.3 Primers ............................................................................................................................................................. 16

10.4 Control reaction ............................................................................................................................................ 16

10.5 Evaluation of PCR products ....................................................................................................................... 17

10.6 PCR product cloning .................................................................................................................................... 17

10.7 PCR Product sequencing ............................................................................................................................ 17

10.8 Evaluation of sequence data ..................................................................................................................... 18

10.9 Sequence analysis/comparison with reference sequences in public databases ................... 18

11 Test report ...................................................................................................................................................... 18

Annex A (informative) Examples of universally available primers........................................................ 20

A.1 Prokaryotic primers specific to cyanobacteria ................................................................................. 20

A.1.1 PCR _16S rDNA amplification ................................................................................................................... 20

A.1.2 Sequencing ...................................................................................................................................................... 20

A.2 Eukaryotic primers more general (microalgae; Labirynthulids; seaweeds) .......................... 20

A.2.1 PCR _18S rDNA amplification ................................................................................................................... 20

A.2.2 PCR primers for COX1 gene ....................................................................................................................... 20

A.2.3 PCR primers for tufA gene ......................................................................................................................... 21

A.2.4 PCR primers for rbcL ................................................................................................................................... 21

Annex B (informative) Scientific literature that may be used for identification ................................ 22

Annex C (informative) Examples of DNA extraction methods for PCR .................................................. 24

Annex D (informative) Example of a standard polymerase chain reaction (PCR) setup ................ 25

Bibliography ................................................................................................................................................................. 26

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European foreword

This document (prEN 17477:2020) has been prepared by Technical Committee CEN/TC 454 “Algae and

algae products”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.
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1 Scope

This document specifies a method for the detection and identification of microalgae, macroalgae

(seaweed), cyanobacteria and Labyrinthulomycetes by using morphological methods and/or molecular

methods.

The morphological methods in this document are applicable to harvested wet biomass and to harvested

dried unground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that

have been grown and/or harvested for further processing and/or use.

The molecular methods in this document are applicable to harvested wet biomass and to harvested

dried and/or ground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes

that have been grown and/or harvested for further processing and/or use.

This document describes a toolbox, consisting of several identification methods that can be chosen

according to the applicability and purpose of the identification:

— morphological methods based on observation and referring to scientific literature on taxonomy:

— macroscopic observation;
— light microscopic observation;
— molecular methods of sequencing and blasting of sequences:
— 16S-rDNA sequencing;
— 18S-rDNA sequencing;
— rbcL DNA sequencing;
— ITS sequencing;
— COX 1 gene sequencing;
— tufA gene sequencing.

This document does not deal with genetic purity of the biomass or quantification of the identified taxa.

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 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — General requirements and definitions

FprEN 17399:2019, Algae and algae-based products or intermediates - Terms and definitions

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3 Terms and definitions

For the purposes of this document, the terms and definitions in FprEN 17399:2019, ISO 24276 and the

following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
16S-rDNA sequencing

process of determining the sequence of nucleotides in a complete or partial gene coding for the 16S-

ribosomal ribonucleic acid

Note to entry 1: The largest amount of 16S rDNA gene sequencing work concerns prokaryotes.

Note to entry 2: DNA is deoxyribonucleic acid.
3.2
18S-rDNA sequencing

process of determining the sequence of nucleotides in a complete or partial gene coding for the 18S-

ribosomal ribonucleic acid

Note to entry 1: The largest amount of 18S rDNA gene sequencing work concerns eukaryotes.

Note to entry 2: DNA is deoxyribonucleic acid.
3.3
alignment

process or result of matching up the nucleotide residues of two or more biological sequences to achieve

maximal levels of identity
3.4
BLAST

sequence comparison algorithm optimized for speed used to search sequence databases for optimal

local alignments to a query

Note to entry 1: It directly approximates alignments that optimize a measure of local similarity, the maximum

signal pair (MST) score.
3.5
blasting of sequences
sequence comparison against commonly used gene sequence databases
3.7
COX1 gene sequencing
process of determining the sequence of nucleotides in the COX1 gene
3.8
detection
discovering the target organism/microorganism using a special method
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3.9
DNA extraction
separation of DNA from the other components in a test sample
Note to entry 1: Adapted from ISO 24276:2006
3.10
DNA sequence
order of nucleotides within a deoxyribonucleic acid molecule
3.12
GenBank
comprehensive public database of genetic reference sequences

Note 1 to entry: GenBank is part of the International Nucleotide Sequence Database Collaboration, which

comprises the DNA DataBank of Japan (DDBJ), the European Nucleotide Archive (ENA), and GenBank at National

Center for Biotechnology Information (NCBI). These three organizations exchange data on a daily basis.

