Title: P1250095200OJmEo
1New methods for the determination of DNA in food
Prof. Rosangela Marchelli Dipartimento di Chimica
Organica e Industriale Facoltà di
Agraria Università di Parma
2Rapid microbiology
1) Direct test directly performed on the
specimen (e.g. PCR) 2) Rapid methods (after a
short enrichment step) 3) Culture
identification methods
3Ribosomal DNA and RNA as targets for microbial
identification
- rDNA sequencing platforms for microbial
identifcations are commercially available - - Cellular Abundance of rRNA relates to an
organisms growth rate and is a good reason for
using rRNA as a target (direct identification
with fluorescent probes with no need of
amplification)
4Peptide Nucleic Acids (PNAs) for rapid
microbiology
Nielsen PE, Egholm M, Berg RH, Buchardt
O. Science 1991 254 1497-1500
DUPLEX WITH COMPLEMENTARY DNA Watson-Crick base
pairing High stability and selectivity
TRIPLEX PNADNAPNA Watson-Crick and Hoogsteen
base pairing High stability Strand invasion in
double strand DNA
5DUPLEX STABILITY RNA-PNA gt PNA-DNA gtgtDNA-DNA
15-mer Tm 72C Tm 69C Tm 53C
POINT-MUTATION RECOGNITION PNA-DNA gt DNA-DNA
15-mer Tm drop with a mismatch Tm 69C -gt
56C Tm 53C -gt 49C
M. Egholm, O. Buchardt, L. Christensen, C.
Behrens, S.M. Freier, D.A. Driver, R.H. Berg,
S.K. Kim, B. Norden, P.E. Nielsen, Nature, 1993,
365, 566-568.
6DNA identification with PNA probes
Food Authenticity
Microbiology
GMOs
Food allergens
7Thiazole orange-PNA (PCR and FISH)
PNA beacon (PCR and FISH)
LightUp probes (LightUp Technologies, Goteborg,
Sweden) LightSpeed probes (Boston probes,
Bedford, MA, USA)
H. Stender, M. Fiandaca, J.J. Hyldig-Nielsen, J.
Coull, PNA for rapid microbiology, J.
Microbiol. Methods, 2002, 48, 1-17.
8PNA-FISH (Fluorescence in situ hybridization)
Fluorescence microscopy
Flow citometry
Advantages of PNA- vs DNA-FISH
- Enhanced hybridization rates
- Resistance to nucleases
- Ability to penetrate recalcitrant biological
structures such as the gram-positive cell walls
on account of the neutral and hydrophobic
character of PNAs
9Four-color multiplex PNA FISH E. coli Salmonella
enterica P. aeruginosa Staphylococcus aureus
DEAC-labeled PNA probe targeting S. enterica rRNA
-gt (blue) Cy5-labeled PNA probe targeting E. coli
16S rRNA -gt (red) Cy3-labeled PNA probe targeting
S. aureus 16S rRNA -gt (yellow) fluorescein-labeled
PNA probe targeting P. aeruginosa 16S rRNA -gt
(green)
The cells were subsequently mounted and examined
by fluorescence microscopy using selective filter
sets. Images obtained with each of the four
filters were then combined electronically (kindly
provided by Dr. H. Perry-O'Keefe, Boston Probes).
10PNA FISH
Fluorescein-PNA against Pseudomonas
Cy3-PNA against Eubacteria
Fluorescein-PNA against Staphylococcus
Fluorescein-PNA against Salmonella
Fluorescein-PNA against Escherichia
DAPI stained
E. Coli
S. enterica
P. aeruginosa
S. aureus
11Applications to Food Diagnostics currently
performed at the Department of Organic and
Industrial Chemistry of the University of Parma
PNA
CD
BIACORE
MS
HPLC
UV and FLUORESCENCE
CZE
MICROARRAYS
12Solid phase PNA synthesis
X O, NH
13MICROARRAYS
1. Probe deposition
2. Hybridization with the target
14Target Roundup Ready Soybean 79mer Cy5
15GMO detection
Multiplex PCR
A. Germini et al., J. Agric. Food Chem. 2004, 52
(11), 3275-3280
16PNA chip for GMOs
Development of a PNA array platform for the
detection of GMOs in food. Andrea Germini,
Stefano Rossi, Alessandro Zanetti, Roberto
Corradini, Corrado Fogher, Rosangela Marchelli.
Journal of Agricultural and Food Chemistry 2005,
53, 3958-3962.
