DNA Isolation Kit Research Articles

MICROBIAL DIVERSITY OF COASTAL WATERS OF ODESA BAY OF THE BLACK SEA

The aim of the study was to determine the biodiversity of the prokaryotic microbiota of the coastal water of Odesa Bay of the Black Sea with the help of metagenomics analysis. Methods. Sample of sea water was taken in the area of Нydrobiological station of Odesa I. I. Mechnikov National University (46°26'28.2N 30°46'20.0 E). Sample of sea water was taken from the horizon of 100 cm and filtered through 0.22 μm membrane filters (Sartorius). Total DNA was isolated from the collected microorganisms using the PowerWater DNA isolation kit (catalog no.14900-50- NF) according to the manufacturer's instructions (MO BioLaboratories). Total DNA extraction has been performed according to the manufacturer's instructions (MO BioLaboratories) from collected microorganisms using the PowerWater DNA isolation kit (catalog no.14900-50-NF). To determine the microbial diversity with the help of metagenomic analysis, there were carried out targeted sequencing performed on the Illumina MiSeq platform after amplify v4 region of the 16S rRNA gene. The obtained sequences in the fastq format were analyzed in the miniconda3 shell with used next programs: Fastqcv.0.11.2., Trimmomatic, Cutadapter, SPAdes, Centrifuge and Krona. Results. As a result of the metagenomics 16S rRNA gene analysis of the coastal waters of Odesa Bay about 261 197 annotated sequences were defined. The representatives of main departments of the domain Bacteria were identified: Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Cyanobacteria, Tenericutes, Spirochaetia, Fusobacteria, Thermotogae, Deinococci, Aquificae , Chlorobia, Chloroflexi ,Verrucomicrobia, Deferribacteres, Planctomycetes, Chlamydiia, Acidobacteria, Nitrospirae, Ignavibacteriae, Thermodesulfobacteria, Dictyoglomia, Elusimicrobia, Synergistia, Fibrobacteria, Gemmatimonadetes. The most common departments of Bacteria domain were Proteobacteria (68.0%) and Bacteroidetes (19.0%). The representatives of Gammaproteobacteria (63.0%) and Alphaproteobacteria (31.0%) were dominated among the Proteobacteria. The presence of small amount of representatives of the Archaea domain (less than 0.5%) as the classes Euryarchaeota, Thaumarchaeota, Crenarchaeota was shown. Conclusion. A comparative analysis of the presented results and those that were obtained during previous studies of the biological diversity of microbial communities in the Black Sea estuaries and in the region of the Zmiiniy Island made it possible to determine the main differences in their composition. As it was shown, microbial communities of the coastal water of Odesa Bay, water of the Khadzhybei estuary and in the region of the Zmiiniy Island is manifold and is represented mainly by the members of Proteobacteria and Bacteroidetes while representatives from Cyanobacteria phylum is dominate in the Dry and Dniester estuaries.

Effects of diet on the childhood gut microbiome and its implications for atopic dermatitis

Effects of diet on the childhood gut microbiome and its implications for atopic dermatitis

The GAPDH partial gene of durik-durik (Syzygium sp.) from Riau Province of Indonesia

GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase enzyme is one of housekeeping genes which is often used as an internal conntrol in gene expression analysis. This study is aimed to isolate the GAPDH gene from durik-durik (Syzygium sp.). Fresh leaves of Syzygium sp. were collected from Kajuik Lake which is located in Langgam District, Pelalawan Regency, Riau Province, Indonesia. The methods included total DNA isolation using plant DNA isolation kit (Qiagen), polymerase chain reaction, electrophoresis, and sequencing. Data was analyzed using BioEdit7 and MEGA6 softwares, and BLAST program. The DNA partial sequence of GAPDH gene was obtained from Syzygium sp. with the length of 623 bp. The DNA sequence was predicted consisting of 2 exons and 3 introns and encoding 68 amino acids. The DNA sequence of Syzygium sp. GAPDH gene had 74%-89% similartiy to one of other plant species, but none was member of Syzygium genus. The deduced amino acid sequence had 100% similartiy to one of Tropaeolum minus dan Mentha spicata plants. The GAPDH partial gene obtained in this study is the first one reported from Syzygium genus and this sequence can be a basis for isolating of GAPDH gene from other species in Syzygium genus.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.

