Genomic DNA Purification Research Articles

Analysis of ANRIL gene polymorphism rs4977574 association with kidney cancer development in Ukrainian population.

ANRIL (Antisense Non-coding RNA in the INK4 Locus, also known as CDKN2B-AS1) – 3.8-kb long non-coding RNA transcribed from the antisense strand of INK4b-ARF-INK4a gene cluster. It is known that ANRIL overexpression is associated with development of oncological pathologies of different localization. In addition, there are a number of studies devoted to role of ANRIL genetic polymorphism in emergence and progression of tumors, including tumors of genitourinary system. The aim of the study was to check the possible association between ANRIL gene polymorphism rs4977574 and kidney cancer development in representatives of Ukrainian population. Whole venous blood of 101 patients with clear cell renal cell carcinoma (CCRCC) (42 women and 59 men) and 100 patients without oncology history (34 women and 66 men) was used in the study. DNA from blood white cells was extracted using GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, USA). Genotyping of rs4977574 ANRIL gene polymorphic locus was performed using real-time polymerase chain reaction (real-time PCR) method in the presence of TaqMan assay C_31720978_30. The mathematical data were processed using the SPSS software package (version 17.0). P values <0.05 were considered as statistically significant. It was found that difference in rs4977574-genotype distribution between patients with CCRCC and control persons was absent in general group (P=0.216). At the same time, the statistical analysis stratified by gender showed that both in female and male subjects rs4977574-genotypes frequency also did not differ significantly between comparison groups (P=0.526 and P=0.160, respectively). However, after adjusting for age, body mass index, and smoking habits statistically significant association between rs4977574 ANRIL gene polymorphism and risk of kidney cancer development was detected in male subjects under superdominant inheritance model (P=0.049). It was revealed that heterozygotes (AG-genotype) have 2.17-fold higher risk of CCRCC development (95% CI=1.005-4.695) compared to patients with AA- and GG-genotypes. In summary, this is the first report about ANRIL gene polymorphisms association with kidney cancer. Obtained results revealed that rs4977574 is related to kidney cancer risk only in Ukrainian men. Male individuals with AG-genotype have higher risk of CCRCC development compared to AA- and GG-genotypes carriers.

P0097IDENTIFICATION OF TWO NOVELS SINGLE NUCLEOTIDE VARIANTS OF THE COMPLEMENT GENES IN A PATIENT WITH THE ASSOCIATION OF PREGNANCY ATYPICAL HEMOLYTIC UREMIC SYNDROME AND C3 GLOMERULOPATHY

