Evaluation of the microbial wet-vacuum system (M-Vac®) for DNA sampling from rough, porous substrates, and its compatibility with fully automated platforms.

DNA retrieval methods traditionally used during forensic evidence recovery including swabbing and tape lifting, can have limited effectiveness when used on porous, rough substrates such as bricks and carpet. This is possibly due to the DNA material being dispersed and unreachable for surface sampling techniques. In this evaluation we investigated the effectiveness of the Microbial Wet-Vacuum System (M-Vac®; M-Vac® Systems, Inc., Sandy, UT), as it has been reported to retrieve greater amounts of DNA material from challenging exhibits. A four-stage evaluation was conducted, starting with seeding carpet and brick substrates with a known donor's saliva in two dilutions and comparing the DNA recovery of tape lifting, swabbing, and the M-Vac®. A victim struggle scenario on carpet was then mimicked to compare trace DNA recovery by each method. Two mock scenarios were also conducted; a shirt was submerged in a creek bed for a period of five days to sample for the wearer's DNA, and a car boot was sampled to assess the possibility of recovering a victim's DNA amongst background DNA from the usual car occupants. Finally, the compatibility of the M-Vac® sampling process was optimised for the fully automated DNA lysis and extraction platforms used in the NSW (Australia) jurisdiction by comparing filter subsampling methods. The results from the study were mixed. For bricks, none of the collection methods were effective in retrieving DNA. On carpet, the M-Vac® retrieved the greatest quantities of DNA from the saliva-seeded samples, however, tape lifts outperformed all methods for 'touch' DNA recovery. The M-Vac® retrieved the greatest amount of DNA from the t-shirt recovered from a creek bed as it was able to retrieve the embedded DNA. The final mock case car boot scenario resulted in greater victim DNA recovery from tape lifts, with the M-Vac® more likely to recover mixtures too weak and/or complex to be interpreted. Finally, operational considerations regarding the compatibility of the M-Vac® system with fully automated DNA lysis and extraction are discussed. Considering the substantial time and cost to deploy the M-Vac®, it is recommended to be utilised in casework only after swabbing and tape lifting methods have failed to yield sufficient DNA material, where the substrate properties would likely benefit from the M-Vac's® niche capabilities for retrieving embedded DNA, and low levels of background DNA may be anticipated.

Touch DNA recovery from unfired and fired cartridges: Comparison of swabbing, tape lifting and soaking.

Over the recent few years, several DNA collection techniques and methodologies have been published for the recovery of DNA from fired cartridge cases. In this study, swabbing, the DNA collection technique currently used in our jurisdiction (NSW, Australia), was compared with tape lifting and soaking to assess DNA recovery rates, DNA quality and profile quality. Brass .22LR and 9mmP cartridges were used as they are the most commonly encountered in our jurisdiction. The cartridges (n = 107) were loaded into cleaned firearm magazines by three volunteers of unknown shedder status, to mimic routine casework sample types. Half of the handled cartridges were fired whilst the other half were kept unfired. STR genotypes were produced at both 29 and 30 PCR cycles to evaluate which improved handler allele detection. DNA recovery rates showed that swabbing recovered significantly less DNA than tape lifting and soaking. Whilst there were no significant differences between tape lifting and soaking, tape lifting, on average, yielded more DNA than soaking. The calibre of ammunition had no influence on DNA recovery and in line with expectations, firing was found to decrease DNA recovery for all three sampling techniques. Assessment of DNA quality showed no evidence of PCR inhibition in any of the samples for this study. However, degradation indices showed that most samples were slightly to moderately degraded. Fewer handler alleles were detected from both fired tape lifted and soaked cartridges than unfired cartridges. Whilst 30 amplification cycles allowed for the detection of slightly more handler alleles, no statistically significant differences were found between 29 and 30 PCR cycles. Nonetheless, 50% of the profiles from unfired soaked cartridges that were non-uploadable after 29 cycles were uploadable after 30 cycles. Furthermore, 83% of profiles from unfired cartridges that were tape lifted were uploadable onto our jurisdiction's database at both 29 and 30 PCR cycles. All magazine controls, despite cleaning, contained some level of background DNA. Furthermore, increasing the number of PCR cycles to 30 also increased the detection of non-handler alleles in DNA profiles. Our results suggest tape lifting yields more uploadable profiles from unfired and fired cartridge cases than swabbing but also more adventitious (non-handler) alleles. However additional research will be needed to evaluate the full potential of this method.

DNA recovery from unfired and fired cartridge cases: A comparison of swabbing, tape lifting, vacuum filtration, and direct PCR.

The ability to recover trace DNA from fired cartridge cases can help establish important leads regarding the handler of the ammunition. Over recent years, several DNA recovery techniques for fired ammunition have been published. Three techniques of significant interest include tape lifting, direct PCR, and vacuum filtration. This study aimed to compare these to the swabbing method currently employed in our jurisdiction. Brass and nickel cartridges of five different calibres were spiked with 20ng of saliva and subject to DNA collection using all four DNA recovery methods. Unfired and fired cartridges were tested to examine the effects of firing. Swabbing recovered a greater quantity of DNA than vacuum filtration while no significant differences were found between swabbing and tape-lifting. The calibre of ammunition had no effect on DNA recovery. Firing significantly reduced DNA yield from nickel cartridges, while unfired brass cartridges returned less DNA than unfired nickel cartridges. PCR inhibition was not observed in any samples, although degradation indices suggested that most samples were slightly or moderately degraded. Analysis of profiles showed that swabbing and tape lifting resulted in greater numbers of alleles from fired nickel and brass cartridges compared to direct PCR. Samples from nickel cartridges were found to have a greater number of uploadable profiles than samples from brass cartridges. In addition, three mixed profiles were obtained from the single source spiked cartridges as well as evidence of pre-existing DNA on uncleaned cartridges and contaminating alleles on cleaned cartridges. Our results suggest that tape-lifting can be a suitable alternative to swabbing, but that caution must be taken when interpreting profiles from fired cartridge cases as small amounts of DNA not associated with the handling of the cartridges may be present.

