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

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.

Fig 1. Relative positions of LAMP primers within the cytochrome c oxidase gene of D. repens.

The first steps of the LAMP reaction are also depicted. i) Primers F3/B3, and DIRF2/DIRB2 (parts of DIRFIP and DIRBIP, respectively), recognize and anneal to their respective complementary targets. The DNA synthesis starts and the strand displacement activity of the DNA polymerase let the strand synthesized from DIRF2 and DIRB2 to be detached from the template strand. ii) The displaced neo-synthesized strands bring, at their termini, DIRF1c and DIRB1c that iii) will self-anneal to the DIRF1 and DIRB1 loci, respectively, thus forming a secondary structure with a loop. DIRB3/DIRF3 and DIRBIP/DIRFIP (specifically with the portions DIRB2 and DIRF2) hybridize with the complementary regions of the strand. The synthesis and the displacement of strand occur again, catalyzed by the same DNA polymerase. iv) the 3’ and 5’ termini, bearing DIRF1/DIRB1 and DIRB1c/DIRF1c, respectively, self-anneal to the complementary loci harbored by the same strand, which, in turn, will form the typical dumbbell structure. The DNA polymerase will catalyze the DNA synthesis starting from the 3’ terminus, displacing the 5’ terminus, and producing a concatamer which will be the basis for following the amplification steps.

Fig 2. Specificity of the ReT-LAMP.

The curves represent the amplification signals expressed as ΔRn, after subtraction of the ROX reference dye fluorescence. a) and b) Amplification curves from ReT-LAMP carried out with DNA from larvae of previously identified species. The curves from species different from D. repens are overlapped because of the lack of an appreciable signal. c) Amplification curves from DNA extracted from biological samples (mosquitoes and canine blood).

Fig 3. Specificity of the PI-LAMP.

The micro-tubes containing the reaction mixture with propidium iodide have been exposed to UV after incubation. The positive reactions that returned an amplification product are evidenced by a bright fluorescence. The reaction have been performed by using purified DNA from larvae of 1) Acanthocheilonema reconditum; 2) Acanthocheilonema sp.; 3)Angiostrongylus vasorum; 4) Brugia sp.; 5) Cercopithifilaria sp.; 6) Dirofilaria immitis; 7) Loa; 8) Mansonella perstans; 9) Spirocerca lupi; 10) Wuchereria bancrofti; 12) and 13) Dirofilaria repens; 14) D. repens positive mosquito pool; 15) D. repens negative mosquito pool; 16) D. repens positive canine blood; 17) D. repens negative canine blood. The sample 11 is a negative sample with water instead of DNA.

Fig 4. Results of the sensitivity assay.

a) ReT-LAMP. The curves represent the ΔRn after subtraction of the ROX reference dye fluorescence. The amount of D. repens DNA for each reaction is indicated near the respective curve. *The curves of the reaction with 15 ag of DNA and of negative control are overlapping. b) PCR. M: AmpliSize Molecular Ruler, 50–2,000 bp Ladder (BioRad Laboratories, Milan, Italy). c) PI-LAMP. The bright fluorescence indicates the positivity of the reaction.