oSIST prEN ISO 22344:2026

• Abstract:

  This document specifies a method for the taxonomic identification of single crustaceans to the genus or species level using DNA barcoding. It allows the identification of a large number of commercially important crustacean species. ; This method was validated on raw crustaceans. Laboratory experience indicates additional applicability to processed crustacean products, e.g. cold smoked, hot smoked, salted, frozen, cooked, fried and deep-fried samples.; The described method is usually unsuitable for the analysis of highly processed foods, e.g. tins of crustaceans, with highly degraded DNA where the fragment lengths are not sufficient for amplification of the targets. Furthermore, it is not applicable for complex seafood products containing mixtures of two or more crustacean species.; The identification of crustacean species is carried out by PCR amplification of either a shorter or longer segment of the mitochondrial 16S rRNA gene, or a segment of the cytochrome c oxidase I gene (cox1, syn. COI) or any combination of the three markers, followed by sequencing of the PCR products and subsequent sequence comparison with entries in databases.

• Status: Not Published
• Public Enquiry End Date: 21-Sep-2026
• ICS: 07.100.30 - Food microbiology
• ICS: 35.040.50 - Automatic identification and data capture techniques
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 09-Jul-2026
• Due Date: 26-Nov-2026
• Ref Project: prEN ISO 22344 - Molecular biomarker analysis - DNA barcoding of crustaceans and products derived from crustaceans using defined mitochondrial 16S rRNA and cytochrome c oxidase I gene segments (ISO/DIS 22344:2026)