Impact of JAK2V617F Mutation Burden on Clinial Presentation and Survival in ET Patients

To better define the effect of JAK2V617F mutant allele burden on clinical presentation of patients with essential thrombo cythamia (ET), especially thrombosis. Two ml of heparin anti-coagulated bone marrow was collected from 229 ET cases, who were diagnosed and treated in the First People's Hospital of Yunnan Province during 2013.10 to 2016.12. and then the mononuclear cells were separated by Red Blood Cell Lysis Buffer, genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen the JAK2V617F mutation, then the JAK2V617F mutation burden was detected by real-time polymerase chain reaction (RT-PCR) in 120 patients with JAK2V617F mutation. Meanwhile, these samples were sequenced in order to verify the accuracy of the PCR screewing. ET patients with thrombotic events had significantly higher JAK2V617F allele burden than those without thrombosis (23.2% vs 14.2%) ( P<0.05). Meanwhile, ET patients showed increased JAK2V617F allele burden in the group with higher leukocytosis (WBC > 10×109/L) (P<0.001) and hemoglobin (> 150 g/L) (P<0.05). JAK2V617F mutation burden in 17 patients with splenomegaly was higher than that in 45 patients without splenomegaly (28.1% vs 11.8%) (P<0.05). but the JAK2V617F mutation burden was regatively correlated with platelet count (P<0.05). On the other hand, no correlation was found between JAK2V617F mutation burden and sex (P > 0.05). Univariate analysis showed that the JAK2V617F allele burden did not affect survival. Multivariable analysis showed that prognostic variable including WBC counts, hemoglobin level, age, sex, and splenomegaly not affected survival, (P > 0.05). The clinical presentations of ET patients, such as WBC counts, hemoglobin level and splenomegaly, are influenced by the JAK2V617F mutation burden. ET patients with thrombotic events has significantly higher JAK2V617F allele burden than those in ET palients without thrombosis.JAK2V617F mutation burden has no relations with sex and age..

Investigation of Detection Limits and the Influence of DNA Extraction and Primer Choice on the Observed Microbial Communities in Drinking Water Samples Using 16S rRNA Gene Amplicon Sequencing.

In recent years, 16S rRNA gene amplicon sequencing has been widely adopted for analyzing the microbial communities in drinking water (DW). However, no comprehensive attempts have been made to illuminate the inherent method biases specifically relating to DW communities. In this study, we investigated the impact of DNA extraction and primer choice on the observed microbial community, and furthermore estimated the detection limit of the 16S rRNA gene amplicon sequencing in these experimental settings. Of the two DNA extraction kits investigated, the PowerWater DNA Isolation Kit resulted in higher yield, better reproducibility and more OTUs identified compared to the FastDNA SPIN Kit for Soil, which is also commonly used within DW microbiome research. The use of three separate primer-sets targeting the V1-3, V3-4, and V4 region of the 16S rRNA gene revealed large differences in OTU abundances, with some of the primers unable to detect entire phyla. Estimations of the detection limit were based on bacteria-free water samples (1 L) spiked with Escherichia coli cells in different concentrations [101–106 cells/ml]. E. coli could be detected in all samples, however, samples with ∼101 cells/ml had several contaminating OTUs constituting approximately 8% of the read abundances. Based on our findings, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination with PCR amplification of the V3-4 or V4 region for DW samples if a broad overview of the microbial community is to be obtained.

Outbreak of Choanephora Stem Rot Caused by Choanephora cucurbitarum on Quinoa (Chenopodium quinoa) in China.

