A Laboratory Developed Assay for Clade II Human Mpox Virus on the Panther Fusion Open Access System.

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.

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