MULTIPLEX ASSAY DESIGN

This thesis explores the design of assays for the multiplexed detection of various infectious diseases. A single-pot, 8-plex end-point PCR reaction for the detection of carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant Enterococcus (VRE) and colistin resistance has been developed in this thesis. The resulting amplicons can be distinguished based on the principles of size, melting-state transitions, and sequence-specific probes. The problem of false-negative methicillin-resistant Staphylococcus aureus (MRSA) calls by commercial assays was also explored. A novel target for the identification of Staphylococcus aureus was identified and validated. It is expected to reduce misclassification of MRSA and be more robust to emerging sequence variations of MRSA. A deeper exploration into the theme of developing assays tolerant to emerging sequence variations was undertaken. This resulted in the discovery of a new oligonucleotide primer design principle that enhances tolerance to primer base pair mismatches, yet at the same time retains the specificity of the assay.

Author dc:creator

• MUHAMMAD NADJAD BIN ABDUL RAHIM

• diagnostics, antimicrobial resistance, multiplex, PCR, MRSA, CPE, VRE, COVID-19