SLOVENSKI STANDARD
01-september-2026
Analiza molekularnih biomarkerjev - Črtno kodiranje DNK rakov in rakovih
proizvodov z uporabo definiranih segmentov genov mitohondrijske 16S rRNA in
citokroma c oksidaze I (ISO/DIS 22344:2026)
Molecular biomarker analysis - DNA barcoding of crustaceans and products derived from
crustaceans using defined mitochondrial 16S rRNA and cytochrome c oxidase I gene
segments (ISO/DIS 22344:2026)
Untersuchung auf molekulare Biomarker - DNA-Barcoding von Krustentieren und
Produkten aus Krustentieren anhand definierter mitochondrialer 16S rRNA und
Cytochrom-c-Oxidase-I-Genabschnitte (ISO/DIS 22344:2026)
Analyse de biomarqueurs moléculaires - Codes-barres d’ADN de crustacés et de
produits à base de crustacés à l’aide de segments définis de gènes mitochondriaux
d’ARNr 16S et de cytochrome c oxidase I (ISO/DIS 22344:2026)
Ta slovenski standard je istoveten z: prEN ISO 22344
ICS:
07.100.30 Mikrobiologija živil Food microbiology
35.040.50 Tehnike za samodejno Automatic identification and
razpoznavanje in zajem data capture techniques
podatkov
67.120.30 Ribe in ribji proizvodi Fish and fishery products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 22344
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
DNA barcoding of crustaceans and
Voting begins on:
products derived from crustaceans
2026-06-30
using defined mitochondrial 16S
Voting terminates on:
rRNA and cytochrome c oxidase I
2026-09-22
gene segments
Analyse de biomarqueurs moléculaires — Codes-barres d’ADN de
crustacés et de produits à base de crustacés à l'aide de segments
du gène mitochondriaux du 16S rRNA et de la cytochrome c
oxidase I
ICS: 67.120.30; 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
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IS THEREFORE SUBJECT TO CHANGE
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INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
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USER PURPOSES, DRAFT INTERNATIONAL
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NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 22344:2026(en)
DRAFT
ISO/DIS 22344:2026(en)
International
Standard
ISO/DIS 22344
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
DNA barcoding of crustaceans and
Voting begins on:
products derived from crustaceans
using defined mitochondrial 16S
Voting terminates on:
rRNA and cytochrome c oxidase I
gene segments
Analyse de biomarqueurs moléculaires — Codes-barres d’ADN de
crustacés et de produits à base de crustacés à l'aide de segments
du gène mitochondriaux du 16S rRNA et de la cytochrome c
oxidase I
ICS: 67.120.30; 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2026
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
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BE CONSIDERED IN THE LIGHT OF THEIR
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POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
or ISO’s member body in the country of the requester.
NATIONAL REGULATIONS.
ISO copyright office
RECIPIENTS OF THIS DRAFT ARE INVITED
CP 401 • Ch. de Blandonnet 8
TO SUBMIT, WITH THEIR COMMENTS,
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NOTIFICATION OF ANY RELEVANT PATENT
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Published in Switzerland Reference number
ISO/DIS 22344:2026(en)
ii
ISO/DIS 22344:2026(en)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms. 3
5 Principle . 4
6 Reagents and materials . 4
6.1 General .4
6.2 Thermostable DNA polymerase. .4
6.3 PCR reaction buffer (including MgCl2 or with separate MgCl2 solution).4
6.4 dNTP mix (dATP, dCTP, dGTP and dTTP). .4
6.5 Oligonucleotides. .5
6.6 Agarose. .5
6.7 DNA size standard. .5
7 Apparatus . 5
7.1 General .5
7.2 UV-spectrophotometer or fluorometer, .5
7.3 Thermocycler. .5
7.4 Gel electrophoresis device. .5
7.5 Gel documentation system. .5
7.6 DNA sequencer. .5
8 Procedure . 5
8.1 Sample preparation .5
8.2 DNA extraction .6
8.3 PCR .6
8.3.1 General .6
8.3.2 PCR setup .6
8.3.3 PCR controls .7
8.3.4 Thermal cycling .7
8.4 Evaluation of PCR products . .8
8.5 Evaluation of the PCR results .8
9 Sequencing . 9
9.1 Sequencing of PCR products .9
9.2 Evaluation of sequence data .9
9.3 Comparison of the sequence with public databases .10
9.3.1 General .10
9.3.2 Sequence comparison of 16S rRNA and / or cox1 amplicon sequences with
GenBank .10
9.3.3 Sequence comparison of cox1 amplicon sequences with BOLD .11
10 Interpretation of database query results .11
11 Validation status and performance criteria .12
11.1 Collaborative study for the identification of crustacean species based on sequence
analysis of the shorter 16S rRNA gene segment (310 bp) . 12
11.2 Collaborative study for the identification of crustacean species based on sequence
analysis of the longer 16S rRNA gene segment (520 bp) and cox1 (660 bp) . 13
12 Test report .16

iii
ISO/DIS 22344:2026(en)
Annex A (informative) Practical laboratory experiences with the amplifiability of 16S
rRNA gene (310bp), 16S rRNA gene (520bp) and cox1 (660 bp) segments from tested
crustacean species . 17
Bibliography . 19

iv
ISO/DIS 22344:2026(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
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with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO [had/had not] received notice of
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Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
Technical Committee 34, Food products, Subcommittee SC 16, Horizontal methods for molecular biomarker
analysis, in collaboration with the European Committee for Standardization (CEN) Technical Committee
CEN/TC 460, Food authenticity, in accordance with the Agreement on technical cooperation between ISO and
CEN (Vienna Agreement).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

v
ISO/DIS 22344:2026(en)
Introduction
Food safety is a key aspect in terms of consumer protection. In the last three decades, globalization has
taken place in the trade of food. Seafood trade channels are becoming steadily longer and more complicated,
so sophisticated traceability tools are needed to ensure food safety. Correct food labelling is a prerequisite
to ensure safe seafood products and fair trade as well as to minimize illegal, unreported and unregulated
(IUU) fishing. Seafood products are increasingly being processed in export countries. That makes the
identification of species by morphological characteristics impossible.
The development of harmonized and standardized protocols for the authentication of crustacean products
is necessary to establish reliable methods for the detection of potential food fraud.

