Desenvolvimento de um método de PCR específico para detecção de Aspergillus Flavus aflatoxigênico em grãos brasileiros

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

In general, foods are extremely susceptible to contamination by mycotoxin-producing fungi that affect human and animal health, with major losses common in agricultural production in Brazil. Aflatoxins are considered the most important group of mycotoxins, because of their high toxicity, hepatic-cancer-inducing properties, and common occurrence in various grain foods. Brazilian grains such as peanut (Arachis hypogaea), Brazil nut (Bertholletia excelsa) and cashew nut (Anacardium occidentale L.) are extensively affected by contamination with aflatoxigenic fungi such as Aspergillus flavus. Contamination by A. flavus affects exportation, leading to large economic losses in countries such as Brazil. Detection of toxins and microbial contamination involves control systems such as HACCP (Hazard Analysis and Critical Control Points) which is a preventive system that aims to produce safe food by using diagnostic tools, whether morphological, molecular or chromatographic. In order to develop a robust molecular diagnosis system for incorporation in control systems such as HACCP (Hazard Analysis and Critical Control Points), the present work was conducted. Thin layer chromatography (TLC) was used to characterize 63 isolates of A. flavus, isolated from Brazil nut and cashew nut in seven Brazilian localities. Whilst most isolates were aflatoxigenic, aflatoxins were absent in 14 isolates. Aflatoxin B1 was reported for the first time in isolates from A. occidentale. RAPD analysis of genetic diversity, based upon coded data generated with 32 RAPD primers, revealed considerable genetic diversity between isolates. No correlation was observed with flatoxigenicy, such that primers tested were concluded as inappropriate for application in RAPD SCAR-derived methods for diagnosis for aflatoxin-producing strains. Nuclear ribosomal DNA (rDNA) intergenic spacer regions ITS 1 and 2 (Internal Transcribed Spacer) and a region of the small subunit (SSU) mitochondrial DNA rRNA gene cluster (mtDNA SSU rDNA) were used as candidates for specific detection of A. flavus in a robust multiple PCR (Polymerase Chain Reaction) system, because of conservation and abundance in target DNA. In addition to the design of specific primers and confirmation of specificity for the species A. flavus and genus Aspergillus, this paper presents two internal amplification control systems (IACs) for verification of the reliability of the PCR detection systems. Both detection methods showed detectability limits for purified DNA from A. flavus in the region of picogram quantities. Analysis of genetic diversity between the three species of A. flavus, A. parasiticus and A. oryzae, based upon comparison of GenBank sequences for the biosynthetic pathway genes aflR, aflP and aflQ, revealed that A. flavus shows greater similarity to A. oryzae than A. parasiticus, with greater divergence between the genes aflR and aflP. Thirteen primers were designed for complete coverage for the three genes, and PCR products were sequenced in 63 aflatoxigenic and non-aflatoxigenic isolates of A. flavus. Partial analysis of these genes in representative aflatoxin-producing and non-producing isolates showed six haplotypes of 11 SNPs (Single Nucleotide Polymorphism), where one among them is the most common.

ASSUNTO(S)

aspergillus flavus aspergillus flavus rdna its rdna its pcr ciencias pcr micotoxinas diversidade mycotoxins diversity aflatoxina mtdna ssu rdna aflatoxin mtdna ssu rdna rapd grãos - contaminação - brasil rapd

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