Identification and environmental detection of Rhodococcus species by 16S rDNA-targeted PCR

Abstract

. Bacteria of the genus Rhodococcus can degrade a wide range of organic pollutants and catalyse many useful biotransformations. There is a need for improved tests to identify Rhodococcus species. PCR-based methods for species identification offer advantages in terms of speed and accuracy over traditional methods and can allow direct detection of microbes in environmental samples., PCR tests, using primers targeted at species-specific sequences in the 16S rRNA gene, were successfully developed for R. globerulus, R. erythropolis, R. opacus and R. ruber. These tests gave positive results with all or most strains of target species but did not generally cross-react with other species. Cases of apparent cross-reaction were shown to be due to prior misclassification of strains of R. opacus as R. erythropolis and of strains of R. ruber as R. rhodochrous. A simple and rapid method for the extraction and purification of DNA from soil was developed and successfully applied to the PCR detection of indigenous R. erythropolis in an environmental sample. Cell lysis in the samples was achieved by lysozyme and sarkosyl treatment, aided by freeze-thaw cycles. Removal of humic compounds inhibitory to PCR was accomplished by CTAB treatment with solvent extraction and, if necessary, passage of extracts through Sepharose CL-6B in a spun-column format. Extracts prepared using a tris-EDTA buffer were much clearer than those prepared using a sodium phosphate buffer, indicating lower levels of humic compounds. A detection limit of 10 4 cfu g −1 of soil was achieved and the use of a secondary PCR allowed detection of 1 cfu g −1 .

Culture-Based and Non-Growth-Dependent Detection of the Burkholderia cepacia Complex in Soil Environments

“…We thus directly extracted DNA from soils and evaluated the extracts for the presence of Bcc DNA by two independently developed sets of 16S rDNA-directed PCR assays. PCR assays targeting 16S rDNA genes have been used in other studies of soil bacterial populations (3,12,37). Advantages to using these sequences as a PCR target include the high copy number of rrn genes (six copies distributed on the three or four replicons typical of most strains of Bcc) (25).…”

Activity Assay of Bacterial Mixed Culture in Biological Treatment of Organochloropesticides

“…[3] Bacteria of the genus Rhodococcus can degrade a wide range of organic pollutants and catalyse many useful biotransformations. [4] Chemical pollutants which can be degraded by Rhodococci range from simple hydrocarbons, which are used as sole carbon sources in the isolation of Rhodococci [8] , through chlorinated hydrocarbons, aromatic hydrocarbons and nitroaromatics to chlorinated polycyclic aromatics such as polychlorinated biphenyls (PCBs) The two isolates, Streptomyces sp. Y7 and Rhodococcus isolated from organochlorine pesticides (OPs) contaminated soil was selected for their capacity to grow in presence of Propachlor as only carbon source; after growing at 30°C, the isolates recorded 54% and 60% respectively consumption of pesticide after 72h.…”

Detección y caracterización por métodos fenotípicos y moleculares de mycolata formadores de espumas en estaciones depuradoras de aguas residuales domésticas con sistemas de fangos activos.

“…El Un ciclo de extensión final (74ºC -10 min.) finaliza con la reacción de amplificación (Bell et al, 1996 erythropolis (Bell et al,1999) que ha sido aislada en muestras de espuma. R.…”

“…En este caso el producto amplificado es un Los ciclos de la reacción de amplificacion son los mismos que para el caso de R. equi (Bell et al, 1999). Los productos generados en las reacciones de amplificación de analizaron como en los casos anteriormente descritos.…”

“…Los inciadores Re1 y Re2 propuestos por Bell et al (1999) son específicos para R. erythropolis. Se ha demostrado que esta especie es responsable de la formación de espumas en sistemas de fangos activos (Soddell et al, 1998).…”