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 mortality and morbidity worldwide, particularly meningitis. Efficient 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-effective EvaGreen-based real-time PCR to simultaneously 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 simultaneously detect N. meningitidis and S. pneumoniae in a single tube from cerebrospinal fluid. In a single-tube reaction, melting curve analysis was used to differentiate the amplicons of each pathogen. Analytical sensitivity and specificity of the assay were conducted by reference bacterial 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 offers a rapid, affordable, and appropriate diagnostic tool to identify the main cause of bacterial meningitis.