Evaluation of commercial DNA extraction methods for biosecurity applications

Sorelle Bowman, Paul Roffey, Dennis McNevin, Michelle E. Gahan

    Research output: Contribution to journalArticle

    1 Citation (Scopus)

    Abstract

    An essential starting point when using molecular methods to identify bacterial biosecurity agents is an efficient extraction procedure that can extract DNA from Gram-positive and Gram-negative bacteria, lyse bacteria and remove inhibitors. ChargeSwitch gDNA mini bacteria kit (Invitrogen), QIAamp DNA extraction kit (Qiagen) with and without bead-beating, and Isolate II Genomic DNA kit (Bioline) were assessed for DNA extraction from Gram-positive (Bacillus thuringiensis) and Gram-negative (Escherichia coli) culture and environmental wipe samples. DNA was quantified using fluorometry, spectrophotometry and real-time polymerase chain reaction (PCR), and correlation between methods examined. In general, ChargeSwitch resulted in the highest DNA yield, however it was more expensive, did not remove environmental inhibitors or lyse all bacteria. Silica-based methods were efficient at lysing bacteria, removing inhibitors and generating sufficient DNA for downstream applications. Bead-beating added additional time and costs but did not significantly increase yields. There was limited correlation between DNA quantifications determined using fluorometry, spectrophotometry and real-time PCR. Results show a range of methods should be considered when developing extraction protocols for biosecurity applications with the optimal method dependant on sample type and starting material amount. Isolate II is recommended for extraction from culture or wipe samples, particularly with small quantities commonly encountered in biosecurity scenarios
    Original languageEnglish
    Pages (from-to)1-15
    Number of pages15
    JournalAustralian Journal of Forensic Sciences
    Volume48
    Issue number4
    DOIs
    Publication statusPublished - 2016

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    DNA
    Bacteria
    Fluorometry
    Spectrophotometry
    Real-Time Polymerase Chain Reaction
    Bacillus thuringiensis
    Gram-Negative Bacteria
    Silicon Dioxide
    Escherichia coli
    Costs and Cost Analysis

    Cite this

    Bowman, Sorelle ; Roffey, Paul ; McNevin, Dennis ; Gahan, Michelle E. / Evaluation of commercial DNA extraction methods for biosecurity applications. In: Australian Journal of Forensic Sciences. 2016 ; Vol. 48, No. 4. pp. 1-15.
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    abstract = "An essential starting point when using molecular methods to identify bacterial biosecurity agents is an efficient extraction procedure that can extract DNA from Gram-positive and Gram-negative bacteria, lyse bacteria and remove inhibitors. ChargeSwitch gDNA mini bacteria kit (Invitrogen), QIAamp DNA extraction kit (Qiagen) with and without bead-beating, and Isolate II Genomic DNA kit (Bioline) were assessed for DNA extraction from Gram-positive (Bacillus thuringiensis) and Gram-negative (Escherichia coli) culture and environmental wipe samples. DNA was quantified using fluorometry, spectrophotometry and real-time polymerase chain reaction (PCR), and correlation between methods examined. In general, ChargeSwitch resulted in the highest DNA yield, however it was more expensive, did not remove environmental inhibitors or lyse all bacteria. Silica-based methods were efficient at lysing bacteria, removing inhibitors and generating sufficient DNA for downstream applications. Bead-beating added additional time and costs but did not significantly increase yields. There was limited correlation between DNA quantifications determined using fluorometry, spectrophotometry and real-time PCR. Results show a range of methods should be considered when developing extraction protocols for biosecurity applications with the optimal method dependant on sample type and starting material amount. Isolate II is recommended for extraction from culture or wipe samples, particularly with small quantities commonly encountered in biosecurity scenarios",
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    Evaluation of commercial DNA extraction methods for biosecurity applications. / Bowman, Sorelle; Roffey, Paul; McNevin, Dennis; Gahan, Michelle E.

    In: Australian Journal of Forensic Sciences, Vol. 48, No. 4, 2016, p. 1-15.

    Research output: Contribution to journalArticle

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