Quinoa (Chenopodium quinoa Willd.) has been cultivated for centuries as an important subsidiary grain crop. In early August 2015 and 2016, Choanephora stem rot symptoms were observed on plants of Quinoa cultivar Longli 1 in a field with 0.3 ha planting area in Yanqing district, Beijing, China. This disease caused about 70% yield loss of quinoa. Disease symptoms initially appeared as water-soaked spots on the stem that turned into brown to dark brown lesions covering the whole stem, often resulting in the infected part breaking off the rest of the plant. Serious infections often caused the plant to wither and die under high humidity. The infected tissues produced an abundance of sporangiophores bearing brown to black sporangiola. To isolate the causal agent, sections of diseased stem were surface disinfected with sodium hypochlorite, placed on acidified potato dextrose agar (APDA), and incubated at 25°C with a 12-h photoperiod. Two isolates were obtained (ZD61 and ZD62). The colonies of both isolates grew quickly on PDA and were initially white with abundant aerial mycelia. The mycelia later turned pale yellow and produced abundant sporangiophores bearing sporangiola. Sporangiophores were smooth, hyaline, and aseptate, erect, unbranched, and apically dilated to form a clavate vesicle, which produced dichotomously branched and distally clavate secondary vesicles bearing monosporous sporangiola. Monosporous sporangiola were broadly ellipsoid, indehiscent, pediculate, brown to dark brown, with distinct longitudinal striations, and were 7 to 15 μm wide and 11 to 24 μm long by measuring 30 sporangiola. Sporangia contained few or multiple spores. They were also spherical, initially white to yellow, and pale brown to dark brown at maturity and measured 63 to 160 μm. Single-celled sporangiospores were aseptate, ellipsoid, and brown to pale brown, with multiple hyaline polar appendages and striations on the wall. They were 6 to 11 μm wide and 9 to 22 μm long by measuring 50 spores. The morphological and cultural characteristics of both isolates were identical to those of Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (Kirk 1984). Genomic DNA was extracted from the isolates with the DNA Isolation Kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) regions of the two isolates were sequenced using primers ITS1 and ITS4 (White et al. 1990). The two resulting sequences were submitted into NCBI (GenBank MG650193 and MG650194), which were 99% identical to known C. cucurbitarum strains (GenBank KY080447, KY080446, KU877802, and KR873353) according to a BLAST analysis. Pathogenicity tests were conducted on hypocotyls of 4-week-old quinoa seedlings inoculated with 2-day-old mycelia plugs. Control plants were inoculated with sterile APDA plugs. Plants were incubated under humid conditions for 3 days and then transferred to a greenhouse (28°C). After 4 to 5 days, lesions were evident on the stems of inoculated plants but not on control plants. After 7 days, all inoculated plants had developed brown stem lesions and were wilted. Additionally, C. cucurbitarum was reisolated from the inoculated plants, fulfilling Koch’s postulates. The fungus C. cucurbitarum reportedly has a wide host range. In China, C. cucurbitarum was recently observed on Moringa oleifera causing seed pod rot (He et al. 2017). However, to our knowledge, this is the first report of C. cucurbitarum causing stem rot on C. quinoa in China or globally.

Evaluation of commercial kits for purification of circulating free DNA

Analysis of liquid biopsies and the identification of non-invasive biomarkers for the diagnosis and prognosis of solid tumors has grown exponentially over the last few years. This has led to an increasing number of commercial kits optimised for the purification of circulating free (cf) DNA and RNA/miRNA from biofluids such as plasma, serum and urine. To optimise and standardise current practices we sought to evaluate the performance of spin column-based and magnetic bead-based commercial kits. The following commercial cfDNA purification kits were analysed in this study: QIAamp circulating nucleic acid kit (Qiagen, Germany); Plasma/serum cell-free circulating DNA Purification midi kit (Norgen Biotek, Canada); QIAamp minElute ccfDNA mini kit (Qiagen); Maxwell RSC ccfDNA plasma kit (Promega, USA); MagMAX cell-free DNA isolation kit (Applied Biosystems, USA); and NextPrep-Mag cfDNA isolation kit (Bioo Scientific, USA). Extracted DNA from the plasma of healthy individuals, either nonspiked or spiked with DNA fragments or cfDNA, was evaluated for recovery using either a BioRad Experion or ddPCR analysis. This study represents the first to use a comprehensive size distribution of spiked-in DNA fragments to evaluate commercial cfDNA kits. The commonly used spin column-based Qiagen QIAamp circulating nucleic acid kit was found to be the most consistent performing kit across the two evaluation assays employed. The Qiagen QIAamp minElute ccfDNA mini kit represented the best performing magnetic bead-based kit and provides an alternative based on lower cost/sample with a simpler workflow than spin column-based kits.

