Biomedical Engineering Reference
In-Depth Information
employed by a number of research groups in the detection of afl atoxigenic
fungi. The fi rst PCR-based detection system for a mycotoxin producing
fungus was published by Tang et al. (1993) who described detection of
A.
fl avus
by nested PCR in human bronchoalveolar lavages. Clearly focussed
on evaluation of toxigenic properties of
Aspergillus
species from section
Flavi were two PCR assays published by Geisen (1996) and a parallel
publication by Shapira et al. (1996). Both papers can be regarded as the
starting point for PCR-based diagnosis of mycotoxigenic fungi. Authors
used sequences of the three genes involved in the biosynthesis of afl atoxins
in
A. fl avus, A. parasiticus
and
A. versicolor
to design their specifi c primers.
The assay published by Geisen (1996) made use of the three primer pairs
in a multiplex PCR in which it was demonstrated that
A. sojae
and
A.
oryzae
, of which are essentially identical with
A. fl avus
but typically do
not produce afl atoxins, for lack of
nor1
gene.
Using a multiplex PCR approach by targeting three aflatoxin
biosynthetic genes namely norsolorinic acid reductase (
nor
1), versicolorin
A dehydrogenase (
ver
1) and sterigmatocystin O-methyltransferase (
omt
A)
(Geisen, 1996), was able to observe a triplet banding pattern in afl atoxin
producing strains of
Aspergillus fl avus
,
A
.
parasiticus
and sterigmatocystin
producing strains of
A
.
versicolor
. Normally in a multiplex PCR system, the
specifi city is increased because a number of genes can be detected in one
reaction simultaneously. However, there are problems with the specifi city
of this particular system, because, while it does appear to distinguish
between afatoxigenic
Aspergillus
species and other non-afl atoxigenic food
related species such as
Penicillium
,
Fusarium
,
Byssochlamys
and
Geotrichum
spp., it cannot distinguish between afl atoxigenic and non-afl atoxigenic
A
.
fl avus
strains. Another specifi city problem associated with the method is on
false positives obtained with
Penicillium
roqueforti
which appears to have
cross-reaction with PCR positive
nor
1 and
ver
1 homologues. Shapira et al.
(1996) outlined a similar diagnostic PCR method, again for the detection
of afl atoxigenic fungi. In this case the target genes were the
omt
1, the
ver
1
gene and the regulatory gene
apa
-2 (since renamed
afl
R) which regulates
expression of the afl atoxin biosynthetic gene cluster (Chang et al. 1993).
Specifi c PCR products were obtained only with DNA from
A
.
parasiticus
with all three primer pairs, with the
afl
R primers failing to give a positive
signal with other
Aspergillus.
This probably refl ects the subtle sequence
differences between the
afl
R gene in
A
.
parasiticus
(for which the primers
were designed) and the
afl
R gene in
A
.
fl avus
(Chang et al. 1995). Despite
this no amplifi cation products were obtained from
Fusarium
,
Penicillium
and
non-afl atoxigenic
Aspergillus
species. Mayer et al. (2003) used sequences of
the
nor
1 gene to set up primers and a probe for a TaqMan™real-time PCR
assay with which
A. fl avus
was quantifi ed in contaminated food samples
