Development of a multiplex, PCR-based genotyping assay for African and Asian elephants for forensic purposes.

Wildlife crimes and the threats they present to elephant populations raise the need to develop and implement DNA-based methodology as an aid for wildlife forensic investigations and conservation efforts. This study describes the development of a tetra-nucleotide repeat STR multiplex, genotyping assay that will identify Asian elephant (Elephas maximus) and African elephant (Loxodonta africana) DNA. The assay targets six tetra-nucleotide STRs and two sex-typing markers simultaneously in both genera of elephants, a first for elephant genotyping assays. The developed assay has potential application in wildlife investigations to associate a biological sample to a particular individual elephant and additionally in conservation science for population management.

ELEquant: a developmental framework and validation of forensic and conservation real-time PCR assays.

A framework for the development and validation of a qPCR assay for species identification and DNA quantification for conservation and forensic purposes is presented. Elephants are commonly poached for their ivory tusks, which is the primary driving force behind their endangered status. In addition to poaching and trade, habitat loss due to logging and mining has also resulted in loss of elephants. Crimes against animals can be deterred and/or further prosecution sought through testing with forensic genetic techniques. The creation of novel genetic assays can greatly impact wildlife forensic science investigations in identifying the species. Molecular genetic techniques can help enforce conservation efforts; however, they must be properly developed and validated to be of evidentiary quality for court systems. African and Asian elephant buccal cells were used as model in this work. The assay provides a method to differentiate biological fluids of both genera of elephants simultaneously. It can be used for identification of elephant derived products and presents valuable quantification for optimized further testing, such as microsatellite detection.

Southern South Australian groundwater microbe diversity.

Groundwater is increasingly used globally for domestic, industrial and agricultural production. While many studies have focused on groundwater as a resource, the diverse ecosystems within are often ignored. Here, we assess 54 Southern South Australian groundwater microbial communities from the populated part of the state to assess their status and dynamics in isolated groundwater systems. We observed a strong site-to-site individuality in groundwater bacterial communities, likely due to the isolated nature of groundwater bodies leading to unique ecosystems. Rank abundance analysis indicates bacterial diversity is maintained even at low abundances and that the distribution fits classical ecological models for strong competition in resource-limited environments. Combined, our data indicates that despite overrepresentation of pollutant-associated bacterial orders in and around the Adelaide metropolitan area, microbial communities remain diverse and show little evidence of converging on a common pollutant-effected community.

Microbial micropatches within microbial hotspots.

The spatial distributions of organism abundance and diversity are often heterogeneous. This includes the sub-centimetre distributions of microbes, which have 'hotspots' of high abundance, and 'coldspots' of low abundance. Previously we showed that 300 µl abundance hotspots, coldspots and background regions were distinct at all taxonomic levels. Here we build on these results by showing taxonomic micropatches within these 300 µl microscale hotspots, coldspots and background regions at the 1 µl scale. This heterogeneity among 1 µl subsamples was driven by heightened abundance of specific genera. The micropatches were most pronounced within hotspots. Micropatches were dominated by Pseudomonas, Bacteroides, Parasporobacterium and Lachnospiraceae incertae sedis, with Pseudomonas and Bacteroides being responsible for a shift in the most dominant genera in individual hotspot subsamples, representing up to 80.6% and 47.3% average abundance, respectively. The presence of these micropatches implies the ability these groups have to create, establish themselves in, or exploit heterogeneous microenvironments. These genera are often particle-associated, from which we infer that these micropatches are evidence for sub-millimetre aggregates and the aquatic polymer matrix. These findings support the emerging paradigm that the microscale distributions of planktonic microbes are numerically and taxonomically heterogeneous at scales of millimetres and less. We show that microscale microbial hotspots have internal structure within which specific local nutrient exchanges and cellular interactions might occur.

Using synthetic oligonucleotides as standards in probe-based qPCR.

Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming, costly, necessitate a specific skill set, and pose a contamination risk. Using a targeted synthetic oligonucleotide, such as a gBlocks® Gene Fragment, overcomes these drawbacks and provides researchers an accurate and quick solution to standard development. Here, we demonstrate that using a gBlocks fragment as a standard provides comparable sensitivity, reliability, and assay performance to a purified amplicon standard.

Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality.

