Multiplexed Recombinase Polymerase Amplification Assay to Detect Intestinal Protozoa

Zachary Crannell, Alejandro Castellanos, Gayatri Nair, Rojelio Mejia, A. Clinton White, Rebecca Richards-Kortum

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1610-1616
Number of pages7
JournalAnalytical Chemistry
Volume88
Issue number3
DOIs
StatePublished - Feb 2 2016

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Protozoa
Recombinases
Amplification
Assays
DNA
Thermal cycling
Microscopic examination
Polymerase chain reaction
Pathogens
Genes
Parasites
Availability

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Multiplexed Recombinase Polymerase Amplification Assay to Detect Intestinal Protozoa. / Crannell, Zachary; Castellanos, Alejandro; Nair, Gayatri; Mejia, Rojelio; White, A. Clinton; Richards-Kortum, Rebecca.

In: Analytical Chemistry, Vol. 88, No. 3, 02.02.2016, p. 1610-1616.

Research output: Contribution to journalArticle

Crannell, Zachary ; Castellanos, Alejandro ; Nair, Gayatri ; Mejia, Rojelio ; White, A. Clinton ; Richards-Kortum, Rebecca. / Multiplexed Recombinase Polymerase Amplification Assay to Detect Intestinal Protozoa. In: Analytical Chemistry. 2016 ; Vol. 88, No. 3. pp. 1610-1616.
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