ArticleActa parasitologica2025
Optimization of a Conventional Assay into SYBR Green Real-Time PCR for the Detection of the Nc5 Segment from Neospora caninum.
Article in Acta parasitologica, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
purposeThe aim of the present study was to establish a SYBR Green-based real-time PCR assay for detection of the Nc5 segment from the Neospora caninum genome.
methodsThe oligonucleotides sequences targeting the Nc5 gene previously reported and designed in-house were validated. Two Primer sets were evaluated and tested in four different combinations. The NP7/NP10 assay was selected and reaction conditions optimized. Efficiency, analytical sensitivity, precision and specificity were assessed. The assay was evaluated in triplicate, in three independent PCR runs performed by two technicians to generate robust results.
resultsThe standard curve determined by tenfold serial dilutions (1 to 1 × 10-
conclusionThe protocol had high specificity, confirmed by melting curve analysis and no cross-reactions with other tested microorganisms. The SYBR Green-based PCR protocol standardized in this study is a highly sensitive and specific method, reproducible and applicable for the detection of N. caninum in different biological samples.
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