Efficient DNA Extraction Method Research Articles

Efficiency comparison of DNA extraction kits for analysing the cockle gut bacteriome

Cockles play a vital ecological role and provide valuable ecosystem services globally. However, the performance, production, and safe consumption of cockles are significantly influenced by their gut-associated bacteriome. Accurate understanding of gut-bacteriome interactions, and surveillance of pathogenic bacteria loads in cockles, rely on efficient DNA extraction methods that yield high-quality and representative bacterial DNA. Despite this importance, reliable extraction methods for cockles are currently overlooked. Therefore, we evaluated the performance of five DNA extraction kits (E.Z.N.A.® Soil DNA; FastDNA® Spin; DNeasy PowerSoil Pro; QIAamp PowerFecal DNA; ZymoBIOMICS™DNA Miniprep) in terms of DNA quality, yield, bacterial community structure (analysed by using denaturating gradient gel electrophoresis; DGGE), and bacteriome composition (analysed by 16S rRNA gene sequencing) in Cerastoderma edule gut. The DNeasy kit provided the highest purity and quantity of bacterial DNA, while the PowerFecal and Zymo kits exhibited reduced extraction efficiency. DGGE profiles revealed significant variability between the tested kits (R = 0.512; mean P = 0.011), but the FastDNA kit under-represented the bacterial community in cockles’ gut. Based on alpha diversity, the DNeasy kit outperformed the others and successfully detected all abundant genera found with the alternative kits. Our findings indicate that the DNeasy kit is an efficient DNA extraction method, enabling a molecular representation of the gut-associated bacteriome in C. edule. These results contribute to the development of effective techniques for studying the cockle gut bacteriome and its ecological implications.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Automated sepsis detection with vancomycin- and allantoin-polydopamine magnetic nanoparticles

Rapid and accurate identification of the bacteria responsible for sepsis is paramount for effective patient care. Molecular diagnostic methods, such as polymerase chain reaction (PCR), encounter challenges in sepsis due to inhibitory compounds in the blood, necessitating their removal for precise analysis. In this study we present an innovative approach that utilizes vancomycin (Van) and allantoin (Al)-conjugated polydopamine (PDA)-coated magnetic nanoparticles (MNPs) for the rapid and automated enrichment of bacteria and their DNA extraction from blood without inducing clumping and aggregation of blood. Al/Van-PDA-MNPs, facilitated by IMS, eliminate the need for preliminary sample treatments, providing a swift and efficient method for bacterial concentration and DNA extraction within an hour. Employing Al/Van-PDA-MNPs within an automated framework has markedly improved our ability to pre-concentrate various Gram-negative and Gram-positive bacteria directly from blood samples. This advancement has effectively reduced the detection threshold to 102 colony-forming unit/mL by both PCR and quantitative PCR. The method's expedited processing time, combined with its precision, positions it as a feasible diagnostic tool for diverse healthcare settings, ranging from small clinics to large hospitals. Furthermore, the innovative application of nanoparticles for DNA extraction holds promising potential for advancing sepsis diagnostics, enabling earlier interventions and improving patient outcomes.

An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1pg/µl while root gDNA remained amplifiable at 10pg/µl. Genomic DNA obtained was also suitable for sequencing. This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Spore-DNA localization and extraction efficiencies of Bacillus subtilis for accurate results in quantitative real-time polymerase chain reaction.

Mechanical bead disruption is an efficient DNA extraction method from spore cells for subsequent quantification of the spore population by quantitative polymerase chain reaction（qPCR）. In this study, to validate spore DNA localization and extraction efficiencies, the fractionated DNA included the total DNA（tDNA）extracted from spore cells and intracellular（iDNA）and extracellular DNA（eDNA）extracted from fractionated spores through chemical decoating and alkaline lysis buffers, each followed by bead disruption. Furthermore, alkaline lysis buffer-treated spore cells were intensively washed three and five times after each centrifugation to determine how the amount of DNA is affected by repeated centrifugation. This process was achieved through fractionated spore pellet and suspension treatments with propidium monoazide xx（PMAxx）before mechanical bead disruption. Three fractionated and extracted DNAs were assessed with qPCR. The amount of eDNA was higher than that of iDNA, and closer to tDNA levels in the qPCR assay. These results indicted the following: 1）amount of eDNA was more than iDNA and responsible for majority of amount of tDNA through the combination method involving alkaline lysis buffer and bead disruption, 2）lysis buffer partially eliminated the eDNA fragments through multiple washing steps, but it was not largely independent of the number of times centrifugation was performed.

