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A Loop-Mediated Isothermal Amplification (LAMP) Assay for Strongyloides stercoralis in Stool That Uses a Visual Detection Method with SYTO-82 Fluorescent Dye
An assay to detect Strongyloides stercoralis in stool specimens was developed using the loop-mediated isothermal amplification (LAMP) method. Primers were based on the 28S ribosomal subunit gene. The reaction conditions were optimized and SYTO-82 fluorescent dye was used to allow real-time and visual detection of the product. The product identity was confirmed with restriction enzyme digestion, cloning, and sequence analysis. The assay was specific when tested against DNA from bacteria, fungi and parasites, and 30 normal stool samples. Analytical sensitivity was to < 10 copies of target sequence in a plasmid and up to a 10−2 dilution of DNA extracted from a Strongyloides ratti larva spiked into stool. Sensitivity was increased when further dilutions were made in water, indicative of reduced reaction inhibition. Twenty-seven of 28 stool samples microscopy and polymerase chain reaction positive for S. stercoralis were positive with the LAMP method. On the basis of these findings, the assay warrants further clinical validation.
Author Notes
Financial support: Matthew R. Watts received financial assistance through an Australian NHMRC Postgraduate Scholarship. This project was partially funded through the Centre for Infectious Diseases and Microbiology Public Health, Institute of Clinical Pathology and Medical Research, Westmead Hospital.
Authors' addresses: Matthew R. Watts, Gregory James, Andrew N. Ginn, Alexander C. Outhred, Fanrong Kong, Jonathan R. Iredell, Sharon C-A. Chen, and Rogan Lee, Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research Westmead, Westmead Hospital, Westmead, New South Wales, Australia, E-mails: watts.idmicro@gmail.com, greg.james@swahs.health.nsw.gov.au, andrew.ginn@sydney.edu.au, aouthred@gmail.com, fanrong.kong@swahs.health.nsw.gov.au, jonathan.iredell@sydney.edu.au, sharon.chen@swahs.health.nsw.gov.au, and rogan.lee@swahs.health.nsw.gov.au. Yasmin Sultana, Department of Zoology, University of Dhaka, Dhaka, Bangladesh, E-mail: ysultana@du.ac.bd. Jaco J. Verweij, Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, The Netherlands, E-mail: j.verweij@elisabeth.nl.