Abstract
Abstract: :
Purpose: To improve identification and speciation of bacteria involved in ocular infection using polymerase chain reaction (PCR) and denatured gradient gel electrophoresis (DGGE). Methods: DNA was extracted from 9 species of bacteria known to cause bacterial keratitis, including Bacillus, E. coli, H. influenza, Nocardia, Proteus mirabilis, S. aureus, S. epidermidis, S. pneumoniae, and S. viridans. Identification was confirmed by PCR with the use of universal primers spanning the conserved nucleotide region of eubacterial 16S rDNA. The PCR products were used to construct 16S rDNA clone libraries for sequencing. Sequencing results were entered into the NCBI database and each one corresponded with its correct organism. After confirming the identification of these nine species of bacteria, the bacteria were reamplified with primers primarily designed for its use in denaturing gradient gel electrophoresis (DGGE). These primers were constructed based on the conserved region present in eubacteria, with one of them attached to a GC clamp. DGGE/PCR products were run through an 8% acrylamide gel, with a gradient of 20–55% made up of formamide and urea, for 5 hours at 150V. The separated bands were viewed with ethidium bromide staining and UV transillumination. Results: The generated DGGE/PCR products were all approximately the same size at 200 bp. After running these products in the DGGE gel, these similarly–sized bands were distinctly located in the gel depending on its DNA sequence. All bands could be confidently distinguished from one another except between the pairs of S. aureus/S. epidermidis and S. viridans/ S. pneumoniae. Conclusions: The DGGE analysis has the potential to be a useful diagnostic tool due to its rapidity compared to the current regimen of cloning, isolating plasmids, restriction digesting, and sequencing PCR products. Although DGGE should be able to distinguish between specific species of bacteria with a single nucleotide difference, it could not discriminate some organisms at the species level. DGGE is a rapid method in identifying most bacteria at the genus level, which may prove to be valuable in ophthalmic diagnoses.
Keywords: microbial pathogenesis: clinical studies • bacterial disease • cornea: clinical science