Open AccessDevelopment of a molecular detection method for naphthalene degrading pseudomonads
Author(s) -
Silva Margarida C.,
Moré Margret I.,
Batt Carl A.
Publication year - 1995
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.1995.tb00179.x
Subject(s) - biology , pseudomonas putida , ethidium bromide , polymerase chain reaction , gel electrophoresis , microbiology and biotechnology , bacteria , dot blot , pseudomonas , alkaline lysis , detection limit , nucleic acid , plasmid , sodium dodecyl sulfate , southern blot , dna , chromatography , gene , biochemistry , chemistry , genetics , dna vaccination
A combined polymerase chain reaction amplification and reverse dot blot assay was designed for the detection of bacterial genes from soil and sediments. Total nucleic acids were directly extracted from soil using a lysozyme/sodium dodecyl sulfate/freeze‐thaw method followed by rapid purification through gel electrophoresis. DNA was amplified using a highly stringent polymerase chain reaction with primers directed against the nahR regulatory gene present in plasmid NAH7 of Pseudomonas putida G7. The resulting amplification product was detected colorimetrically by reverse dot blot with an improved sensitivity ten‐fold greater than traditional ethidium bromide staining after gel electrophersis. A lower limit of 10 3 , P. putida G7 cfu (g soil) −1 was detected. This method was successfully employed to detect indigenous naphthalene‐degrading bacteria from subsurface sediment collected from different locations of a naphthalene‐contaminated site. Similar approaches could be developed for other soil‐borne genetic markers.