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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
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Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa
Zachary Crannell1,2,3,4,5, Alejandro Castellanos-Gonzalez1,2,3,4,5, Gayatri Nair1,2,3,4,5
11980 West Getaway Trail, Flagstaff, Arizona 86005, United States.
Analytical Chemistry
|December 17, 2015
Summary
This study introduces a novel multiplex recombinase polymerase amplification (RPA) assay for simultaneous detection of Giardia, Cryptosporidium, and Entamoeba DNA. This isothermal method offers a sensitive, accessible alternative to traditional diagnostics for diarrheal protozoa.
Area of Science:
- Molecular Biology
- Parasitology
- Diagnostic Development
Background:
- Diarrheal protozoa like Giardia, Cryptosporidium, and Entamoeba cause significant global health burdens.
- Traditional stool microscopy for diagnosis is insensitive, missing many cases.
- Current sensitive DNA-based methods (PCR) require expensive equipment, limiting accessibility.
Purpose of the Study:
- To develop and validate a multiplex recombinase polymerase amplification (RPA) assay for simultaneous detection of Giardia, Cryptosporidium, and Entamoa.
- To establish a sensitive, isothermal DNA-based diagnostic tool for common diarrheal protozoa.
- To overcome limitations of existing diagnostic methods for these parasitic infections.
Main Methods:
- Development of a multiplex RPA assay targeting DNA from Giardia, Cryptosporidium, and Entamoa.
- Characterization of the assay using synthetic DNA targets to determine limits of detection.
- Validation of the assay with DNA extracted from live parasites spiked into stool samples.
Main Results:
- The multiplex RPA assay successfully detected and differentiated DNA from all three targeted protozoa.
- Limits of detection were established for synthetic targets (403-425 gene copies/reaction) and spiked stool samples (6-444 parasites/reaction).
- The assay demonstrated comparable sensitivity to singleplex assays, with slight increases in detection limits.
Conclusions:
- This proof-of-concept multiplex RPA assay provides a sensitive and accessible diagnostic tool for common diarrheal protozoa.
- The isothermal nature of RPA eliminates the need for thermal cycling equipment, broadening diagnostic accessibility.
- The assay platform is adaptable for detecting a wide range of other pathogens by redesigning primer and probe sequences.