Abstract Background and Aims Advances in the past 2 decades have shown atypical hemolytic uremic syndrome (aHUS) to be a disorder of the alternative pathway of complement. Most aHUS cases involve sequence variations in genes encoding complement proteins The term pregnancy associated aHUS (P-aHUS) refer to the thrombotic microangiopathy (TMA) that result from uncontrolled complement activation during pregnancy or the postpartum period. P-aHUS is a devastating systemic disease, with high maternal mortality and morbidity rates, in the pre-eculizumab era. The term ‘C3 glomerulopathy’ (C3GN) encompasses a heterogeneous spectrum of immune-mediated nephropathies that share a common pathological feature, glomerular deposition of C3. This entity may progress to advanced stages of chronic kidney disease and shares a common genetic risk factors with aHUS. Even more, some authors even suggest that C3GN and aHUS represent two forms of a disease spectrum with a common pathogenic principle. Here, we report this rare association and describe the family genetic variants that could cause it Method We describe the clinical and laboratoy data of a patient with the association of aHUS and C3 Glomerulopathy. A genetic study was performed and her available relatives were also screened for mutations/polymorphisms in aHUS-associated complement genes. After extracting gDNA from whole blood (Wizard Genomic DNA Purification Kit, Promega), PCR products of coding sequences and intronic flanking regions of complement genes were sequenced by ABI PRISM 310 Genetic Analyzer (Applied Biosystems). In silico analysis for pathogenicity was completed with Polyphen2-HDIV, PhyloP/Phastcons (MutationTaster), SIFT and PANTHER. All the participants provided informed written consent Results In 3/2012 a 27-year-old patient, with no family history, started his current illness one month after his first natural birth, with acute renal failure and microangiopathic hemolytic anemia, demonstrating a severe TMA in a renal biopsy. Laboratory results showed low C3 serum levels, but C4 were normal, haptoglobine was undetectably low, and all ADAMTS13 parameters were normal. Her urine tests showed also glomerular hematuria and proteinuria in the nephrotic range. She was treated with plasmapheresis and fresh frozen plasma with hematological improvement, but hemodialysis was required for more than 3 months. Despite partial recovery of renal function, six months later reappeared anemia and developed severe arterial hypertension, congestive heart failure and progressive renal insufficiency. Diagnosis of aHUS was made and start treatment with eculizumab with progressive recovery of renal function in the following months. Nevertheless the C3 serum levels persisted low and the proteinuria and hematuria did not change even after long term treatment with eculizumab. Retrospectively, her urynalisis before the pregnant showed proteinuria and hematuria, and a revision of a renal biopsy revealed the presence of dominant C3 deposits in the immunofluorescence, and electrondense deposits in the electronic microscopy, suggesting the diagnostic of C3GN. In a genetic study two novel single nucleotide variants were founded (CFH c.575G>A, p.C192Y (exon 5) (NM_000186), predicted to be pathogenic by 4 of 5 available pathogenicity prediction programs; and CFI c.1189G>T, p.V397L (exon 11) (NM_000204), predicted pathogenic by 0 of 6 available pathogenicity prediction programs). (Figure 1) Conclusion We present here the family genetic bases of a patient who developed a C3GN and a aHUS with a different response to treatment with eculizumab. In this case we identified two novel genetic variants in the CFH and CFI genes in a patient with aHUS, who inherited one variant from each parent. Although the CFI variant is predicted to be benign, the CFH variant is predicted to be damaging. It is located in exon 5, which encodes a portion of the factor H protein implicated in binding to C3b

The allele frequency of C825T GNB3 gene in hypotension

Hypotension is signed by the systolic blood pressure less than 90 mmHg and the diastolic less than 60 mmHg. The prevalence of orthostatic hypotension is high, about 10-33%. Genetic polymorphism one of factors influenced the prevalence of blood pressure abnormality. GNB3 gene located at chromosome 12p13 influenced blood pressure by their involved in the second messenger during the transduction pathway. The survey conducted to 54 volunteers (29 normotension and 25 hypotension). The peripheral blood was collected from brachial vein. GNB3 gene were analysis by PCR-RLFP. DNA extraction was processing by GeneJET Genomic DNA Purification Kit. The genotype frequency of CC: CT:TT was 12%:72%:16% in hypotension, while 21%:52%:17% in normotension. The allele frequency of C:T was 48:52% in hypotension and 46,5:53,5% in normotension. The frequency allele of C and T were difference between hypotension and normotension (p=0.005). Concluded that C825T of GNB3 gene associated with hypotension evidence.

Філогенетичний аналіз домінантних мікроорганізмів родів Bacillus i Phyllobacterium, ізольованих з ризосфери ячменю ярого

Goal. To determine the taxonomic position of dominant microorganisms in the rhizosphere of spring barley — the representatives of the genera Bacillus and Phyllobacterium — based on phylogenetic analysis of the nucleotide sequence of the 16S rRNA gene. Methods. To identify the bacteria they used the analysis of the nucleotide sequence of the 16S rRNA gene. Bacterial DNA was extracted from the suspension of bacterial cells using GeneJet Genomic DNA Purification Kit (Thermo Scientific) according to the protocols of the manufacturer. Amplification of the 16S rRNA gene was performed with primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-CGGTTACCTTGTTACGACTT-3′). Results. The taxonomic position is determined by dominant microorganisms in the rhizosphere of spring barley (strains of Phyllobacterium ifriqiyense 1 and Bacillus methylotrophicus 10 from working collection of the Department of ecobiotechnology and biodiversity, NULES of Ukraine) based on phylogenetic analysis of the nucleotide sequence of the 16S rRNA gene. The nucleotide sequence of a fragment of the gene 16S pPHK of the mentioned above strains was registered in the international database GenBank (NCBI) with numbers: MK947049, MK947055, and MK947050, MK947056 respectively. Obtained amplicon in size of ~1500 BP was cut out from the gel and purified using GeneJet PCR Purification Kit (Thermo Scientific). The DNA concentration was determined on spectrophotometer DS11FX+ (DeNovix, USA). The purified PCK-product was sequenced in two directions on the device 3130 «Genetic Analyzer» (Applied Biosystems, USA) using a set of reagents «BigDye Terminator v 3.1 Cycle Sequencing Kit». Conclusions. Analysis of the isolated strains of Phyllobacterium ifriqiyense 1 and Bacillus methylotrophicus 10 for the similarity of the nucleotide sequences of the 16S rRNA gene revealed 99% similarity with sequences of typical representatives of the species concerned. Promising strain Phyllobacterium ifriqiyense 1 and Bacillus methylotrophicus 10 can be successfully introduced in the metagenome of aboriginal groups of the soil as biological agents of microbial preparations. They can provide metabolic functions of biological systems of the rhizosphere of barley, and be practically valuable agents of bioprotector action, induction of systemic resistance of plants against phytopathogens.