Influence of commercial DNA extraction kit choice on prokaryotic community metrics in marine sediment

AbstractCommercial DNA extraction kits are widely used for cultivation‐free surveys of marine sediment. However, the consequences of popular extraction‐kit choices for sequence‐based biological inferences about marine sedimentary communities have not previously been exhaustively assessed. To address this issue, we extracted DNA from multiple paired subsamples of marine sediment using two popular commercial extraction kits (MO BIO Laboratories PowerSoil® DNA isolation kit and MP Biomedicals FastDNATM Spin Kit for Soil). We report comparisons of (1) total DNA yield, (2) extract purity, (3) gene‐targeted quantification, and (4) post‐sequencing ecological inferences in near‐seafloor (&lt; 1 meter below seafloor [mbsf]) and subsurface (&gt; 1 mbsf) marine sediment. In near‐seafloor sediment, the MP Biomedicals FastDNATM Spin Kit for Soil exhibits higher extraction yields, higher 16S rRNA gene loads, higher taxonomic diversity, and lower contaminant loads. In subseafloor sediment, both kits yield similar values for all of these parameters. The MO BIO Laboratories PowerSoil® DNA isolation kit generally co‐extracts less protein with the DNA in both near‐seafloor and subseafloor sediment. For samples from all depths, both kits exhibit similar depth‐dependent community richness patterns, taxonomic composition, and ordination‐based similarity trends. We conclude that, despite kit‐specific differences in extract yields, purity and reagent contaminant loads, ecological inferences based on next‐generation sequencing of DNA extracted using these popular commercial kits are robustly comparable, particularly for subseafloor sediment samples.

Investigation of Streptococcus pyogenes virulence factors and typing by multiple locus variable number tandem repeat fingerprinting (MLVF) method

Streptococcus pyogenes is an important bacterial pathogen that colonizes the throat and skin of human beings and causes a wide variety of diseases ranging from mild infections like pharyngitis, tonsillitis and impetigo to severe invasive infections such streptococcal toxic shock syndrome, septicemia, and necrotizing fasciitis, and produces a wide variety of virulence factors. The aim of this study was to investigate the antibiotic resistance, virulence genes; [pyrogenic exotoxin genes (speA, C, G, H, I, J, K, L, M, smeZ and ssa), deoxyribonuclease genes (sdaB, spd3, sdc ve sdaD), protease genes (speB, spyCEP ve scpA) and inhibitor genes (mac and sic)] of S.pyogenes strains isolated from throat cultures of patients with symptomatic tonsillo-pharyngitis and typing by multiple locus variable number tandem repeat fingerprinting (MLVF) method. One hundred and fifty S.pyogenes isolates were identified by conventional methods and streptococcus group A latex kit (Biomerieux, France). Antibiotic susceptibility tests were performed by Kirby-Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute. DNA isolation was performed by using a commercial DNA isolation kit (Qiagen, Germany) in accordance with manufacturer's recommendations. The virulence genes were determined by multiplex PCR. MLVF method was performed with multiplex PCR using specific primers for repeated sequences within bacterial genome. All of the S.pyogenes isolates were susceptible to penicillin G, cefotaxime, ceftriaxone, chloramphenicol, clindamycin, erythromycin, levofloxacin, vancomycin and linezolid. Among streptococcal pyrogenic exotoxin genes the most frequent gene was smeZ (90.0%) followed by speG (88.0%), speC (58.7%), ssa (42.7%), speA (33.3%), speJ (24.0%), speK (18.7%), speH (14.0%), speI (13.3%), speL and speM (9.3%). Of the DNase genes, sdaB was detected in all strains (100%), spd3, sdc, sdaD genes were determined as 64.7%, 36.0%, 24.7% respectively. Protease genes (speB, spyCEP, scpA) and mac gene from the inhibitor genes were positive in all strains, and sic gene was positive in only 3 (2.0%) of the isolates. Thirty-two different patterns that contained two or more isolates were determined by MLVF analysis. Ninety one isolates were included in any of the 32 different patterns, while 59 isolates were defined as sporadic isolates. In conclusion, S.pyogenes isolates collected from throat cultures of patients with symptomatic tonsillo-pharyngitis in Konya/Turkey were susceptible to all antibiotics studied and have carried a very high rate of virulence factors. However the isolates were mostly clonally unrelated and sporadic. This study is the first report in Turkey, in which S.pyogenes isolates were typed by the MLVF method and a large number of virulence factors were investigated.

