PCR-RFLP using a SNP on the mitochondrial Lsu-rDNA as an easy method to differentiate Tuber melanosporum (Perigord truffle) and other truffle species in cans.

Canned truffle products labeled Tuber melanosporum, the famous Perigord truffle, may contain other less tasty and cheaper truffle species. To protect consumers from fraud, a PCR DNA-based method was used to unequivocally identify the nature of the product. Several rapid and simple cell lysis procedures, used in conjunction with a commercially available DNA purification kit, were evaluated for their effectiveness in recovering DNA from canned truffle. In parallel, a marker for T. melanosporum was tested on the mitochondrial rDNA. These two techniques were then combined to differentiate T. melanosporum from other truffle species like T. aestivum, T. brumale or T. indicum up to the legal threshold in canned products. These findings not only allow a comparison of the effectiveness of the different DNA extraction methods but also provide a preliminary indication of the specificity and sensitivity of the detection with the mitochondrial marker that might be attainable for truffle species in a quantitative PCR-based analysis method.

Rapid molecular typing of Tuber melanosporum, T. brumale and T. indicum from tree seedlings and canned truffles.

We have developed new DNA extraction and purification procedures for investigation of mycorrhized seedlings and canned truffles. Use of these procedures on approximately 100 mg initial material enabled good sample representation. For mycorrhized seedlings, Taq polymerase inhibitors were discarded irrespective of tree species. In routine analysis we systematically used consensus primers ITS1/ITS4 to check the absence of Taq polymerase inhibitors and the presence of fungus DNA. Positive response with ITS validates other positive or negative PCR results. Absence of amplification with ITS prevents validation of other results. For canned truffles, DNA harvested from ascocarps sterilized for one and a half hours at 115 degrees C was amplified with specific primers. We have developed consensus primers, named R12/F12, to check for the presence of amplifiable fungus DNA and the absence of Taq polymerase inhibitors. Here also, positive response with consensus R12/F12 validates other positive or negative PCR results. We have developed one primer pair specific for T. brumale and another specific for T. melanosporum. We can then characterize these two taxa, which enables the use of "truffle or truffled" French designations. We can also characterize T. indicum, the Asiatic black truffle that might fraudulently be sold as T. melanosporum and T. brumale. These three specific primer pairs were used independently of DNA extraction from tree seedlings or canned truffles. Our process is specific, sensitive, convenient, and quick.