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.
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A duplex real-time PCR has been assessed for discrimination between D. immitis and D. repens and their quantification in blood samples and mosquitoes.
“Recently, a duplex real-time PCR has been assessed for the discrimination between D. immitis and D. repens and their quantification in blood samples and mosquitoes [130].”
From Vector-borne helminths of dogs and humans in Europe (2013) · cites this paper as [130]
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- Authors
- Latrofa, M. S., Dantas-Torres, F., Annoscia, G., Genchi, M., Traversa, D., & Otranto, D.
- Affiliations
- University of Bari Aldo Moro (IT)
- University of Milan (IT)
- University of Teramo (IT)
- Corresponding author
- Otranto, Domenico
- Venue
- Veterinary Parasitology
- Publisher
- Elsevier BV
- Published
- 2012-04
- Volume
- 185
- Issue
- 2-4
- Pages
- 181-185
- ISSN
- 0304-4017 (ISSN-L)
- OpenAlex
- W2051126021
- Cited by
- 47
- References
- 41
- Citations by year
- 2026: 1 · 2025: 4 · 2024: 4 · 2023: 1 · 2022: 4 · 2021: 4 · 2019: 3 · 2018: 1 · 2017: 8 · 2016: 2 · 2015: 4 · 2013: 7 · 2012: 4
- FWCI
- 2.38
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Veterinary Parasitology (publishedVersion)
- PubMed (publishedVersion)
- CINECA IRIS Institutional Research Information System (University of Bari Aldo Moro) (submittedVersion)
- Funders
- University of Melbourne
- Topics
- Health Sciences › Medicine › Infectious Diseases › Parasitic Diseases Research and Treatment
- Health Sciences › Veterinary › Small Animals › Helminth infection and control
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Research on Leishmaniasis Studies
- Keywords
- Dirofilaria immitis, Dirofilaria repens, canine heartworm disease, filarial infection in mosquitoes, duplex real-time PCR, species differentiation, cytochrome c oxidase subunit I, ITS2, microfilariae, adult worms, Aedes albopictus, Aedes aegypti, Culex pipiens, molecular detection, disease surveillance, dogs
- MeSH terms
- Animals, Dirofilaria immitis, Dirofilariasis, Dog Diseases, Dogs, Culicidae, Species Specificity, Dirofilaria repens, Real-Time Polymerase Chain Reaction
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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