Conclusions

Fungi responsible for food spoilage cause significant economic losses for food manufacturers. In order to minimize food spoilage and be able to predict the quality and shelf-life of a particular food, a better understanding of the mechanisms underlying food spoilage is essential. To achieve this goal, there is a need for the development of direct detection methods for spoilage organisms including fungi. The main aim being to develop a rapid, sensitive, robust, and specific method to use directly on the sample for the detection of food spoilage organisms. Rapid identification of spoilage organisms is of profound importance to the food industry. This will enable intervention with appropriate measures to prevent serious economic losses. Rapid molecular techniques are valuable tools for screening foods and feeds for fungi. Among these, hybridization techniques have only rarely been used for direct detection of fungi, although novel developments in this field, including the application of PNA probes, and microarray techniques are encouraging. Amplification-based methods are frequently applied for direct detection of spoilage organisms. Most PCR-based detection methods target part of the ribosomal RNA gene cluster, although SCAR markers are also widely used. For the detection of mycotoxin producing fungi, the most direct procedure is targeting of the mycotoxin biosynthetic genes. Such approaches have been used recently for the detection of a number of fungi-producing mycotoxins such as ATs, patulin, PR toxin, and trichothecenes, and projects for the detection of fungi producing ochratoxins and fumonisins are in progress. The recent development of real-time PCR methodology has made it possible to quantify the amount of organisms in foods and feeds.

Sensitivity of a molecular method depends not only on the detection system, but also to a great extent on the food matrix. In order to increase sensitivity, adequate protocols have to be established in order to discern potential PCR inhibitors. The speed, accuracy, and reliability of detection methods can greatly be enhanced by enrichment procedures including the use of magnetic particles coated with specific binding proteins (antibodies or lectins); by optimizing the DNA extraction protocol; or by using internal standard DNA in the PCR reaction. One of the main drawbacks of PCR-based detection methods is that DNA from dead cells can also be amplified leading to false positive results. This limitation can be addressed by including a propagation step prior to PCR, by using mRNA as template in NASBA or RT-PCR reactions. On the other hand, this disadvantage is only relative, since detection of dead mycotoxin producing fungi in a food matrix can serve as a prediction for mycotoxin contamination of the food.

The detection of microorganisms in food is still a time-consuming method particularly in the case of large-scale testing. The increasing availability of molecular kits for the detection of food-borne pathogenic bacteria is a step toward standardization of the molecular techniques. The development of similar kits for fungi causing food spoilage would be a further necessary step for the authorization of molecular methods as accepted detection methods in food microbiology.

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