vi
DRAFT International Standard ISO/DIS 22344:2026(en)
Molecular biomarker analysis — DNA barcoding of
crustaceans and products derived from crustaceans using
defined mitochondrial 16S rRNA and cytochrome c oxidase I
gene segments
1 Scope
This document specifies a method for the taxonomic identification of single crustaceans to the genus or
species level using DNA barcoding. It allows the identification of a large number of commercially important
crustacean species.
This method was validated on raw crustaceans. Laboratory experience indicates additional applicability to
processed crustacean products, e.g. cold smoked, hot smoked, salted, frozen, cooked, fried and deep-fried
[1]
samples .
The described method is usually unsuitable for the analysis of highly processed foods, e.g. tins of
crustaceans, with highly degraded DNA where the fragment lengths are not sufficient for amplification of
the targets. Furthermore, it is not applicable for complex seafood products containing mixtures of two or
more crustacean species.
The identification of crustacean species is carried out by PCR amplification of either a shorter or longer
[2] [3] [4]
segment of the mitochondrial 16S rRNA gene , , , or a segment of the mitochondrial cytochrome c
[5]
oxidase I gene (cox1, syn. COI) or any combination of the three markers, followed by sequencing of the PCR
[6][7]
products and subsequent sequence comparison with entries in databases .
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 16577:2022, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
ISO 20813:2019, Molecular biomarker analysis — Methods of analysis for the detection and identification of
animal species in foods and food products (nucleic acid-based methods) — General requirements and definitions
ISO 22949-1:2021, Molecular biomarker analysis — Methods of analysis for the detection and identification of
animal species in food and feed products (nucleotide sequencing-based methods) — Part 1: General requirements
3 Terms and definitions
For the purposes of this document, the following terms and definitions / terms and definitions given in
ISO 16577:2022 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

ISO/DIS 22344:2026(en)
3.1
alignment
sequence alignment
arrangement of nucleic acid sequences or protein sequences according to regions of similarity
Note 1 to entry: Alignment is a process or result of matching up the nucleotide residues of two or more biological
sequences to achieve maximal levels of identity (3.3).
[SOURCE: ISO 16577:2022, 3.7.18 – modified, Note 1 to entry added, alternative name added]
3.2
FASTA format
text-based format for representing either nucleotide sequences or amino acid (protein) sequences, in which
nucleotides or amino acids are represented using single-letter codes
Note 1 to entry: A sequence in FASTA format begins with a single-line description, followed by lines of sequence data.
The description line (defline) is distinguished from the sequence data by a greater-than (“>”) symbol at the beginning.
It is recommended that all lines of text be shorter than 80 characters in length.
Note 2 to entry: An example sequence in FASTA format is:
Figure 1
Note 3 to entry: Blank lines are not allowed in the middle of FASTA input. Sequences are represented in the standard
IUB/IUPAC amino acid and nucleic acid codes, with these exceptions:
— lower-case letters are accepted and are mapped into upper-case;
— a single hyphen or dash can be used to represent a gap of indeterminate length.
It is common to end the sequence with an “*” (asterisk) character and to leave a blank line between the description and
the sequence.
[SOURCE: ISO 16577:2022, 3.1.2, modified – Last sentence in Note 1 to entry removed, another example is
used in Note 2 to entry, 3rd bullet point in note 3 to entry deleted]
3.3
identity
extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an
alignment (3.1)
Note 1 to entry: Identity is often expressed as a percentage.
Note 2 to entry: In the sequence database of the Barcode of Life (BOLD), the term "similarity" is used instead of identity.
[8]
[SOURCE: ISO 17174:2024 , 3.3]

