Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa
| Autor: | Zachary Austin Crannell, Gayatri Nair, A. Clinton White, Rebecca Richards-Kortum, Rojelio Mejia, Alejandro Castellanos-Gonzalez |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
Cryptosporidium
Recombinase Polymerase Amplification 02 engineering and technology medicine.disease_cause Polymerase Chain Reaction 01 natural sciences Analytical Chemistry law.invention Entamoeba chemistry.chemical_compound law parasitic diseases medicine Humans Giardia lamblia Multiplex Polymerase chain reaction biology Chemistry 010401 analytical chemistry DNA Protozoan 021001 nanoscience & nanotechnology biology.organism_classification Molecular biology Healthy Volunteers 0104 chemical sciences Intestines Protozoa 0210 nano-technology Nucleic Acid Amplification Techniques Applications of PCR DNA |
| Zdroj: | Analytical Chemistry. 88:1610-1616 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences. |
| Databáze: | OpenAIRE |
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