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

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Abstract

A real-time PCR system directed against the nor-1 gene of the aflatoxin biosynthetic pathway as a target sequence has been applied to detect an aflatoxinogenic A. flavus strain in plant-type foods like maize, pepper and paprika. The system is based on the TaqMan® fluorescent probe technology. The copy numbers of the nor-1 gene were compared to conventional cfu data obtained from the same set of samples. In general, a good correlation between nor-1 gene copy number and the cfu data was observed; however, the nor-1 copy numbers were always higher. It was shown that the system is specific for nor-1 containing species.

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.

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