Optimized DNA Extraction Method Research Articles

Multicenter comparative study of Enterocytozoon bieneusi DNA extraction methods from stool samples, and mechanical pretreatment protocols evaluation

Nowadays, the use of qPCR for the diagnosis of intestinal microsporidiosis is increasing. There are several studies on the evaluation of qPCR performance but very few focus on the stool pretreatment step before DNA extraction, which is nevertheless a crucial step. This study focuses on the mechanical pretreatment of stools for Enterocytozoon bieneusi spores DNA extraction. Firstly, a multicenter comparative study was conducted evaluating seven extraction methods (manual or automated) including various mechanical pretreatment. Secondly, several durations and grinding speeds and types of beads were tested in order to optimize mechanical pretreatment. Extraction methods of the various centers had widely-varying performances especially for samples with low microsporidia loads. Nuclisens® easyMAG (BioMérieux) and Quick DNA Fecal/Soil Microbe Microprep kit (ZymoResearch) presented the best performances (highest frequencies of detection of low spore concentrations and lowest Ct values). Optimal performances of mechanical pretreatment were obtained by applying a speed of 30 Hz during 60 s with the TissueLyser II (Qiagen) using commercial beads of various materials and sizes (from ZymoResearch or MP Biomedicals). Overall, the optimal DNA extraction method for E. bieneusi spores contained in stool samples was obtained with a strong but short bead beating using small-sized beads from various materials.

An optimal genomic DNA extraction method for shoots of four Dendrocalamus species based on membership function analysis.

High-quality genomic DNA extraction is fundamental for the study of gene cloning and expression in plants. Therefore, this study evaluated several methods for extracting genomic DNA from shoots of four Dendrocalamus species to determine the optimal technique. Genomic DNA was extracted using three different methods: a commercial DNA extraction kit method, a modified cetyltrimethylammonium bromide method and a sodium dodecyl sulfate method. A membership function analysis was employed to compare these methods. The results demonstrated that the commercial DNA extraction kit method was the most effective and comprehensive approach for extracting genomic DNA from shoots of four Dendrocalamus species. Furthermore, this study provided valuable insights into optimizing techniques for extracting genomic DNA in other bamboo species.

Comparison of DNA Extraction Methods for the Detection of Canned Tuna Species with DNA Mini-Barcoding

Tuna is susceptible to species mislabeling due to its high demand, quick rate of production, and wide range of price points. DNA barcoding, a sequencing-based technique, allows for the detection of species mislabeling by targeting a standardized region of DNA. A mitochondrial control region (CR) DNA barcode has been found to be capable of species discrimination for tuna, but it is challenging to recover the entire DNA fragment from canned tuna. While a short fragment of CR, referred to as a “mini-barcode,” has shown some success with canned tuna species identification, more research is needed to improve identification rates. The objective of this study was to determine the optimal DNA extraction method for species identification of canned tuna using CR mini-barcoding. Four commercial DNA extraction kits were compared using a sample set of 24 different cans of tuna labeled as albacore, light tuna, skipjack, or yellowfin. All samples were tested in duplicate. The greatest success was found using the Qiagen DNeasy Blood and Tissue Kit and the Qiagen DNeasy Mericon Food Kit, which resulted in species identification for 42% of the samples. In comparison, the MP Biomedicals FastPrep-24 + Machery-Nagel NucleoSpin Tissue Kit resulted in species identification for 30% of the samples and the Qiagen DNeasy Blood and Tissue Kit + PowerClean Pro Cleanup Kit resulted in species identification for 21% of the samples. Overall, the top-performing DNA extraction methods for use with CR mini-barcoding of canned tuna products were determined to be the DNeasy Blood and Tissue Kit and the DNeasy Mericon Food Kit.

Comparative study of extraction and analysis of DNA from different temperatures using phenol chloroform method