ISO/DIS 22344:2026(en)
3.4
introgressed DNA
allele from one species incorporated in the gene pool of another, divergent species
Note 1 to entry: Introgression has usually happened via hybridization and backcrossing of individuals belonging to
different species.
[8]
[SOURCE: ISO 17174:2024 , 3.4]
3.5
query
sequence (or other type of search term) that is compared to entries in a database
[8]
[SOURCE: ISO 17174:2024 , 3.5]
3.6
query coverage
percentage of the query (3.5) covered by alignment (3.1) to the database sequence
[8]
[SOURCE: ISO 17174:2024 , 3.6]
3.7
specificity
analytical specificity
diagnostic specificity
ability of a detection method to distinguish the specific organism or pathogen from other organisms,
whether related or not, and the extent to which the analysis can distinguish known or unknown variants of
the organism
Note 1 to entry: Specificity is a term that describes the same phenomenon as selectivity but in a different way; while
selectivity is applied to analytical chemistry and physics, specificity is applied to organisms and pathogens.
[SOURCE: ISO 16577:2022, 3.3.76]
4 Symbols and abbreviated terms
For the purposes of this document, the following symbols and abbreviations apply:
16S rRNA gene for the 16S ribosomal RNA
gene
B not A (cytosine, guanine or thymine)
bp base pair
C cytosine
cox1, syn cytochrome c oxidase I gene
COI
DNA deoxyribonucleic acid
dNTP deoxyribonucleotide triphosphate
G guanine
H not G (adenine, cytosine or thymine)
IUU illegal, unreported and unregulated
K keto group base (guanine or thymine)

ISO/DIS 22344:2026(en)
PCR polymerase chain reaction
R purine base (adenine or guanine)
rRNA ribosomal ribonucleic acid
SNP single nucleotide polymorphism
T thymine
W weak base pairing (adenine or thymine)
Y pyrimidine base (cytosine or thymine)
5 Principle
DNA is extracted from crustaceans or crustacean products applying a suitable method. Segments of
approximately 310 and/or 520 bp of the 16S rRNA gene and/or 660 bp of the COI gene are amplified by PCR.
For the amplification of the three gene segments three sets of primer pairs are used. Some of the primers
include ambiguous bases to increase the number of species detected by the three methods. The nucleotide
sequences of the PCR products are determined by a suitable DNA sequencing method, e.g. Sanger sequencing.
The determined sequences are evaluated by comparison to sequence entries in databases, thus allowing the
assignment to a crustacean species or genus according to the degree of identity with available sequences.
The decision whether one or both of the 16S rRNA gene segments or the cox1 segment or all three are used
for crustacean identification depends on the declared or suspected crustacean species, especially the
applicability of the PCR method for the crustacean species and the availability of comparative sequences in
the public databases.
6 Reagents and materials
6.1 General
During the analysis, unless otherwise stated, use only reagents of recognized molecular biology grade
and distilled, demineralized water or water of equivalent purity, according to ISO 20813:2019. Laboratory
organization shall follow ISO 20813:2019.
6.2 Thermostable DNA polymerase.
1)
6.3 PCR reaction buffer (including MgCl2 or with separate MgCl2 solution).
PCR reaction buffer can also be part of a commercial PCR master mix.
6.4 dNTP mix (dATP, dCTP, dGTP and dTTP).
dNTPs can also be part of a commercial PCR master mix.
1) During the collaborative study, the laboratories used the Maxima® Hot Start PCR MasterMix (2×) (Fermentas) for the
amplification of the 310 bp-16S rRNA gene segment . For the amplification of the 520 bp-16S rRNA gene segment and for
cox1, the HotStarTaq Mastermix (2x) (Qiagen) was recommended. In addition to the HotStarTaq polymerase Kit, other
mastermixes and polymerases were successfully used. Maxima® Hot Start PCR Master Mix (2×) of Fermentas GmbH
(ready-to-use PCR buffer solution including thermostable DNA polymerase) and HotStarTaq Mastermix (2×) (Qiagen)
are examples of suitable products available commercially. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of these products.

