Citation

Bowman S, Roffey P, McNevin D, Gahan ME. Aust. J. Forensic Sci. 2016; 48(4): 407-420.

Copyright

(Copyright © 2016, Australian Academy of Forensic Sciences, Publisher Informa - Taylor and Francis Group)

DOI

10.1080/00450618.2015.1106585

PMID

unavailable

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.

Language: en