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Landesamt für Verbraucherschutz Sachsen-Anhalt
Fachbereich Lebensmittelsicherheit
Workshop Species Identification 14.04.15
Species Identification
Dietrich Mäde
Workshop Species Identification 14.04.15 Dietrich Mäde
This is how the press sees us….
superficially
Workshop Species Identification 14.04.15 Dietrich Mäde
Legal requirements for species identification
• Reg. (EC) No 178/2002 - Art 8 - Protection of consumers' interests -
prevention of:
(a) fraudulent or deceptive practices;
(b) the adulteration of food; and
(c) any other practices which may mislead the consumer.
• Reg. (EU) No. 1196/2011 - Art 7 - Fair information practices
• Reg. (EU) No. 1196/2011 - Art 22 - Quantitative indication of ingredients
• Reg. (EU) No 1379/2013 – Art 35 (1) (a): Indication of commercial
designation of the species and its scientific name
Workshop Species Identification 14.04.15 Dietrich Mäde
Fraudulent Practices
• Fatstock prices (rounded)
– Pig 1,45 €/kg
– Cattle (Cow) 3,30 €/kg
– Turkey 1,40 €/kg
– Lambs 5,00 €/kg
– Horses 0,50 €/kg
– mechanically deboned meat
(chicken or turkey) ~ 1€/kg
4
Workshop Species Identification 14.04.15 Dietrich Mäde
Horsemeat scandal
• Quelle: Süddeutsche Zeitung vom 17.202013
Workshop Species Identification 14.04.15 Dietrich Mäde
Press release
of 07/04/2015
6
“Dutch authorities took 167 samples from his meat supplies in
February 2013 and 35 tested positive for horse DNA, the court
in Den Bosch heard.”
Workshop Species Identification 14.04.15 Dietrich Mäde
Turkey DNA in salad with sausage
• turkey at ~1 %
level
• not indicated
• technologically not
necessary
• some producers
were striking than
others
Workshop Species Identification 14.04.15 Dietrich Mäde
Methods for Species Detection
• Immunological methods
– ELISA
– Agargel precipitation (old)
• Electrophoreses
– isoelectric focussing
• molecular methods
– universal PCR and subsequent species determination
by RFLP or Sequencing
– species or “group of species” specific PCR / real-time
PCR with verification by RFLP, probes or sequencing
8
Workshop Species Identification 14.04.15 Dietrich Mäde
Immunological methods
• ELISA
– detection of muscle specific
glycoproteins
– Sensitivity ~1 %
– false negative results in
some canned foods (F>12 -
(temperature > 134 °C)
– sometimes very helpful
when PCR fails
9
Workshop Species Identification 14.04.15 Dietrich Mäde
Electrophoresis
• Electrophoresis
– detection of myoglobin
– sensitivity 5% - 10%
– not applicable for highly
processed food
– method of choice for milk,
milk products and cheese
10
po
rk
beef
10892
10895
10898
ho
rse
me
at
mu
tton
10892
10895
10898
ho
rse
me
at
mu
tton
Workshop Species Identification 14.04.15 Dietrich Mäde 11
bovine γ2 casein ovine γ2 casein
bovine γ3 casein ovine γ3 casein
1. cow milk std
2. sample of mislabelled
„feta“ cheese
3. sheep cheese with
cow´s milk added
4. ewe's milk std
5. ewe's milk with 1 %
cow´s milk
6. sheep cheese with
cow´s milk added
7. cow milk std
8. sample of mislabelled
„feta“ cheese
Electrophoresis of milk products
Workshop Species Identification 14.04.15 Dietrich Mäde
Molecular methods - Qualitative PCR
• Often extremely sensitive
– mitochondrial DNA is present in
hundreds of copies per cell
resp. skeletal muscle fiber
sycytium
– sensitivity drops in highly acidic
and heated food (some canned
soups)
• Interpretation of results
sometimes risky
– traces from other food in artisan
production
• QM is highly demanding
– multiple extraction negative
controls necessary (e.g. every
10 extractions)
12
widely used for
qualitative species
detection
Workshop Species Identification 14.04.15 Dietrich Mäde
• conserved universal mitochondrial sequences with
some species specific differences
– differences are made visible by RFLP or DNA sequencing
(sheer products mostly)
• cyt b can be used as amplification control for
subsequent specific assays
– check for inhibition and for DNA degradation
Species detection in the cyt b-gene
13
DNA-extraction
cyt b PCR – diagnostic rxn & amplification ctrl
restriction analysis DNA-sequencing
Workshop Species Identification 14.04.15 Dietrich Mäde
cyt b-PCR – detection of pig by RFLP with AluI
conserved conserved
PCR-product is 359 bp long.
