ChapterDx MLSTnext MPXV NGS assay amplifies and sequences 21 MPXV loci, and provides genotyping results to the subtype level. The assay is designed for tracking the source of infection (viral surveillance). This assay can differentiate MPXV from other mammalian poxviruses, can distinguish MPXVs at different lineages,  and can also discriminate MPXVs at the same lineage such as those responsible for 2022 outbreak. The assay requires low amounts of nucleic acid material, has a relatively fast turnaround time, and is suitable for low, medium and high-throughput sample scales. The assay has a simple and user-friendly workflow and is easy to automate. The analysis software is included in the kit. 

ChapterDx MPXV NGS assay consists of the following steps:

1. DNA extraction (not provided)

2. Multiplex PCR

3. Pooling and purification

4. Next-generation sequencing

In the PCR reaction, both amplification and indexing/barcoding of each specimen occurs simultaneously. After PCR reaction, amplicons for different samples are pooled in one tube. The following procedures deal with only one tube regardless of how many samples. After the cleanup step, the library is sequenced on an Illumina sequencing platform. The entire workflow takes about 24 hours, of which about 3 hours is benchwork/incubation time and 17 hours of sequencing. It takes less than half hour to complete data analysis.

• Analyze sequences at 21 most polymorphic loci spanning poxvirus genome

• Loci are at diverse sites including intergenic regions

• Unaffected by ongoing mutations

• Differentiate MPXVs from poxviruses infecting other mammals

• Distinguish MPXVs at different lineages

• Discriminate MPXVs at same lineage

• Single-tube and single-step PCR reaction 

• User-friendly,  easy-to-automate workflow

• Cost-efficient and suitable for low, medium and high throughput scales

• Data analysis software is included

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