The extraction of DNA from biological samples is a fundamental process in forensic investigations, providing invaluable information for the identification and profiling of individuals involved in criminal activities. Saliva is a commonly encountered biological fluid at crime scenes and can serve as a rich source of DNA evidence. This article focuses on the extraction of DNA from saliva samples and explores the methods and techniques employed in forensic investigations to optimize DNA recovery and maximize profiling success. Saliva samples were collected in saline in a swab tube and half of the samples were kept at 4C and half at Room Temperature (RT) to understand the temperature variance. After that, DNA extraction was done using the phenol-chloroform method. The quality and quantity of the extracted DNA are assessed using established techniques such as Gel electrophoresis and qPCR amplification. The results of the experiments reveal the efficiency and reliability of the DNA extraction method for saliva samples. DNA was successfully extracted from the set conditions. Although the DNA bands obtained from 4oC were more prominent compared to the samples kept at RT. The identification of optimal DNA extraction methods for saliva samples can contribute to the development of standardized protocols and guidelines for forensic DNA analysis. In conclusion, this article sheds light on the extraction of DNA from saliva samples for its use in forensic investigation. The outcomes of this study have the potential to improve the efficiency and effectiveness of forensic investigations. Further research in this field is warranted to explore emerging technologies and refine existing methodologies for DNA extraction from saliva samples in forensic contexts.

Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. Effective treatment of melioidosis requires rapid detection of B. pseudomallei infection. Previous studies demonstrated that real-time PCR for the diagnosis of melioidosis in blood samples was insensitive. We optimized the DNA extraction method and evaluated real-time PCRs with an internal control for the detection of B. pseudomallei in plasma samples and compared the performance of real-time PCRs based on six target genes. The lower limit of detection (LOD) of TTS1-orf2, BPSS0745, BPSS1187, and BPSS1498 real-time PCRs was ≤10 genome equivalent (GE)/reaction, while the LOD of BPSS0087 and BPSS1492 was 100 GE/reaction. In the initial phase, we evaluated four targets (TTS1-orf2, BPSS0745, BPSS1187, BPSS1498) for the diagnosis of melioidosis using 87 plasma samples from 38 bacteremic-melioidosis patients and 49 other infection patients at a hospital in Northeast Thailand. Among these, BPSS1187 exhibited the highest sensitivity of 76.3% and specificity of 100%. A second evaluation phase was conducted at two hospitals in Northeast Thailand to compare two real-time PCRs between BPSS1187 and widely used TTS1-orf2 targets. A total of 705 clinical samples were collected from 421 suspected melioidosis cases; 77 of them were subsequently confirmed as melioidosis by culture using one or more specimens. The sensitivities of BPSS1187 and TTS1-orf2 were 89.6% and 79.2%, respectively (P = 0.004), and the specificities were 96% and 99%, respectively. The sensitivities of these two assays in plasma samples were 84.6% and 67.7%, respectively (P = 0.007). Therefore, the BPSS1187 real-time PCR was sensitive and specific for early diagnosis of melioidosis. IMPORTANCE Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, an environmental Gram-negative bacterium. Early detection of B. pseudomallei infection is crucial for successful antibiotic treatment and reducing mortality rates associated with melioidosis. Bacteria culture is currently used to identify B. pseudomallei in clinical samples, but the method is slow. Therefore, there is a need for more accurate and sensitive molecular-based diagnostic methods that can detect B. pseudomallei in all sample types, including samples from blood. We developed an optimal DNA extraction method for B. pseudomallei from plasma samples and used an internal control for real-time PCR. We evaluated six PCR target genes and identified the most effective target for the early detection of B. pseudomallei infection in patients. To prevent delays in the treatment of melioidosis that can lead to fatal outcomes, we recommend implementing this new approach for routine early detection of B. pseudomallei in clinical settings.

Grinding Beads Influence Microbial DNA Extraction from Organic-Rich Sub-Seafloor Sediment.

Sub-seafloor sediment is the largest microbial habitat on Earth. The study of microbes in sub-seafloor sediment is largely limited by the technical challenge of acquiring ambient microbial DNA because of sediment heterogeneity. Changes in the extraction method, even just by one step, can affect the extraction yields for complicated sediment samples. In this work, sub-seafloor sediment samples from the Baltic Sea with high organic carbon content were used to evaluate the influence of different grinding beads on DNA extraction. We found that the grinding beads can affect the DNA extraction from the organic-matter- and biosiliceous-clay-rich samples. A mixture of 0.5-mm and 0.1-mm grinding beads exhibited higher DNA yields and recovered more unique taxa than other bead combinations, such as Stenotrophomonas from Gammaproteobacteria and Leptotrichia from Fusobacteria; therefore, these beads are more suitable than the others for DNA extraction from the samples used in this study. This advantage might be magnified in samples with high biomass. On the contrary, the use of only small beads might lead to underestimation for certain Gram-positive strains. Overall, the discovery of abundant widespread deep biosphere clades in our samples indicated that our optimized DNA extraction method successfully recovered the in situ microbial community.

