title: A loop-mediated isothermal amplification (LAMP) assay for Strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye.
abstract: 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.
authors: Centre for Infectious Diseases and Microbiology Public Health; Pathology West - Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia; Marie Bashir Institute for Infectious Diseases and Biosecurity and Centre for Research Excellence in Critical Infection, University of Sydney, New South Wales, Australia; Department of Zoology, University of Dhaka, Dhaka, Bangladesh; Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
authors: Matthew R
authors: MR
authors: Watts
authors: Gregory
authors: G
authors: James
authors: Yasmin
authors: Y
authors: Sultana
authors: Andrew N
authors: AN
authors: Ginn
authors: Alexander C
authors: AC
authors: Outhred
authors: Fanrong
authors: F
authors: Kong
authors: Jaco J
authors: JJ
authors: Verweij
authors: Jonathan R
authors: JR
authors: Iredell
authors: Sharon C-A
authors: SC
authors: Chen
authors: Rogan
authors: R
authors: Lee
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018509:DNA, Helminth / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology*; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017171:Strongyloides stercoralis / Q000302:isolation & purification*; D013322:Strongyloidiasis / Q000175:diagnosis
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D005456:Fluorescent Dyes