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A Simple DNAzyme-Based Fluorescent Assay for Klebsiella pneumoniae
M Monsur Ali1, Anatoly Slepenkin2, Ellena Peterson2
1Biointerfaces Institute, McMaster University, 1280 Main St. W., Hamilton, ON, L8S 4K1, Canada.
Abstract:
Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA-RNA chimeric substrate in the presence of K. pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 105 CFUs mL-1 . Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K. pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K. pneumoniae.
Insights
A new DNAzyme fluorescent paper sensor offers rapid and cost-effective detection of Klebsiella pneumoniae. This simple assay can identify the pathogenic bacteria in clinical samples without complex equipment.
Area of Science:
- Biotechnology
- Molecular Biology
- Analytical Chemistry
Background:
- Pathogenic bacteria, including Klebsiella pneumoniae, present a significant public health risk.
- Rapid, cost-effective, and sensitive detection methods for bacteria are crucial for timely intervention.
- Current detection methods often involve complex procedures and specialized equipment, limiting their accessibility.
Purpose of the Study:
- To develop a novel DNAzyme-based fluorescent paper sensor for the detection of Klebsiella pneumoniae.
- To establish a rapid, simple, and cost-effective assay for bacterial identification.
- To evaluate the performance of the developed sensor using clinical bacterial isolates.
Main Methods:
- In vitro selection was employed to generate a DNAzyme specific for Klebsiella pneumoniae.
- A DNAzyme-based fluorescent paper sensor was created by immobilizing the DNAzyme on a paper substrate in a 96-well format.
- The sensor utilizes a fluorogenic DNA-RNA chimeric substrate that is cleaved by the DNAzyme in the presence of Klebsiella pneumoniae, releasing a fluorescent signal.
- The limit of detection (LOD) was determined to be 10^5 Colony Forming Units (CFUs) per milliliter.
- The sensor's specificity and efficacy were tested against 20 clinical bacterial isolates.
Main Results:
- The DNAzyme-based fluorescent paper sensor successfully detected Klebsiella pneumoniae in clinical samples.
- The assay demonstrated a limit of detection of 10^5 CFUs/mL.
- The sensor produced a positive fluorescence signal for Klebsiella pneumoniae isolates, irrespective of their origin or antibiotic resistance profile.
- The assay proved to be simple, rapid, and cost-effective, requiring minimal sample preparation and no complex equipment.
Conclusions:
- The developed DNAzyme-based fluorescent paper sensor is a promising tool for the rapid and cost-effective identification of Klebsiella pneumoniae.
- This assay offers a simplified alternative to conventional bacterial detection methods, enhancing accessibility for practical applications.
- The sensor's ability to detect Klebsiella pneumoniae regardless of drug resistance is a significant advantage for clinical diagnostics.
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