The present study reports the applicability of a multiplex PCR for the simultaneous detection and differentiation of common filarioids infecting dogs, i.e., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum and Cercopithifilaria sp. Amplicons of different sizes (i.e., 170 bp, 480 bp, 590 bp and 300 bp, respectively) of regions within the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were amplified on a single-step multiplex PCR using a mix of species-specific forward primers coupled with a single reverse primer. Experiments were carried out by amplifying genomic DNA extracted from blood or skin samples test-positive for microfilariae (mff). The number of mff present in each blood sample was quantified (from 800 to 25,000 mff/ml for A. reconditum and D. repens, respectively) and mixed blood samples were tested for the simultaneous detection of DNA from these mff. Specific amplicons for blood-circulating mff of A. reconditum, D. immitis and D. repens and for those whose adults are localized in skin (i.e., A. reconditum and Cercopithifilaria sp.) were simultaneously detected on agarose gel up to a dilution of 250 mff/ml for D. repens. The specific identity of the amplicons was confirmed by sequencing. The multiplex PCR assay reported herein represents a new tool for the molecular detection and differentiation of canine filarioids in blood and skin samples.

A multiplex PCR for the simultaneoous detection of species of filarioids infesting dogs.

LATROFA, MARIA STEFANIA;WEIGL, STEFANIA;DANTAS TORRES, FILIPE;ANNOSCIA, GIADA;OTRANTO, Domenico
2012

Abstract

The present study reports the applicability of a multiplex PCR for the simultaneous detection and differentiation of common filarioids infecting dogs, i.e., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum and Cercopithifilaria sp. Amplicons of different sizes (i.e., 170 bp, 480 bp, 590 bp and 300 bp, respectively) of regions within the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were amplified on a single-step multiplex PCR using a mix of species-specific forward primers coupled with a single reverse primer. Experiments were carried out by amplifying genomic DNA extracted from blood or skin samples test-positive for microfilariae (mff). The number of mff present in each blood sample was quantified (from 800 to 25,000 mff/ml for A. reconditum and D. repens, respectively) and mixed blood samples were tested for the simultaneous detection of DNA from these mff. Specific amplicons for blood-circulating mff of A. reconditum, D. immitis and D. repens and for those whose adults are localized in skin (i.e., A. reconditum and Cercopithifilaria sp.) were simultaneously detected on agarose gel up to a dilution of 250 mff/ml for D. repens. The specific identity of the amplicons was confirmed by sequencing. The multiplex PCR assay reported herein represents a new tool for the molecular detection and differentiation of canine filarioids in blood and skin samples.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11586/135037
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? 22
  • Scopus 47
  • ???jsp.display-item.citation.isi??? 45
social impact