Mitochondrial DNA (mtDNA) can provide a means for forensic identity testing when genotyping of nuclear DNA (nuDNA) targets is not possible due to degradation or lack of template. For degraded samples, an indication of the quantity and quality of mtDNA is essential to allow selection of appropriately sized targets for hypervariable region (HVR) analysis, which may conserve sample and resources. Three human-specific mtDNA targets of increasing length (86, 190 and 452 base pairs) were amplified by singleplex quantitative real-time PCR (qPCR), capable of providing an index of mtDNA degradation from fragment length information. Quantification was achieved by preparation of a standard curve for each target, using a purified mtDNA standard containing all three targets of interest, which produced a linear, accurate and precise result from 1×108 to 10 copies. These novel assays demonstrated excellent sensitivity, specificity and reproducibility in line with the minimum information for qPCR experiments (MIQE) guidelines. Further, a separate inhibition control reaction was included to guide sample clean-up and ensure the validity of degradation assays. This protocol assists the selection and analysis of appropriately sized targets to maximize the chance of obtaining an informative result in downstream assays like sequencing.

Microbial composition analyses by 16S rRNA sequencing: A proof of concept approach to provenance determination of archaeological ochre.

Many archaeological science studies use the concept of "provenance", where the origins of cultural material can be determined through physical or chemical properties that relate back to the origins of the material. Recent studies using DNA profiling of bacteria have been used for the forensic determination of soils, towards determination of geographic origin. This manuscript presents a novel approach to the provenance of archaeological minerals and related materials through the use of 16S rRNA sequencing analysis of microbial DNA. Through the microbial DNA characterization from ochre and multivariate statistics, we have demonstrated the clear discrimination between four distinct Australian cultural ochre sites.

Host-specific associations affect the microbiome of Philornis downsi, an introduced parasite to the Galápagos Islands.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact on affected hosts. Here, we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galápagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data, we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp., respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harboured similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unravelling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies.

Complete Genome Sequences of the Endophytic Streptomyces sp. Strains LUP30 and LUP47B, Isolated from Lucerne Plants.

The complete genome sequences of two endophytic Streptomyces sp. strains, LUP30 and LUP47B, were analyzed. These strains were isolated from surface-sterilized roots of lucerne plants from South Australia and were found to promote the growth of the rhizobial partner in vitro and significantly increased nodulation and nitrogen fixation in lucerne plants.

A proof of principal study on the use of direct PCR of semen and spermatozoa and development of a differential isolation protocol for use in cases of alleged sexual assault.

Sexual assault samples are some of the most common samples encountered in forensic analysis. These samples can require a significant time investment due to differential extraction processes. We report on the first record of successful direct amplification of semen for STR analysis. Neat seminal fluid, dilutions ranging from 1:5 to 1:160 and GEDNAP samples were successfully amplified using a direct method. A mild differential isolation technique to enrich spermatozoa was developed and successfully implemented to separate and directly amplify a mixture of semen and female epithelial cells. Aliquots of samples subjected to the differential isolation protocol were stained with Haemotoxylin and Eosin for sperm scoring. Samples stained after PCR showed a complete lack of intact spermatozoa demonstrating that the cells are lysed during the PCR process. This paper demonstrates the potential to incorporate direct PCR in cases of sexual assault to more rapidly obtain results and achieve a higher sensitivity.

Complete Genome Sequences of the Endophytic Streptomyces Strains EN16, EN23, and EN27, Isolated from Wheat Plants.

The complete genome sequences of three endophytic Streptomyces species were compared. Strains EN16, EN23, and EN27 were isolated from surface-sterilized roots of wheat plants from South Australia. In field trials, these strains are effective in suppressing fungal root diseases of wheat when added as spore coatings to wheat seed.

Optimization of Diamond Nucleic Acid Dye for quantitative PCR.

Here, we evaluate Diamond Nucleic Acid Dye (DD) for use in quantitative PCR (qPCR) applications. Although DD is a commercially available stain for detection of DNA separated by gel electrophoresis, its use as a detection dye in qPCR has yet to be described. To determine if DD can be used in qPCR, we investigated its inhibitory effects on qPCR at concentrations ranging 0.1-2.5×. Serial dilution of DNA was used to determine the efficiency, sensitivity, and linearity of DD-generated qPCR data in comparison to other commonly used fluorescent dyes such as SYBR Green (SG), EvaGreen (EG), and BRYT Green (BG). DD was found to be comparable with other dyes for qPCR applications, with an R2 value >0.9 and an efficiency of 0.83. Mitochondrial DNA (mtDNA) target signals were successfully produced by DD over a DNA dilution range of ~28 ng- 0.28 pg, demonstrating comparable sensitivity to the other dyes investigated. Cq values obtained using DD were lower than those using EG by almost 7 cycles. We conclude that Diamond Nucleic Acid Dye is a cheaper, less toxic alternative for qPCR applications.