Abstract 5138: Comparative profiling for bacterial inhabitance in pancreatic ductal adenocarcinoma and matched adjacent normal tissues

Abstract Objective: Composition of resident microbes of the human body is associated with different disease manifestations. The microbiome in bodily fluids and different organs is altered with diverse health outcomes including cancer. Microbiome composition is a potential source for identifying biomarkers in cancers. Studies have shown the association of specific bacterial communities with the occurrence of pancreatic ductal adenocarcinoma (PDAC). However, investigations for the altered microbiota in an individual with the emergence of the disease are not studied. So, this study presents a comparative profiling for bacterial inhabitance in cancer tissues with that of matched adjacent normal tissues. This association will enable discovery of potential microbial biomarker(s) for PDAC. Methods: Fresh frozen cancer and matched adjacent normal (N= 20 each) tissues were collected from PDAC patients. Genomic DNA was extracted by MO BIO PowerSoil DNA Isolation kit and enriched using NEBNext Microbiome DNA Enrichment kit. Next-generation sequencing (NGS) method, pyrosequencing of 16S rRNA was used to assess the diversity of the tissue-associated microbiota. Operational Taxonomic Units (OTUs) were defined at &amp;gt;97% similarity followed by taxonomic classification using BLASTn against a curated GreenGenes/NCBI/RDP derived database (bTEFAP®). The Bray-Curtis coefficient was used to quantify the compositional dissimilarity between microbiome profiles. Statistical analysis was done using “R” and NCSS 2010. Alpha and beta diversity analyses were conducted to cluster samples by their microbiome profiles. Overall associations between PDAC and microbiome compositions were assessed based on weighted or unweighted UniFrac distances using PERMANOVA. Results: 16S taxonomic profiling reveal 10 and 11 dominant bacterial genera associated with normal and cancer lesion, respectively. Prominent genera that are only found in cancer lesions include Bacteroides, Enhydrobacter, Lautropia, Mycobacterium, and Phascolarctobacterium, whereas Fusobacterium, Acinetobacter, Propionibacterium, and Pseudomonas are the genera with high relative abundance in both cancer lesion and adjacent normal tissues. The most prevalent bacterial species in all observed samples are Propionibacterium acnes and Pseudomonas pseudoalcaligenes. We found Fusobacterium nucleatum in 4 samples (2 each in tumor and adjacent normal) in our preliminary work (10% prevalence). The PCoA multidimensional plot showed clustering of “tumor” samples, indicating the microbiota of these specimens is distinct from normal adjacent tissues. Conclusion: This study suggests a plethora of distinct bacterial community structure in PDAC lesions that is significantly different from that of matched adjacent normal tissues. Additionally, it suggests that the tissue-associated microbiota can serve as a useful biomarker for PDAC. Citation Format: Sheema S. Khan, Pratik Banerjee, Saini Setua, Daleniece Higgins, Satish Kedia, Yu Jiang, Meena Jaggi, Subhash Chauhan. Comparative profiling for bacterial inhabitance in pancreatic ductal adenocarcinoma and matched adjacent normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5138.

Genetic variations among three major ethnic groups in Nigeria using RAPD.