Rapid and efficient DNA extraction method from high oily content seeds:

Ricinus communis seeds harbor high oil and polyphenolics contents that hinder DNA extraction. Here, a rapid and efficient protocol for isolating total DNA from R. communis seeds was developed. The current method implies the use of repeated cycles of freeze/heat shock for the seed tissue to lyse the cells. DNA isolated with this protocol was successfully used as a template for PCR amplification of the internal transcribed spacer (ITS) region of the rRNA encoding gene that widely used for molecular identification of different plant species. As far to our knowledge, this study is the first one that report the efficient use of freeze/heat shock repeated cycles for isolation of a high-quality DNA from plant cells. The current protocol would support the subsequent analysis for seed lot purity analysis.

Comparative Study of Genomic DNA Extraction Methods for Common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is one of the significant grains used in the human diet, accounting for half of all grain legumes consumed globally. To enhance production, conventional breeding and molecular approaches have been used so far. An efficient and rapid genomic DNA extraction method is required for these molecular approaches. The aim of this study was to compare and optimize an efficient and rapid DNA extraction protocol for common bean. Modified cetyl trimethylammonium bromide (CTAB) and potassium chloride (KCL) extraction methods were used. The mean DNA yield per nanoliter was 209 µg from modified CTAB and 150.3 µg from the KCL method. The concentration of gDNA was significantly (P< 0.05) higher for the KCL method, which was 5.01 µg/µl and 2.09 µg/µl for the CTAB method. The obtained DNA was also pure, with an absorbance ratio at 260 nm to an absorbance of 280 nm (A260/280) of 1.75-2.23 for the KCL method and 1.86-2.09 for the modified CTAB method. Gel electrophoresis separation was used to evaluate the quality of the total DNA extracted by the present protocols. The results showed that intense bands close to the gel wells were obtained from both extraction methods. DNA isolated with the two methods was successfully used for PCR-based downstream analysis, which includes random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. In this study, it took approximately 150 minutes for KCL and 240 minutes for the CTAB for whole process. In contrast to the CTAB method, the KCL method uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. Therefore, it is more convenient and economical than the traditional technique.

Comparative analysis indicates a simple protocol for DNA extraction of the aromatic plant Lippia alba

An efficient method of genomic DNA extraction that provides high quality and yield is a crucial pre-requisite and limiting factor in plant genetic analysis. However, pure genomic DNA can be challenging to obtain from some plant species due to their sugar and secondary metabolite contents. Lippia alba is an important aromatic and medicinal plant, chemically characterized by the presence of tannins, flavonoids, anthocyanins, and essential oils, which interfere with the extraction of pure genomic DNA. In this scenario, optimizing the extraction methods and minimizing the effects of these compounds are necessary. This study compares six plant DNA extraction protocols based on the CTAB method. The quality and quantity of DNA samples obtained were determined by physical appearance by electrophoresis in agarose gels and spectrophotometry. The results highlight the difficulty in obtaining pure and clear bands for all tested methods, except for the polyvinylpyrrolidone (PVP)-based protocol created by our team, which was the better option for obtaining high-quality genomic DNA of L. alba. We conclude that adding PVP-40 into DNA extraction buffers can optimize the DNA extraction of L. alba and indicate this protocol for DNA extraction from other aromatic plants.

An efficient, fast and inexpensive method for genomic DNA extraction of fish tissue.