bands can be made visible by electrophoresis
AluI recognizes agct at position 114 - 117 and cuts there
ag ct
conserved conserved variable (species specific differences)
ag ct
fragment A 115 bp fragment B 244 bp
Workshop Species Identification 14.04.15 Dietrich Mäde
Imaging of cyt b-PCR and restriction
fragments on a agarosegel
verification of ampfliability of the
extracted DNA:
all samples result in a 359 bp
PCR product
separation of the restriction
fragments after cutting with AluI
and verification of species specific
gel bands
Sus scrofa f. domestica
Workshop Species Identification 14.04.15 Dietrich Mäde
Adaptation of the cyt b-primers to species
groups
• Primer mismatches in some
species: Galliformes,
Cervidae, Equidae, Bovidae
compared to humans, mouse
and pig resulting in reduced
sensitivity
– competitive assay
• Adaptations of primers at the
same molecular region to the
taxonomic orders or families
can overcome this
16
primers for Galliformes →
higher sensitivity for turkey
and chicken in mixed products
Workshop Species Identification 14.04.15 Dietrich Mäde
Conventional species specific PCR
• established for cattle and
sheep
• we use of single copy genes
to allow quantitative
estimations (year 2000)
– GnRH gene
• verification necessary, e.g.
by RFLP
• 1 % w/w as positive control
– 1 % beef in 99 % pork
– 1 % mutton in 99 % beef
17
Workshop Species Identification 14.04.15 Dietrich Mäde
Analysis of fish by
mitochondrial PCR
and RFLP resp. DNA
sequencing
• Official method in
Germany
• high amount of
fraudulent practices
esp. in restaurants
• amplification of
mitochondrial genes
and comparision with
Genbank or other
databases
18
Solea solea
mislabelledsamples
n=12
Workshop Species Identification 14.04.15 Dietrich Mäde
Practical cases – special applications
19
068
16
Pa
sta
Pe
nn
e B
olo
gn
ese
068
20
Sp
ag
hetti B
olo
gn
ese
06818 m
inced b
eef
with
hors
e m
eat
068
16
min
ced
bee
f
with
hors
e m
eat
hors
e c
ontro
l
Workshop Species Identification 14.04.15 Dietrich Mäde
Species detection of teeth
Request for species identification: Pig or Rat?
• cyt b-PCR:
– porcine DNA detected
– rat DNA not detectable
20
Workshop Species Identification 14.04.15 Dietrich Mäde
Analysis of a finger nail
21
• sample preparation is crucial: All DNA molecules on the surface need
to be removed or destroyed
• sequence analysis of ZFX/ZFY revealed that is was a man
Workshop Species Identification 14.04.15 Dietrich Mäde
>gb|DQ309764.1| Canis familiaris cytochrome b (cytb) pseudogene, partial sequence;
nuclear copy of mitochondrial gene
Length=356
Score = 187 bits (101), Expect = 7e-45
Identities = 106/111 (95%), Gaps = 0/111 (0%)
Strand=Plus/Minus
Query 1 CATGGTAGGACGTAACCTATGAATGCTGTGGCTATGGTTGCAAAKAAGAGAATAATTCCA 60
|||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||
Sbjct 335 CATGGTAGGACGTAACCTATGAATGCTGTGGCTATGGTTGCAAAGAAGAGAATAATTCCA 276
Query 61 ATATTTCATGTTTCTATGAATACRTAGGRGCCGTAGTATAAYCYTCATCCT 111
||||||||||||||||||||||| |||| |||||||||||| | |||||||
Sbjct 275 ATATTTCATGTTTCTATGAATACGTAGGAGCCGTAGTATAACCTTCATCCT 225
Detection of dog meat in a restaurant
22
Further sequence analysis of ZFX/ZFY revealed that is was a male dog
Workshop Species Identification 14.04.15 Dietrich Mäde
further cases
• forensic assays differentiation between animal or
human blood
– criminologists found blood stained trousers
– Ovine blood or human blood?