A loop-mediated isothermal amplification (LAMP) based assay for the rapid and sensitive group-specific detection of fumonisin producing Fusarium spp

Fumonisins are mycotoxins that contaminate maize and maize-based food products, and feed. They have been associated with nerve system disorders in horses, pulmonary edema in swine as well as neural tube defects and esophageal cancer in humans. The fum1 gene codes for a polyketide synthase involved in the biosynthesis of fumonisins. It is present in the genomes of all fumonisin producing Fusarium spp. Reliable detection of fum1 can provide an estimate of the toxicological potential of cultures and food sources. Therefore, a fum1 specific LAMP assay was developed and tested with purified DNA of 48 different species from the Fusarium fujikuroi species complex (FFSC). The fum1 gene was detected in 22 species among which F. fujikuroi, F. globosum, F. nygamai, F. proliferatum, F. subglutinans and F. verticillioides were the most prominent fumonisin producers. None out of 92 tested non-Fusarium species showed cross reactions with the new assay. The lowest limit of detection (LOD) was 5 pg of genomic DNA per reaction for F. fujikuroi, F. nygamai and F. verticillioides. Higher LODs were found for other LAMP positive species. Apart from pure genomic DNA, the LAMP assay detected fumonisin-producers when 103 conidia/reaction were used as template after mechanical lysis. LAMP-results were well correlated with FB1 production. This is the first report on fumonisin production in strains of F. annanatum, F. coicis, F. mundagurra, F. newnesense, F. pininemorale, F. sororula, F. tjataeba, F. udum and F. werrikimbe. Usefulness of the LAMP assay was demonstrated by analyzing fumonisin contaminated maize grains. The new LAMP assay is rapid, sensitive and reliable for the diagnosis of typical fumonisin producers and can be a versatile tool in HACCP concepts that target the reduction of fumonisins in the food and feed chain.

Abstract P3-08-50: Genome-wide association study predict prognosis of hormone receptor positive metastatic breast cancer in premenopausal women with endocrine therapy

