16S rDNA-Based Identification of Bacteria from Conjunctival Swabs by PCR and DGGE Fingerprinting

PURPOSE. Establishment of a new molecular biology technique for the identification of multiple bacteria from the ocular environment, which can be applied supplementarily to cultivation in cases of severe bacterial infections. METHODS. From 60 human conjunctivae (29 with purulent and 31 with nonpurulent conjunctivitis), swabs were taken and DNA was extracted. Fragments of 200 bp, spanning the V3 region of the eubacterial 16S rDNA, were amplified by polymerase chain reaction (PCR) and separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were excised and directly sequenced, or 16S rDNA clone libraries were constructed and clones were screened by DGGE. Sequences were compared with sequences of known bacteria listed in the EMBL database. Furthermore, the results were compared with results obtained from conven