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Phlebotomine sand flies constitute a group of haematophagous insects of great medical and veterinary importance. Despite the significant knowledge on the biology and behavior of adult sand flies in the wild, there is little information available on the ecology of their larval stages due to difficulties of detecting them in the soil. So far, investigations on sand fly breeding sites have been based on methods to catch emerging adults or on microscopic examination of the soil for the presence of immatures, neither of which is very precise or efficient for studying immatures in the field. Thus, there is a need for a better method to detect, identify and quantify immatures in soil samples. To address this issue we developed a novel molecular genetic approach for the detection of sand fly larval DNA within soil samples. Different numbers of live larvae of Phlebotomus papatasi and P. tobbi (alone or in combination), were mixed with soil and analyzed to identify and quantify the sand flies. By modifying already existing soil DNA extraction protocols in combination with a real-time PCR using species-specific primers, we successfully detected as little as one larva/40 ml of soil. We could also distinguish the two species of sand flies. This method should be very useful for increasing our knowledge of sand fly larval ecology in the field, and thus help develop more efficient, targeted control strategies against Phlebotomus sand flies.

Preservation variance of soil DNA is neglected in the literature, and exceptional cases exaggerate amplification capabilities. This study sought to amplify a short mitochondrial fragment (212 bp) specific to Sus scrofa domesticus from the soil surrounding decomposing pig remains from an open-air locale. Samples collected above the body at incremental distances after 145 days of initial placement yielded pig DNA. A secondary sampling was collected in 2017, approximately 768 days after burial. Inhibition tests corroborated that pig DNA was no longer present in the soil resulting in a loss of original DNA between 145 and 768 days. The results provide evidence that genetic material leaches out radially from the source and DNA fragments longer than 200 bp do not persist in soil for a relatively short timeframe in western Montana. The conclusions support the collection of soil in crime scene investigation procedures within the first few months of decomposition.

Quantitative real-time PCR (qPCR) analysis for accurate quantification of targeted microbial genes is compromised by the presence of co-extracted inhibitors from soil samples. Dilution of DNA extracts is a commonly-used method to reduce levels of inhibition. However, the applications of dilution method are mostly empirical, and need to be further elaborated. Here, we propose a dilution model to re-evaluate dilution as a method to eliminate qPCR inhibition. We found that DNA extracts without dilution or with a minor dilution (e.g., 10-fold) resulted in qPCR inhibition for most of studied soils. However, excessive dilution (e.g., 200- or 400-fold) caused an overestimation of the quantified gene copy numbers. Only under a moderate dilution range could qPCR inhibition be efficiently eliminated, which has been well captured by our proposed dilution model. The pre-testing of qPCR inhibition for determining the appropriate dilution range for extracted DNA samples aids accurate quantification of nucleic acids in soils.

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.

linA is a gene encoding γ-hexachlorocyclohexane dehydrochlorinase identified in a γ-HCH-degrading Sphingobium japonicum UT26 that had been isolated from soil of an experimental field treated with γ-HCH. The presence of linA-homologous nucleotide sequences and their sequence diversity was investigated by linA-targeted PCR amplification of DNA obtained from different soils in the field and in Japan. linA-homologous sequences were obtained from almost all the soils some of which had never been treated with γ-HCH. Deduced amino acid sequences suggested that they can be regarded as fragments of linA that encode LinA. Some common, natural function of LinA in soil was suggested., Microbes and environments. 2007, 22(4), p.399-404

A real-time polymerase chain reaction (PCR) assay was developed to quantify simultaneously the biocontrol agent Colletotrichum coccodes (DAOM 183088) and soil compounds inhibitory to PCR. The external control used in this assay was spiked at known concentrations in soil DNA extracts and amplified in real-time PCR with its own primer set. A comparison between the estimated quantities of the external control and the known quantities added to the extracts allowed an estimation of the PCR efficiency on a sample per sample basis in 18 extracts of soil DNA analyzed, originating from the bioherbicide field-release trials. All 18 extracts tested positive for the presence of inhibitory compounds, but with substantial variability in the magnitude of PCR efficiency (from 12% to 82%) from 1 g of soil sample to another, even when soil DNA extracts had been diluted. This variability demonstrates quantitatively the heterogeneity of soil with regards to content in PCR-inhibitory compounds. The differences in amplificatio...

This paper describes a efficient screening gene nitrilase from contaminated soil from Lombok gold mine industry. DNA was extracted directly from soil using the soil DNA isolation kit based on enzymatic, chemical and mechanical lysis. The existence of nitrilase gene in soil sample can be identified by nitrilase gene amplification using H1F˗H1R primer. BLASTN analysis result revealed that the nitrilase gene fragment which was amplified by H1F˗H1R primer has a high homology with Rhodococcus rhodochrous strain tg1˗A6 nitrilase gene. These amplification and DNA fragment sequencing results indicated that nitrilase gene existence on soil sample can be identified by metagenomic approach Keywords: Metagenomics; nitrilase gene; Rhodococcus rhodochrous; soil.

The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method.

DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent.

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.