Abstract Introduction: According to phase II clinical trial of endocrine treatment for hormone receptor positive (HR+) metastatic breast cancer(MBC) after tamoxifen treatment in premenopausal women, fulvestrant plus goserelin (F+G) has better clinical outcome than goserelin (G) alone in premenopausal women, in terms of time to progression (TTP). However, aromatase inhibitor (AI) with goserelin (A+G) is not superior to goserelin alone. Among three treatment arms, difference of overall survival (OS) was not observed. Accompanied with this clinical trial, we conducted further genotyping to identify alleles at some genomic loci associated with clinical outcome of endocrine treatments in premenopausal women with HR+ MBC. Methods: This prospective genotyping study included patients enrolled in a randomized phase II clinical trial of fulvestrant plus goserelin versus anastrozole plus goserelin versus goserelin alone for HR+, human epidermal growth factor receptor 2(HER2)-negative tamoxifen-pretreated premenopausal women with recurrent or metastatic breast cancer (KCSG BR10-04). To isolate genomic DNA from peripheral blood leukocytes, we used the Wizard Genomic DNA Purification Kit according to the manufacturer’s instructions (Promega, WI) and Axiom Precision Medicine Research Array (PMRA) (ThermoFisher, CA) was used to genotyping array. Survival analyses for time-to-progression (TTP) and overall survival (OS) were performed using the Kaplan-Meier method. To determine the association between clinical outcomes and single nucleotide polymorphisms (SNPs), we used an R package for genome-wide survival analysis (gwasurvivr). Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for univariate and multivariate analyses. Finally, SNPs with adjusted p-value <0.05 were selected and used for survival analysis. Results: Of 138 participants, genomic DNA was collected and extracted from 127 participants. After quality control (QC), 103 genomic DNA were finally selected for genotyping (35 in F+G, 36 in A+G and 32 in G group). Patients with age under 40 were 34 (33%) and visceral metastases were observed in 50 patients (49%). Medial TTP was 16.3 months (95% confidence interval (CI): 13.3, 19.3) and median OS was 59.6 months (95% CI: 41.1, 78.1). Among 523,289 SNPs, 76 SNPs were associated with TTP (adjusted p-value <0.05, respectively). Of 76 SNPs, 47 were in coding lesions including ELF3, ARHGEF3, FOXP2, NTRK2, XPO5 and PI3KC2A. Other 29 were in non-coding lesions. In terms of OS, 13SNPs were associated and eleven were in coding lesions (ZMYM4, GRM7, ERC2, LEKR1, C3orf700, CEP135, ZNF827, LOC285692, DNAH11, FLJ42102 and LOC283440). Conclusions: Genetic polymorphisms influenced the clinical outcome of HR+ MBC patients treated with anti-hormonal therapy. Further studies on the functional mechanisms relating to these SNPs in these genes are warranted. Citation Format: Ji-Yeon Kim, Seock-Ah Im, Kyung Hae Jung, Keun Seok Lee, Joohyuk Sohn, Jee Hyun Kim, Hae Hyun Jung, Minjoo Lee, Young-Hyuck Im. Genome-wide association study predict prognosis of hormone receptor positive metastatic breast cancer in premenopausal women with endocrine therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-50.

Improved DNA extraction technique from blood clot for diagnosis of Trypanosoma evansi in camels

The detection of Trypanosoma evansi genetic material in clinical samples is considered an important diagnostic tool. When blood clots can be a valuable source of genetic information. In our paper, Fourty blood samples were collected from camels at Cairo abbatoir in plane tubes. Blood clots were dispersed by scissor and treated by lysis buffer and proteinase-K, then DNA was extracted according to protocoal of Gene JET Whole Blood Genomic DNA Purification Mini Kit. the quality and integrity of the extracted DNA were corroborated by agarose gel electrophoresis and conventional PCR. Results confirmed that DNA purified from clotted blood can be used in further amplifications. Moreover 16 out of 40 samples (40%) were positive in PCR with bands at the expected molecular weight (164pb). The objective of the present study was to improve and develop a more sensitive method to recover T. evansi DNA from clot samples.

Screening porcine DNA in collagen cream cosmetic products

Pig derivatives such as collagen are commonly added as an ingredient in cosmetics to improve appearance and skin health. To ensure cosmeceutical products comply with halal regulations in Muslim countries, the development of a quick, valid, practical, and economical method to detect the presence of porcine DNA is necessary. The aim of this study was to detect the presence of pork DNA from cosmetic product. Genomics DNA from highly processed cosmetics cream products and raw meat (as positive control) were isolated by using Wizard Genomic DNA purification kit from Promega. Five cosmetics cream samples that labeled as collagen cream were purchased through the online store. One of the products is declared contains piggy collagens, one is halal and other three are unknown source. Polymerase chain reaction (PCR) assay was performed to amplify the fragment of the 12S rRNA gene by a set of species-specific primer which produces amplicons length 387 bp in porcine DNA. The result showed the presence of porcine DNA which was isolated from raw pork, cream cosmetics that contain piggy collagens and cream hands that contains collagen from unknown source using commercially PCR MyTaq™ DNA polymerase kit and a set of species-specific primer with an annealing temperature of 44.4 ºC. The band produced from this PCR was the highest intensity. The success of the amplification of porcine DNA shows that this method is practical, easy and efficient for routine product analysis for halal authentication in undeclared and declared of the porcine material presence in the product. Hence, consuming cosmetic cream contains porcine DNA is prohibited according to the Islamic view in Malaysia.

