TY - JOUR
T1 - A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment
AU - García-Mena, Jaime
AU - Cano-Ramirez, Claudia
AU - Garibay-Orijel, Claudio
AU - Ramirez-Canseco, Sergio
AU - Poggi-Varaldo, Héctor M.
N1 - Funding Information:
Acknowledgements The work described in this paper was partially supported by CINVESTAV-IPN, Accesolab México, BioRad Méx-ico, 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 Neurocien-cias) 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.).
PY - 2005/6
Y1 - 2005/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=21344448830&partnerID=8YFLogxK
U2 - 10.1007/s00253-004-1795-z
DO - 10.1007/s00253-004-1795-z
M3 - Artículo
C2 - 15586279
SN - 0175-7598
VL - 67
SP - 524
EP - 531
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 4
ER -