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. 2016 Dec 1;9(1):624.
doi: 10.1186/s13071-016-1913-7.

An isothermal DNA amplification method for detection of Onchocerca volvulus infection in skin biopsies

Ole Lagatie  1 , Michelle Merino  2 , Linda Batsa Debrah  3 , Alexander Y Debrah  4 , Lieven J Stuyver  2
Affiliations
Free PMC article

An isothermal DNA amplification method for detection of Onchocerca volvulus infection in skin biopsies

Ole Lagatie et al. Parasit Vectors. .
Free PMC article

Abstract

Background: 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.

Methods: An isothermal, loop-mediated amplification method has been designed using the mitochondrial O. volvulus cox1 gene as a target.

Results: 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%.

Conclusions: 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.

Keywords: DNA; Diagnostic; Isothermal; LAMP; Onchocerca volvulus; Onchocerciasis; River blindness; Skin biopsy; cox1.

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Figures

Fig. 1
Fig. 1
Design of a LAMP assay targeting O. volvulus cox1. a Alignment of partial gene sequences of cytochrome c oxidase subunit 1 from O. volvulus, W. bancrofti, L. loa, B. malayi and O. ochengi. b Primer set targeting O. volvulus cox1. c Species-specific LAMP assay targeting O. volvulus cox1. c Dilution series of gBlocks containing the cox1 fragment of O. volvulus, W. bancrofti, L. loa and B. malayi were used as template in the LAMP assay using real-time fluorescent dye detection. All samples were analyzed in duplicate. Dilution indicated with an asterisk had detectable signal in only one of both duplicates
Fig. 2
Fig. 2
O. volvulus cox1 assay discriminates between O. volvulus and O. ochengi. a Dilution series of gBlocks containing the cox1 fragment of O. volvulus and O. ochengi were used as template in the O. volvulus cox1 LAMP assay using real-time fluorescent dye detection. b Melting point determination of the amplicons generated from different dilutions of O. volvulus and O. ochengi template. c Melting curves of the amplicons generated from O. volvulus and O. ochengi template
Fig. 3
Fig. 3
Comparison of results obtained by LAMP, O-150 qPCR and microscopic detection of microfilariae in skin snips. Comparison of results obtained by O. volvulus cox1 LAMP and O-150 qPCR for 146 subjects. Samples that were positive in microscopic examination are indicated in blue (n = 9). For each quadrant the total number of samples is indicated, as well as the number of microscopy positive samples in parentheses
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