Evaluation of commercial DNA extraction methods for biosecurity applications

Bowman, S; Roffey, P; McNevin, D; Gahan, ME

Evaluation of commercial DNA extraction methods for biosecurity applications

Bowman, S Roffey, P McNevin, D

Gahan, ME

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Publisher:: TAYLOR & FRANCIS LTD
Publication Type:: Journal Article
Citation:: Australian Journal of Forensic Sciences, 2016, 48, (4), pp. 407-420
Issue Date:: 2016-07-03

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Field	Value	Language
dc.contributor.author	Bowman, S
dc.contributor.author	Roffey, P
dc.contributor.author	McNevin, D https://orcid.org/0000-0003-1665-3367
dc.contributor.author	Gahan, ME
dc.date.accessioned	2022-01-31T22:30:53Z
dc.date.available	2022-01-31T22:30:53Z
dc.date.issued	2016-07-03
dc.identifier.citation	Australian Journal of Forensic Sciences, 2016, 48, (4), pp. 407-420
dc.identifier.issn	0045-0618
dc.identifier.issn	1834-562X
dc.identifier.uri	http://hdl.handle.net/10453/154000
dc.description.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.
dc.language	English
dc.publisher	TAYLOR & FRANCIS LTD
dc.relation.ispartof	Australian Journal of Forensic Sciences
dc.relation.isbasedon	10.1080/00450618.2015.1106585
dc.rights	info:eu-repo/semantics/closedAccess
dc.subject.classification	Legal & Forensic Medicine
dc.title	Evaluation of commercial DNA extraction methods for biosecurity applications
dc.type	Journal Article
utslib.citation.volume	48
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - CFS - Centre for Forensic Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Mathematical and Physical Sciences
utslib.copyright.status	closed_access	*
dc.date.updated	2022-01-31T22:30:52Z
pubs.issue	4
pubs.publication-status	Published
pubs.volume	48
utslib.citation.issue	4

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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http://hdl.handle.net/10453/154000