A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides |
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Authors: | Jonathan Scauflaire Marie Godet Mélanie Gourgue Charlotte Liénard Françoise Munaut |
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Institution: | 1. Université catholique de Louvain, Earth and Life Institute, Laboratory of Mycology, Croix du Sud 3 bte L7.05.06, B-1348 Louvain-la-Neuve, Belgium;2. Centre Hospitalier Universitaire Mont-Godinne, Laboratoire de Biologie Médicale, Secteur Chimie, Avenue Dr Thérasse 1, B-5530 Yvoir, Belgium;3. Université catholique de Louvain, Earth and Life Institute, Laboratory of Mycology, Mycothèque de l''Université catholique de Louvain (BCCM™/MUCL), Croix du Sud 3 bte L7.05.06, B-1348 Louvain-la-Neuve, Belgium |
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Abstract: | Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize. |
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Keywords: | Calmodulin Fusarium sp Hybridization probe LightCycler® instrument Maize Multiplex real-time PCR Translation elongation factor 1α |
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