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New genus-specific primers for the PCR identification of members of the genera Pseudonocardia and Saccharopolyspora

Centro de Investigación Básica de España, Merck Sharp and Dohme Research Laboratories, Josefa Valcárcel 38, 28027 Madrid, Spain

Author for correspondence: OlgaGenilloud. Tel: +3491 3210 568. Fax: +34 91 3210 614. e-mail: olga_genilloud{at}merck.com

ABSTRACT

Members of the family Pseudonocardiaceae are difficult to identify on the basis of their micromorphology only. The biochemical characterization of each new isolate is a painstaking and time-consuming task which cannot always be undertaken when handling large numbers of strains as is the case in natural product screening programmes. In this study, two sets of genus-specific oligonucleotides were designed which allow rapid detection of members of the genera Pseudonocardia and Saccharopolyspora by means of PCR-specific amplification. The genus specificity of these primers was validated on a wide range of collection strains and the primers were subsequently used to study group of 106 wild-type isolates that possessed morphological characteristcs of the family. Out of this group, 51 strains could be identified as members of the genus Pseudonocardia and only nine isolates could be assigned to the genus Saccharopolyspora. The diversity indicated by whole-cell fatty acid profiles of both wild-type and reference strains was compared with that identified using the oligonucleotide primers. The partial 16S rDNA sequencig of representative wild-type strains was used to validate their genus assignment by PCR-specific amplification. This study shows the industrial usefulness of the application of these direct identification tools as well as the complementary use of two sources of data, PCR-specific amplification results and fatty acid composition, to assess the diversity of a microbial population.

Key Words: Pseudonocardiaceae • PCR primers • 16S rDNA sequences

The GenBank accession numbers for the sequences reported in this paper are AF061976, AF061977 and AF064968-AF064983.

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