Genetically, every individual is unique; this may stem from inheritance, geographical locations, and/or environmental interactions. This study examined the possibility of developing a cheap and easy-to-use marker that can distinguish among the three ethnic groups in Nigeria using RAPD-PCR. Five RAPD primers, OPA1-3 and OPC1-2, were randomly selected and used to amplify DNA samples isolated from blood of eighteen human subjects representing the three major ethnic groups in Nigeria (six subjects each). Genomic DNAs were extracted using DNA isolation kit, RAPD-PCR amplification was performed and gel electrophoresis was done. Genetic similarity between the band polymorphism was evaluated as frequencies of occurrence and the phylogenetic tree constructed. Three of the five primers show various polymorphisms; the highest frequency band for primer OPA1 is 50% while that of primer OPA2 is 100% and for OPC2 is 83.33%. Although OPA2 has common bands in majority of the samples few of the bands are ethnic group specific. Bands 471 and 435 bp are specific for the Hausa ethnic group at 66.67% frequency. Similarly, in primer OPC2, band 320 can be used to distinguish the Hausas from the other two ethnic groups. Analysis of variance (ANOVA) and test for homogeneity showed that there is no significant difference in the polymorphism between and among the groups. In conclusion this research has given an insight into the possibility of developing RAPD primers that could be used to distinguish people of different ethnic groups.

Comparison of Some Plant DNA Extraction Methods

DNA isolation is a routine procedure when performed in laboratory environment, yet in the field it may still remain problematic. This is especially true of some crop species bred for useful metabolites that may also hinder DNA extraction. Here we compare the efficiency of DNA extraction protocols and commercial DNA isolation kits when used on samples from Helianthus and Allium. Since extraction of DNA is known to be compromised by co-extraction of PCR-inhibiting metabolites, the isolation of DNA was followed by PCR as a testing procedure for the isolation step. The MagnoPrime Fact and MagnoPrime Uni DNA isolation kits were better suited for field work due to faster processing times and smaller required amount of starting material (20 mg fresh/0.5 mg dry). In all cases the subsequent PCR managed to amplify the DNA fragments of interest well enough to be useful in further research.

Prevalence of alpha actinin-3 gene (ACTN3) R577X and angiotensin converting enzyme (ACE) insertion / deletion gene polymorphisms in national and amateur Turkish athletes

Studies to date showed the importance of alpha- actinin-3 (ACTN3) R577X and angiotensin converting enzyme (ACE) ID polymorphisms on determining athletic performance. Therefore, in this study, we aimed to examine polymorphisms given to Turkish athletes and compare them with sedentary individuals. Genomic DNAs were extracted from peripheral blood by using commercially available DNA isolation kit (Macherey-Nagel, NucleoSpin®, Germany). For this study, a total of 84 volunteers (23 national athletes, 27 amateur athletes and 34 sedentary controls) was recruited. ACE ID genotypes were determined by conventional polymerase chain reaction, and ACTN3 R577X polymorphisms by polymerase chain reaction- restriction fragment length polymorphism methodology. In ACTN3 R577X polymorphism, RX was the dominating genotype, and we detected no RR genotype in national athletes. (no RR genotype was detected in national athletes) X allele is more frequent in national athletes and R allele was more frequent in both amateur athletes and control group. II genotype was more frequent in national athletes and in control group, whereas DD genotype was more frequent in amateur athletes for ACE ID polymorphism. When we consider alleles, D allele was found more frequently in amateur athletes and control group whereas I allele was more frequent in national athletes in ACE ID polymorphism. For ACTN3, X allele was superior to R allele. ACTN3 R577X and ACE ID polymorphisms were important biomarkers in determining athletic performance. However, our results in Turkish athletes suggest that ACE D allele and ACTN3 X alleles may be beneficial to athletes potentially, regardless of the distance they perform.

Reponse to Send et al. telomere length in newborns is related to maternal stress during pregnancy.

