Abstract
We report on a new colorimetric DNA detection method that takes advantage of the power of polymerase chain reaction (PCR) and the simplicity of the classic litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in the increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of the bacterium Clostridium difficile that are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients.
Highlights
polymerase chain reaction (PCR) has become a widely adopted technique in clinical laboratories, it has not become commonly used point-of-care or field tools
C. difficile is a gram-positive, spore-forming anaerobic bacterium that has been identified as the leading cause of diarrhea in developed countries and pseudomembranous colitis in humans[14]
The incidence and mortality of C. difficile infection (CDI) has increased dramatically over the past years and CDI has become a common problem in hospitals across North America, Europe and some regions of Asia[15]
Summary
Dingran Chang 1, Kha Tram[2], Ben Li1, Qian Feng[2], Zhifa Shen[1], Christine H. We describe a strategy for adopting the same litmus test for the detection of PCR amplicons through the use of a set of specially modified DNA primers and urease. To achieve colorimetric detection of PCR products, we firstly conjugated urease with an oligonucleotide as reported in a previous method (see details in the methods)[13] This conjugated urease-DNA (UrD) is used to hybridize to the extended binding region found in the reverse primer. The sensitivity of the PCR-litmus assay was evaluated by testing genomic DNA isolated from serially diluted C. difficile stocks containing a known number of cells (Fig. 3a). The featured PCR-litmus test can be extended to the detection of other gene targets by changing the primers, and we envision that this method can find wide applications in diverse fields
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