Fringed Spiderflower (Cleome rutidosperma) Is a New Host for Purple Coneflower Witches’ Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan

Fringed spiderflower (Cleome rutidosperma) is an annual herbaceous plant that grows as a weed. Originally from tropical Africa, fringed spiderflower has been introduced and is becoming widely distributed at low altitudes in Taiwan. In June 2019, symptoms such as small leaves, witches’ broom, floral virescence, and phyllody were observed on fringed spiderflowers grown at the agricultural experiment station in Wufeng, Taichung City, Taiwan. These disease symptoms are similar to those associated with purple coneflower (Echinacea purpurea) witches’ broom disease caused by a 16SrII-A subgroup phytoplasma identified in the same area (Chang et al. 2015; Tseng et al. 2012). Leaf samples of spiderflowers exhibiting disease symptoms were collected for western blotting and PCR assays. Total proteins were extracted from plant samples and examined by western blotting using the polyclonal antibody raised against immunodominant membrane protein (Imp, accession no. KPN61379) of purple coneflower witches’ broom phytoplasma. A specific signal of expected size (19 kDa) for Imp was detected in the symptomatic leaf samples but not in healthy leaf samples. For PCR analysis, the total DNA of each sample was extracted using the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) and used as a template in nested PCR with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). DNA extracted from leaves sampled on healthy plants were used as a negative control. An amplified DNA fragment of 1.2 kb specific for 16S ribosomal RNA (16S rRNA) obtained from symptomatic leaf samples was gel purified and sequenced. The nucleotide sequence was deposited into GenBank (accession no. MN213634). BLAST analysis revealed that MN213634 is identical to the nucleotide sequence of GenBank accession LKAC01000001.1 (complement [31093 to 32611]) of phytoplasma associated with purple coneflower witches’ broom disease. Using iPhyClassifier, the virtual restriction fragment length polymorphism pattern of the 16S rDNA fragment (accession no. MN213634) confirmed that the phytoplasma detected in symptomatic fringed spiderflower belongs to the 16SrII-V subgroup. Subsequent PCR and DNA sequencing assays further confirmed that the genes encoding the putative secreted proteins (accession nos. WP_054598519 and KPN61138) identified in purple coneflower witches’ broom phytoplasma were also found in samples of symptomatic fringed spiderflowers. Our findings revealed that the fringed spiderflower is a new weed plant host for purple coneflower witches’ broom phytoplasma. This result expands our knowledge of the natural host range of 16SrII-V subgroup strains.

Comparison of 12 DNA extraction kits for vertebrate samples

Comparison of 12 DNA extraction kits for vertebrate samples Martincová, I. Aghová, T. Abstract Obtaining high quality DNA extractions is a crucial step for molecular biology research projects. At present, numerous protocols are available for vertebrate tissue extractions. In the present study we compared eleven column–based protocols and one HotSHOT protocol using similar conditions (i.e., type of sample, weight of starting material). We evaluated time of extraction, quality and quantity of DNA yield, and price of extraction for a single sample. Based on our analysis, the most successful kits for producing DNA with the highest concentration and purity are the JetQuick® Genomic DNA Purification Kit (Genomed) and the NucleoSpin® Tissue (Macherey–Nagel). Nevertheless, it is highly recommended to test various extraction kits with specific samples to find the optimal kit in all aspects of time, quality and cost for a particular project.

Magnetic-silica nanoshell for extraction of fungal genomic DNA from Rhizopus oryzae

The main objective of the present article describes an established genomic DNA purification technique improved in vitro by using magnetic nanoparticles as a stable phase adsorbent. Stöber technique was used to synthesis Fe3O4@SiO3 nanoshell, and described using transmission electron microscopy (TEM) and dynamic light scattering. The quality and quantity of purified DNA were verified by using agarose gel electrophoresis and some PCR strategies such as universal-primed UP-PCR, RAPD patterns. Up to 50 µg of DNA was obtained from a slight quantity of one hundred mg of fungal mycelium. Fungal DNA produced with the aid of this technique was applied as a fungal DNA template for PCR method to amplify UP-PCR, RAPD provided replicable profiles. DNA purified from fungal mats was 100 % pure enough to generally be used at high concentrations for PCR technique. The end result confirmed that Fe3O4@SiO3 nanoparticles of ∼140 nm diameter with concentration of 0.4 mg/ml produced premium quality and amount of isolated DNA produced from fungal mats. The obtained DNA was free from any polluting proteins, polysaccharides and coloured dyes. Magnetic nanoparticles (MNP)-mediated genomic DNA extraction is pretty simple (loose from filtration and centrifugation), speedy (30 min), and ecofriendly (not contain any toxic chemical compounds).

