Degola, F. and Berni, E. and Dall'Asta, C. and et al, .
(2007)
A multiplex RT-PCR approach to detect aflatoxigenic strains
of Aspergillus flavus.
Journal of Applied Microbiology, 103.
pp. 409-417.
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Abstract
Aims: To develop a multiplex reverse transciption-polymerase chain reaction
(RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonproducing
strains of Aspergillus flavus.
Methods and Results: The protocol was first optimized on a set of strains
obtained from laboratory collections and then validated on A. flavus strains
isolated from corn grains collected in the fields of the Po Valley (Italy). Five
genes of the aflatoxin gene cluster of A. flavus, two regulatory (aflR and aflS)
and three structural (aflD, aflO and aflQ), were targeted with specific primers
to highlight their expression in mycelia cultivated under inducing conditions
for aflatoxins production. 48-h-old cultures expressed the complete set of the
genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex
PCR) was also performed in parallel using chromosomal DNA extracted from
the same set of strains to correlate the integrity of the genes with their expression.
Conclusions: We show that a good correlation exists between gene expression
of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production,
except for one strain that apparently transcribed all the relevant genes
but did not produce aflatoxin in the medium.
Significance and Impact of the Study: This is the first example of the application
of a combination of multiplex PCR and RT-PCR approaches to screen a
population of A. flavus for the presence of aflatoxigenic and nonaflatoxigenic
strains. The proposed protocol will be helpful in evaluating the risk posed by
A. flavus in natural environments and might also be a useful tool to monitor
its presence during the processing steps of food and feed commodities.
| Item Type: |
Article
|
| Additional Information: |
Francesca Degola is a recipient of a post-graduate fellowship
from SIQUAL (Laboratorio Regionale di Qualita`
e Sicurezza degli Alimenti), which has partially funded
this research. |
| Uncontrolled Keywords: |
aflatoxin, Aspergillus flavus, maize, multiplex
polymerase-chain reaction, multiplex reverse
transcriptase-polymerase chain reaction, wild
strains. |
| Author Affiliation: |
Department of Genetics, Biology of Microorganisms, Anthropology, Evolution, University of Parma, Parma, Italy |
| Subjects: |
Plant Protection > Pesticides |
| Divisions: |
General |
| Depositing User: |
Mr Siva Shankar
|
| Date Deposited: |
04 Oct 2012 10:30 |
| Last Modified: |
04 Oct 2012 10:30 |
| Official URL: |
http://dx.doi.org/10.1111/j.1365-2672.2006.03256.x |
| URI: |
http://eprints.icrisat.ac.in/id/eprint/8250 |
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