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Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay
Noelia Sardiñas1, Covadonga Vázquez, Jessica Gil-Serna
1Departamento de Microbiologia III, Facultad de Biologia, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Abstract:
Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies.
Insights
A new PCR method accurately detects Aspergillus parasiticus, a key aflatoxin producer, in food. This rapid assay identifies contamination early in the food chain, ensuring consumer safety.
Area of Science:
- Food Microbiology
- Molecular Biology
- Mycology
Background:
- Aspergillus parasiticus is a significant cause of aflatoxin contamination in food and beverages.
- Aflatoxins pose serious health risks to humans and animals.
- Accurate and rapid detection methods are crucial for food safety.
Purpose of the Study:
- To develop a specific and sensitive PCR protocol for detecting Aspergillus parasiticus.
- To validate the assay's specificity against related fungal species.
- To assess the assay's utility in routine food safety analyses.
Main Methods:
- Design of primers targeting the internal transcribed spacer region of the rDNA unit (ITS1-5.8S-ITS2 rDNA).
- Development and optimization of a conventional PCR assay.
- Testing specificity with various fungal species, including Aspergillus flavus.
- Validation using artificially contaminated wheat flour with known spore concentrations.
Main Results:
- The developed PCR assay demonstrated high specificity for Aspergillus parasiticus.
- The assay could detect spore concentrations as low as 10(6) spores/g without prior sample incubation.
- Effective discrimination from closely related species like Aspergillus flavus was achieved.
Conclusions:
- A specific and sensitive PCR protocol for Aspergillus parasiticus detection has been established.
- The assay is suitable for direct detection in food commodities.
- This method can be integrated into Hazard Analysis and Critical Control Points (HACCP) strategies for enhanced food safety.