3.13
ITS sequencing

process of determining the sequence of nucleotides in the internal transcribed spacer (ITS)

3.14
macroscopic observation
identification with the naked eye, based on taxonomic keys
3.15
microscopic observation

identification with magnification by using magnifying glasses, binoculars or microscopes, based on

taxonomic keys
3.16
molecular identification method

set of tools that rely on the comparison of the nucleic acid sequences of DNA obtained from an

organism/microorganism using the PCR (polymerase chain reaction)-based method with

public/documented data of known (micro)organisms

Note to entry 1: The data obtained using the respective follow-up tools like gene sequencing can be compared

with sequences of known species accessible via public databases (see 3.4).

Note to entry 2: These methods allow detection of low concentrations of DNA, non-viable organisms.

3.17
morphological identification method
identification method based on morphological characteristics
3.18
positive PCR control

known positive (identified) sample representating the DNA-sequence of the (micro)organism under

study

Note to entry 1: This control is used to demonstrate that the PCR reagents are working as intended.

Note to entry 2: Adapted from ISO 24276:2006
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3.19
rbcL DNA sequencing

process of determining the sequence of nucleotides (A, T, C, and G) in the gene that codes for the large

subunit of the protein ribulose-1,5-bisphosphate carboxylase/oxygenase
3.20
Sanger sequencing
cycle sequencing method using fluorescent-labelled dideoxynucleotides
3.21
strain identification
determination of the genus and preferably also of the species name of a strain
3.23
tufA gene sequencing
process of determining the sequence of nucleotides in the tufA gene
4 Abbreviations
A Adenosine
BLAST Basic Local Alignment Search Tool
C Cytosine
COX1 Cytochrome c oxidase subunit 1
DDBJ DNA Data Bank of Japan
DNA Deoxyribonucleic acid
ENA European Nucleotide Archive
G Guanine
GenBank GenBank at National Centre for Biotechnology Information (NCBI)
ITS Internal transcribed spacer
NCBI National Centre for Biotechnology Information
PCR Polymerase chain reaction
rbcL Ribulose-1,5-bisphosphate carboxylase/oxygenase
rDNA Ribosomal DNA
RNA Ribosomal ribonucleic acid
T Thymine
tufA Elongation factor TU
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5 Reagents
5.1 Reagents for morphological methods
5.1.1 General

Either of the following reagents shall be used when examining biomass for morphological identification

ensuring a certain amount of salts in the reagent, so that when examining the sample, bursting or

shrinking of cells is prevented. Distilled water shall not be used.
5.1.2 Tap water

Normal tap water usually contains sufficient amount of salts to prevent bursting of cells and can be used

to prepare a sample for microscopic observation.
5.1.3 Culture medium

Culture medium is used for culturing the organism to be identified contains sufficient amount of salts to

prevent bursting of cells and can be used to prepare a sample for microscopic oberservation.

5.1.4 Phosphate-buffered saline (PBS)

PBS is a commercially available water-based salt solution and it contains sufficient amount of salts to

prevent bursting of cells and thus, can be used to prepare a sample for microscopic oberservation

5.2 Reagents for molecular methods
5.2.1 Thermostable DNA polymerase

Thermostable DNA polymerase is a critical player in replicating the target DNA. It is an enzyme that is

derived from a thermophilic bacterium and functions at high temperature. It is used in the polymerase

chain reaction. Thermostable DNA polymerases are commercially available and should be used as

directed by the manufacturer’s protocol.
5.2.2 PCR reaction buffer

PCR reaction buffers are generally sold with the thermostable DNA polymerase (5.2.1). PCR reaction

buffers can include MgCl or the can come with a separate MgCl solution.
2 2
5.2.3 Deoxynucleoside triphosphate mix (dNTPs)

A deoxynucleoside triphosphate mix (dNTPs) consists of four basic nucleotides—dATP, dCTP, dGTP,

and dTTP. They are the building blocks of new DNA strands. These four nucleotides are typically added

to the PCR reaction in equimolar amounts for optimal base incorporation. dNTPs are commercially

available.
5.2.4 Primer

A primer is a synthetic DNA oligonucleotide of approximately 15–30 bases. They are designed to bind

(via sequence complementarity) to sequences that flank the region of interest in the template DNA.

