Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of Dirofilaria repens from Biological Samples

Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The...

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Bibliographic Details
Published in:PLoS neglected tropical diseases 2016-06, Vol.10 (6), p.e0004789-e0004789
Main Authors: Raele, Donato Antonio, Pugliese, Nicola, Galante, Domenico, Latorre, Laura Maria, Cafiero, Maria Assunta
Format: Article
Language:English
Subjects:
Animals
Biology and Life Sciences
Computer Simulation
Culicidae
Deoxyribonucleic acid
Diagnosis
Dirofilaria repens - genetics
Dirofilaria repens - isolation & purification
Dirofilariasis - diagnosis
Dirofilariasis - parasitology
DNA
DNA, Helminth - isolation & purification
Dog Diseases - diagnosis
Dog Diseases - parasitology
Dogs
Electron Transport Complex IV - genetics
Electron Transport Complex IV - metabolism
Funding
Gene amplification
Gene Expression Regulation, Enzymologic
Insect Vectors
Medicine and Health Sciences
Methods
Models, Biological
Molecular diagnostic techniques
Mosquitoes
Nucleic Acid Amplification Techniques - methods
Parasites
Parasitic diseases
Real time
Research and Analysis Methods
Roundworm infections
Sensitivity and Specificity
Social Sciences
Time Factors
Tropical diseases
Zoonoses
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Description
Summary:Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic.
ISSN:1935-2735
1935-2727
1935-2735
DOI:10.1371/journal.pntd.0004789