Biomedical Engineering Reference
In-Depth Information
method.
A. ochraceus
and
A. westerdijkiae
were only recently dismembered
into two species, the primer-pairs designed until now are not specifi c either
to
A. ochraceus
or to
A. westerdijkiae
, i.e., they recognize both species. The
second primer pair AoLC35-12L/AoLC35-12R was found to detect OTA
producing fungi
A. carbonarius
,
A. melleus
,
A. ochraceus
,
A. sulfureus, P.
verrucosum
and citrinin producing
P. citrinum
and
Monascus ruber
.
In
A. carbonarius
, fi ve different Keto synthase domain sequences (AcKS9,
AcKS10, Ac12RL3, AcLC35-4 and AcLC35-6) were identifi ed by Atoui
et al. (2006). Two different species specifi c PCR detection systems were
developed based on primers designed from AcKS10 (Selm et al
.
2008) and
Ac12RL3 (Atoui et al
.
2007) and the method proved to be highly specifi c,
yielding amplifi cation only in
A. carbonarius
strains, the main OTA producer
in grapes. In the genus
Penicillium
, a PCR method for differentiation and
detection of two ochratoxigenic
Penicillium
species,
P. nordicum
and
P.
verrucosum
, has been developed. It is based upon two genes of the OTA
biosynthetic pathway, namely the OTA polyketide synthase gene (
otapksPN
)
and a nonribosomal peptide syntethase gene (
otanpsPN
) from
P. nordicum
(Geissen et al
.
2004, Farber et al. 2004).
P. verrucosum
gives consistently
only a positive reaction with the primers for the otanpsPN gene, whereas
P. nordicum
is positive for both genes. The PCR reaction is negative with
all other food related fungal species tested. This system has been used to
analyze 62
Penicillium
strains isolated from cured meat products or ripening
rooms.
RT-PCR have proven useful in monitoring and quantifi cation of OTA
fungal producers in many food commodities (Giessen et al
.
2004, Farber et
al
.
2004, Mule et al. 2006, Selma et al
.
2008 and Atoui et al. 2007). One of the
major motivations for the development of PCR based detection systems in
many publications is the prospect of using this kind of analysis to estimate
OTA concentrations in sample material. One might therefore anticipate
that assays based on OTA biosynthetic genes might better fi t that purpose
as compared to systems based on genes unrelated to their biosynthesis.
By using real-time PCR, a positive correlation between OTA content and
DNA quantity has been indicated for
P.
nordicum
and
A. ochraceus
(Farber
et al. 2004, Kennedy et al. 199) and more recently, in
A. carbonarius
(Selma
et al. 2008, Atoui et al. 2007). Such a correlation has been established with
quantitative real-time PCR on mycotoxin biosynthesis genes (Geisen et al.
2004) or when using primers targeted sequences of housekeeping genes
(Mule et al. 2006). Currently, RT- PCR quantifi cation of
A. carbonarius
in
grapes is clearly the best alternative to conventional methods in order
to investigate the relation between OTA producers and OTA content
with regards to food safety (Atoui et al.
2006). All the systems described
above are based on PCR with genomic DNA as template to estimate OTA
concentrations in sample materials. Given the fact that biosynthesis of
