In order to address a global need for rapid, cost-effective, sensitive, and specific assays for many pathogens, CDC scientists have developed a broad-use, multiplexed RT-PCR assay. This comprehensive assay covers numerous pathogens that are common causes of infection in neonates and also important to food-safety. Specifically, this assay (and respective probes, primers, and kits) is capable of detecting one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Toxoplasma gondii, Moraxella catarrhalis, Escherichia coli, Shigella, Staphylococcus aureus, Pneumocystis jirovecii, Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Ureaplasma spp., Bartonella spp., Streptococcus agalactiae, and Neisseria meningitidis in a biological sample.