ISO/DIS 22344:2026(en)
6.5 Oligonucleotides.
NOTE The abbreviations of the DNA bases in Table 1, Table 2 and Table 3 are based on the recommendations
for unambiguous, uniform, and consistent nomenclature, published by the International Union of Pure and Applied
[9]
Chemistry (IUPAC).
[2][3]
Table 1 — Oligonucleotides for amplification of the shorter 16S rRNA gene region (310 bp)
Name DNA Sequence of oligonucleotide
16S-312F 5‘-GRA GGC TTG TAT GAA TGG TTG-3‘
16S-312R-1 5’-AAR WAR ATW ACG CTG TTA-3’
[4]
Table 2 — Oligonucleotides for amplification of the longer 16S rRNA gene region (520 bp)
Name DNA Sequence of oligonucleotide
16SAR 5’-CGC CTG TTT ATC AAA AAC AT-3’
16SBR 5’-CCG GTC TGA ACT CAG ATC ACG T-3’
[5]
Table 3 — Oligonucleotides for amplification of the cox1 gene region (660 bp)
Name DNA Sequence of oligonucleotide
LoboF1 5’-KBT CHA CAA AYC AYA ARG AYA THG G-3’
LoboR1 5’-TAA ACY TCW GGR TGW CCR AAR AAY CA-3’
6.6 Agarose.
6.7 DNA size standard.
7 Apparatus
7.1 General
In addition to standard laboratory equipment, the following apparatus should be used.
7.2 UV-spectrophotometer or fluorometer,
to determine the concentration of DNA.
7.3 Thermocycler.
7.4 Gel electrophoresis device.
7.5 Gel documentation system.
7.6 DNA sequencer.
8 Procedure
8.1 Sample preparation
The test portion used for DNA extraction shall be representative of the laboratory sample. In samples that
consist of processed materials (e.g. convenience foods), single crustacean pieces shall be separated and
analysed. For the analysis of samples composed of several pieces (e.g. bags with different crustaceans), test

ISO/DIS 22344:2026(en)
portions for every putative crustacean species are taken and analysed separately. To minimize the risk of
amplifying adhering contaminants, test sample material shall not be taken from the surface of the laboratory
sample, see also ISO 20813:2019.
8.2 DNA extraction
[10]
General instructions and measures specified in ISO 21571:2005 should be followed for the extraction of
[10]
DNA from the test sample. For example, the DNA extraction methods specified in ISO 21571:2005 , Annex
A, can be used. Commercial kits can be used for the extraction and purification of DNA if their applicability
for the extraction of DNA from crustaceans has been experimentally confirmed.
8.3 PCR
8.3.1 General
The primers used for amplification of segments of the mitochondrial 16S rRNA gene and cox1 are universal
[2][3][4]
primers that not only amplify DNA from crustaceans but also from a range of other taxa . The primer
[2][3]
pair 16S-312F/16S-312R-1 for the 310 bp-16S rRNA gene segment is optimized for shrimp species . It is
currently known that the primer pair 16S-312F/16S-312R-1 does not react with samples labelled as common
[3] [11]
shrimp / brown shrimp (Crangon crangon) , . The primers 16SAR/16SBR for the 520 bp 16S rRNA
gene segment are universal primers and can be used additionally for the identification of echinoderms,
[4]
vertebrates, insects, gastropods, fish and many more taxa . The primer pair LoboF1/R1 amplifies a
fragment of the 5´end of cox1 and is designed for a broad spectrum of marine organisms belonging to Phyla
[5]
like Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes .
8.3.2 PCR setup
The method was validated for a total volume of 25 µl (shorter 16S rRNA gene segment) or 20 µl (longer 16S
rRNA gene segment and cox1) per PCR. The reagents given in Table 4, Table 5 and Table 6 shall be used for
the 16S rRNA gene PCRs and the cox1 PCR, respectively.
Reagents shall be thawed completely and centrifuged briefly before usage. A PCR reagent mixture is
prepared containing all PCR components in the given concentrations except for the DNA extract or the
controls. The amount of PCR mixture prepared depends on the total volume per PCR and the total number of
reactions including a sufficient pipetting reserve.
Positive PCR results are expected when using a DNA concentration of approximately 1 ng DNA per μl of the
final solution (shorter 16S rRNA gene segment [25 ng] and cox1 [20 ng]) or 0,5 ng DNA per µl of the final
solution (longer 16S rRNA gene segment [10 ng]).
To improve the PCR result, the DNA quantity can be increased (e.g. to increase the yield of PCR product) or
decreased (e.g. to avoid PCR inhibition).
Table 4 — Components for the shorter 16S rRNA gene segment PCR (310 bp fragment)
Reagent (stock solution) Final composition in the reaction solution
PCR buffer 1 ×
a
MgCl 1,5 mmol/l
a
dNTP mix 0,2 mmol/l for each dNTP
Primer 16S-312-F 500 nmol/l
Primer 16S-312-R 500 nmol/l
Hot-start DNA Polymerase 0,5 units to 1 unit
Water Add to obtain final volume of 25 µl
Sample DNA About 1 ng/µl
a
Use reagent only if not already included in the PCR buffer.

