Journal: Microorganisms

Article Title: Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1–9

doi: 10.3390/microorganisms12050932

Figure Lengend Snippet: Schematic representation of multiplex PCR-xMAP workflow. Nucleic acid is extracted in an automated extraction robot (i). The resulting eluent is then split across two wells and combined with biotinylated primers from multiplex reaction mix A in one well (for the detection of AHSV serotypes -1, -3, -5, and -7) and multiplex reaction mix B in the second well (for the detection of AHSV serotypes -2, -4, -6, -8, and -9) (ii). This mixture is then denatured, followed by the addition of qPCR mastermix (iii) and then RT-PCR (iv). The resulting amplified product is added to a 96-well plate preloaded with microspheres (v). Hybridisation of amplified product (if present) to microspheres occurs during an incubation step at 50 °C. Streptavidin, R-Phycoerythrin Conjugate (SAPE) is then added to each well and binds to the amplified product (if present) that is attached to the microsphere (vi). Finally, detection of both SAPE and microsphere is performed using the Bio-Plex 200 instrument (vii). Schematic created with Biorender.com.

Article Snippet: The plate was then placed in a Bio-Plex 200 instrument (Luminex corp., Hertogenbosch, The Netherlands) set to 50 °C, where it was incubated for 20 min prior to the detection of hybridised amplification product ( vii).

Techniques: Multiplex Assay, Extraction, Reverse Transcription Polymerase Chain Reaction, Amplification, Hybridization, Incubation