Primers are used for PCR and sequencing reactions. For example, during PCR reaction, DNA polymerase

extends the primers from their 3′ ends. As such, the primers’ binding sites shall be unique to the vicinity

of the target with minimal homology to other sequences of the input DNA to ensure specific amplication

of the intended target.
See Annex A for PCR and sequencing primers recommendations.
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5.2.5 Agarose gel
Use an agarose gel of suitable concentration (e.g. 1 % to 2 % (w/v))
5.2.6 DNA Ladder

The DNA ladder is the molecular weight size marker. It is a set of standards that are used to identify the

approximate size of a molecule such as PCR product run on a gel during electrophoresis. DNA ladders

are commercially available.
6 Apparatus
6.1 General

The laboratory shall use properly maintained equipment according to the manufacturer's instructions

and should use the requirements given in ISO/IEC 17025. In addition to standard laboratory

equipment, specific apparatus is described in the individual standards.

Where available, calibration should be routinely performed on equipment where performance could

impact the data produced. Apart from the usual equipement, the following equipment is required.

6.2 Apparatus for morphological identification methods

6.2.1 Low-magnifying optical system, such as a magnifying glass or a dissecting microscope

For the morphological investigation of details, which are necessary for the identification of macroalgae

or tissues, a commercially available magnifying glass or a dissecting microscope might be sufficient.

These have lenses with magnifying factors between 0.65x and 5x. In the case of a dissecting microscope

complemented by the magnifying factor of the eyepiece (usually 10x) or lens in the light path of the

camera (often 10x as well). This results in a total magnification between 6.5x and 50x. Use as directed in

the apparatus’ manual.
6.2.2 Light microscope

For the morphological investigation of details of microscopic organisms, a commercially available light

microscope, optionally equipped with different contrasting and/or fluorescence units can be used.

These usually have front lenses with magnifying factors between 4x and 100x complemented by the

magnifying factor of the eyepiece (usually 10x) or lens in the light path of the camera (often 10x as

well). This results in a total magnification between 40x and 1000x. Optional contrasting equipment

such as phase contrast or Differential Interference Contrast (DIC, also called Nomarski contrast) or also

dark field mode might be useful depending on the taxonomic group to be studied. Use as directed in the

apparatus’ manual.
6.2.3 Scientific literature, on taxonomy

Choose scientific literature according to the organism to be identified. A selection is given in Annex B.

This literature might carry a dichotomous or polytomous key to guide you through the identification

process. Additional drawing or photos could help in identifying the correct taxon.

6.2.4 Microscopic slide

Microscopic slides are composed of glass and are commercially available in the typical size of

76 × 26 mm (length x width) and a thickeness of 1 mm to accommodate the x/y-table of a microscope.

Glass quality is optimized not to interfere with the optical light path and the front lenses. Before use, the

microscope slide should be cleaned so that no particles can interfere with the microscope details of the

organism to be investigated.
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6.2.5 Microscopic cover glass

Microscopic cover glasses are composed of glass and are commercially available in various sizes and

forms (round, squre, rectangular). For broad applications, square cover glasses with a size of

18 × 18 mm and a thickeness of 0,13-0,16 mm are recommendable. The thickeness of the cover glass is

important if higher magnifications are applied, such when using lenses > 50x magnifying factor; theses

usually are immersion objectives usually used with immersion oil. The thickeness of cover glass is

optimized for the light path providing an optimal vision of the organism studied.

6.3 Apparatus for molecular identification methods
6.3.1 Thermocycler

Thermocycler is a device used to amplify DNA or RNA samples by the polymerase chain reaction. The

thermocycler is programmed to raise and lower the temperature of the samples in a holding block

allowing for denaturation and reannealing of samples with various reagents. Amplified genetic products

can be used in many downstream applications such as cloning, sequencing, expression analysis and

genotyping.
6.3.2 Gel electrophoresis devic
...

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