Genetic characterization of farmed and wild populations of African catfish (Clarias gariepinus Burchell, 1822) using the random amplified polymorphic marker

The genetic characterization of Clarias gariepinus was conducted in this study. Thirty (30) C. gariepinus specimens were collected, fifteen (15) each from the wild and farms in northeastern Nigeria for their genetic relatedness and diversity using the RAPD markers. DNA extraction from the blood sample was performed using the Gene Jet Genomic DNA Purification Kit. Five primers were used in employing PCR and amplified 402 RAPD bands from the four strains of Clarias gariepinus. Three hundred and six (76.12%) bands were polymorphic while 86 (21.39%) were monomorphic. The percentage of polymorphism obtained from farmed and wild populations ranged from 58 (47.3%) to 69 (75.9%), respectively. The polymorphic bands per loci within the populations ranged from 67.9% to 82.0%. The mean numbers of inbreeding coefficient (FIS) were 0.083 and 0.053 in the farmed and wild populations. Gene diversity values within farmed and wild populations (Ht) were 0.4522 and 0.4018. The mean genetic differentiation (FST) ranged between (0.203) in the farmed and 0.129 in the wild populations. The analysis of molecular variance revealed that there was 96% of genetic variance within the population and 4% among the population. The genetic identity and distance of four populations were 0.9490 and 0.1038, respectively. The phylogenetic measure has shown that the four strains were divided into two clusters at approximately 0.089 similarity levels. The result indicated a significant level of genetic variation and minimal dendrogram separation in Clarias gariepinus in northeastern Nigeria. Conclusively, this information will be a useful tool for the genetic and breeding program of Clarias gariepinus.

ПРИМЕНЕНИЕ МЕТОДА NGS ДЛЯ ОЦЕНКИ СИМБИОТИЧЕСКОЙ МИКРОФЛОРЫ РУБЦА СЕВЕРНЫХ ОЛЕНЕЙ РОССИЙСКОЙ АРКТИКИ

The method of next generation sequencing (NGS) allows deeply analyze the composi-tion of the microorganisms of the rumen of ruminants. Sampling of the contents of the rumen was carried out in the winter-spring period during the slaughter of reindeer in the territory of the Murmansk region - in the agricultural complex “Tundra” of the Lovozero district. 12 samples of rumen con-tent were taken. Samples of rumen contents intended for molecular genetic studies were frozen immediately after selection at -20 ° C and then placed for long-term storage in a freezer. Total DNA was extracted from the samples using the Genomic DNA Purifica-tion Kit (Fermentas Inc., Lithuania), in ac-cordance with the manufacturer's recommen-dations. The amount of DNA extracted was measured using a Qubit® 2.0 fluorometer (Life Technologies, USA). The extracted total DNA was stored at -20 ° C for further analysis. Evaluation of the bacterial community of the rumen was carried out by NGS sequencing on the next generation sequencing platform MiSeq (Illumina, USA) using primers for 16S rRNA. Sequencing was carried out us-ing reagents for library preparation (Illumina, USA). The processing of the reads (readings of DNA), including overlapping, quality filtra-tion (Q30), and primer trimming, were per-formed using the Illumina bioinformatics platform. Quality control and data analysis was carried out in accordance with the QIIME2 ver.2019.10 program (https://docs.qiime2.org). NGS samples of rumen content of reindeer in the Murmansk region made it possible to describe the bacterial biodiversity patterns of the microbial community of the reindeer rumen and to reveal a number of pathogenic groups of bacteria. Thus, the use of the NGS method for the analysis of the microbial communities of the reindeer rumen is inter-esting for identifying various pathogens and characterizing the digestive system and the health of the animal as a whole.

