Molecular detection of Nocardia: development and application of a real-time PCR assay in sputum and bronchoalveolar lavage fluid samples.

Abstract

The diagnosis of pulmonary nocardiosis remains challenging. Rapid detection of Nocardia is of primary importance for early diagnosis and precise treatment of nocardiosis. In this study, our objective was to develop and validate a new TaqMan real-time PCR (qPCR) assay for rapidly detecting Nocardia spp. in respiratory samples. Based on published sequence data, primers in a conserved region of the 16S rRNA gene and a probe within that region that was specific for Nocardia were designed. The distinction effect of the qPCR assay was assessed between Nocardia and other respiratory-associated bacteria. Furthermore, the specificity and sensitivity of the assay were evaluated in respiratory clinical samples (n = 205), compared to the results of 16S rRNA gene amplicon sequencing and clinical diagnosis. The qPCR assay exhibited high specificity, sensitivity, repeatability, and reproducibility. The limit of detection of standard plasmid DNA was 3 × 102 copies/mL. Additionally, the qPCR assay was applied to the direct detection of 205 clinical respiratory samples. The specificity and sensitivity of the qPCR were all 100% compared to 16S rRNA gene amplicon sequencing, as well as 98.4% and 100% compared to clinical diagnosis respectively. The qPCR yielded results within 3h of sample processing, compared to several days for culture, significantly reducing turnaround time. The results suggest that the new qPCR assay developed in this study provides reliable and rapid detection of Nocardia spp. in the respiratory tracts and is expected to reduce the time required for diagnosing and treating nocardiosis.