LLNL Summer 2007 Internship Experience

Since the 2001 anthrax attacks involving the US postal service, there have been increased efforts to study more advanced methods of decontamination and detection of viable Bacillus anthracis before and after decontamination efforts. Current methods for sample processing and viability analysis are low throughput ({approx}30-40 per day) requiring several manual steps, with confirmed results obtained days later. The group I am working with has developed more rapid, high throughput methods using automation to process surface samples combined with a time-course real-time Polymerase Chain Reaction (PCR) approach to determine the presence of viable B. anthracis spores. This process is referred to as Rapid Viability (RV)-PCR. These methods based on an observable change in PCR response during culturing showed detection of low numbers of bacterial pathogens in hours compared to days required for conventional culture analysis. In this project, we are studying detection limits, growth inhibition and PCR inhibition of a modified real-time PCR-based automated method of detecting B. anthracis Sterne (non-infectious variant) in various environmental samples containing levels of background debris expected during sampling. In order to decrease the detection limit, additional clean-up steps are employed. Since B. anthracis spores are very resilient to solvents, ethanol treatment can also be used to kill other bacteria (vegetative cells) in the sample. Finally, dilution of the sample may be useful to dilute out contaminants. Using commercially available robotics (Figure 1), each of these treatment steps can be automated, allowing processing of 100-200 swabs per day, with quantitative results obtained within 24 hours. Automation also reduces the risk of pathogens since no manual liquid handling steps and no plating or centrifugation is required. Traditional viability analysis uses manual steps for sample processing including performing dilutions, plating onto solid media, counting colonies and confirming the presence of B. anthracis using biochemical tests. The RV-PCR approach uses specific detection via real-time PCR so that additional verification of the pathogen is unnecessary. The RV-PCR method is based on a significant shift in real-time PCR response curve over time ({Delta}Ct), but also is dependent on Ct{sub 0} and Ct{sub final} (Figure 2). Criteria were developed to accurately distinguish live cells from dead spores by testing with thousands of samples containing low levels (1-10) of live spores in background of 106 dead spores and/or background debris and high populations of non-target bacteria. Finally, a Most Probable Number (MPN) method was combined with the RV-PCR approach to yield a quantitative method to estimate the number of spores in the sample. In this study, the automated MPN RV-PCR method has been optimized to accommodate high amounts of debris from real-world samples