Identifying and distinguishing bacterial strains using Real Time PCR and Microarrays
Introduction to Bacterial Identification
Accurate and definitive microorganism identification, including bacterial identification and pathogen detection, is essential for correct disease diagnosis, treatment of infection and trace-back of disease outbreaks associated with microbial infections. Bacterial identification is used in a wide variety of applications including microbial forensics, criminal investigations, bio-terrorism threats and environmental studies.
Challenges in Bacterial Identification
Traditional methods of bacterial identification rely on phenotypic identification of the causative organism using gram staining, culture and biochemical methods. However, these methods of bacterial identification suffer from two major drawbacks. First, they can be used only for organisms that can be cultivated in vitro. Second, some strains exhibit unique biochemical characteristics that do not fit into patterns that have been used as a characteristic of any known genus and species.
In the past decade or so, molecular techniques have proven beneficial in overcoming some limitations of traditional phenotypic procedures for the detection and characterization of bacterial phenotypes. Several non-culture based methods have emerged in the past 15 years.
Real time PCR and microarrays are currently the most commonly employed molecular techniques. Real time PCR is highly sensitive and allows quantitation of bacteria at a species level. Microarray based bacterial identification relies on the hybridization of preamplified bacterial DNA sequences to arrayed species-specific oligonucleotides. Each probe is tagged with a different colored dye which fluoresces upon hybridization. See how microarray technology works...
Real Time PCR Based Bacterial Identification
Using a DNA based assay, one can easily detect bacterial strains directly from clinical samples or from small amounts of cultured bacterial cells, thus improving the sensitivity and decreasing the time required for bacterial identification. PCR has been particularly useful in this regard, which relies on primer sequences designed to facilitate bacterial identification at any level of specificity: strain, species or genus.
In recent years, real-time PCR methods have been developed and described for the rapid detection and identification of several bacterial strains. Real-time PCR is a promising tool for distinguishing specific sequences from a complex mixture of DNA and therefore is useful for determining the presence and quantity of pathogen-specific or other unique sequences within a sample. Real-time PCR facilitates a rapid detection of low amounts of bacterial DNA accelerating therapeutic decisions and enabling an earlier adequate antibiotic treatment.
Microarray Based Bacterial Identification
Microarrays combines the potential of simultaneous bacterial identification and speciation. This method is versatile and makes it possible to detect and discriminate different bacterial samples on a single slide. The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. DNA microarray-based approach is used for the quick detection and identification of bacteria using species-specific oligonucleotide probes designed for specific regions of various targeted genes.
AlleleID® automatically designs oligos for microarray and real time PCR assays for pathogen detection, bacterial identification, taxa discrimination and cross species research by combining ClustalW alignments with probe design.