ISO/DIS 22344:2026(en)
Table 5 — Components for the longer 16S rRNA gene segment PCR (520 bp fragment)
Reagent (stock solution) Final composition in the reaction solution
PCR buffer 1 ×
a
MgCl 1,5 mmol/l
a
dNTP mix 0,2 mmol/l for each dNTP
Primer 16SAR 500 nmol/l
Primer 16SBR 500 nmol/l
Hot-start DNA Polymerase 1 unit
Water Add to obtain final volume of 20 µl
Sample DNA About 0,5 ng/µl
a
Use reagent only if not already included in the PCR buffer.
Table 6 — Components for the cox1 PCR (660 bp fragment)
Reagent (stock solution) Final composition in the reaction solution
PCR buffer 1 ×
a
MgCl 2,0 mmol/l
a
dNTP mix 0,2 mmol/l for each dNTP
LoboF1 500 nmol/l
LoboR1 500 nmol/l
Hot-start DNA Polymerase 1 unit
Water Add to obtain final volume of 20 µl
Sample DNA About 1 ng/µl
a
Use reagent only if not already included in the PCR buffer.
Mix the PCR reagent mixture, centrifuge briefly and split into the individual reactions. Pipette the DNA
extracts to be examined or the PCR controls (see 8.3.3) into the different reaction solutions.
For further information on PCR controls, see also ISO 20813:2019.
8.3.3 PCR controls
In addition to the reaction setups for the samples to be analysed, an PCR reagent control and an extraction
blank control in accordance with ISO 20813:2019 shall be included.
A positive DNA target control (see ISO 20813:2019 ) can be used to demonstrate the ability of the PCR to
amplify the target sequence. As positive control material, genomic DNA extracted from a known crustacean
species or an available plasmid containing the target sequence can be used.
If a sample shows no amplification in all three targets, an inhibition control reaction should be performed
to exclude an inhibition of the PCR as the cause (see ISO 20813:2019). This can be done either by dilution of
sample DNA or by using an internal inhibition control assay.
Additional PCR controls can be used, see ISO 20813:2019.
8.3.4 Thermal cycling
Transfer the reaction setups into the thermal cycler and start the temperature-time programme. The
temperature-time programmes as outlined in Tables 7 - 9 have been successfully used in the collaborative
studies.
NOTE The use of different reagent conditions and thermocyclers can require specific optimization. For example,
the time for initial denaturation depends on the thermostable DNA polymerase used. Other conditions may be used
provided they produce the same results.

