Paul Roffey

Recovery and identification of bacterial DNA from illicit drugs

Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002-2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated. Methods based on the ChargeSwitch(®)gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS). An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch(®) method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed. This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug users as well as enabling forensic links between seizures to be examined.

Comparison between magnetic bead and qPCR library normalisation methods for forensic MPS genotyping

Massively parallel sequencing (MPS) is fast approaching operational use in forensic science, with the capability to analyse hundreds of DNA identity and DNA intelligence markers in multiple samples simultaneously. The ForenSeq™ DNA Signature Kit on MiSeq FGx™ (Illumina) workflow can provide profiles for autosomal short tandem repeats (STRs), X chromosome and Y chromosome STRs, identity single nucleotide polymorphisms (SNPs), biogeographical ancestry SNPs and phenotype (eye and hair colour) SNPs from a sample. The library preparation procedure involves a series of steps including target amplification, library purification and library normalisation. This study highlights the comparison between the manufacturer recommended magnetic bead normalisation and quantitative polymerase chain reaction (qPCR) methods. Furthermore, two qPCR chemistries, KAPA® (KAPA Biosystems) and NEBNext® (New England Bio Inc.), have also been compared. The qPCR outperformed the bead normalisation method, while the NEBNext® kit obtained higher genotype concordance than KAPA®. The study also established an MPS workflow that can be utilised in any operational forensic laboratory.

An overview of biosecurity in Australia

Threats of natural and deliberate biological agent release are very real and are of serious concern within Australia and internationally, having the potential to substantially impact human, plant and animal health as well as agriculture, trade relations, infrastructure and the economy. Biosecurity programmes and policies are essential for counteracting these threats and enabling a nation to effectively prevent and/or respond to the natural, accidental and deliberate release of a biological agent. The major biosecurity focus in Australia is the protection against invasive animal, plant and insect species; however, more recently, the threat of microbiological agents has become a priority for Australia’s national security. Australia’s biosecurity capabilities are maintained through a coordinated effort, involving national, state and local government and non-government agencies. Communication and collaboration between all parties, at both a national and international level, is the key to an effective response. The threat from biological agents is a very real concern and Australia must maintain its preparedness in order to ensure its capacity to respond to biological threats. This review provides a contemporary overview of biosecurity in Australia and the role of forensic science in the investigation of biosecurity incidents.

Species identification using high resolution melting (HRM) analysis with random forest classification

Abstract Species identification is an important facet of forensic investigation. In this study, human and non-human species (cow, chicken, pig, sheep, cat, dog, rabbit, fox, kangaroo and wombat) were assayed on the ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) to rapidly screen for their species of origin using the high resolution melt (HRM) analysis targeting the 16S rRNA gene. Classification of HRM difference profiles using the onboard ViiA 7 software resulted in a classification accuracy of <20%. Derivative profiles (temperature versus negative first derivative of fluorescence, –dF/dT) were classified using random forest algorithms supplemented by bagging and boosting, with either a randomly partitioned test set or a variety of folds of cross-classification, in addition to a range of trees and variables. Random forest classification with bagging conditions (constructed over 500 trees) was found to considerably outperform the ViiA 7 software for species differentiation with 100% classification accuracy for biological material from humans, domestic pets (cat and dog) and consumable meats (chicken and sheep) with an average classification accuracy of 70% across all species.

Application of a mobile laboratory for real time response to incidents

With an increase in requirements for rapid identification of chemical and biological agents in cases of CBRNE incidents the Australian Federal Police have utilised the concept of a mobile laboratory (Mobilab). The vehicle was manufactured by ENG (Concord, CA, USA) and equipped with an Agilent GC-MS/DFPD for identification of chemical warfare agents. A class 2 biohazard cabinet (Germfree) and glove box (Plas-Labs) are utilised for sample preparation for suspected biological agents. Presumptive identification of biological agents is performed using portable rapid analysis systems and microscopic techniques. The concept of operation and deployment options are described and discussed.

Evaluation of commercial DNA extraction methods for biosecurity applications

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.