ELISA Real-time PCR

Anwendungen der real-time PCR
in der Lebensmittelanalytik

„stacked crops“
Source: International Service for the Acquisition of Agri-biotech Applications (ISAAA).
http://www.isaaa.org
144 GM events, representing 24 crops, have been approved worldwide.
33 GM crops are commercialised worldwide.

GM crops approved in the EU
http://ec.europa.eu/food/dyna/gm_register/index_en.cfm
Validated method:
• Sample homogenization
• DNA extraction
• PCR (reagents, primer sequences, temperature program,
negative controls, positive controls…)

Legal regulations for GMO labeling
Source: E. Michelini et al., Anal. Bioanal. Chem. 392 (2008) 355-367.

Labeling regulation in the EU
Food and feed which contains, consists of, or is produced from GMOs must
carry a label which refers to the presence of GMOs.
Labeling is not required for food/feed (ingredient) in a proportion lower than 0.9%
if its presence is technically unavoidable.

Specificity of the PCR assays
Plant DNA Promoter Enhancer Structural gene Terminator Plant DNA
1. Screening
2. Gene-specific
P35S (35S promoter from Cauliflower mosaic virus)
tNOS (nopaline synthase terminator from Agrobacterium tumefaciens)
3. Construct-specific
4. Event-specific

Typical scheme in routine GMO analysis
Food product
Screening
Quantification
Less than 0.9%
No labeling required
negative
positive
Identification/
Authorised?
yes no
More than 0.9%
Labeling required

Quantification
Threshold cycle
Event MON810 Reference maize
log gene copy number

Selection of target sequences for screening
Source: H.-U. Waiblinger et al., Anal. Bioanal. Chem. 396 (2010) 2065-2072

Lebensmittelallergie
IgE
vermittelt
Lebensmittelunverträglichkeitsreaktionen
Lebensmittelallergien
(Immunologische
Reaktionen)
Nicht-toxische Toxische
Nicht IgE
vermittelt
Lebensmittelintoleranzen
(Nicht immunologische
Reaktionen)
Enzymatisch Pharmakologisch Undefiniert

Lebensmittelallergene
• Körperfremde Glycoproteine
• Molekulargewicht von 5 – 70 kDa
• Stabilität gegenüber Säure und Proteasen, Hitze
Die meisten allergenen Lebensmittel enthalten mehrere allergene
Proteine.
z.B. Corylus avellana: Cor a 1, …Cor a 11
Sesamum indicum: Ses i 1, Ses i 2, Ses i 3
Papaver somniferum: ???

Kennzeichnungspflichtige Zutaten
DIRECTIVE 2006/142/EC, Official Journal of the European Union, 2006,
L 368/110
• Glutenhaltiges Getreide
• Krustentiere
• Eier
• Fisch
• Erdnüsse
• Sojabohnen
• Milch und Milcherzeugnisse
• Nüsse
• Sellerie
• Senf und Senfsamen
• Sesam
• Schwefeldioxid und Sulfite (Konzentration > 10 mg/kg oder 10 mg/L)
• Lupine
• Weichtiere

Analytical methods – state of the art
(B-NSB)/B max [%]
1,0
0,8
0,6
0,4
0,2
0,0
Immunoanalytical methods DNA based methods
10-4 10-3 10-2 10-1 100 101 102 103 104
lupine protein concentration [mg/L]
Immunoassay
Immunosensor
Immunoblot
Lateral flow test
real-time PCR

Vergleich ELISA – Real-time PCR
ELISA Real-time PCR
Apparative Kosten gering hoch
Reagentien Verfügbarkeit der reproduzierbar
Antikörper herstellbar
Spezifität hoch hoch
Nachweisgrenze 0.0001-0.001% 0.001 – 0.01%
Matrixinterferenzen abhängig vom Format Hemmung der
Polymerase
Prozessierung Denaturierung des höhere Stabilität
Proteins der DNA
Kontaminationsgefahr gering hoch

Rapid Alert System for Food and Feed
(RASFF)
when a food or feed presenting a serious health risk is on the
market and when rapid action is required

Alert notifications in 2009

Culture-based methods
Sampling
Preenrichment in
non-selective medium
Selective enrichment
Isolation on
selective plates
Serological/biochemical
confirmation
real-time PCR
Sampling
Short preenrichment
DNA isolation
real-time PCR

eal-time PCR:
+ faster
vs.
+ less laborious
+ more sensitive
+ more specific

Species identification for detecting meat adulteration
Species Target gene LOD Reference
Beef, pork
Phosphodiesterase,
myostatin
Beef, pork, chicken, lamb, turkey Mitochondrial cytochrome b
Duck Mitochondrial cytochrome b
Beef, pork
Beef, pork, chicken, lamb, goat,
duck, turkey
Mitochondrial tRNA
ATPase sub. 8
Cyclic guanosine
monophosphatase,
phosphodiesterase,
ryanodine receptor,
interleukin-2 precursor,
myostatin
Beef Growth hormone
(Ballin et al., Meat Science 83 (2009) 165)
0.1% w/w (raw)
Laube et al.
(2003)
0.1% w/w (raw) Dooley et al.
(2004)
0.0001% w/w,
based on DNA
dilutions (raw)
Hird et al.
(2005)
0.1% w/w (heated) Furniere et al.
(2006)
0.1% w/w (heated) Laube et al.
(2007)
0.01% w/w, based
on DNA dilutions
(raw)
Brodmann et
al. (2003)

Quantification
Difficult to correlate the DNA concentration to the meat content
expressed as weight/weight (w/w):
• meat products frequently consist of different tissue types
• in most cases the tissue composition is not known
• different tissue types contain different concentrations of DNA
• the tissue type influences the extraction yield

Influence of the tissue type on the extraction
yield
Source: Laube et al., Int. J. Food Sci. Technol. 42 (2007) 336-341

DNA material
Single copy genomic DNA
• one copy per cell high LOD
• constant copy number enables quantification
Mitochondrial DNA
• most cells contain multiple copies low LOD
• variation on copy numbers in different tissues
not suitable for quantification