Analysis of physical evidence is typically a deciding factor in forensic casework by establishing what transpired at a scene or who was involved. Forensic geoscience is an emerging multi-disciplinary science that can offer significant benefits to forensic investigations. Soil is a powerful, nearly 'ideal' contact trace evidence, as it is highly individualistic, easy to characterise, has a high transfer and retention probability, and is often overlooked in attempts to conceal evidence. However, many real-life cases encounter close proximity soil samples or soils with low inorganic content, which cannot be easily discriminated based on current physical and chemical analysis techniques. The capability to improve forensic soil discrimination, and identify key indicator taxa from soil using the organic fraction is currently lacking. The development of new DNA sequencing technologies offers the ability to generate detailed genetic profiles from soils and enhance current forensic soil analyses. Here, we discuss the use of DNA metabarcoding combined with high-throughput sequencing (HTS) technology to distinguish between soils from different locations in a forensic context. Specifically, we provide recommendations for best practice, outline the potential limitations encountered in a forensic context and describe the future directions required to integrate soil DNA analysis into casework.

To the Editor:E. M. Geigl (2001xInadequate use of molecular hybridization to analyze DNA in Neanderthal fossils. Geigl, EM. Am J Hum Genet. 2001; 68: 287–290Abstract | Full Text | Full Text PDF | PubMed | Scopus (6)See all References2001) challenges the results published by Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) on the genomic differentiation of Neanderthals and modern humans, deduced from Southern hybridization. She is convinced that Scholz et al. presented artifacts resulting from coextracted DNA of soil organisms. However, I do not feel that Geigl has provided any evidence for her “contamination hypothesis.” Because of space limitations, I will mention only the most important aspects supporting my point of view, but the list is far from complete.Geigl claims that the indirect evidence for DNA survival (e.g., amino acid racemization data) given by Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) is not sufficient to prove authenticity of aDNA samples. It is certainly a very difficult task to exclude contamination of samples. For this reason, researchers usually apply a variety of methods to assess the preservation of DNA by biochemical means. However, none of these methods is able to prove conclusively the authenticity of DNA; these methods can merely indicate whether or not it is likely that a certain sample is contaminated with contemporary DNA. Surprisingly, Geigl focuses exclusively on the biochemical data and does not even mention the most convincing evidence that supports the authenticity of the ancient DNA extracts used by Scholz et al.—namely, the experiments themselves. The reindeer and mammoth controls do not yield any hybridization signal. Why should we assume that the controls are not contaminated with soil DNA when all hominid samples are? Why would contemporary human and chimpanzee DNA exclusively cross-hybridize with soil DNA coextracted from hominid fossils but not do so with soil DNA coextracted from other fossils? Contemporary human DNA extracted from blood gives basically the same hybridization pattern that the DNA extracted from a human fossil gives. Furthermore, contemporary chimpanzee DNA hybridizes exclusively to hominid ancient DNA extracts (and not to reindeer and mammoth) but yields signals that are clearly different from those obtained with human DNA. Thus, the results of the experiments make phylogenetic sense. In particular, the results of the hybridization of chimpanzee DNA to the ancient DNA extracts are crucial for the conclusions drawn by Scholz et al. It therefore remains to be resolved why Geigl claims that “chimpanzee DNA, with 99% homology to modern human DNA, fails to hybridize.” Furthermore, the electrophoretic separation of the ancient DNA extracts prior to blotting will separate DNA of high molecular weight (expected to be contaminating contemporary DNA) from DNA of low molecular weight (expected to be authentic ancient DNA). Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) did not observe any hybridization signal with high–molecular-weight DNA.Rather than discussing the evidence just mentioned, Geigl attempts to prove that the data presented by Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) result from coextracted contemporary soil DNA by mere theoretical considerations and artificial experimental set-ups. However, her calculation that under the hybridization conditions used by Scholz et al. only sequences diverging by 30%–40% would be distinguishable is meaningless. Such calculations refer to contemporary DNA and do not apply to ancient DNA, which is of low molecular weight and is highly degraded. It also seems unlikely to me that coextracted inhibitors prevent an enzymatic labeling of ancient DNA but will cause a preferential labeling of contaminating DNA. If such inhibitors are coextracted, they will certainly inhibit the labeling of all DNAs. In addition, it is not justified to conclude from hybridization signals that look similar to those of Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) but were obtained with mixtures of fungal, bacterial, plant, and human DNA that the procedure of Scholz et al. “allows for the identification of the presence of common microorganismal DNA in the sample far better than it allows for the distinguishing of closely related species.” One can design an almost unlimited number of analogous experiments, but none of them will be a proof of Geigl's “contamination hypothesis.”To conclude, I believe that it is certainly worthwhile to exploit the power of Southern hybridizations in ancient DNA research. However, a discussion on the pros and cons should be based on appropriate scientific evidence rather than on unsupported assumptions.

A method for determining soil RNA and DNA concentrations after their extraction from soil is described. The determination was carried out with colorimetric methods specific for the sugar moiety of the nucleic acids. Various extraction procedures were tested when nucleic acids were added to soil either as pure reagents or as internal standards. A procedure involving three extraction steps gave the highest yields. Soil samples were extracted twice with 0.5 N HC104, neutralized with 2 n NaOH and the extraction continued after the addition of 0.1 m Na4P2O4 at pH 7.2. In the third step the soil sediment was extracted with 0.1 m Na4P2O7 at pH 7.2. Low molecular weight ribose and deoxyribose-containing compounds were separated from the nucleic acids on the basis of their solubility characteristics in the acid medium of the supernatant obtained after the first extraction step. Approximate recoveries of organic P in the nucleic acids were found to range from 3.2 to 6.5% of the total soil organic P. Nucleic acid-N ranged from 2.0 to 4.0% of the total N. The organic C content of the soils used was lower than 2% but when soils richer in organic matter are used, extracted humic molecules are likely to interfere during the colorimetric determination.