ISO/DIS 22344:2026(en)
Table 7 — Temperature-time program for the shorter 16S rRNA gene segment PCR (310 bp
fragment)
Step Parameter Temperature Time Cycles
Initial DNA denaturation and activation of the
1 95 °C 15 min 1
hot-start DNA polymerase (if used)
Denaturation 95 °C 60 s
2 Amplification Annealing 50 °C 60 s 35
Elongation 72 °C 60 s
3 Final elongation 72 °C 10 min 1
Table 8 — Temperature-time program for the longer 16S rRNA gene segment PCR (520 bp fragment)
Step Parameter Temperature Time Cycles
Initial DNA denaturation and activation of the
1 95 °C 5 min 1
hot-start DNA polymerase (if used)
Denaturation 94 °C 60 s
2 Amplification Annealing 55 °C 60 s 35
Elongation 72 °C 60 s
3 Final elongation 72 °C 5 min 1
Table 9 — Temperature-time program for the cox1 PCR (660 bp fragment)
Step Parameter Temperature Time Cycles
Initial DNA denaturation and activation of the
1 95 °C 5 min 1
hot-start DNA polymerase (if used)
Denaturation 94 °C 30 s
2 Amplification Annealing 54 °C 90 s 40
Elongation 72 °C 60 s
3 Final elongation 72 °C 5 min 1
After the PCR thermal cycling is finished, follow with the evaluation of the PCR products or store samples in
the refrigerator until further analysis.
8.4 Evaluation of PCR products
The PCR product should be assessed for quality and its quantity estimated (e.g. by agarose gel
electrophoresis).
Gel electrophoresis of DNA in an agarose gel is a standard technique in molecular biology. Therefore, only
general conditions that need to be adapted to each laboratory are suggested.
A volume of 1 µl to 10 µl of each PCR product is separated in, for example, an agarose gel of suitable
concentration (e.g. a mass fraction of 1 % to 2 % per volume) and evaluated with a gel documentation
system. In one lane, an appropriate DNA size standard is included for comparison.
8.5 Evaluation of the PCR results
For the shorter 16S rRNA gene segment a product of approximately 310 bp, for the longer 16S rRNA
gene segment a PCR product of approximately 520 bp, and for the cox1 gene segment a PCR product of
approximately 660 bp should be clearly visible after gel electrophoresis. If PCR products obtained from a
sample show a single band in the gel, the (remaining) PCR reaction mixture can be purified directly using
a suitable commercial kit. If more bands are present, slice the appropriate band from the gel prior to
purification.
ISO/DIS 22344:2026(en)
No amplicons should be visible for the PCR reagent control and the extraction blank control. For the positive
DNA target control, PCR products of the expected size should be visible.
The 16S rRNA gene PCRs and the cox1 PCR can show positive or negative results for the amplification of the
target sequence(s).
Depending on the outcome of the PCR, the next step is to evaluate the following:
— If the sample is positive for one or more targets (16S rRNA gene segments and/or cox1), sequencing of all
positive PCR products is the next step (see 9.1).
— If the sample is negative for all three targets, the control results are acceptable, and the sample showed
no inhibition (8.3.3) it is possible that one of the following cases applies:
— The primer sets do not sufficiently anneal to the target sequence of the DNA extracted from the
shrimp species under analysis. In this case, species identification of the sample is not possible with
this method and analyses with further universal primer pairs (e.g. other cox1-specific primer sets)
[10]
can follow the tests (see ISO 22949-1:2021, 7.5.2.2) ISO 21571:2005 .
— The DNA extracted was degraded or not of sufficient quantity or quality for PCR.
NOTE 1 For some crustacean species, the described primer pairs do not amplify the target sequences. Examples are
listed in Table A.1.
NOTE 2 Additional universal primer pairs are described in the literature [e.g. LCO1490/HCO2198 for the
[12]
mitochondrial cox1 barcode (660 bp) ]. Verified or validated barcoding methods are listed in the Barcoding Table of
[13]
Animal Species (BaTAnS) . The current version of the BaTAnS can be obtained via the webpage www .bvl .bund .de.
9 Sequencing
9.1 Sequencing of PCR products
Sequencing of PCR products is carried out in-house or outsourced according to the method available for the
testing laboratory.
A commonly applied standard procedure is Sanger sequencing (a cycle sequencing method) using
fluorescence-labelled dideoxynucleotides. For sequencing, the primers used for the generation of the
amplicons serve as sequencing primers.
DNA fragments from the sequencing reaction are subsequently separated by means of a DNA sequencer, e.g.
using capillary electrophoresis. Fluorescence signals are recorded and analysed with the device software
[10]
(see ISO 22949-1:2021, 7.5.2.5) ISO 21571:2005 .
9.2 Evaluation of sequence data
The sequence trace data (or electropherogram) shall be checked visually to ensure the sequence reaction has
worked appropriately, and base calling is accurate. In case of misassigned nucleotides to electropherogram
peaks, sequences should be edited using appropriate software and evaluating the fluorescence peak data.
Based on experience, the length of the determined sequence should be in general approximately 80 % of the
expected read length.
A sequence analysis should be preferably performed of both DNA strands. These complementary/overlapping
sequences should be assembled into a consensus sequence. This serves as an important way of checking the
accuracy of the sequence and can help remove any ambiguous bases.
The sequences of the primers are excluded from the determined sequences before the comparison to
database sequences. The consensus sequence resulting from the forward and the reverse reaction should be
used for sequence comparison with public databases.

