Development and Proof-of-Concept Analytical Validation of a HotStart Loop-Mediated Isothermal Amplification (LAMP)...

Macacine alphaherpesvirus 1 (McHV-1, commonly known as Monkey B Virus) is a fatal zoonotic pathogen naturally prevalent in macaques. In this study, we developed and conducted a proof-of-concept analytical validation of a HotStart Loop-Mediated Isothermal Amplification (LAMP) assay targeting a conserved, moderately GC-rich (61-62%) region of the UL1 gene in McHV-1. The reaction was optimized at 70 °C using engineered HotStart Bst 3.2/4.2 DNA Polymerase, tracked via real-time fluorescence and closed-tube visual colorimetric detection (pre-added L-HNB dye) without post-amplification tube opening to reduce the risk of post-amplification aerosol carry-over contamination. The assay achieved a preliminary practical analytical limit of detection (analytical LOD) of 100 copies/reaction (3/3 replicates) within a 15-30 min diagnostic cutoff window, matching the practical sensitivity of reference qPCR. Strict analytical specificity was demonstrated against a comprehensive panel of human herpesviruses (HHV-1 through HHV-5) and host macaque genomic DNA. As a single-target analytical prototype, this visual HotStart LAMP platform provides a rapid, equipment-free molecular tool with promising potential for McHV-1 surveillance in resource-limited settings and macaque breeding facilities upon further clinical validation.