Efficient differentiation of Nocardia farcinica, Nocardia cyriacigeorgica and Nocardia beijingensis by high-resolution melting analysis using a novel locus

Accurate identification of Nocardia species remains a challenge due to the complexities of taxonomy and insufficient discriminatory power of traditional techniques. We report the development of a molecular technique that utilizes real-time PCR-based high-resolution melting (HRM) analysis for differentiation of the most common Nocardia species. Based on a novel fusA-tuf intergenic region sequence, Nocardia farcinica , Nocardia cyriacigeorgica and Nocardia beijingensis were clearly distinguished from one another by HRM analysis. The limit of detection of the HRM assay for purified Nocardia spp. DNA was at least 10 fg. No false positives were observed for specificity testing of 20 non-target clinical samples. In comparison to established matrix-assisted laser desorption/ionization-time of flight MS, the HRM assay improved the identification of N. beijingensis . Additionally, all the products of PCR were verified by direct sequencing. In conclusion, the developed molecular assay allows simultaneous detection and differentiation of N. farcinica , N. cyriacigeorgica and N. beijingensis with high sensitivity and specificity.