Translating Bacterial Detection by DNAzymes into a Litmus Test

Abstract

Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world.

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].…”

“…Although this property will make RFDs immediately useful for fluorescence-based assays, there is increasing interest in developing simpler, colorimetric assays that are more suitable for point-of-care applications. We have recently developed an approach to take advantage of the classic litmus test for colorimetric detection of bacteria using RFDs [80]. Our expanded litmus test platform is illustrated in Figure 6.…”

“…Since this test takes advantage of magnetic separation that is easy to implement and pH-sensitive dyes or pH papers that are cheap and widely available, it is well suited for point of care applications. We have validated this approach using E. coli-detecting probe RFD-EC1 [80]. The key to this expanded litmus test is the use of cleavagebased RFD probes, and therefore, it can be applied for the detection of any target for which an RFD can be derived.…”

A Catalytic DNA Activated by a Specific Strain of Bacterial Pathogen

“…[6] RFD-EC1 has since been used in acolorimetric assay for the field detection of E. coli and in amicrofluidic-based counting assay for the rapid detection of E. coli in blood. [7,8] Although RFD-EC1 is highly specific for E. coli with minimal crossactivity to other bacterial species, [6] it non-discriminatively recognizes all E. coli strains. [8] Theg oal of this study was to investigate whether it was possible to derive strain-specific RFDs by in vitro selection.…”

Highly Specific Recognition of Breast Tumors by an RNA-Cleaving Fluorogenic DNAzyme Probe

“…[30][31][32][33][34][35][36][37][38][39] Some DNAzymes have also been used for the development of biosensors. [40][41][42][43] For example, DNAzymes that bind to specific metal ions have been developed and used to detect toxic metal ions such as Pb (II), 44 Cu (II), 45,46 Hg (II). 47 For the past 10 years, Li and colleagues have developed a biosensing platform named "RNA-cleavage fluorogenic DNAzyme (RFD)".…”