PCR-RFLP on β tubulin and calmodulin gene as a tool for rapid identification of the most important species of Aspergillus
2018
Ćurčić, Nataša | Krulj, Jelena (https://orcid.org/0000-0001-7348-3961) | Bočarov-Stančić, Aleksandra | Perović, Jelena (https://orcid.org/0000-0003-3550-755X) | Marić, Boško (https://orcid.org/0000-0003-4264-8086) | Kojić, Jovana (https://orcid.org/0000-0002-8816-9892) | Bodroža-Solarov, Marija (https://orcid.org/0000-0001-8151-6276)
In recent decade numerous molecular techniques have focused on the identification of different fungi species. Due of its reproducibility, speed, high sensitivity and specificity, PCR based test have been used to identify the most important Aspergillus species. Some of the Aspergillus species are capable to producing a wide range of toxin among which aflatoxin are the most important in food safety. The aim of this study was to validate one PCR-RFLP based test to discriminate Aspergillus at the species level. Genomic DNAs from all tested Aspergillus strains were extracted and PCR was performed to amplify parts of β tubulin and calmodulin gene. Using the calmodulin primer pair (cmd5/cmd6), a 475-595 base pair fragment was successfully amplified. Amplification of a part of the β tubulin gene was performed by using the primer pairs (Bt2a/Bt2b) and generated PCR product ranging in size from 415 to 580 bp. This PCR product were digested with restriction enzyme AlwI (BspPI) who had one or two restriction sites in tested Aspergillus species (Aspergillus flavus, A. ochraceus, A. nidulans, A. versicolor, A. candidas and A. tamari). Restriction enzyme AlwI had no restriction site for A. nidulans. The RFLP pattern of AlwI for tested Aspergillus was species-specific and none of species generated products with similar sizes. We can conclude that RFLP test on β tubulin gene provided rapid identification of the most important species of Aspergillus.
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