Determination of the Optimal Bacterial DNA Extraction Method to Explore the Urinary Microbiota.

Recent advances in molecular biology have been successfully applied to the exploration of microbiota from various fluids. However, the urinary microbiota remains poorly explored, as its analysis requires specific technical considerations. Indeed, urine is a low microbial biomass environment, in which the representativity of each bacterium must be respected to obtain accurate data. Thus, sensitive extraction methods must be used to obtain good quality DNA while preserving the proportions between species. To address this, we compared the efficiency of five extraction methods on artificial urine samples spiked with low amounts of four bacteria species. The quality of the DNA obtained was further evaluated by different molecular biology approaches, including quantitative PCR and amplicon-based next-generation sequencing (NGS). Although two extraction methods allowed DNA of sufficient quality for NGS analysis to be obtained, one kit extracted a larger amount of DNA, which is more suitable for the detection of low-abundant bacteria. Results from the subsequent assessment of this kit on 29 human clinical samples correlated well with results obtained using conventional bacterial urine culture. We hope that our work will make investigators aware of the importance of challenging and adapting their practice in terms of the molecular biology approaches used for the exploration of microbiota.

Development of an Efficient Method to Extract DNA from Refined Soybean Oil

Recently, soybean oil has become the most consumed genetically modified (GM) vegetable oil globally. To monitor products derived from GM sources, DNA-based analysis methods are being widely adopted, particularly to control the authenticity of several food products including oil. Considering that DNA isolation from foodstuffs is the first step in the detection of GM organisms, we compared five different methods to extract high quality DNA from refined soybean oil. We used ultraviolet spectrophotometry, nested polymerase chain reaction (PCR), and real-time PCR to evaluate DNA yield and purity, and the sensitivity of the optimized method was evaluated. According to our results, the most effective DNA extraction method was the cetyltrimethylammonium bromide–NucleoSpin Food Kit method. Using this method, 21–225 DNA copies were isolated from 1 mL soybean or blended oils, and the minimum ratio of soybean oil that could be detected from a mixture of soybean and peanut oil was 5%. To summarize, we report a novel method that combines pre-concentrating oils and commercial kits. This optimized DNA extraction method advances the traceability of soybean oils for authenticity issues and transgenic detection.

A long-amplicon quantitative PCR assay with propidium monoazide to enumerate viable Listeria monocytogenes after heat and desiccation treatments

The objective of this study was to develop a qPCR method for specific enumeration of viable Listeria monocytogenes in food processing facilities and heat treated products. Primers specific for L. monocytogenes were designed to amplify a short (199 bp) or long (1561 bp) fragment of the listeriolysin (hly) gene. The short- and long-amplicon qPCR methods with and without propidium monoazide (PMA) treatment of the cells were tested for their ability to discriminate between viable (no heat) and heat-killed cells (90 °C, 10 min). The PMA-qPCR methods were subsequently used to assess the survival of L. monocytogenes during desiccation (33% RH, 15 °C) on stainless steel surfaces for ten days with and without prior biofilm formation. The long-amplicon qPCR method had a limit of quantification (LOQ) of 1.32 log CFU/reaction (efficiency 92%, R2 = 0.991), while the LOQ for the short-amplicon qPCR method was 1.44 log CFU/reaction (efficiency 102%, R2 = 0.991). PMA was essential for detection of viable cells, and the long-amplicon PMA-qPCR significantly (p < 0.05) reduced the signal from heat-killed cells compared to the short-amplicon method. L. monocytogenes survival during desiccation without biofilm formation was accurately enumerated with the long-amplicon PMA-qPCR method. However, when L. monocytogenes had formed biofilm prior to desiccation, the long-amplicon PMA-qPCR accurately measured the log fold inactivation but underestimated the number of viable cells even with use of an optimized DNA extraction method. This long-amplicon PMA-qPCR method can aid in the detection and enumeration of viable L. monocytogenes cells to further the understanding of its survival and persistence in food processing facilities. The developed method was demonstrated to work on both heat and desiccation treated cells and highlights the importance of amplicon size in viability-qPCR.

