Improved specificity of a probe-based real-time PCR for the detection of Dirofilaria immitis in canine blood
Abstract
Background: Dirofilaria immitis is a vector-borne filarioid nematode distributed worldwide and endemic across most of North America. In dogs, it is considered the most clinically significant parasite and a known cause of acquired cardiomyopathy. Clinically, canine heartworm disease can manifest acutely as caval syndrome and chronically as heart failure, often resulting in death. Different types of diagnostic tests, such as antigen-detection and microfilariae-detection tests (MFDTs), including modified Knott's test and molecular assays, may be used to diagnose D. immitis infection. Although some molecular tools, such as real-time PCR (qPCR), exhibit high accuracy, cross-reactivity with other Dirofilaria species found in companion animals remains a concern. Following our laboratory's previously published protocol, cross-reactivity was found with Dirofilaria striata isolated from a subcutaneous nodule of a cat from Texas. The objectives of this study were to: (i) evaluate the performance of three newly designed probe-based qPCR assays for detecting D. immitis and (ii) assess overall detection with the newly designed protocols compared with the previously published protocol and other diagnostic tests.
Methods: We designed three new probes using accessioned D. immitis cytochrome c subunit 1 sequences from various countries worldwide to increase reliability. These new probes were then tested with genomic DNA extracted from D. immitis and D. striata. Following this, we further…
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