Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction

• Published in: Journal of biomedical nanotechnology Vol. 15; no. 7; p. 1598
• Main Authors: Liu, Hongna, Heflin, Kathryn, Han, Jian, Conover, Matt, Wagner, Leslie, Bertrand, Jeff, Ewing, Phillip, Lu, Stanley, Clemmons, Scott, Watts, John, Li, Song
• Format: Journal Article
• Language: English
• Published: United States 01.07.2019
• Subjects: Gram-Positive Bacteria; Multiplex Polymerase Chain Reaction; Pathology, Molecular; Reproducibility of Results
• ISSN: 1550-7033
• DOI: 10.1166/jbn.2019.2783

We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 10 -1.7 × 10 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.