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PCR assay for identification of A. carbonarius and A. japonicus

The visual inspection of the aligned calmodulin partial gene sequences readily identified unique regions within the amplified fragment, (e.g. in the range of 350-420 base pairs) that has been exploited in the development of generic PCR-based assay for the detection of A. carbonarius and A. japonicus/aculeatus.

Using the genetic sequence variation founded in this region, all primers were designed to operate at high annealing temperatures (58°C), thereby preventing the co-amplification of non specific target of DNA. In calmodulin sequences, we observed 99,98 % identity for strains of A. carbonarius, and 99,40 % identity for strains of A. japonicus and A. aculeatus analysed. Specific primer pairs amplified in A. carbonarius a fragment of 371 bp length and in A. japonicus a fragment of 583 bp length.

To determine the diagnostic value of the primer set in a large scale, experiments were started to test its usability on a large number of isolates: 30 strains each of the target species, 42 strains of other closely related species within the black aspergilli and others main fungal/yeast species occurring on grapes as Botrytis cinerea, Saccharomyces cerevisae and other toxigenic fungi such as some Fusarium species. Aspergillus japonicus strains were not amplified by A. carbonarius-specific PCR primers (CARBO1/2), and A. carbonarius strains were not amplified by A. japonicus -specific PCR primers (JAPO1/2).

None of the others Aspergillus section Nigri were amplified by the two sets of primers with the exception of the four strains of A. aculeatus which were amplified by the specific primers JAPO1/2. No product was amplified from any fungal/yeast tested. The primers were also tested against others main fungal/yeast species occurring on grapes as Botrytis cinerea, Saccharomyces cerevisae and other toxigenic fungi such as some Fusarium species, without any amplifications

Contacto

Giuseppina MULÈ, (Researcher)
Tel.: +39-080-5929329
Fax: +39-080-5929374
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