Rebecca D Villa, Michael A Pentella, Jeffrey L Benfer

In May 2022, an exported mpox case was confirmed in the United Kingdom, eventually evolving into a multi-country outbreak. In response, the Centers for Disease Control and Prevention recommended the Non-variola Orthopoxvirus Generic Real-Time PCR Test for public health labs to test clinical samples; however, this method is not clade specific and was originally designed as a low-volume screening assay. Therefore, aspects of the test (timeliness, labor costs, and workflow efficiency) required improvement if high-volume testing were to become necessary. Here, we describe the process for developing and optimizing a laboratory developed assay (LDA) for detecting clade II mpox virus using the automated Panther Fusion platform and Open Access software. Various concentrations of reagents in a primer-probe mix were tested to optimize the LDA. The LDA was validated using 10 previously characterized positive and 10 negative human mpox samples, resulting in 95% accuracy and 100% precision. The LDA resulted in 100% specificity among previously tested HSV1, HSV2, and VZV positive human samples. Several spiked media extensions (Aptima Multitest Swab Transport Media, RTM4, an in-house Universal Transport Medium, and dry swabs) were also validated and achieved 98% accuracy and 100% precision across all collection media types. The assay's LOD was calculated to be 1.475 copies/reaction, and the PCR efficiency resulted in 89.87% (slope: -3.5911, R2: 0.9947). The methods described in this paper can be applied to the rapid optimization and development of LDAs for many possible pathogens of public health importance.