Molecular Tools for the Identification of Tuber melanosporum in Agroindustry

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• 作者：Nathalie Sé ; jalon-Delmas ; Christophe Roux ; Monique Martins ; Michel Kulifaj ; Guillaume Bé ; card ; and Robert Dargent
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2000
• 出版时间：June 2000
• 年：2000
• 卷：48
• 期：6
• 页码：2608 - 2613
• 全文大小：121K
• 年卷期：v.48,no.6(June 2000)
• ISSN：1520-5118

文摘

Tuber melanosporum Vitt., Tuber magnatum Pico, and Tuber uncinatum Chat. can be differentiatedby their morphological characters. Fraud problems have arisen recently with the importation toEurope of truffles from China. T. melanosporum is morphologically very close, but distinct from theChinese species [Tuber indicum (Cooke and Massee) and T. himalayense BC (Zhang and Winter)].We have optimized molecular tools to unequivocally identify T. melanosporum. DNA extraction fromascocarps of black truffles is not straightforward. Problems to obtain pure DNA are due to highcontents of phenolic compounds, melanine, and various polymers (proteins, polysaccharides, etc).These compounds coprecipitate with the DNA during extraction and strongly inhibit the PCRreaction. We have developed an efficient and reliable protocol for DNA extraction from truffleascocarps. It was used successfully for DNA extraction from mycorrhizal root tips as well as fromcanned preparations of T. melanosporum. Several approaches to identify T. melanosporum by PCRwere developed. Two specific primers for T. melanosporum were designed after comparison of theITS region of this species with those of three Chinese fungi. They proved to be efficient to specificallydetect the presence of T. melanosporum by PCR. The mycorrhizal status of trees inoculated with T.melanosporum but unable to produce truffles was confirmed in a single-step PCR reaction. Amultiplex PCR approach was also developed with three sets of primers (including a specific one forChinese truffles) to detect, in one PCR reaction, the presence of any other Tuber species mixedwith T. melanosporum ascocarps. This optimized protocol, in association with the specific primerswe designed, is applicable to quality control in the truffle industry from the production stages tofinal commercial products.Keywords: Alcalase; Black truffles; Chinese truffles; BLOTTO (Bovine Lacto Transfer TechniqueOptimizer); DNA extraction; ITS (internal transcribed spacer) sequence; PCR specific detection