DNA extraction is an essential step for many genetic techniques like PCR and other molecular analyses. Based on the method of extraction and type of tissue used, the quality of extracted DNA for genetic studies varies. An appropriate extraction method is evaluated by the high concentration and purity of DNA. Thus, this study aimed to find a more efficient and effective method of DNA extraction from fish tissues and compare it to commercially available kits. A total of 200 fish tissue samples were extracted using each method and then validated with restriction enzymes and PCR amplification. The result revealed that the mean quantity of the isolated genomic DNA, when measured by Nanodrop for grass and common carp, was estimated at (624.41 ± 34.51) µg/ml and (651.27 ± 46.31) µg/ml, respectively, and the purity of this DNA was about (1.83 ± 0.04) and (1.88 ± 0.03) respectively, as compared to commercial extraction kits. Furthermore, gel electrophoresis was performed on the PCR-ready DNA, and the results were confirmed with restriction enzymes and PCR amplification. Based on results obtained from restriction enzymes and PCR analysis, it was determined that no significant inhibitors existed for the enzymes that were used in molecular biology reactions. As a result, this technique provides an efficient and versatile alternative to the traditional method for obtaining bulk amounts of highly qualified DNA from fish tissue and can be easily used for subsequent analyses such as PCR and several molecular experiments on other fish species.

A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples

A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.

Laboratory and field evaluation of polymerase chain reaction assays for diagnosis of zoonotic tuberculosis in bovine post mortem samples

The diagnosis of zoonotic tuberculosis revolves around detection of pathogen by gold standard culture method. In the live animals, tuberculin testing and interferon gamma assays are the options with their own limitation of specificity and cost effectiveness respectively. Clinical examination along with the affordable methods can lead to a definitive diagnosis which could help in decision making for not only the individual animal but also for the whole farm. During post-mortem examination, the post-mortem lesions with visible tubercle and acid fast staining give a fair idea of tuberculosis infected carcass for tuberculosis. The molecular testing of tissue samples with proven mycobacterial genus, Mycobacterium tuberculosis complex (which includes M. bovis also) and M. bovis species-specific primers can add to the diagnostic strength for bovine tuberculosis. In this regard, we evaluated sequential PCRs for mycobacterial genus specific primer (hsp65), M. tuberculosis complex specific primer (IS6110 & IS1081) and M. bovis specific primer (RD4 and 500 bp) in spiked and field tissue samples of cattle. The research was carried out in 2019-20 in Division of Animal Health for framing out a diagnostic confirmation protocol for TB infected cattle on tissue samples. All the PCR assays were almost equally efficient in detection of M. bovis in spiked samples, if supported by an efficient DNA extraction method which is the foremost limiting factor. It was concluded that a combination of minimum of two assays can give a definitive result along with the support of the clinical history or post mortem outcome of the animals.

A Review of Environmental DNA Field and Laboratory Protocols Applied in Fish Ecology and Environmental Health

Environmental DNA (eDNA) has been used in research relevant to fish ecology such as species diversity and conservation studies, threatened and invasive species monitoring, and analyses of population structure and distribution. How to choose the optimal laboratory protocols on the basis of the research targets is the first question to be considered when conducting an eDNA study. In this review, we searched 554 published articles using the topic subject ((eDNA or environmental DNA) and (fish)) within the time span 2011–2021 via Thompson Reuters Web of Science (WoS) and China National Knowledge Infrastructure (CNKI) literature databases, and screened 371 articles related to eDNA research on fish ecology. These articles were categorized into “article (334)”, “review (36)”, and “letter (1)” based on the type, and “article” was divided into “article (method research)” and “article (eDNA application)” in line with the study objectives. The experimental methods adopted in each study were reviewed, and advantages and disadvantages of the main protocols were analyzed for each step. We recommend a set of optimal protocols for regular eDNA-based fish diversity detection and present the following suggestions for water sample collection and subsequent sample processing and experiments. Sample size is suggested to be 2 L regardless of the type of water bodies; three water replicates are recommended per sampling site, and water collection sites should be designed to cover various water layers and micro-habitats within research areas. Filtration is the best method for collecting eDNA from the larger water samples; 0.45 μm glass fiber/glass microfiber (GF) filters and mixed cellulose acetate and nitrate (MCE) filters are recommended for use, and MCE filters are suitable for use in turbid waters; pre-filtration (>10 μm filtering membranes) can be used to prevent clogging. Freezing temperature storage can slow eDNA degradation, and this is the optimal way to store DNA no matter what filtering method is applied. The Qiagen DNeasy Blood and Tissue DNA extraction kit was the most economical and efficient DNA extraction method compared to other commercial kits. The 12S rRNA gene is the first choice for detecting interspecies variation in fishes, and five 12s primer sets, Ac12S, MDB07, Mi-Fish, Vert-12SV5, and Teleo, are recommended. The TruSeq DNA PCR-free LT Sample Prep kit and NEBNext DNA Library Prep Master Mix Set for the 454 kit can be chosen. The Illumina HiSeq platform can obtain sufficient data depth for fish species detection. QIIME and OBITools are independent software packages used for eDNA sequences analysis of fishes, and bioinformatic analyses include several indispensable steps such as filtering raw reads, clustering filtered reads into molecular operational taxonomic units (MOTUs) or amplicon sequence variants (ASVs), and completing taxon annotation. Contamination, inhibition, lack of reference DNA data, and bioinformatic analysis are key challenges in future eDNA research, and we should develop effective experimental techniques and analysis software regarding these aspects. This review intends to help eDNA beginners to quickly understand laboratory protocols applied in fish ecological research; the information will be useful for the improvement and development of eDNA techniques in the future.