Development of a method to extract protozoan DNA from black soil

ObjectivesMicroorganisms in environmental samples are identified by sequential screening, isolation, and culture steps, followed by the verification of physiological characteristics and morphological classification. Isolation and purification of Amoebae from soil samples is extremely complex, laborious, and time-consuming and require considerable expertise for morphological evaluation. PCR testing of soil DNA seems to be an effective means for protozoa habitat screening. In this study, we added Acanthamoeba sp. (MK strain) to soil and developed a method of extracting protozoan DNA from the soil. MethodsSoil allophane is a known DNA adsorbing substance that inhibits the PCR reaction. After comparing the soil properties and allophane contents of 7 soil samples, we attempted to combine multiple cell disruption and DNA purification methods to design an optimal soil DNA extraction method that can be used for downstream PCR analysis. ResultsWe compared five different crushing/refining methods. Amplification of the gene was confirmed by Acanthamoeba specific PCR in protocol V where the concentration of Acanthamoeba in soil (1.0 × 102/g) was the detection limit of PCR. ConclusionThe soil DNA extraction method following protocol V allows DNA amplification of protozoa, including Amoeba, which is difficult to cultivate, thus simplifying the investigation of protozoa habitats and genetic analyses.

Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform: Chelex-100 (in-house and commercialized) and MagPurix TB DNA Extraction Kit

Tuberculosis (TB), caused by infections of the Mycobacterium tuberculosis (MTB) complex, is the ninth leading cause of death worldwide, and several molecular approaches for MTB species identification and the detection of mutations associated with drug resistance have been developed to date. We previously developed a diagnostic assay for drug susceptibility testing that can detect mutations conferring resistance to anti-TB drugs using allele-specific primer extension on a microsphere-based platform for multiplex polymerase chain reaction. The aim of the present study was to optimize this diagnostic assay based on the evaluation of three methods for extracting mycobacterial DNA from clinical samples. Mycobacterial DNA of 81 samples was digested and decontaminated by N-acetyl-l-cysteine-2% NaOH and then extracted using three methods: “in-house” 5% Chelex-100 chelating resin, InstaGene Matrix, and MagPurix TB DNA Extraction Kit. The former two methods are manual extraction methods, whereas the MagPurix TB DNA Extraction Kit is an automated extraction method used with the MagPurix 12 s automated nucleic acid purification system. The extracted DNA was then subjected to our diagnostic assay, and the results were compared among methods. The magnetic bead method exhibited a higher extraction efficiency and resulted in greater diagnostic efficacy than the two resin-based methods with respect to both target gene detection and acid-fast bacilli smear grades. Therefore, the MagPurix TB DNA Extraction Kit is the optimal MTB DNA extraction method for our diagnostic assay of TB drug susceptibility testing.

Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta.

Background and Aim:A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.Materials and Methods:Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.Results:Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme-Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/µl and 161 ng/µl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A260 nm/A280 nm) and 2.3 (A260 nm/A230 nm) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at −80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as Prevotella ruminicola, Streptococcus lutetiensis, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Selenomonas ruminantium.Conclusion:To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment.

Reptile-associated Borrelia species in the goanna tick (Bothriocroton undatum) from Sydney, Australia

BackgroundKnowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp.ResultsWe collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98–99%), while the remaining OTUs belonged to Proteobacteria (51–81%), Actinobacteria (6–30%) and Firmicutes (2–7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa.ConclusionsTwo distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species.

Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

Objectives: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. Methods: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. Results: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. Conclusions: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

Genomic DNA Isolation from High Polyphenolic Content Grewia asiatica L. Leaf Without Using Liquid Nitrogen

Grewia asiatica (Grewoideae family) is also known as Phalsa, used for the medicinal purpose has a high content of polysaccharides and polyphenols together with flavonoids and other secondary metabolites which obstructs their pure genomic DNA extraction. Although, there are various good quality DNA extraction protocols, extracting DNA from a high phenolic content plant is a challenging task. The present study describes an efficient and rapid DNA isolation protocol by optimizing cetyl trimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP) and sodium dodecylsulphate (SDS) cocktail specifically for the extraction of DNA from polyphenolic leaf of G. asiatica without using liquid nitrogen. Extracted genomic DNA has high purity index (1.95) examined by A260/A280 ratio. The presence of good quality DNA was also evident by gel electrophoresis. The optimized DNA extraction method is efficient for both fresh and old tough leaves with high polyphenolic contents. The achievement of this method in extracting high quality of genomic DNA demonstrated the broad applicability of this method.

GENOMIC DNA EXTRACTION METHOD FROM MATURE LEAF OF LAI (Durio kutejensis Becc.)