We were initially very enthusiastic about the recent report by Send and colleagues [1], particularly given its consistency with previous studies [2]. However, more careful reading revealed several critical concerns with significant relevance to the field. First, although the authors report sex differences in cord blood telomere length (TL), they fail to account for this in their analytic models. A significant difference by sex in the primary outcome (i.e., TL) necessitates, at the very least, controlling for sex analytically in all models. This is especially important given the robust evidence of sex differences in prenatal programming across species and offspring outcomes [3, 4]. Furthermore, the authors fail to report the race/ethnicity frequencies of their cohort, aside from Caucasians, and do not control for race in analyses, despite reported race differences in TL [5]. The second concern is of TL measurement methodology. While it is important that the authors address TL differences resulting from DNA isolation kit, the actual comparison of samples is unclear. The authors report longer cord blood TL with the Chemagen module isolated DNA relative to the Qiagen GmbH, however only 14 DNA samples were isolated using Qiagen GmbH. It is unknown if the same cord blood samples were extracted using both methods and then assayed or if a simple t-test was conducted. Secondly, the authors report significant batch effects for the qPCR assays, yet fail to offer explanation. If 128 samples were repeated on two separate occasions, perhaps a correction factor could be used. In addition, only a coefficient of variation (CV) was reported for the 128 repeated samples, no CV was reported for the entire TL database, and no threshold of quality was reported, a standard quality control outcome. Furthermore, while the authors residualized newborn TL for batch effects, the same standard of genomic DNA from the K562 cell line was used and therefore an indication of the batch variability may be determined by differences in the standard curves suggesting that the batch effect was the result of user error or in reagents (which is unlikely) used between plates. In short, although the conceptual framework for the report by Send and colleagues [1] has merit, the methodology regarding TL measurements and the failure to control for infant sex, given significant differences, is puzzling. Defining the true relationship between TL and maternal prenatal stress has substantial significance, both for our understanding of the biological mechanisms through which early adversity is embedded and because telomere attrition across the life span is related to initial TL [6]. With the explosive growth in telomere studies, researchers need to ensure the most stringent methodologic assays and statistical analyses, ideally using significant sample sizes and longitudinal data to protect against false positive findings and intra-assay variability.

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

BackgroundPolymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process.Methodology/Principal findingsWe assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately.Conclusions/SignificanceThis is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

PCR diagnostics of Chlamydia trachomatis in asymptomatic infection by women.

Chlamydial infection is often asymptomatic. The lack of symptoms may result in the infection developing into a chronic. Chlamydial infections of the genitourinary system in women can lead to serious complications like PID, fallopian tubes infertility, ectopic pregnancy, and chronic pelvic pain. An infection of the genitourinary system does not cause any lasting immune resistance and does not protect against re-infection. The aim of this research was to conduct tests for Chlamydia trachomatis on healthy women without any genital system symptoms and to estimate the frequency of asymptomatic infections. During preventive examinations a cervical smear was obtained from the patients n = 100. The aver-age age of the patients was 24.86 ± 3.15. The swabs were sampled by gynecologists. During the examinations Geneproof PathogenFree DNA isolation Kit and GeneProof Chlamydia trachomatis PCR kit which detects 16S rRNA conservative coding sequence, conservative region of cryptic plasmid DNA, including deletion mutation in cryptic plasmid (Swedish variant). Swabs were sampled from 100 women aged 18-32 who had no symptoms of chlamydial genitourinary system infection. Within the study group 4% of women received a positive result, i.e. 4/100. The study confirmed asymptotic infection in 4% of women. In own research it was not possible to confirm cor-relation between the presence of Chlamydia trachomatis and the number of partners or the number of sexual intercourses.

First Report of Alternaria arborescens Species Complex Causing Leaf Blotch and Associated Premature Leaf Drop of ‘Golden Delicious’ Apple Trees in the Netherlands

