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Fluorescence-Enhancement Based Detection and Identification of Bacterial Contaminants

  • Author(s): Upadhyayula, Srigokul
  • Advisor(s): Vullev, Valentine I
  • et al.

Due to the considerable challenges for reliable and expedient detection of virulent vegetative bacterial, bacterial endospores and other hazardous biological agents, accidental exposure is a principal cause of fatality due to wound infection or slow diagnosis. Staining techniques, due to their simplicity, form a set of important tools for pathogen monitoring and control. The staining assays, such as immunoassays, however, provide only Boolean information: i.e., stain vs. does not stain. As a result, only the species being sought may have a chance to be identified, leaving key determining factors for the diagnosis quite susceptible to human error.

Our principle motivation for this project is to expand the current analytical techniques beyond their Boolean nature, and to address the apparent need for simple, expedient and inexpensive assays. We describe the development of an assay system based on the dynamics of fluorophore uptake that will serve as a robust platform for detection of both vegetative and endospore bacterial contaminants. The kinetics of fluorescence staining depends on the coatings and protein content, which reflect the species phenotype. Hence, such kinetic measurements gain access to genetic information, in less than a minute, without conducting amplification procedures such as PCR.

We have demonstrated for the first time that the kinetics of emission enhancement, caused by cell uptake of fluorophores, provides statistically significant discernibility between different and closely related vegetative bacteria and bacterial endospore species respectively. We observed that the time course of the fluorescence signal provides a unique species-specific signature that can prove indispensible for the identification of dangerous and life-threatening spores.

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