Lai ( Durio kutejensis Becc.) is an indigeneous germplasm of Kalimantan which has some superiorities compared to its close-relative, durian ( Durio zibethinus Murr.). Genetic exploration of lai is important to support its breeding program. According to rapid development in molecular biology, genetic exploration effort of lai will be easier. One of significant step in any molecular biology activities is DNA isolation to produce high quality DNA for further analysis. Leaves of lai, as other perennial crop, contain of high concen-tration of polysaccharides and polyphenol which will be co-extracted with the DNA. These compounds can interfere enzymes activities in subsequent molecular analysis. The aim of this study was to establish an optimal and effective DNA extraction method to obtain high-quality DNA from mature leaf of lai. An established extraction buffer and its modification were used in this study. The result showed that modification 4 could produce high quality DNA, and was considered to be the most effective DNA extraction method for mature leaf of lai.

Optimized DNA extraction method from skeletal remains using different typing methodologies in forensics

In forensics DNA typing is usually based on STRs, but sometimes challenging samples demand the development of optimized DNA extraction methodologies. In addition, when DNA is severely degraded or in small amounts, results can be inconclusive. Thus another approaches, as miniSTRs, which consist on STRs redesigned to be more sensitive and generate smaller amplicons, are recommended. This study aimed to test an optimized, less time-expensive DNA extraction methodology for human bones combined with STRs and Mini-STRs on samples with previous inconclusive results. Our findings showed an efficiency improvement on both typing methodologies, which suggests that the optimized extraction method is promising.

An enhanced analytical procedure to discover table grape DNA adulteration in industrial musts

The need of accurate and reliable methods for DNA isolation and plant species identification in foodstuffs is of great importance, especially in the protection of high added value products. Fresh foods, which are not subjected to any modifications, are suitable for many kind of analysis; for processed products, such as musts, wines, olive oils, and pasta, the situation may be more complicated due to DNA fragmentation and, in the worst case, by its degradation. This work aimed to establish an exhaustive and reproducible analytical procedure for table grape DNA tracing in industrial musts. Three different DNA extraction methods were initially compared and DNA was tested in PCR for its suitability for the amplification reaction of microsatellite markers or simple sequence repeats (SSRs). An optimized DNA extraction method for microsatellite amplification was developed and adapted for industrial musts. Two SSR-based molecular methods, High Resolution Melting and capillary electrophoresis, were tested and the markers VrZAG62 and VrZAG79 were found to be the most informative. High Resolution Melting analysis, here applied for the first time on musts, proved to be the method of choice for a preliminary screening using four cultivars chosen as references and different DNA mixtures prepared in laboratory. Capillary electrophoresis, providing allele size, allowed a fine genotyping of musts in comparison with reference cultivars. The LOD6 of a single grape cultivar in mixture with other varieties was also determined at 2.5 ng. Merging the information of the two molecular analyses applied to real samples, we demonstrated that is possible to discover case of musts adulterated with table grapes, and we propose our procedure in controlling musts quality and origin certification.

Measurement of 8-Oxo-7,8-Dihydro-2?-Deoxyguanosine in Human Semen and Urine by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry with On-Line Solid-Phase Extraction: Comparison with a Commercial Available Enzyme-Linked Immunosorbent Assay

This study aimed to assess the correlation between 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dGuo) in semen and urine, and to compare the analytical methods of the isotope-diluted liquid chromatograph-tandem mass spectrometry (LC-MS/MS) coupled with an on-line Solid-Phase Extraction (SPE) and commercial Enzyme- Linked Immunosorbent Assay (ELISA) used for detecting 8-oxo-dGuo as an oxidative DNA damage marker. Semen and urine samples were simultaneously collected from 85 apparently healthy human subjects. An optimized DNA extraction method was employed to extract DNA from sperm while minimizing oxidation of DNA. All of the biological samples were analyzed by LC-MS/MS and ELISA. All of the biological samples were detected with 8-oxodGuo. ELISA consistently detected two to three times higher 8-oxodGuo levels in urine samples than LC-MS/MS. However, there was no significant correlation between measurements of 8-oxo-dGuo levels in urine and semen. In conclusion, the LC-MS/MS coupled with an SPE was a sensitive method to detect and quantify 8-oxo-dGuo in human sperm and urine. Urinary 8-oxo-dGuo may not be a reliable marker for detecting oxidatively damaged DNA in sperm.

Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing

To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.