Technical note: Polyvinylpyrrolidone (PVP) and proteinase-K improve the efficiency of DNA extraction from Japanese larch wood and PCR success rate

Illegal distribution of timber disrupts the timber market and depletes forest resources. DNA markers are used to verify the legal distribution of wood. However, it is difficult to obtain the quantity and quality of DNA suitable for genetic analysis because of the physicochemical properties of wood; therefore, an efficient wood DNA extraction method is required. In this study, to prepare an efficient DNA extraction method from Japanese larch (Larix kaempferi) wood, we investigated the ability of polyvinylpyrrolidone (PVP) and proteinase-K to improve DNA extraction efficiency and PCR success rate. It was found that the addition of PVP resulted in a significant increase in the DNA concentration of the treatment group compared to that of the control group, while the purity (A260/A280) showed no difference. Moreover, the addition of proteinase-K significantly increased both the DNA concentration and purity of the treatment group compared to those of the control group. Further analysis showed that the PCR success rate of psbC (approximately 350 bp) was higher than 90% in the control, PVP treatment, and proteinase-K treatment groups. However, in the PCR success rate of rbcL (approximately 1.3 kb) was higher in the proteinase-K and PVP treatment groups than in the control group. The addition of PVP and proteinase-K increased the success rate of PCR amplification for long regions by preventing DNA damage caused by phenolic compounds and proteins in the wood. The results of this study can thus develop DNA extraction methods to identify the species and origin of woods.

Detection of Nudibranch Chromodoris quadricolor associated microbiome using two DNA extraction methods

DNA extraction with high quality is critical to all molecular genetic analyses. However, obtaining DNA from microbes associated with animals is challenging. Despite the availability of various DNA extraction kits in the markets, no studies were conducted to date to evaluate their potential for the invertebrates such as nudibranch, one of Mollusca. This study compared the Quick—DNA Fungal/Bacterial Miniprep Kit and CTAB methods and tested them across four samples of Chromodoris quadricolor gut and skin tissues. The universal bacterial primers 331f and 797r and the animal-specific primers LCO1490-JJ and HCO2198-JJ were used to amplify the 16S rRNA gene and partial mitochondrial cytochrome oxidase I gene using extracted DNA as a template. The DNA and PCR products’ quality and concentration were verified with agarose gel and Nanodrop, respectively. The two methods' quality assessed using the deep pyrosequencing of the 16S rRNA gene in capturing a more diverse microbiome. The highest yield and purity (over 1000 ng / µL) were obtained with the CTAB method, while it was not exceeded 260 ng/µL with Quick DNA kit and display high purity. Also, 16S rRNA community amplicon sequencing revealed that the CTAB way could catch more diverse bacterial groups. The most efficient method of DNA extraction was CTAB, as it achieved both high concentration and purity

Development and Application of a Fast Method to Acquire the Accurate Whole-Genome Sequences of Human Adenoviruses.