Stygofauna enhance prokaryotic transport in groundwater ecosystems.

More than 97% of the world's freshwater reserves are found in aquifers, making groundwater one of the most important resources on the planet. Prokaryotic communities in groundwater underpin the turnover of energy and matter while also maintaining groundwater purity. Thus, knowledge of microbial transport in the subsurface is crucial for maintaining groundwater health. Here, we describe for the first time the importance of stygofauna as vectors for prokaryotes. The "hitch-hiking" prokaryotes associated with stygofauna may be up to 5 orders of magnitude higher in abundance and transported up to 34× faster than bulk groundwater flow. We also demonstrate that prokaryotic diversity associated with stygofauna may be higher than that of the surrounding groundwater. Stygofauna are a newly recognized prokaryotic niche in groundwater ecosystems that have the potential to transport remediating, water purifying and pathogenic prokaryotes. Therefore, stygofauna may influence ecosystem dynamics and health at a microbial level, and at a larger scale could be a new source of prokaryotic diversity in groundwater ecosystems.

Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.

An assessment of the subjectivity of sperm scoring.

Current histological investigation of vaginal swabs after alleged sexual assault includes the scoring of spermatozoa (0, + to ++++) and the recording of visible tails. It is a method that is universally employed. Despite this method being used for 40 years, there has never been a study investigating its suitability for forensic science. Here, we investigate the reproducibility and subjectivity of sperm scoring among different investigators. Dilutions of seminal fluid were randomly distributed onto 20 slides, stained with haematoxylin/eosin and assessed by 37 investigators, over 2 years. Slides were assessed for levels of spermatozoa and the presence of tails. Each slide was scored by a minimum of 25 investigators. On no slide was there a consensus between all scores. Standard deviation remained below 1, but relative standard deviation (RSD) ranged from 6 to 105% in a positive correlation as the average score decreased. Spermatozoa were not observed 56 times (9.6%) and 27 investigators (73%) did not observe spermatozoa on at least one slide. Spermatozoa with tails were observed on every slide by at least 10 examiners, but as the average score of the slide decreased, so did the observation of tails. The current sperm scoring method is highly subjective with a particularly high %RSD in slides with low overall sperm counts. Moreover, the recording of tails does not add value to the current technique of sperm scoring. Further research might improve the objectivity of sperm scoring and the reliability of recording of tails.

Properties of nucleic acid staining dyes used in gel electrophoresis.

Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen™ and Diamond™ Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.

Molecular identification of python species: development and validation of a novel assay for forensic investigations.

Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question.

Sensitivity and specificity of presumptive tests for blood, saliva and semen.

PURPOSE: Despite their wide use, the limits of presumptive tests can be poorly understood. The aim of this study was to investigate the specificity and sensitivity of conventional, as well as innovative, presumptive tests for blood, semen and saliva. METHODS: We investigated Kastle-Meyer (KM) and leucomalachite green (LMG) tests for blood with regard to their sensitivity and specificity in the presence of oxidizing (hypochlorite) and anti-oxidizing (ascorbic acid) agents. The suitability and specificity of the red starch paper (RSP) test for saliva was assessed. Finally, the inhibitory effect of detergent on the acid phosphatase (AP) test for semen was investigated along with possible cross reactions to tea stains. RESULTS: Our results confirm previous findings of higher sensitivity and specificity of the KM test compared to LMG test for blood. Contrary to previous studies, no statistically significant difference was observed in the sensitivity of the tests between dry and wet stains. The novel RSP test was found to successfully detect saliva. We demonstrated that acid phosphatase (AP) testing for semen is possible on used RSP. A common multipurpose detergent had an inhibitory effect on AP tests. False positive results were obtained from tea stains. Testing different sorts of tea (black, green and herbal teas) revealed that only Camellia varieties produce positive result with the AP test, due to AP being present in the plants. CONCLUSIONS: From our results we conclude that specific knowledge of each test, including substances that may affect the test outcome, is imperative to ensure correct interpretation of presumptive test results.

Identification multiplex assay of 19 terrestrial mammal species present in New Zealand.

An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species-specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species-specific fragments, making the assay self-confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost-effective manner.

Recovery of human DNA profiles from poached deer remains part 2: improved recovery protocol without the need for LCN analysis.

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis. Samples from 10 deer (40 samples total - one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5pg±43.2pg, 31× greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpFℓSTR® SGM Plus® kit at 30cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690×10(-05) to 2.2706×10(-14). The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents.