Biomedical Engineering Reference
In-Depth Information
IMPORTANCE OF METABOLIC PATHWAY GENES
IN PCR METHOD
Molecular methods mostly PCR based have been employed for specifi c
identifi cation of many fungal species and also strain typing. A reliable
identifi cation of species or strain does not always provide a straightforward
and consistent approach to identify a toxin producing fungal strain.
Delineating the molecular pathways involved in biosynthesis of toxin has
proved to be highly valuable on one hand to understand the dynamics
involved in toxin production by a specifi c fungal species or strain and
for growth and environmental conditions that are necessary for toxin
production. On the other hand, the metabolic pathway genes involved
turn out to be more relevant and pertinent that can be exploited to
identify the toxigenic potential of fungal species and their strains. Many
mycotoxin biosynthetic path way genes are present within gene clusters
and some of these appear to have undergone horizontal transfer from one
species to another and are now present in several species (Niessen et al.
2007). Regions of homology within mycotoxin biosynthetic gene from the
different species can be used to develop primers to detect the presence of
the relevant mycotoxigenic species. This strategy was successfully applied
for afl atoxin producers (Shapira et al., 1996), trichothecene-producing fungi
(Paterson et al. 2006, Ramana et al. 2011), fumonisin-producing
Fusarium
species Gonzalez et al. 2004) and also for producers of patulin (Paterson
et al. 2006). PCR based detection has been applied as an alternative assay
replacing cumbersome and time consuming microbiological and chemical
methods for detection and identifi cation of the most serious pathogenic
and mycotoxin producers in the fungal genera
Fusarium, Aspergillus
and
Penicillium
. The main advantage of pathway gene targeting is that genes
are highly specifi c for the particular chemotype of the group which has
the ability to produce mycotoxin or to cause disease.
Over the past decade there has been great improvement in development
of nucleic acid based techniques and diagnostics of fungal pathogens.
The benefi ts of these developments offer potential for rapid, specifi c and
sensitive tests. The use of fungal biosystematics and molecular techniques
has paved the way for fungal phylogeny and evolution. Prior DNA sequence
database of fungi is necessary for the design of pairs of oligonucleotide
primers to confer the necessary degree of specifi city. The genes which
are involved in secondary metabolite synthesis are highly specifi c to the
respective fungi. The molecular sequence analysis of such genes would
clearly reveal the relationship among the fungal species based on their
clustering in the phylodendrogram. Amplicons thus generated can be
sequenced and the variability among such sequences can be exploited to
design specifi c primer pairs for the target pathogen. Designing of metabolic