Genomic insights into the carbohydrate catabolism of Cairneyella variabilis gen. nov. sp. nov., the first reports from a genome of an ericoid mycorrhizal fungus from the southern hemisphere.

This paper describes a novel species of ericoid mycorrhizal fungus from Australia, Cairneyella variabilis, Midgley and Tran-Dinh, gen. nov. sp. nov. The genome of C. variabilis was sequenced and a draft genome assembled. The draft genome of C. variabilis is 52.4 Mbp in length, and to our knowledge, this is the first study to present a genome of an ericoid mycorrhizal fungus from the southern hemisphere. Using the SignalP and dbCAN bioinformatic pipelines, a study of the catabolic potential of C. variabilis was undertaken and showed genes for an array of degradative enzymes, most of which appear to be secreted from the hyphae, to access a suite of different carbon sources. Isolates of C. variabilis have been previously shown to utilise cellulose, carboxymethyl cellulose (CMC), cellobiose, xylan, pectin, starch and tannic acid for growth, and in the current study, putative enzymes for these processes were revealed. These enzymes likely play key roles in nutrient cycling and other edaphic processes in heathland environments. ITS phylogenetic analyses showed C. variabilis to be distinct from the fungi of the "Hymenoscyphus ericae aggregate".

Genetic population structure of the ectomycorrhizal fungus Pisolithus microcarpus suggests high gene flow in south-eastern Australia.

Pisolithus are ectomycorrhizal fungi that associate with roots of numerous plant species in natural and plantation forests worldwide. Despite the fact that Pisolithus spp. are present in plantation forests in many countries, knowledge of the genetic population structure of Pisolithus spp. remains limited. In this study, we have tested the hypothesis that a propensity for long-distance spore dispersal in Pisolithus microcarpus, along with the widespread distribution of potential eucalypt and acacia plant hosts in south-eastern Australia facilitates gene flow that limits population differentiation. Five polymorphic simple sequence repeat markers were used to investigate the population structure of P. microcarpus. Isolates were grouped according to geographical origin and isolate genotypes were analysed among the geographical populations. Pairwise F (ST) estimates indicated limited genetic differentiation among the geographical populations. Analysis of molecular variance revealed that most of the genetic variation present was within geographical populations, with only 1.3% of the genetic variation among P. microcarpus geographical populations. This was particularly pronounced for four geographical populations within a ca 7,000 km(2) area New South Wales, which were each separated by < 100 km and appeared to be genetically homogeneous. The lack of population structure is suggested to be due to a high degree of gene flow, via basidiospores, between the New South Wales geographical populations.

Ectomycorrhizal mycobionts of Pisonia grandis on coral cays in the Capricorn-Bunker group, Great Barrier Reef, Australia.

The diversity of ectomycorrhizal mycobionts of Pisonia grandis (Nyctaginaceae) from coral cays in the Capricorn-Bunker group, Great Barrier Reef, Australia, was examined. Only two ectomycorrhiza morphotypes (brown and black) were identified in soil from seven cays and DNA from both morphotypes was subjected to ITS-RFLP and sequence analysis. The brown morphotype was present in soil from all cays but the black morphotype was only observed in soil from three cays. ITS-RFLP analysis showed that the brown and black morphotypes were formed by different fungal taxa, with the RFLP pattern for the black morphotype being consistent with that of the culture previously obtained from black ectomycorrhizal roots on Heron Island. Comparison with the GenBank database revealed that closest matches to both morphotypes were sequences for various Thelephoraceae (Basidiomycota), but the brown and black morphotypes had only 80% sequence similarity to each other. Neighbour-joining analysis of these sequences with sequences for other Thelephoraceae grouped the brown and black morphotypes in a well-supported clade with several Tomentella species, suggesting that both belong to this genus. The data are discussed in relation to ectomycorrhizal fungal diversity and the coral cay habitat.

Development of markers for simple sequence repeat-rich regions that discriminate between Pisolithus albus and P. microcarpus.

Inter-simple sequence repeat PCR (ISSR-PCR) was used to develop markers for simple sequence repeat-rich (SSR) regions for investigation of genetic relatedness of Pisolithus isolates collected from eastern mainland Australia. Primers were designed to amplify ten SSR-rich regions and these were used to screen 14 Pisolithus isolates. Two amplified loci showed size polymorphisms among the isolates (regarded as polymorphic), two were monomorphic for all isolates, while the remainder amplified alleles for only some isolates. UPGMA analysis of the alleles for each isolate at each locus together with ITS-RFLP analysis, separated the isolates into groups. These two groups appear to correspond to isolates that ITS sequence data have previously separated as P. albus and P. microcarpus.