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A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment

  • Applied Genetics and Molecular Biotechnology
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Abstract

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 μg–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

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Acknowledgements

The work described in this paper was partially supported by CINVESTAV-IPN, Accesolab México, BioRad México, and Kirkegaard & Perry Laboratories, USA. We thank Maria Guadalupe Aguilar-Gonzalez from the DNA Sequencing Unit (Departamento de Genética y Biología Molecular) and Carolina Miranda-Brito (Departamento de Fisiología, Biofísica y Neurociencias) for technical assistance, Daniel Castro-Roa for critical review of the manuscript, and Rosa Maria Cruces for clerical assistance. We wish to thank Dr. Ian Reid, formerly with PAPRICAN, Canada, for the kind donation of the strain of Trametes versicolor to one of the authors (H.M.P.-V.).

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Authors and Affiliations

  1. Departamento de Genética y Biologia Molecular, CINEVISTAV-IPN, P.O. Box 14-740, México DF, 07000, Mexico

    Jaime García-Mena & Claudia Cano-Ramirez

  2. Departamento de Biotechnología y Bioingeniería, CINEVISTAV-IPN, P.O. Box 14-740, México DF, 07000, Mexico

    Claudio Garibay-Orijel, Sergio Ramirez-Canseco & Héctor M. Poggi-Varaldo

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  1. Jaime García-Mena
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  2. Claudia Cano-Ramirez
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  3. Claudio Garibay-Orijel
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  4. Sergio Ramirez-Canseco
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  5. Héctor M. Poggi-Varaldo
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Correspondence to Jaime García-Mena.

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García-Mena, J., Cano-Ramirez, C., Garibay-Orijel, C. et al. A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment. Appl Microbiol Biotechnol 67, 524–531 (2005). https://doi.org/10.1007/s00253-004-1795-z

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  • DOI: https://doi.org/10.1007/s00253-004-1795-z

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