– 2 methods used to verify the result: cyt b PCR and
species specific PCR
It was ovine blood.
• detection of mouse material in canned tomato
sauce
– The superior sensitivity of the cyt b PCR was very useful
as the DNA was partially degraded
– Verified by RFLP and DNA sequencing
23
Workshop Species Identification 14.04.15 Dietrich Mäde
Qualitative species detection - summary
Advantages
• cheap
• applicable to all species
• useful for very sensitive
analysis if necessary
• very helpful for food of a
single ingredient
– meat
– fish
– plant
24
Disadvantages
• too sensitive (in general)
– further quantification
required, or ELISA as
alternative method
• target taxon need to be
specified before
– If You do not look for
horsemeat, You won´t see
it if present in minor traces
as adapted cyt b primers or
specific assays are
necessary
Workshop Species Identification 14.04.15 Dietrich Mäde
Quantitative species detection by real-time PCR
• Quantitative real-time PCR
– detection of species
specific gene sequences
– limited sensitivity 0,1% • depends on amount and
quality of extracted DNA
– quantitative analysis is
most reliable as copy
number quantification
• DNA-content is tissue
specific fat<skeletal
muscle=connecting
tissue<entrails
25
Workshop Species Identification 14.04.15 Dietrich Mäde
Quantitative digital PCR
• droplet digital PCR (BioRad,
Raindance)
• digital PCR Chip (Fluidigm, Life
Technologies)
• digital PCR on a special microplate
(constellation system by
Formulatrix)
• allows absolute quantification
• no need for standard curves
• resistant to PCR inhibitors
26
Workshop Species Identification 14.04.15 Dietrich Mäde
1st trial with the Constellation system from
Formulatrix (01.04.2015, not optimised)
27
species cattle pig sheep chicken turkey horse
525 580 1375 981 623 978
491 570 1442 1236 1128 1073
539 641 1287 897 1090 1031
582 704 1350 992 1000 978
533 545 843 1168 991 1118
552 598 1315 1201 957 1039 mean 537 606 1269 1079 965 1036
copies (by real-time PCR 2013)
500 500 1000 1000 1000 1000
deviation 7 % 21 % 27 % 8 % 4 % 4 %
Workshop Species Identification 14.04.15 Dietrich Mäde
Is quantification possible?
• single copy genes need to be chosen
• target taxon PCR systems should be as specific
as possible
– cross reactions between different equine species
• generic PCR systems should amplify mammals
and birds
• mass per mass is difficult to establish
– different tissue types contain different DNA amounts
• copy per copy approach overcomes this issue
28
Workshop Species Identification 14.04.15 Dietrich Mäde
DNA content of different tissue types
29
Microscopic pictures: Liebich, HG: Funktionelle Histologie der Haussäugetiere. Stuttgart, New York: F.K. Schattauer Verlagsgesellschaft mbH 1999
Workshop Species Identification 14.04.15 Dietrich Mäde
DNA content of different tissue types
30
Microscopic pictures: Liebich, HG: Funktionelle Histologie der Haussäugetiere. Stuttgart, New York: F.K. Schattauer Verlagsgesellschaft mbH 1999
tissue type DNA content
(mean and range)
skeletal muscle 100 (70-130) µg/g
connective tissue 100 (50-200) µg/g
fat tissue 40 (10-150) µg/g
liver 700 (600-800) µg/g
spleen 700 (550-850) µg/g
Workshop Species Identification 14.04.15 Dietrich Mäde
Why copy per copy?
• there is a different DNA content in different tissues
• composition of meat products and sausages is
highly heterogeneous
– different quantities of meat, fat, and entrails
• relatively independent from the quality of standard
material
– standard material need to be made available
Proposal: Laboratories should not determine the
very exact amount of ingredients, the point is that
every laboratory gets the same results.
