Case Studies
Nucleic Acid
The Opportunity
Radix BioSolutions and The Institute for Advanced Technologies at the University of Texas at Austin partnered to transition technologies developed at the University of Texas at Austin. The long-term objective of this project is was provide a fieldable system composed of a sampler and pre-sample preparation module and a transduction platform. The envisioned system combines pathogenicity island sequences with a multiplexed assay platform to develop a rapid, extensive infectious pathogen sensor.
The Challenge
Present biological agent detection systems tend to rely on the detection of single, or a few, DNA sequences, antigens or antibodies. The objective for this work was to use identified pathogenicity islands to completely define the critical domains of a set of virulence genes of key pathogens, and to use this information to design and build a prototype multiplexed detection system that would rapidly screen and detect every virulent mutant of that pathogen or pathogen class, and give no false alarms. This detection system would have hundreds of probes (multiplexed), so that false positives and false negatives will be determined immediately. The system was to be rugged, lightweight, and low-cost. A side benefit of this type of multiplexed detection is that it may inform investigators if a particular pathogen is man-made, if it is a new emergent infectious disease, or the source of the strain.
The enteric pathogens included a large number of related, but distinct organisms. Identification of the organisms had been based on antigenic or biochemical analysis. While this approach is effective, it may have failed to identify potential pathogens if they were antigenic variants or differed in metabolic pathways. An alternative approach to rapid detection and identification of pathogens is to develop methods that detect genes encoding virulence factors associated with a particular syndrome rather than detecting bacteria that may or may not have these virulence factors.
The Solution
We have applied this approach to rapid identification of pathogens in the E. coli/Shigella group. These pathogens contain the same “core” sequences but have additional regions of DNA encoding virulence determinants that appear to have been inserted into the chromosomes (as pathogenicity islands and prophages) or are carried on extrachromosomal elements (plasmids). It is the particular set of virulence factors encoded by a member of this group that determines pathogenic potential. By identifying the specific factors present in a member of this group, we can determine its potential to cause disease and the type of disease. This could lead to more rapid identification of the pathogen and determination of the proper course of treatment. As new pathogenicity islandswere discovered or mutations determined, new microsphere sets carrying these sequences were added to the current assay kit, thus seamlessly updating the kit.
The Results
Experiments have shown that the instrument is capable of detecting multiple pathogens at less than 1000 genome copies per amplified sample. An initial pathogenicity island multiplexed assay was developed that examined three different sequences, two for pathogenicity islands and one for a common junction area insertion site. Data demonstrate a 3.5-7 fold signal to noise ratio using as little as 1uL of PCR product for detection.
The use of pathogenicity islands can be used as indiscriminant identifiers of pathogens thus providing a genomic fingerprint to identify potential of pathogens. The use of the Luminex xMAPª system provides for a rapid high throughput screening system which can be modified by adding additional gene sequences as needed to current sequences or immunoassays.