{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:10:00Z","timestamp":1785409800787,"version":"3.56.0"},"reference-count":41,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T00:00:00Z","timestamp":1776816000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82202593"],"award-info":[{"award-number":["82202593"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["82202593"],"award-info":[{"award-number":["82202593"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100014764","name":"Beijing Medical Award Foundation","doi-asserted-by":"publisher","award":["YXTL-2024-0002-0600"],"award-info":[{"award-number":["YXTL-2024-0002-0600"]}],"id":[{"id":"10.13039\/501100014764","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Government funded clinical medical talents training project","award":["ZF2024001"],"award-info":[{"award-number":["ZF2024001"]}]},{"name":"Hebei Province Medical Science Research Project","award":["2024021"],"award-info":[{"award-number":["2024021"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>In this study, we developed a multiplex one-tube nested real-time fluorescent quantitative PCR (mONRT-PCR) assay integrated with a portable, fully automated nucleic acid point-of-care testing (POCT) platform for the detection of Haemophilus influenzae (H. influenzae), Listeria monocytogenes (L. monocytogenes), and Cryptococcus neoformans (C. neoformans) in cerebrospinal fluid (CSF). The assay enables nested amplification within a closed system using conventional primers and probes, thereby reducing operational complexity and minimizing contamination risk. Analytical evaluation demonstrated limits of detection of 100 copies\/\u03bcL for H. influenzae and L. monocytogenes, and 101 copies\/\u03bcL for C. neoformans using recombinant plasmids, as well as 10\u22127 to 10\u22126 ng\/\u03bcL using genomic DNA. No cross-reactivity was observed when tested against a panel of 17 common non-target microorganisms encountered in clinical microbiology laboratories. In simulated CSF samples, the assay maintained detectable amplification at low pathogen concentrations. When implemented on the POCT platform, detection limits reached 5, 10, and 50 CFU\/mL for the three pathogens, respectively. Clinical evaluation using 43 CSF samples showed almost perfect agreement with conventional qPCR (\u03ba = 0.861, p &lt; 0.001). Notably, additional C. neoformans detections were observed by mONRT-PCR-POCT compared with qPCR, suggesting improved sensitivity under clinical conditions. The assay yielded results within approximately 1 h and 47 min. These findings indicate that the proposed assay provides a rapid, sensitive, and integrated approach for meningitis pathogen detection, while maintaining a practical balance between analytical performance and operational simplicity. Further validation in larger cohorts is warranted.<\/jats:p>","DOI":"10.3390\/pathogens15050456","type":"journal-article","created":{"date-parts":[[2026,4,22]],"date-time":"2026-04-22T10:38:14Z","timestamp":1776854294000},"page":"456","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Multiplex One-Tube Nested Real-Time PCR Assay for the Point-of-Care Testing of Infectious Meningitis"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-7014-0277","authenticated-orcid":false,"given":"Duoxiao","family":"Zhang","sequence":"first","affiliation":[{"name":"Graduate School, Hebei North University, Zhangjiakou 075000, China"},{"name":"Department of Clinical Laboratory, Hebei General Hospital, Shijiazhuang 050051, China"},{"name":"Hebei Key Laboratory of Molecular Medicine, Shijiazhuang 050051, China"},{"name":"Hebei Clinical Research Center for Laboratory Medicine, Shijiazhuang 050051, 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