title: Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis.
abstract: Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species.
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano Via Celoria 10, Milano 20133, Italy. Electronic address: francesca.albonico@unimi.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Albonico
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano Via Celoria 10, Milano 20133, Italy. Electronic address: monica.loiacono@unimi.it.
authors.forename: Monica
authors.initials: M
authors.lastname: Loiacono
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano Via Celoria 10, Milano 20133, Italy. Electronic address: gloria.gioia@unimi.it.
authors.forename: Gloria
authors.initials: G
authors.lastname: Gioia
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano Via Celoria 10, Milano 20133, Italy. Electronic address: claudio.genchi@unimi.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Genchi
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano Via Celoria 10, Milano 20133, Italy. Electronic address: marco.genchi@unimi.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Genchi
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano Via Celoria 10, Milano 20133, Italy. Electronic address: michele.mortarino@unimi.it.
authors.forename: Michele
authors.initials: M
authors.lastname: Mortarino
mesh_terms: D000818:Animals; D018509:DNA, Helminth / Q000097:blood / Q000235:genetics*; D004183:Dirofilaria immitis / Q000235:genetics*; D060348:Dirofilaria repens / Q000235:genetics*; D004184:Dirofilariasis / Q000175:diagnosis* / Q000469:parasitology; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D018509:DNA, Helminth
keywords: Canine peripheral blood; Dirofilaria immitis; Dirofilaria repens; Real-time PCR–HRMA