The whole-genome sequencing (WGS) of human adenoviruses (HAdVs) plays an important role in identifying, typing, and mutation analysis of HAdVs. Nowadays, three generations of sequencing have been developed. The accuracy of first-generation sequencing is up to 99.99%, whereas this technology relies on PCR and is time consuming; the next-generation sequencing (NGS) is expensive and not cost effective for determining a few special samples; and the third-generation sequencing technology has a higher error rate. In this study, first, we developed an efficient HAdV genomic DNA extraction method. Using the complete genomic DNA instead of the PCR amplicons as the direct sequencing template and a set of walking primers, we developed the HAdV WGS method based on first-generation sequencing. The HAdV whole genomes were effectively sequenced by a set of one-way sequencing primers designed, which reduced the sequencing time and cost. More importantly, high sequence accuracy is guaranteed. Four HAdV strains (GZ01, GZ02, HK35, and HK91) were isolated from children with acute respiratory diseases (ARDs), and the complete genomes were sequenced using this method. The accurate sequences of the whole inverted terminal repeats (ITRs) at both ends of the HAdV genomes were also acquired. The genome sequence of human adenovirus type 14 (HAdV-B14) strain GZ01 acquired by this method is identical to the sequence released in GenBank, which indicates that this novel sequencing method has high accuracy. The comparative genomic analysis identified that strain GZ02 isolated in September 2010 had the identical genomic sequence with the HAdV-B14 strain GZ01 (October 2010). Therefore, strain GZ02 is the first HAdV-B14 isolate emergent in China (September 2010; GenBank acc no. MW692349). The WGS of HAdV-C2 strain HK91 and HAdV-E4 strain HK35 isolated from children with acute respiratory disease in Hong Kong were also determined by this sequencing method. In conclusion, this WGS method is fast, accurate, and universal for common human adenovirus species B, C, and E. The sequencing strategy may also be applied to the WGS of the other DNA viruses.

Development of an on-spot and rapid recombinase polymerase amplification assay for Aspergillus flavus detection in grains

Aspergillus flavus is one of the main contaminants in grains, and most strains of A. flavus can produce aflatoxins (AFs), which poses a great threat to food safety and human health. Early detection of food contaminated by A. flavus can effectively reduce the harm of AFs to human health and prevent further spread of A. flavus. In this study, a sensitive and rapid method for on-spot detection of A. flavus in grains, based on recombinase polymerase amplification (RPA), has been developed. Three RPA primer pairs were designed, and then a suitable primer pair was selected. A simplified and efficient DNA extraction method for on-spot detection was developed and applied. Two temperatures, ambient temperature (AT, 26 ± 1 °C) and body temperature (BT, 36 ± 1 °C), were provided for the detection. The reaction time and primer concentration were optimized at AT and BT, respectively. And a fluorescent DNA dye was used for the visual detection. The whole detection-assay can be completed within 20 min at BT and 30 min at AT, respectively. The sensitivity evaluation proved that the limit of detection (LOD) of the RPA-assay was 101 conidia/g. The specificity of the assay was equal to the real-time PCR. The new RPA-assay also had great potential in other fields′ applications.

Impact of DNA extraction efficiency on the sensitivity of PCR-based plant disease diagnosis and pathogen quantification

PCR and qPCR are important methods for plant disease diagnosis and quantification of pathogen populations in host tissues or soil. For most plant diseases, DNA extracted from infected tissue or soil is a prerequisite for PCR or qPCR. The efficiency of DNA extraction should have a direct impact on the sensitivity of PCR and qPCR systems. In this study, plant pathogen DNA extraction efficiencies were evaluated based on three plant disease systems and three DNA extraction methods. DNA of bacterial (Pectobacterium atrosepticum), protist (Plasmodiophora brassicae) and fungal (Botrytis cinerea) pathogens was extracted and aliquots were prepared. Aliquots of the DNA were mixed with healthy tissues of the corresponding host plant and DNA was extracted again from the mixture. The resultant mixed DNA, as well as the original pathogen DNA, were analysed by qPCR to assess the quantities of the pathogen DNA. Extraction efficiency was calculated based on the qPCR Cq values of the mixed DNA and the original pathogen DNA for each extraction method on each pathogen. Our results indicated the pathogen DNA extraction efficiencies were generally less than, or near, 50%. This result called attention to the importance of: 1) including the DNA extraction efficiency in the evaluation and announcement of new qPCR diagnostic systems, and 2) developing and using high efficient DNA extraction methods in plant disease diagnosis and pathogen quantification.

