Detection of Bacteria Using Fluorogenic DNAzymes

Abstract

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-toresults (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR-and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment. 2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection. 5DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions. [6][7][8] These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 10 16 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment). 9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications. 6-8Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools. [17][18][19][20][21][22][23][24][25][26][27][28][29] RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.More recently, we developed a method of isolating RFDs for bacterial detection. 5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifyi...

A review on detection methods used for foodborne pathogens

“…This process includes a DNA RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by fluorophore (F) and a quencher (Q). There will be a cleavage where the separation of fluorophore and quencher will lead to increase in fluorescence intensity which makes bacterial detection easy and rapid 43 . Epitope tags which confer specific properties, including affinity for resins or antibodies or detection by fluorescence microscopy, are useful for biochemical and cell biological investigations.…”

A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection

“…Detection sensitivity (for immunoassay) and tendency to generate false-positive results (for PCR) are also issues of concerns. For these considerations, we recently began to examine the utility of RNA-cleaving fluorogenic DNAzyme (RFD) probes for bacterial detection [ 12 , 13 , 14 ]. RFDs can be isolated from random-sequence DNA pools to perform three linked functions: ligand binding, catalysis and fluorescence generation.…”

In vitro selection of RNA-cleaving DNAzymes for bacterial detection

“…Over the past decade, we have isolated many RFDs and examined their catalytic and signaling properties [38,39,[67][68][69][81][82][83][84][85][86][87][88][89][90]. More recently, we began to develop RFDs that that can be activated in the presence of a specific bacterium, such as Escherichia coli and Clostridium difficile [72,80,82,[91][92][93][94][95][96].…”