title: Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.
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.
authors.affiliation: 1980 West Getaway Trail, Flagstaff, Arizona 86005, United States.|Department of Internal Medicine, University of Texas Medical Branch , 301 University Boulevard, Galveston, Texas 77555.0435, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Department of Pediatrics, Section of Tropical Medicine; Department of Medicine, Section of Infectious Diseases; and National School of Tropical Medicine, Baylor College of Medicine 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , Mary Moody Northen, Room 4.230, 301 University Boulevard, Galveston, Texas 77555-0435, United States.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Crannell
authors.affiliation: 1980 West Getaway Trail, Flagstaff, Arizona 86005, United States.|Department of Internal Medicine, University of Texas Medical Branch , 301 University Boulevard, Galveston, Texas 77555.0435, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Department of Pediatrics, Section of Tropical Medicine; Department of Medicine, Section of Infectious Diseases; and National School of Tropical Medicine, Baylor College of Medicine 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , Mary Moody Northen, Room 4.230, 301 University Boulevard, Galveston, Texas 77555-0435, United States.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Castellanos-Gonzalez
authors.affiliation: 1980 West Getaway Trail, Flagstaff, Arizona 86005, United States.|Department of Internal Medicine, University of Texas Medical Branch , 301 University Boulevard, Galveston, Texas 77555.0435, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Department of Pediatrics, Section of Tropical Medicine; Department of Medicine, Section of Infectious Diseases; and National School of Tropical Medicine, Baylor College of Medicine 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , Mary Moody Northen, Room 4.230, 301 University Boulevard, Galveston, Texas 77555-0435, United States.
authors.forename: Gayatri
authors.initials: G
authors.lastname: Nair
authors.affiliation: 1980 West Getaway Trail, Flagstaff, Arizona 86005, United States.|Department of Internal Medicine, University of Texas Medical Branch , 301 University Boulevard, Galveston, Texas 77555.0435, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Department of Pediatrics, Section of Tropical Medicine; Department of Medicine, Section of Infectious Diseases; and National School of Tropical Medicine, Baylor College of Medicine 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , Mary Moody Northen, Room 4.230, 301 University Boulevard, Galveston, Texas 77555-0435, United States.
authors.forename: Rojelio
authors.initials: R
authors.lastname: Mejia
authors.affiliation: 1980 West Getaway Trail, Flagstaff, Arizona 86005, United States.|Department of Internal Medicine, University of Texas Medical Branch , 301 University Boulevard, Galveston, Texas 77555.0435, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Department of Pediatrics, Section of Tropical Medicine; Department of Medicine, Section of Infectious Diseases; and National School of Tropical Medicine, Baylor College of Medicine 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , Mary Moody Northen, Room 4.230, 301 University Boulevard, Galveston, Texas 77555-0435, United States.
authors.forename: A Clinton
authors.initials: AC
authors.lastname: White
authors.affiliation: Department of Bioengineering, Rice University , Houston, Texas 77030, United States.|1980 West Getaway Trail, Flagstaff, Arizona 86005, United States.|Department of Internal Medicine, University of Texas Medical Branch , 301 University Boulevard, Galveston, Texas 77555.0435, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Department of Pediatrics, Section of Tropical Medicine; Department of Medicine, Section of Infectious Diseases; and National School of Tropical Medicine, Baylor College of Medicine 1709 Dryden Road, No. 1700, Houston, Texas 77030, United States.|Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch , Mary Moody Northen, Room 4.230, 301 University Boulevard, Galveston, Texas 77555-0435, United States.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Richards-Kortum
mesh_terms: D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000032:analysis* / Q000235:genetics*; D004747:Entamoeba / Q000235:genetics / Q000302:isolation & purification*; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification*; D064368:Healthy Volunteers; D006801:Humans; D007422:Intestines / Q000469:parasitology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction
chemical_list: D016054:DNA, Protozoan