title: Comparison of point-of-care-compatible lysis methods for bacteria and viruses.
abstract: Nucleic acid sample preparation has been an especially challenging barrier to point-of-care nucleic acid amplification tests in low-resource settings. Here we provide a head-to-head comparison of methods for lysis of, and nucleic acid release from, several pathogenic bacteria and viruses-methods that are adaptable to point-of-care usage in low-resource settings. Digestion with achromopeptidase, a mixture of proteases and peptidoglycan-specific hydrolases, followed by thermal deactivation in a boiling water bath, effectively released amplifiable nucleic acid from Staphylococcus aureus, Bordetella pertussis, respiratory syncytial virus, and influenza virus. Achromopeptidase was functional after dehydration and reconstitution, even after eleven months of dry storage without refrigeration. Mechanical lysis methods proved to be effective against a hard-to-lyse Mycobacterium species, and a miniature bead-mill, the AudioLyse, is shown to be capable of releasing amplifiable DNA and RNA from this species. We conclude that point-of-care-compatible sample preparation methods for nucleic acid tests need not introduce amplification inhibitors, and can provide amplification-ready lysates from a wide range of bacterial and viral pathogens.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA, United States. Electronic address: erinkh@uw.edu.
authors.forename: Erin K
authors.initials: EK
authors.lastname: Heiniger
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA, United States.
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Buser
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA, United States.
authors.forename: Lillian
authors.initials: L
authors.lastname: Mireles
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA, United States.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA, United States.
authors.forename: Paula D
authors.initials: PD
authors.lastname: Ladd
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA, United States.
authors.forename: Barry R
authors.initials: BR
authors.lastname: Lutz
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Yager
mesh_terms: D001886:Bordetella pertussis / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D009161:Mycobacterium / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques; D009975:Orthomyxoviridae / Q000302:isolation & purification; D019095:Point-of-Care Systems; D012136:Respiratory Syncytial Viruses / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral
keywords: AudioLyse; DNA; Low resource settings; POC; RNA; Sample preparation