Development of a quadruplex PCR amplicon next generation sequencing assay for detection and differentiation of spp.

The genus includes a group of species that are associated with a wide range of mammalian species, including human. It is challenging to detect all species using a single molecular target due to its high genetic diversity. To solve this issue, we developed a quadruplex PCR amplicon sequencing assay using next-generation sequencing (NGS) technology for the detection and differentiation of species. Our objective was to obtain the specific sequences of a minimum of two of the four target genes as confirmation of the identity of a particular species using the assay. Four pairs of primers targeting specific regions on , and were evaluated for their capability of differentiating species individually and collectively by performing singular PCR amplicon sequencing and quadruplex PCR amplicon sequencing. Using the quadruplex PCR amplicon sequencing, 24 reference species were tested, all of which were successfully differentiated by at least two targets. pecies were accurately identified from the artificially mixed DNA templates developed to simulate coinfections. The limit of detection was determined to be 1 fg based on testing a series of 10-fold dilutions of DNA from the species. Testing of high DNA concentrations of 19 non- species showed high specificity with none of the non- species misclassified as . Finally, the assay was evaluated by testing DNA extracts from field-collected body lice () and Norway rats (): was detected and confirmed by three targets in the lice and was detected and confirmed by two targets in the rats. These results demonstrated that species could be accurately and rapidly detected and differentiated into different tissue types using the quadruplex sequencing assay.