A high-throughput multiplex RT-qPCR assay for detecting eight pathogens in experimental mice

This study aimed to establish an efficient multiplex, broad-spectrum real-time quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous screening of eight important pathogens prevalent in experimental rats and mice. These included murine rotavirus, Filobacterium rodentium, Cryptosporidium spp., Bordetella pertussis, Klebsiella oxytoca, Demodex spp., Campylobacter jejuni, and Helicobacter rodentium. This assay was developed to address the throughput, speed, and coverage limitations inherent to conventional detection methods.

Specific primers and TaqMan probes were designed based on conserved genomic sequences of the target pathogens using bioinformatic analysis. These components were integrated using a “three-channel fluorescence grouping” strategy to establish a one-step multiplex RT-qPCR workflow. A comprehensive methodological validation was performed, including assessments of the linear range, limit of detection (LOD), specificity, and robustness.

The assay demonstrated a broad linear range of 10¹–10⁶ copies/μL, excellent amplification efficiency of 91%–109% (R² > 0.99), and a low LOD of 12.5 copies. The coefficient of variation for all technical replicates was <1%. Cross-reactivity testing confirmed high specificity, as amplification was obstructed by potentially interfering DNA from mouse intestinal contents or the Escherichia coli genome. Furthermore, performance remained stable without any pronounced degradation of the reaction mixture after repeated freeze–thaw cycles.

The established octoplex RT-qPCR method is characterized by its simplicity, rapidity, high sensitivity, specificity, and robustness. It enables simultaneous detection of pathogens across categories, including viruses, bacteria, and parasites, thereby providing an efficient and reliable molecular tool for large-scale health monitoring, early disease diagnosis, and microbiological quality control in experimental rats and mice.