17GMO specificity control
Soy Maize Mon810 RR soy Bt11
Bt176 GA21
Development of a PNA array platform for the
detection of GMOs in food. Andrea Germini,
Stefano Rossi, Alessandro Zanetti, Roberto
Corradini, Corrado Fogher, Rosangela Marchelli.
Journal of Agricultural and Food Chemistry 2005,
53, 3958-3962.
18Identification of RR-soybean PCR amplicon by a
PNA probe in AE-HPLC
ss RR-169 bases amplicon
RR-amplicon complementary PNA
RR-amplicon non complementary PNA
Column TSK gel DEAE-NPR (4.6mm ID x 3.5cm),
eluents A TRIS 20 mM in H2O a pH9 B NaCl 1M
in eluent A a pH9, gradient from 50 A to 30A
in 10 min. Temp 35C Detector fluorescence
lecc 550 nm, lem 570 nm (Cy3) .
19PNA BEACONS
20PNA BEACON FOR RR SOYBEAN
PNA-DNA (FULL MATCH)
PNA-DNA (NOT COMPLEMENTARY)
?EC497 nm
?EM520 nm
21HPLC-AE using PNA- BEACON
Beacon - DNA
Negative control
22Uova
Arachidi, soia, altri legumi
Pesce
Allergen detection
Molluschi e crostacei
Frumento e altri cereali
Latte
Tree nuts
Sesamo e altri semi
23Methods for the detection of hidden allergens in
foods
24- Ara h2 Peanut
- Cor a1 Hazelnut
- Pru du 2.1 Almond
PNA probes for allergens
Detection limit 50fmol
PNA probes specificity
25 PCR duplex for the DNA detection of hazelnut and
peanut in commercial products
Hazelnut Cor a 1 (156bp) and Peanut Ara h 2
(201bp) amplicons. Limit of detection 50pg of
DNA.
26 Microarray detection of hazelnut and peanut DNA
in commercial products
Sample 3. Muesli snack
Sample 1. Breakfast cereals
H P A CP
H P A CP
PNA array platform for the detection of traces
of potentially allergenic nuts in foodstuffs.
Stefano Rossi, Elena Scaravelli, Andrea Germini,
Roberto Corradini, Rosangela Marchelli. European
Food Research Technology, in press.
27DNA sequences for olive varieties
Olive oil authenticity
60 120 183 198 345
Biancolilla A A G C G
Canino A R R S G
Carolea A A G C G
Coratina A A G C G
Frantoio A A G C G
Leccino A A G C R
Nocellara Belice A A G C R
Ogliarola leccese T A G C G
Moraiolo A R R C G
Bosana A R R C G
Nocellara Etnea A A G C R
Arbequina A A G C G
SNPs in the actin gene
R A or G S G or C
28Olive variety identification by PNA microarrays
29Chiral Box D-lysine-based PNAs
V. Menchise, G. De Simone, T. Tedeschi, R.
Corradini, S. Sforza, R. Marchelli, D. Capasso,
M. Saviano, C. Pedone, Proc Nat Acad Sci USA,
2003, 100, 12021-12026.
30Match/mismatch selectivity of achiral and chiral
PNAs in the recognition of the CF W1282X mutation
WT-PNA
Mut-PNA
RU(match)/RU(mismatch)
CHIRAL
ACHIRAL
ACHIRAL
CHIRAL
G. Feriotto, R. Corradini, S. Sforza, N. Bianchi,
C. Mischiati, R. Marchelli, R. Gambari Laboratory
Investigation, 2001, 81, 1415-1427 R. Corradini,
G. Feriotto, s. Sforza, R. Marchelli, R.
Gambari Journal of Molecular Recognition, 2004,
17, 76-84.
31Capillary electrophoresis on DNA from human
subjects
dsDNA
Heterozygous subject
Heterozigous
Homozygous subject
M. Chiari, T. Tedeschi, G. Galaverna, S. Sforza,
M. Cretich, R. Corradini, R. Marchelli,
Electrophoresis, 2005, 26, 4310-4316.
32Acknowledgments
Roberto Corradini Stefano Sforza Arnaldo
Dossena Gianni Galaverna Andrea Germini Stefano
Rossi Elena Scaravelli Andrea Faccini Tullia
Tedeschi Filbert Totsingan
33H. Stender, M. Fiandaca, J.J. Hyldig-Nielsen, J.
Coull, PNA for rapid microbiology, J.
Microbiol. Methods, 2002, 48, 1-17.