title: Characterization of Aspergillus species on Brazil nut from the Brazilian Amazonian region and development of a PCR assay for identification at the genus level.
abstract: BACKGROUND
  Brazil nut is a protein-rich extractivist tree crop in the Amazon region. Fungal contamination of shells and kernel material frequently includes the presence of aflatoxigenic Aspergillus species from the section Flavi. Aflatoxins are polyketide secondary metabolites, which are hepatotoxic carcinogens in mammals. The objectives of this study were to identify Aspergillus species occurring on Brazil nut grown in different states in the Brazilian Amazon region and develop a specific PCR method for collective identification of member species of the genus Aspergillus.
  
  
  RESULTS
  Polyphasic identification of 137 Aspergillus strains isolated from Brazil nut shell material from cooperatives across the Brazilian Amazon states of Acre, Amapá and Amazonas revealed five species, with Aspergillus section Flavi species A. nomius and A. flavus the most abundant. PCR primers ASP_GEN_MTSSU_F1 and ASP_GEN_MTSSU_R1 were designed for the genus Aspergillus, targeting a portion of the mitochondrial small subunit ribosomal RNA gene. Primer specificity was validated through both electronic PCR against target gene sequences at Genbank and in PCR reactions against DNA from Aspergillus species and other fungal genera common on Brazil nut. Collective differentiation of the observed section Flavi species A. flavus, A. nomius and A. tamarii from other Aspergillus species was possible on the basis of RFLP polymorphism.
  
  
  CONCLUSIONS
  Given the abundance of Aspergillus section Flavi species A. nomius and A. flavus observed on Brazil nut, and associated risk of mycotoxin accumulation, simple identification methods for such mycotoxigenic species are of importance for Hazard Analysis Critical Control Point system implementation. The assay for the genus Aspergillus represents progress towards specific PCR identification and detection of mycotoxigenic species.
authors.forename: Glaucia E O
authors.initials: GE
authors.lastname: Midorikawa
authors.forename: Maria de Lourdes M
authors.initials: Mde L
authors.lastname: de Sousa
authors.forename: Otniel
authors.initials: O
authors.lastname: Freitas Silva
authors.forename: Jurema do Socorro A
authors.initials: Jdo S
authors.lastname: Dias
authors.forename: Luis I B
authors.initials: LI
authors.lastname: Kanzaki
authors.forename: Rogerio E
authors.initials: RE
authors.lastname: Hanada
authors.forename: Renata M L C
authors.initials: RM
authors.lastname: Mesquita
authors.forename: Rivadalve C
authors.initials: RC
authors.lastname: Gonçalves
authors.forename: Virginia S
authors.initials: VS
authors.lastname: Alvares
authors.forename: Daniela M C
authors.initials: DM
authors.lastname: Bittencourt
authors.affiliation: Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, CEP 70,910-900 Brasília D,F,, Brazil. robertmiller@unb.br.
authors.forename: Robert N G
authors.initials: RN
authors.lastname: Miller
mesh_terms: D001230:Aspergillus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001938:Brazil; D016000:Cluster Analysis; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009754:Nuts / Q000382:microbiology*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S