{"id":{"repo_id":"nus","oai_identifier":"oai:scholarbank.nus.edu.sg:10635/212684"},"canonical_url":"https://search.dev.ndltd.org/etd/nus/oai:scholarbank.nus.edu.sg:10635/212684","repository":{"repo_id":"nus","name":"National University of Singapore","base_url":"https://scholarbank.nus.edu.sg/oai/request"},"display":{"title":"MULTIPLEX ASSAY DESIGN","abstract":"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.","abstract_html":"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. 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