2010 NS-AUA Abstracts

Urinary tract infections (UTI) are among the most common bacterial infections in the United States. The gold standard for diagnosis is urine culture. This provides bacteria identification and sensitivity, but is labor intensive and time-consuming. As such, many UTIs are treated empirically contributing to resistance and cost. There is an urgent need for a diagnostic tool that can rapidly identify uropathogens and their sensitivities. We present the development of a PCR-based assay for Enterobacter cloacae, and evidence for its further use in the context of NanoLantern technology. The NanoLantern is a chip sensor that utilizes DNA hairpins immobilized onto a gold surface. In the presence of complementary (target) DNA, the hairpin stem is opened, exposing a quantifiable fluorophore. A synthetic DNA hairpin, unique to Enterobacter cloacae, was immobilized onto 9 gold coated chips. Baseline fluorescence intensity was recorded. The chip was exposed to synthetic target and then imaged with a fluorescence microscope. Fluorescence intensity was recorded and compared to baseline. PCR, asymmetric PCR and linear after the exponential PCR were used to amplify the target DNA from the bacteria. The amplicon was exposed to the NanoLantern chip and fluorescence intensity was quantified. Pre-synthetic target fluorescence imaging of 9 chips exposed to synthetic DNA resulted in a mean raw count of 665 (SD +- 244). Post-synthetic target fluorescence imaging of 9 chips resulted in a mean raw count of 8197 (SD +- 648), a greater than 12-fold increase in intensity. See Fig. 1. We have demonstrated the feasibility of a novel DNA hairpin probe. Work is ongoing to develop a PCR assay and to begin testing the NanoLantern chip with bacteria derived amplicon and urine samples. Ultimately, these integrated assays will provide rapid analysis, as part of an automated system, to be used in the clinical setting.