title: A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes.
abstract: This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast™ EvaGreen(®) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.
authors: Dipartimento di Sanità Pubblica e Zootecnia, Università degli Studi di Bari, Strada Provinciale per Casamassima km 3, 70010 Valenzano (Bari), Italy.
authors: Maria Stefania
authors: MS
authors: Latrofa
authors: Filipe
authors: F
authors: Dantas-Torres
authors: Giada
authors: G
authors: Annoscia
authors: Marco
authors: M
authors: Genchi
authors: Donato
authors: D
authors: Traversa
authors: Domenico
authors: D
authors: Otranto
mesh_terms: D000818:Animals; D009033:Culicidae / Q000469:parasitology*; D004183:Dirofilaria immitis; D060348:Dirofilaria repens; D004184:Dirofilariasis / Q000097:blood / Q000469:parasitology*; D004283:Dog Diseases / Q000097:blood / Q000469:parasitology*; D004285:Dogs; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013045:Species Specificity