Infectious diseases caused by pathogenic bacteria are a significant burden on public health and the economy. Current methods for identifying pathogenic bacteria are time consuming, particularly with polymicrobial infections which complicate patient diagnosis and treatment. Nucleic acid amplification tests (NAATs) have served to dramatically accelerate bacteria identification in polymicrobial infections. These techniques rely on PCR-based amplification of conserved bacteria genes as well as downstream high-resolution melt (HRM) analysis for broad bacteria identification. Once identified, effective antibiotic treatment and dosage must be determined using an antimicrobial susceptibility test (AST). Pheno-molecular AST combines growth-based phenotyping using antibiotics with NAATs to assess antimicrobial susceptibility. However, there is currently no commercially available platform that simultaneously performs reliable AST and broad bacteria identification in polymicrobial samples.
In a recent publication in Analytical Chemistry, Athamanolap et al. developed the first digital PCR and HRM (dPCR-HRM) platform for broad bacteria ID and pheno-molecular AST toward diagnosis of infectious diseases. The main component of the platform is a Nanoarray capable of digitizing single bacteria DNA molecules for amplification and analysis. The assay utilizes universal PCR primers that flank hypervariable regions of the 16S rRNA gene and EvaGreen® dye to facilitate dPCR and HRM analysis. This allows individual melt curve profiles to be derived from each bacterial species within a polymicrobial sample. A machine learning-based algorithm can then identify each species using a digital melt curve database. The platform simultaneously performs AST by comparing the amount of DNA in control and antibiotic treated samples. The authors validated the platform by correctly identifying both the bacterial species and antimicrobial resistance within a simulated polymicrobial urine sample containing S. aureus and gentamicin-resistant E. coli. The authors envision that the platform can be continually improved and expanded for use in more complex clinical samples, facilitating precision-directed therapy and improving patient outcomes.
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Athamanolap, P. et al. Nanoarray digital PCR with high-resolution melt enables broad bacteria identification and pheno-molecular antimicrobial susceptibility test. Anal. Chem. 91, 12784–12792 (2019).