baumannii, annotated complete genome sequences for 38 E. The combination of 16S rRNA gene detection with species-specific primers can greatly reduce the effort required in screening large populations of unknown organisms, with samples negative for 16S rRNA being excluded from further testing.Į. We also describe a novel bacterial 16S rRNA real-time PCR primer designed from the alignment of 962,279 bacterial 16S rRNA gene sequences, which will amplify a product from the 16S rRNA gene in 93.6% of all bacterial 16S rRNA genes published to date. This primer set will allow researchers to rapidly identify these pathogens directly from culture in less than 90 minutes with a high degree of accuracy and sensitivity. This report adds an additional four real-time PCR primers, designed using a combination of in silico and in vitro methodologies that are highly specific to the four most common nosocomial pathogens. We recently published a highly sensitive and specific real-time PCR assay for the detection of MRSA. A number of conventional PCR and real-time PCR assays have been developed targeting these organisms –, but a comprehensive panel that can target all five clinically important species is lacking. aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA) has been prolific, as befits their status as the most important nosocomial pathogens. pneumoniae –, research on CRE, multi-drug resistant A. Furthermore, using appropriate bioinformatic tools and careful primer design, many of the limitations outlined above can be mitigated considerably.Įven prior to recent global calls to help combat the spread of carbapenem-resistant Enterobacteriaceae (CRE), particularly E.coli and K. However, despite these limitations, molecular methods can provide significant advantages over phenotypic-based methods, including rapid turnaround time, scalability, and high sensitivity. Factors such as allelic variation within target genes, cross-reaction with other species and the lack of experienced molecular technicians have all contributed to their relative scarcity. Furthermore, many research laboratories lack the funds necessary to invest in these instruments, and often rely on labor-intensive biochemical characterization or 16S rRNA sequencing to confirm species type.ĭespite significant advances in molecular biology, molecular methods for species identification have not achieved widespread use, with the possible exception of the Biodefense community. In recent years, the introduction of automated identification instruments has resulted in greater reliability, but discrepancies between the various platforms have been reported –. These methods are time-consuming, can involve multiple biochemical tests, and can be expensive, particularly for fastidious organisms. In clinical microbiology laboratories, traditional culture based techniques remain the primary methodology used for identifying bacterial isolates. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. A comparison of culture and real-time PCR demonstrated 100% concordance. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources.

All primers were tested for specificity in vitro against 50 species of Gram-positive and –negative bacteria. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria.

Primer sequences