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 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.Author Summary: Dirofilaria repens is a filarial nematode which mainly infests the dog, but humans may be occasionally infested, too. The spread of the parasite is mediated by a number of mosquitoes species, which are well recognized as vectors of D. repens. The majority of reports of the disease come from the European Countries, especially those along the Mediterranean basin, but in the last decade several cases have been recorded also from Asian and African Countries, and this led the scientific community to consider such parasitosis an emerging disease. To date, diagnosis is based the morphologic analysis of microfilariae, isolated from the blood of infected hosts, but this may be time-consuming and the identification of parasite requires specialized parasitologists. The here described approach, based on the loop-mediated isothermal amplification (LAMP), allows the detection of D. repens genomic DNA directly from the biological samples, and it may be easily and rapidly performed, producing unequivocal results in less than a hour. We also presented two versions of the assays. The first, a real-time LAMP, is characterized by a very high sensitivity but it requires an expensive real time PCR instrument, while the second, performed with the addition of propidium iodide, does not need such equipment, therefore being very affordable. This makes it suitable to be carried out in field and whenever expensive equipment or specialized personnel lacks.