title: An isothermal DNA amplification method for detection of Onchocerca volvulus infection in skin biopsies.
abstract: Diagnostic procedures for the diagnosis of infection with the nematode parasite Onchocerca volvulus are currently based on the microscopic detection of microfilariae in skin biopsies. Alternative approaches based on amplification of parasitic DNA in these skin biopsies are currently being explored. Mostly this is based on the detection of the O-150 repeat sequence using PCR based techniques.
  
  
  
  An isothermal, loop-mediated amplification method has been designed using the mitochondrial O. volvulus cox1 gene as a target.
  
  
  
  Analysis of dilution series of synthetic DNA containing the targeted sequence show a non-linear dose-response curve, as is usually the case for isothermal amplification methods. Evaluation of cross-reactivity with the heterologous sequence from the closely related parasites Wuchereria bancrofti, Loa loa and Brugia malayi demonstrated strong specificity, as none of these sequences was amplified. The assay however amplified both O. volvulus and O. ochengi DNA, but with a different melting point that can be used to discriminate between the species. Evaluation of this assay in a set of skin snip biopsies collected in an endemic area in Ghana showed a high correlation with O-150 qPCR and also demonstrated a similar sensitivity. Compared to qPCR, LAMP had a sensitivity of 88.2% and a specificity of 99.2%.
  
  
  
  We have developed a sensitive and specific loop-mediated amplification method for detection of O. volvulus DNA in skin biopsies that is capable of providing results within 30 min.
authors.affiliation: Janssen Diagnostics, Janssen R&D, Turnhoutseweg 30, 2340, Beerse, Belgium. olagatie@its.jnj.com.
authors.forename: Ole
authors.initials: O
authors.lastname: Lagatie
authors.affiliation: Janssen Diagnostics, Janssen R&D, Turnhoutseweg 30, 2340, Beerse, Belgium.
authors.forename: Michelle
authors.initials: M
authors.lastname: Merino
authors.affiliation: Kumasi Centre for Collaborative Research into Tropical medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
authors.forename: Linda
authors.initials: L
authors.lastname: Batsa Debrah
authors.affiliation: Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
authors.forename: Alexander Y
authors.initials: AY
authors.lastname: Debrah
authors.affiliation: Janssen Diagnostics, Janssen R&D, Turnhoutseweg 30, 2340, Beerse, Belgium.
authors.forename: Lieven J
authors.initials: LJ
authors.lastname: Stuyver
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001706:Biopsy; D051545:Cyclooxygenase 1 / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D005869:Ghana; D006801:Humans; D008297:Male; D008875:Middle Aged; D024101:Mitochondrial Proteins / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017181:Onchocerca volvulus / Q000235:genetics / Q000302:isolation & purification*; D009855:Onchocerciasis / Q000175:diagnosis* / Q000469:parasitology; D012680:Sensitivity and Specificity; D012867:Skin / Q000469:parasitology*; D013997:Time Factors; D055815:Young Adult
chemical_list: D018509:DNA, Helminth; D024101:Mitochondrial Proteins; D051545:Cyclooxygenase 1
keywords: DNA; Diagnostic; Isothermal; LAMP; Onchocerca volvulus; Onchocerciasis; River blindness; Skin biopsy; cox1