Characterization of a DNAzyme for the detection of Legionella pneumophila in cooling tower water

Abstract

Ineffective bacterial monitoring in water systems represents a danger to public health and can result in costly disease outbreaks. Of interest is Legionella pneumophila, a deadly water-borne bacterial pathogen that causes Legionnaires’ disease - a severe form of pneumonia. The Center for Disease Control stated that reported cases of Legionnaires’ disease have quadrupled since 2000 and ranks L. pneumophila as the number one cause of waterborne disease outbreaks in the United States. This threat is expected to increase given an aging population who are more susceptible to L. pneumophila infection and rising global temperatures that can promote L. pneumophila growth. Presently, Public Health agencies recommend bacterial culturing for the detection of L. pneumophila in environmental samples, however, this process can take up to ten days to complete. Consequently, there is a delay between sample collection and subsequent L. pneumophila detection, creating an opportunity for a Legionnaires’ disease outbreak to occur. There is a great need to develop a field-appropriate device that can provide early-stage detection of L. pneumophila in water as a means of mitigating Legionnaires’ disease outbreaks. We propose the use of DNAzymes for the development of such a device. DNAzymes are small, catalytically-active single-stranded DNA molecules that demonstrate target-specific enzymatic activity. We have successfully isolated an RNA-cleaving fluorescent DNAzyme (RFD) specific for the detection of L. pneumophila using in vitro selection. Thorough characterization of the DNAzyme has revealed key structural features influencing kinetics, specificity and sensitivity. In addition, the ability of the DNAzyme to function in cooling tower water, and conservation of the DNAzyme target across Legionella bacteria, has been investigated. In the future we plan to incorporate this RFD into a field-appropriate paper-based device which would play a key role in managing infectious diseases and preventing large-scale outbreaks.