Apple (Malus domestica) is an important fruit crop in the Netherlands, with a total production area of 7,600 ha in 2016, and 1,600 ha of fruit tree nurseries. For decades, fruit growers and nurseries have reported a leaf blotch, characterized by irregular light brown spots, bordered by a dark brown to purple margin, and premature leaf drop, mainly on ‘Golden Delicious’. The disease causes severe, sometimes almost complete defoliation. Leaf blotch typically appears in early July and is most severe in the latter part of the growing season. In a survey in August 2014, affected ‘Golden Delicious’ leaves were collected from different orchards and taken to the laboratory. Next, 20 leaves per location were rinsed with sterile water, sprayed with 70% ethanol until droplet runoff and rinsed again with sterile water, and tissue sections were excised from lesions using a sterile scalpel and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were transferred to fresh PDA plates. These isolates produced fast-growing colonies of irregular shape, tan brown to black and felty. Sporulation patterns showed long conidiophores with extensive terminal branching. Conidia were ovoid with a tapering apical beak and a size range of 10 to 30 × 5 to 10 μm, with 1 to 5 septa. The isolated fungi were morphologically identical to small spored Alternaria spp. (Simmons 2007). However, small spored Alternaria spp. cannot be identified based on morphological characteristics (Woudenberg et al. 2015). The identity of 12 representative isolates from two different orchards in their 4th growing season, located in the central part of the Netherlands, was determined by multilocus gene sequencing. To this end, genomic DNA was extracted using the UltraClean Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA). Sequences of the ITS region, the endoPG gene, and the anonymous region OPA10-2 locus were amplified and sequenced as described in Woudenberg et al. (2013, 2015) and deposited under GenBank accession numbers MG744449–MG744460 (ITS), MG744473–MG744484 (endoPG), and MG744461–MG744472 (OPA10-2). MegaBLAST analysis revealed that our ITS, endoPG, and OPA10-2 sequences matched with >99 to 100% identity to Alternaria arborescens species complex (SC) isolates in GenBank (AF347033 and KP124400 [ITS], AY295028 and KP124104 [endoPG], and KP124712 and KP124714 [OPA10-2]). Subsequently, Koch’s postulates for three A. arborescens SC isolates were performed in the laboratory on ‘Golden Delicious’ leaves. Surface sterilized leaves were inoculated on the abaxial side with 10 μl of a suspension of 10⁵ conidiospores ml–¹ water, prepared from a 14-day-old PDA culture, after wounding with a needle, with four inoculations per leaf. Inoculated leaves were sealed in a plastic box and incubated in darkness at 20°C. The experiment was carried out in five replicates. Symptoms appeared within 7 days on 100% of the leaves, while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions cultured on PDA morphologically resembled the original isolate from the infected leaves. The identity of the reisolations was confirmed as A. arborescens SC by sequencing. To our knowledge, this is the first report of A. arborescens SC causing leaf blotch and subsequent premature leaf drop of apple cultivars in the Netherlands. Multiple Alternaria species groups are associated with leaf blotch diseases of apple in Australia and Italy (Harteveld et al. 2012; Rotondo et al. 2012). A. arborescens-like isolates are most prevalent in Australia and are mostly associated with leaf blotch symptoms (Harteveld et al. 2012).

Isolating DNA sourced non-invasively from koala scats: a comparison of four commercial DNA stool kits

Genetic sampling from faeces is a useful method for obtaining DNA samples non-invasively. The quantity and quality of DNA isolated from faecal samples is, however, an important factor affecting the success of downstream analyses. Commercial DNA isolation kits offer an efficient and convenient means for recovering DNA, but the kit methodology can influence the quantity and quality of DNA obtained. Comparisons of kit performance for the isolation of DNA from non-invasive sources for ecological studies based on genetic analysis are uncommon in the literature. This study compared the quantity and quality of DNA isolated from surface washings of fresh koala (Phascolarctos cinereus) faecal pellets (scats) using four commercial DNA isolation kits: Axygen® AxyPrep™ MAG Soil, Stool, and Water DNA Kit (AX), Bioline ISOLATE Fecal DNA Kit (BL), Qiagen QIAamp® Fast DNA Stool Mini Kit (QFS), and Qiagen QIAamp® DNA Stool Mini Kit (QS). DNA quantitation, standard PCR and electrophoresis, real time PCR and replicate genotyping using capillary electrophoresis were used to compare the performance of resultant DNA isolates. The performance of DNA isolated from koala scats varied substantially with the DNA kit utilised. All kits provided accurate genotypes but with differing amounts of missing data. Overall, kit AX performed best, providing DNA isolates of higher quantity and quality compared to kit QS, which has previously been thoroughly assessed for genotyping reliability using DNA from koala scats. Given the high variability noted, assessing kit performance is an important way to maximise data quality from non-invasively sourced DNA.

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N2, room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.