ISO/DIS 22344:2026(en)
9.3 Comparison of the sequence with public databases
9.3.1 General
The 16S rRNA gene and cox1 amplicon sequences are evaluated according to the taxon by comparison to
sequence entries in GenBank® (an appropriate database shall be chosen, e.g. core nucleotide BLAST
® [14] 2)
database (core_nt)) by BLAST . For cox1 DNA sequences, the cox1 sequence database of the Barcode of
[7]
Life (BOLD) project is used in parallel. ®
NOTE GenBank ’s core nucleotide BLAST database and (nr/nt) nucleotide collection are non-redundant ®
databases in which identical sequences have been merged into one single entry. GenBank is hosted by the
National Center for Biotechnology Information (NCBI).
Prior to queries in public databases, it is important to gather information about the taxon under investigation,
[15] [16]
e.g. from SeaLifeBase , World Register of Marine Species (WoRMS) NCBI Taxonomy Browser and/or
3)
BOLD Taxonomy section including:
— additional species belonging to the same genus; ®
— presence of declared and related species in GenBank and/or BOLD; ®
— amount of 16S rRNA and / or cox1 sequences of respective species in GenBank and BOLD.
A scientific literature search should be performed to obtain information about the genetic relationship
between and among species within a taxon, the possibility of hybrid shrimp species or the occurrence of
[17]
introgressed mitochondrial DNA in a particular species . ®
FASTA format should be used when pasting sequences into the query boxes of BLAST and BOLD, so that the
query results are displayed together with the names of the sequences.
9.3.2 Sequence comparison of 16S rRNA and / or cox1 amplicon sequences with GenBank
The edited 16S rRNA and/or cox1 amplicon sequences are subjected to a comparison with sequences from
® ®
the nucleotide collection (nr/nt) or the core nucleotide BLAST database (core_nt) of GenBank by BLAST ,
optimized for highly similar sequences (Megablast) to identify the species from which the sequences
originated. The identified target sequences (hits) are displayed as a list. Before assigning a species, re-sort
the hits by maximum identity. The hits are additionally presented as alignments with the query sequence.
When more than 100 hits with ≥ 98 % identity are present, the number of maximal target sequences shall ®
be increased (under algorithm parameters) to identify all relevant taxa. The BLAST tool can also produce
a neighbor-joining tree to graphically display the results of the homology search. Neighbor-joining is a
bottom-up (agglomerative) clustering method for the creation of phylogenetic trees. The query sequence is
highlighted in the tree diagram.
The query result should be documented at the time of comparison and shall include:
— species of sequence entries with identities ≥ 98 % (including gaps);
— degree of id
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