Designing and Development of Simultaneous Detection of Neisseria meningitidis and Streptococcus pneumoniae based on EvaGreen Real-Time PCR

Background: Neisseria meningitidis and Streptococcus pneumoniae are serious causes of invasive infections associated with high mor-tality and morbidity worldwide, particularly meningitis. Eÿcient diagnostic strategies play a crucial role in the management of disease and the prevention of overtreatment. The low sensitivity and time-consuming nature of culture and gram stain methods have led to the demand for alternative methods in clinical laboratories. Objectives: This study aims to design and develop a rapid, sensitive, and cost-e ective EvaGreen-based real-time PCR to simultane-ously detect N. meningitidis and S. pneumoniae. Methods: We designed and evaluated an accurate, reliable, and inexpensive approach based on EvaGreen dye real-time PCR to si-multaneously detect N. meningitidis and S. pneumoniae in a single tube from cerebrospinal fluid. Melting curve analysis was used to di erentiate the amplicons of each pathogen. Analytical sensitivity and specificity of the assay were conducted by reference bacte-rial strains genomes. Besides, in order to clinical validation we used 53 positive CSF samples and 7 negative CSF samples. Results: Our assay demonstrated no amplification curve with non-target microorganisms indicating 100% analytical specificity. In the EvaGreen multiplex assay, the lower limit of detection (LLD) was nine copies/reaction for N. meningitidis and 13 copies/reaction for S. pneumoniae. The clinical validation of positive CSF samples revealed 100% sensitivity and no false positives. The reproducibility and repeatability of tested replicates indicated low intra-assay and inter-assay CVs of less than 1.5%. Conclusions: EvaGreen-based multiplex real-time PCR o ers a rapid, a ordable, and appropriate diagnostic tool to identify the main cause of bacterial meningitis.