Abstract
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.
Highlights
Dirofilariases are parasitic diseases caused by nematodes of the genus Dirofilaria (Nematoda: Onchocercidae) and transmitted by hemathophagous arthropods
This study describes two versions of a novel loop-mediated isothermal amplification assay for a rapid diagnosis of D. repens in dogs and mosquitoes
Two different variants of loop mediated isothermal amplification (LAMP) have been developed: a real-time LAMP and a propidium iodide LAMP (PI-LAMP). They differed for the visualization of results: in the first cases, the amplification is visualized as a curve in a real-time PCR instrument, while the second allows to visualize the amplification as UV fluorescence following the addition on propidium iodide
Summary
Dirofilariases are parasitic diseases caused by nematodes of the genus Dirofilaria (Nematoda: Onchocercidae) and transmitted by hemathophagous arthropods. The subgenera Dirofilaria (Nochtiella) is represented by 22 species including Dirofilaria repens Railliet et Henry, 1911 [1] The latter is a common mosquito–borne parasite of subcutaneous tissues of dogs and others carnivores in the Old World [2], including wolf and fox [3]. D. repens has zoonotic potential: it can infest humans, and it is considered one of the most important vectorborne parasitosis in humans in Europe [6]. Among these hosts, the dog is the most important and it act as a reservoir for the parasite [7,8]. The nematode is not highly pathogenic for the dog, where it usually causes subcutaneous tissue diseases, it is considered of primary veterinary importance due to its zoonotic behavior [10]
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