Discovery of Novel Diagnostic Biomarkers for Common Pathogenic Nocardia through Pan- genome and Comparative Genome Analysis, with Preliminary Validation

Abstract Purpose This study aims to utilize pan-genome and comparative genome analysis techniques to reveal diagnostic biomarkers of considerable importance for common pathogenic Nocardia, thereby facilitating accurate identification of clinical Nocardia infections. Methods Complete or assembled genome sequences of common pathogenic Nocardia and closely related species were obtained from NCBI (https://www.ncbi.nlm.nih.gov/) as discovery and validation sets, respectively. Genome annotation was performed using Prokka software, and pan-genomic analysis and extraction of Nocardia core genes were performed using BPGA software. Comparative genome analysis of these core genes with the validation set gene sequences was then performed using BLAT, with a threshold of 30% amino acid coverage and identity to distinguish specific core genes. Finally, candidate gene-specific primers were designed using Snapgene software and DNA samples were obtained from clinical Nocardia strains and closely related species for validation. Results The analysis identified 18 core genes specific to Nocardia spp., 4 core genes specific to N. farcinica, and 46 core genes specific to N. cyriacigeorgica. After rigorous clinical validation, 1 gene (F6W96_34950) from Nocardia spp. and 5 genes (NCTC10797_02287, NCTC10797_01760, NCTC10797_05842, NOCYR_2299 and C5B73_13220) from N. cyriacigeorgica all showed relatively high specificity, suggesting their potential as promising biomarkers for the diagnosis of Nocardia infections. Conclusion This pioneering research reveals diagnostic biomarkers of considerable significance, with the potential to substantially enhance the precise diagnosis of common pathogenic Nocardia infections, thereby laying the groundwork for innovative diagnostic methodologies in subsequent studies.