Estudo de variabilidade genÃtica de aspergillus flavus como base para o desenvolvimento de PCR multiplex para detecÃÃo de fungos produtores de aflatoxinas em castanha-do-brasil e bastanha de caju

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Aflatoxins represent a group of similar heterocyclic and highly oxygenated substances, which are toxic to man and animals. Products of secondary metabolism in the filamentous fungi Aspergillus flavus, A. parasiticus, A. pseudotamarii and A. nomius, the species A. flavus and A. parasiticus are of greatest economic importance. The most important aflatoxins are grouped as B1, B2, G1 and G2, with letters refering to emitted colour (blue or green) under UV light (365nm), and numbers referring to degree of toxicity. As aflatoxins are carcinogenic, imunosupressive, teratogenic and genotoxic, they are cause for concern worldwide, especially in tropical countries, as well as temperate regions with warm, humid summers. Brazil nut (Bertholletia excelsa H. B. K.) is the first extractivist product of importance for communities living in the Amazon region, and is a product principally destined for the export market, with, for example, 20000 tons of the 28467 tons produced in 2001 destined for Europe and the USA. Recently, however, exports have suffered serious restrictions, as a result of contamination of nuts with aflatoxins, with detected levels exceeding accepted levels in both markets. The official methods for detection of aflatoxins and other mycotoxins are based on chromatography, which can detect the toxin but not the mycotoxigenic fungus. Monitoring the productive chain is not currenly required, as such detection methods for toxins are relatively expensive. In order to resolve this problem, an additional method is required for detection of not only the aflatoxin, but also the aflatoxigenic fungus, applicable for monitoring the entire productive chain. Genetic variability was assessed in 48 isolates of potentially aflatoxigenic and non-aflatoxigenic isolates of A. flavus from B. excelsa and cashew nut (Anacardium occidentale L.) from different geographic regions in Brazil, with phenetic analysis of 140 DNA fragments generated with 11 RAPD primers separating isolates according to host of origin, and isolates from A. occidentale also according to geographic origin. A number of candidate PCR products for the design of primers specific to A. flavus were amplified (nuclear rDNA ITS regions, mtDNA SSU rRNA gene, and β-tubulin gene), and sequence analysis of the amplified rDNA ITS regions, together with Genbank-derived sequences for the rDNA ITS regions for other Aspergillus species and other fungal genera associated with B. excelsa and A. occidentale in Brazil, enabled the design of a potential specific primers pair ASPITSF and ASPITSR. Twenty six potential degenerate primers pairs targetting 10 genes within the aflatoxin biosynthetic pathway were also designed based upon conserved protein sequence motifs in A. flavus, A. parasiticus and A. nomius. Further research is planned for testing and adapting the designed primers in the form of a multiplex PCR system, for molecular detection of aflatoxigenic isolates of A. flavus in contaminated material.

ASSUNTO(S)

pcr multiplo rapd genetica caju metabolism secondary products aspergillus flavus brazil rdna aflatoxinas, castanha do brasil via biosintetica

ACESSO AO ARTIGO

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