K-082. A Universal Method for the Identification of Bacteria Based on General PCR Primers

S. A. Barghouthi;
Al-Quds Univ., Jerusalem, West Bank, PALESTINIAN TERRITORY, OCCUPIED.

There is a growing need for swift and accurate identification of organisms, including bacteria. Genotyping methods appear to have superiority over those based on phenotypic features. A universal method utilizing general PCR primers targeting the 16S rDNA for the prompt and accurate identification of bacteria is described. The procedure requirements were: pure bacterial cultures or DNA, a set of general PCR primers targeting the 16S bacterial rDNA, and access to DNA sequencing and computer assisted DNA sequence alignment (BLAST). The principle of the method is simple; if a pure PCR product can be obtained from an organism (e.g a bacterium), then it can be sequenced and compared to available DNA databases. Confirmation of bacterial identity may follow. In this work, eleven general primers targeting the 16S bacterial rDNA were designed and tested against a number of bacterial isolates (42 isolates). The obtained PCR product was subjected to DNA sequencing. Sequences were obtained for 18 isolates, analyzed (BLAST), and isolates were identified. Although comprehensive analysis of bacterial species is beyond the scope of this study, the obtained results were consistent with the identities of tested isolates. Two approaches were considered in testing the validity of this universal method. First approach depended on the use of known confirmed isolates that were re-identified using the universal method (this work). Second approach was to identify unknown isolates using the universal method, followed by confirmation of isolate identities using other methods (e.g. API, or published specific PCR primers).The resulting nucleotide sequences can be reanalyzed (BLAST) periodically against gene banks as new data accumulate and taxonomies change. This method may allow the identification of species, novel species, variations within species, subspecies, and correction of miss-identities. The method is applicable to clinical bacterial isolates and other bacterial categories such as soil and food bacteria. The approach itself can be applied to other taxa such as protists, nematodes, insects, and others.