Genomics for Hospital-Acquired Infection Diagnosis
Research Focus
Hospital-acquired Staphylococcus aureus infections are leading causes of healthcare-associated mortality, but existing gold-standard diagnosis depends on bacterial culture, which has a days-long turnaround time that delays intervention. In Deborah Hung’s lab, I employed a two step approach to diagnose S. aureus infections from healthy cfDNA: a 107-locus multiplexed PCR amplification of S. aureus-specific genomic targets, and a CRISPR-Cas13 signal detection using SHERLOCK. My lab mentors, Jackson Lirette and Gowtham Thakku, published a similar project for Mycobacterium tuberculosis infections (WATSON), where they built an 18-locus panel that leveraged the IS6110 1-25x multicopy repeat genomic element in TB to increase sensitivity.
Key findings
Preliminary testing on an 18-locus subset pool resulted in an LOD of 0.1 Genome Equivalents per reaction, which was an order of magnitude higher than the WATSON TB LOD of 0.01 GE per reaction, as expected due to the absence of multicopy genomic elements in S. aureus. After increasing the 18-locus multiplex subset to the full 107-locus pool, non-uniform PCR amplification of genomic targets, coupled with differing signal-to-noise ratios of individual CRISPR-Cas13 guide RNAs, resulted in noisy readout.
I addressed rebalancing needs through systematic sequencing-based characterization of the primer pool, so that non-uniform input concentrations of primers could correct for non-uniform amplification of genomic targets.