title: Detection and quantification of schistosome DNA in freshwater snails using either fluorescent probes in real-time PCR or oligochromatographic dipstick assays targeting the ribosomal intergenic spacer.
abstract: Several DNA probes were designed for use in real-time polymerase chain reaction (PCR) assays to target sequence variation within the ribosomal intergenic spacer (IGS) of schistosomes. A sub-section of the IGS (∼300bp) was amplified, with cross-specific primers, after which group-specific fluorescent, locked nucleic acid probes were assessed for their ability to differentiate and quantify DNA from Schistosoma haematobium and Schistosoma mansoni group parasites. A number of fluorescent probe candidates were screened and validated against genomic DNA from adult schistosome worms and laboratory infected freshwater snails. Two fluorescent, locked nucleic acid probes ShaemLNA5 and SmanLNA2, of 20-26bp in length, were identified and found to be effective in providing evidence of infection in field-collected snails. To adapt these real-time PCR assays for more resource-poor laboratory settings, a PCR-restriction fragment length polymorphism (RFLP) assay was developed and primer/probe combinations were modified for use in oligochromatography, a DNA 'dipstick' technology. An appropriate dipstick was developed, inclusive of internal amplification and amplicon migration controls that could be of particular importance for assessing schistosome transmission dynamics. These assays and tools also have future potential for use in detection of schistosome infections in humans and livestock.
authors.affiliation: Wolfson Wellcome Biomedical Laboratories, Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Kane
authors.forename: J Russell
authors.initials: JR
authors.lastname: Stothard
authors.forename: David
authors.initials: D
authors.lastname: Rollinson
authors.forename: Thierry
authors.initials: T
authors.lastname: Leclipteux
authors.forename: Jonathan
authors.initials: J
authors.lastname: Evraerts
authors.forename: Claire J
authors.initials: CJ
authors.lastname: Standley
authors.forename: Fiona
authors.initials: F
authors.lastname: Allan
authors.forename: Martha
authors.initials: M
authors.lastname: Betson
authors.forename: Rehana
authors.initials: R
authors.lastname: Kaba
authors.forename: Pascal
authors.initials: P
authors.lastname: Mertens
authors.forename: Thierry
authors.initials: T
authors.lastname: Laurent
mesh_terms: D000818:Animals; D002845:Chromatography; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D005618:Fresh Water; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D059208:Parasite Load / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012548:Schistosoma haematobium / Q000235:genetics / Q000302:isolation & purification*; D012550:Schistosoma mansoni / Q000235:genetics / Q000302:isolation & purification*; D012908:Snails / Q000469:parasitology*
chemical_list: D018509:DNA, Helminth; D005456:Fluorescent Dyes