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PCR detection of aflatoxin producing fungi and its limitations
1Department of Food Science, Massachusetts Agricultural Experiment Station, University of Massachusetts, Amherst, MA 01003, USA. relevin@foodsci.umass.edu
Abstract:
Unlike bacterial toxins that are primarily peptides and are therefore encoded by a single gene, fungal toxins such as the aflatoxins are multi-ring structures and therefore require a sequence of structural genes for their biological synthesis. There is therefore no specific PCR for any one of the four biologically produced aflatoxins. Unfortunately, the structural genes presently in use for PCR detection of aflatoxin producing fungi are also involved in the synthesis of other fungal toxins such as sterigmatocystin by Aspergillus versicolor and Aspergillus nidulans and therefore lack absolute specificity for aflatoxin producing fungi (Table 1). In addition, the genomic presence of several structural genes involved in aflatoxin biosynthesis does not guarantee the production of aflatoxin by all isolates of Aspergillus flavus and Aspergillus parasiticus. The most widely used DNA target regions for discriminating Aspergillus species are those of the rDNA complex, mainly the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) and the variable regions in the 5'-end of the 28S rRNA gene. Since these sequence regions are unrelated to the structural genes involved in aflatoxin biosynthesis there successful amplification can be used for species identification but do not confirm aflatoxin production. This review therefore presents the various approaches and limitations in the use of the PCR in attempting to detect aflatoxin producing fungi.
Insights
Detecting aflatoxin-producing fungi using PCR is challenging due to gene specificity issues. Current methods for fungal identification do not confirm aflatoxin production, highlighting the need for improved PCR strategies.
Area of Science:
- Mycology
- Molecular Biology
- Food Safety
Background:
- Aflatoxins are fungal toxins with complex structures requiring multiple genes for synthesis, unlike simpler bacterial toxins.
- Current Polymerase Chain Reaction (PCR) methods for detecting aflatoxin-producing fungi lack absolute specificity.
- The genes targeted by PCR are often involved in synthesizing other fungal toxins, leading to false positives.
Purpose of the Study:
- To review the various approaches and limitations of using PCR for detecting aflatoxin-producing fungi.
- To highlight the challenges in achieving specific PCR detection of aflatoxin biosynthesis.
- To discuss the discrepancy between fungal species identification and actual aflatoxin production.
Main Methods:
- Review of existing literature on PCR-based detection of aflatoxin-producing fungi.
- Analysis of the genetic basis of aflatoxin biosynthesis and its comparison with other fungal toxin pathways.
- Evaluation of DNA target regions, including ribosomal DNA (rDNA) complex, internal transcribed spacer regions (ITS1 and ITS2), and 28S rRNA gene.
Main Results:
- No single PCR assay can specifically detect all four biologically produced aflatoxins due to their complex structures.
- Structural genes used in PCR for aflatoxin detection are also involved in sterigmatocystin synthesis, reducing specificity.
- Amplification of rDNA regions identifies fungal species but does not confirm aflatoxin production capability.
Conclusions:
- The genomic presence of aflatoxin biosynthesis genes does not guarantee aflatoxin production in all fungal isolates.
- Current PCR methods for fungal species identification are not sufficient for confirming aflatoxin production.
- There is a critical need for developing more specific and reliable PCR-based methods for detecting aflatoxin-producing fungi.
