Development and analytical validation of a multiplex qPCR assay for Toxoplasma gondii , Treponema pallidum , cytomegalovirus, and herpes simplex virus types 1 and 2 in neonates

Background Congenital and perinatal infections caused by pathogens such as Toxoplasma gondii ( T. gondii ), Treponema pallidum ( T. pallidum ), cytomegalovirus (CMV), and herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are significant causes of neonatal morbidity. Traditional diagnostic methods, such as serological tests or viral cultures, are frequently limited by cross-reactivity, variable accuracy, and prolonged turnaround times. Therefore, this study aimed to design and analytically validate a rapid, highly sensitive, and specific multiplex quantitative real-time polymerase chain reaction (qPCR) assay for the detection of these critical neonatal pathogens. Methods The multiplex qPCR assay was optimized, and analytical performance, including sensitivity, specificity, and efficiency, was evaluated following Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results The assay demonstrated a limit of detection (LOD) of 10 attograms per microliter (ag/µL) for T. gondii, T. pallidum , and CMV, and 1 ag/µL for herpes simplex virus (HSV)-1 and HSV-2. Additionally, the assay showed 100% analytical specificity against 38 organisms. The PCR efficiency exhibited ranged between 95% and 109% for each organism, with a coefficient of variation (CV) of less than 3%. Conclusion This multiplex qPCR assay provides highly sensitive and specific analytical performance for detecting key congenital pathogens. It represents a promising high-throughput tool with the potential to improve the turnaround time of neonatal molecular screening.