Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods
Introduction
Aflatoxins are potent carcinogenic and mutagenic metabolites produced primarily by the fungal species Aspergillus flavus and Aspergillus parasiticus. These species can contaminate several food commodities including cereals (Pittet, 1988), peanuts (Jelinek et al., 1989) and spices (Bartine and Tantaoui-Elaraki, 1997). Conventional methods to detect, quantify and identify these fungi include cultivation and taxonomic identification at the morphological level. This approach, however, is very time consuming and requires taxonomic skills. Alternative rapid methods like the detection of ergosterol, immunological or impedimetric methods have been described De Ruiter et al., 1993, Gourama and Bullermann, 1995, Seitz et al., 1977, but they have the drawback of being unspecific. In general, these methods can hardly differentiate between species. The characterization of the aflatoxin biosynthetic genes (Woloshuk and Prieto, 1998), however, made the application of diagnostic PCR methods for the detection of aflatoxinogenic fungi possible Geisen, 1996, Shapira et al., 1996. The described PCR systems, however, only deliver qualitative results indicating the presence or absence of an aflatoxinogenic fungus. For the estimation of food quality or for the monitoring of the influence of hygienic measures upon the amount of fungi present in a food sample, a reliable rapid quantification system is important.
Recently, a technique based on the real-time PCR approach has been developed to detect and quantify fungal conidia in indoor environments (Haugland et al., 1999) or for spores of the pathogenic fungi Glomus mosseae and Phytophthora infestans (Böhm et al., 1999). Schnerr et al. (2001) described a system directed against the tri5 gene to detect and quantify trichothecene producing Fusaria.
The detection principle is based on a PCR reaction with two specific primers which define the target sequence and an additional internal probe which hybridizes between the primers. This additional hybridization step increases the specificity of the reaction and is required for quantification. The internal probe is 5′-labeled with a fluorogenic dye (FAM) and at the 3′-end is ligated to a quencher (TAM). The quencher reduces the fluorescence of the dye as long as it is in close proximity. During the PCR reaction the hybridized probe is degraded by the 5′3′-exonuclease activity of the Taq polymerase and the fluorescent dye is released. This process increases the fluorescence, which can quantitatively be determined.
The aim of this work was the establishment of a reliable real-time PCR system for quantification of the nor-1 gene and the determination of its correlation to conventional cfu data in various foods.
Section snippets
Strains and culture collection
Strains used in this work were taken from the culture collection of the Federal Research Centre for Nutrition (Karlsruhe, Germany). The strains were routinely grown in malt extract broth (1.05397, Merck, Damstadt, Germany) at 25 °C under shaking conditions (180 rpm) or on malt extract agar plates (1.05398, Merck, Darmstadt, Germany) at the same temperature.
Determination of aflatoxin production
For the determination of aflatoxin production the strains were grown at 30 °C on malt extract agar plates or in broth for 3 to 5 days. One
Primer/probe system for the nor-1 real-time PCR
A conventional non-quantitative PCR reaction with the nor-1 gene as the target sequence has been described (Geisen, 1996). The nor-1 gene codes for the norsolorinic acid reductase, one of the first genes in the aflatoxin biosynthetic pathway (Woloshuk and Prieto, 1998). In the conventional system, the primers nor1 and nor2 have been used and a fragment of 400 bp was produced. For the real-time PCR system, however, the primers have to fulfill specific requirements. First of all, the amplicon
Discussion
The quantification of a fungal contamination is a challenging task because of the nature of the fungal colony, which consists of filamentous mycelial cells and single celled spores. Several methods for the detection or quantification of a fungal contamination have been established, e.g. the determination of the colony forming units (Spangenberg and Ingham, 2000), the measurement of ergosterol content (Seitz et al., 1977), the determination of volatiles (Schnürer et al., 1999) or immunological
Acknowledgements
Zsuzsanna Mayer was supported by a DAAD fellowship. Angelo Bagnara was a recipient of a post-degree scholarship from the University of Messina, Italy. This work was supported by the INCO-Copernikus project ERB IC15-CT98-0901.
References (26)
- et al.
New methods in food mycology
Trends Food Sci. Technol.
(1993) - et al.
Detection of aflatoxinogenic fungi in figs by a PCR reaction
Int. J. Food Microbiol.
(1997) A multiplex PCR reaction for the detection of aflatoxin and sterigmatocystin producing fungi
Syst. Appl. Microbiol.
(1996)- et al.
Detection of molds in foods and feeds: potential rapid and selective methods
J. Food Protect.
(1995) - et al.
Quantitative measurement of Stachybotrys chartrarum conidia using real time detection of PCR products with the TaqMan™ fluorogenic probe system
Mol. Cell. Probes
(1999) - et al.
Aspergillus pseudotamarii, a new aflatoxin producing species in Aspergillus section Flavi
Mycol. Res.
(2001) - et al.
Inhibition of PCR by components of food samples microbial diagnostic assays and DNA-extraction solutions
Int. J. Food Microbiol.
(1992) - et al.
Real Time detection of the tri5 gene in Fusarium species by LightCycler™-PCR using SYBR Green I for continuous fluorescence monitoring
Int. J. Food Microbiol.
(2001) - et al.
Fungal volatiles as indicators of food and feed spoilage
Fungal Genet. Biol.
(1999) - et al.
Comparison of methods for enumeration of yeasts and molds in shredded low-moisture, part-skim Mozzarella cheese
J. Food Protect.
(2000)
The use of reverse transcription polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus 439
Int. J. Food Microbiol.
Genetic organization and function of the aflatoxin B1 biosynthetic genes
FEMS Microbiol. Lett.
On the safety of Aspergillus oryzae: a review
Appl. Microbiol. Biotechnol.
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