Improved autosomal STR typing of degraded femur samples extracted using a custom demineralization buffer and DNA IQ™

Bone samples are utilized as a source of DNA in disaster victim identification and human remains investigations. However, DNA recovery from bones is time consuming and prone to contamination. A logical approach for postmortem identification is to validate efficient DNA extraction methods requiring less bone material using improved molecular kits with less hands-on time and workflows that facilitate faster turn-around time for processing. In this study, we evaluated DNA yield and amplification efficiency of DNA extracts using a new custom bone demineralization buffer (DMB; Promega) and extracted via manual and automated DNA IQ™ workflows. Including the demineralization step, the bone protocol can be completed in ∼4 h and even less with minimal sample handling when automated. Overall, a rapid and simple DNA extraction with improved allele recovery was validated using degraded bone samples exposed to tropical environments and post-disaster recovery as well as adverse conditions of embalming prior to internment.

Deciphering indigenous bacteria in compacted bentonite through a novel and efficient DNA extraction method: Insights into biogeochemical processes within the Deep Geological Disposal of nuclear waste concept

Compacted bentonites are one of the best sealing and backfilling clays considered for use in Deep Geological Repositories of radioactive wastes. However, an in-depth understanding of their behavior after placement in the repository is required, including if the activity of indigenous microorganisms affects safety conditions. Here we provide an optimized phenol:chloroform based protocol that facilitates higher DNA-yields when other methods failed. To demonstrate the efficiency of this method, DNA was extracted from acetate-treated bentonites compacted at 1.5 and 1.7 g/cm3 densities after 24 months anoxic incubation. Among the 16S rRNA gene sequences identified, those most similar to taxa mediating biogeochemical sulfur cycling included sulfur oxidizing (e.g., Thiobacillus, and Sulfurimonas) and sulfate reducing (e.g., Desulfuromonas and Desulfosporosinus) bacteria. In addition, iron-cycling populations included iron oxidizing (e.g., Thiobacillus and Rhodobacter) plus reducing taxa (e.g., Geobacillus). Genera described for their capacity to utilize acetate as a carbon source were also detected such as Delftia and Stenotrophomonas. Lastly, microscopic analyses revealed pores and cracks that could host nanobacteria or spores. This study highlights the potential role of microbial driven biogeochemical processes in compacted bentonites and the effect of high compaction on microbial diversity in Deep Geological Repositories.

Review: Diagnosis of Helicobacter pylori infection.

New imaging techniques are still the topic of many evaluations for both the diagnosis of Helicobacter pylori gastritis and the detection of early gastric cancer. Concerning invasive tests, there were studies on the reuse of the rapid urease test material for other tests, and a novel fluorescent method to be used for histology but with limited sensitivity. Progress occurred essentially in the molecular methods area, especially next-generation sequencing which is applied to detect both H pylori and the mutations associated with antibiotic resistance. For non-invasive tests, a few studies have been published on the validity of breath collection bags, the shortening of the testing time, the performance of different analysers or the added value of citric acid in the protocol. The accuracy of serological immunochromatographic tests is also improving. Multiplex serology detecting antibodies to certain proteins allows confirmation of a current infection. Dried blood spots can be used to collect and store blood without a loss of accuracy. Finally, the serum antibody titer can be useful in predicting the risk of gastric cancer. Several stool antigen tests were evaluated with good results, and a novel test using immunomagnetic beads coated with monoclonal antibodies is potentially interesting. PCR detection in stools can also be effective but needs an efficient DNA extraction method. The use of easyMAG® (bioMérieux) combined with Amplidiag® H pylori+ClariR (Mobidiag) appears to be powerful.