31
Workshop Species Identification 14.04.15 Dietrich Mäde
Advantages of the quantitative approach
• no misinterpretation of traces
– cross contamination at retail
– <0,1 % - adventitious traces
– 0,1 % – 1% - on site checks requested to find out where
the species comes from
– >1% legal action
• tool for verification of the quantitative ingredient
declaration
32
European legislation should be harmonized
Workshop Species Identification 14.04.15 Dietrich Mäde
Limitations of the quantitative real-time PCR
• sampling protocols do not exist
• dynamic range is limited
– reliable quantification between 1 % and 50 %
• cross reactions
– closely related species: Cattle and buffalo, cattle and
Cervidae
• spectral overlap (cross talk) in multiplex assays
– What is crosstalk and what is positive in minor
amounts?
– A cut off value is NOT a scientifically based approach
33
Workshop Species Identification 14.04.15 Dietrich Mäde
Influence of reduced efficiency on Ct values
34
Relationship between the PCR product to initial template:
Nc = N (1+η)ν
Nc is the number of amplified molecules;
N is the initial number of the target molecules;
η is the efficiency of the system; ν is the number of amplification cycles.
In praxi, reduced
efficiency by minor
inhibitions cannot
be excluded.
This will lead to a
shift of the amplifi-
cation curve with
higher ct-valiues.
Workshop Species Identification 14.04.15 Dietrich Mäde
lot size
1000 kg
Are there adventitious traces?
1. Industrial meat products are produced in big lots
2. slaughterhouses are highly specialized – there is not possibility for
adventitious contamination at industrial levels
35
10 kg (true to scale graphic)
• Amounts of <1 % are easily to
be seen in factories
• even small amounts could
have economical effects
Workshop Species Identification 14.04.15 Dietrich Mäde
Standardisation
36
national:
BS, AFNOR, NMKL, DIN, Ö-Norm, off.
methods collection …
European Standar-dization
CEN
International Standardization
ISO
Workshop Species Identification 14.04.15 Dietrich Mäde
Official methods available
• German official method collection
– BVL L 11.00-7: Identification of fish species in raw and
cooked foodstuffs (PCR-RFLP)
– BVL L 10.00-12: Fish species identification by sequence
analysis of cytochrome-b-sequences
– BVL L 12.01-3: Crustacean species determination in raw
crustacean and crustacean products by sequence analysis
of 16S rRNA sequences
– BVL L 06.26/27-2: Detection of horse-specific DNA
sequences in tinned meat products and verification by
restriction analysis
37
Workshop Species Identification 14.04.15 Dietrich Mäde
Further experiences with
method standardisation
• Failed method validation studies:
– Detection of poultry by a adapted cyt b PCR and restriction
analysis
• false positives due to chromosomal pseudogenes in some species
– Quantification of bovine DNA in meat products by real-time
PCR
• to high standard deviation between participants
• Currently under development: Multiplex real-time
PCR method for species identification
– high amount of cross reactions observed
Reliable species identification is highly demanding
38
Workshop Species Identification 14.04.15 Dietrich Mäde
Standardisation projects
within ISO TC 34 SC 16
• Iranian proposals for qualitative methods based on
conventional PCR:
– Species Identification of Meat and meat products by
Multiplex PCR
– Methods of analysis for the detection of Buffalo meat in
meat products
• no proper control for the amount and the quality of extracted DNA
• no molecular verification of results
• Chinese proposal
– Detection of animal derived materials in foodstuffs and
feedstuffs by real-time PCR
• mixture of mitochondrial or nuclear genome sequences – no reliable
quantification possible
39
Workshop Species Identification 14.04.15 Dietrich Mäde
Conclusion
• Urgent need for harmonization of methods for
species identification
• Methodologies are applied for nearly 20 years in
routine diagnostics, however, due to the lack of
standardized principles a comparison of analytical
results is not always guaranteed
• Start of with simple standards and not with complex
multiplex protocols
• Methods should comprise both, sensitive qualitative
protocols and reliable quantitative protocols (the
latter based on the DNA content)
40
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