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

An Effective and Viable DNA Extraction Protocol for FFPE Tissues and its Effect on Downstream Molecular Application

Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues are a readily available source of DNA for retrospective studies including genetic predisposition. FFPE tissues present significant challenges for extraction of pure genomic DNA in adequate amounts due to the harsh fixation conditions and long-term storage associated with samples. The aim of the study is to propose a simple, in-expensive, viable protocol for the extraction of DNA from FFPE tissues and to optimize the same for the detection of 3’UTR miRNA binding site polymorphism in ADAMTS1 gene in colorectal cancer tissues and normal colonic mucosa archival tissues using Sanger sequencing and TaqMan SNP genotyping assay. Five different DNA extraction methods were compared and analyzed. NanoDrop quantification demonstrated that out of the various methods tested, highest quantity and pure DNA was obtained from Modified non heating extraction. When we further applied this method for colorectal cancer tissues and normal colonic mucosa archival tissues the NanoDrop readout indicated that the DNA was intact and pure. Intra samples comparison over a time period of 6 months revealed that the DNA was intact. The samples isolated with Modified non heating extraction were found suitable for downstream processes such as Sanger sequencing and TaqMan SNP genotyping assay. This cost effective and non-arduous protocol can successfully extract even minute quantities of DNA from FFPE tissues and facilitate the downstream molecular analysis of a large number of archival specimens for retrospective studies.

The Effect of Size of Black Cherry Stumps on the Composition of Fungal Communities Colonising Stumps.

We investigated fungal communities colonising black cherry stumps. We tested the hypothesis that black cherry stumps of greater diameter should be characterised by more diverse fungal communities than stumps of smaller diameter. The material for analyses came from Podanin Forest District. DNA was extracted using a Plant Genomic DNA purification kit. The results were subjected to bioinformatic analysis and statistical analysis. The OTU sequences were compared using the BLAST algorithm with reference sequences from the UNITE database. In total, 8192 raw sequences were obtained from samples of black cherry stumps applying the Illumina sequencing technique. The results of the statistical analysis indicate a trend towards increased diversity in bigger black cherry stumps. The dominant share of fungi associated with wood decomposition indicates the progressing process of decomposition in stumps. Identification of the role and functions of the individual components of fungal communities colonising stumps may provide insight into the overall ecology of these organisms and provide a basis for improved plant protection, with a view to limiting the occurrence of black cherries in the future in undesirable locations outside their natural range.

Complete genome sequence and comparative analysis of Streptomyces seoulensis, a pioneer strain of nickel superoxide dismutase.

Streptomyces seoulensis has contributed to the discovery and initiation of extensive research into nickel superoxide dismutase (NiSOD), a unique type of superoxide dismutase found in actinomycetes. Still so far, there is no information about whole genome sequence of this strain. To investigate complete genome sequence and perform bioinformatic analyses for genomic functions related with nickel-associated genes. DNA was extracted using the Wizard Genomic DNA Purification Kit then sequenced using a Pacific Biosciences SMRT cell 8Pac V3 DNA Polymerase Binding Kit P6 with the PacBiov2 RSII platform. We assembled the PacBio long-reads with the HGAP3 pipeline. We obtained complete genome sequence of S. seoulensis, which comprises a 6,339,363bp linear chromosome. While analyzing the genome to annotate the genomic function, we discovered the nickel-associated genes. We observed that the sodN gene encoding for NiSOD is located adjacent to the sodX gene, which encodes for the nickel-type superoxide dismutase maturation protease. In addition, several nickel-associated genes and gene clusters-nickel-responsive regulator, nickel uptake transporter, nickel-iron [NiFe]-hydrogenase and other putative genes were also detected. Strain specific genes were discovered through a comparative analysis of S. coelicolor and S. griseus. Further bioinformatic analyses revealed that this strain encodes at least 22 putative biosynthetic gene clusters, thereby implying that S. seoulensis has the potential to produce novel bioactive compounds. We annotated the genome and determined nickel-associated genes and gene clusters and discovered biosynthetic gene clusters for secondary metabolites implying that S. seoulensis produces novel types of bioactive compounds.

Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing

BackgroundNontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques provide new opportunities to sequence and construct complete bacterial genomes faster and at a lower cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to its slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA with no additional clean-up steps.ResultsOur DNA extraction method was compared to 6 other methods with variations in timing of mechanical disruption and enzymatic digestion of the cell wall, quantity of matrix material, and reagents used in extraction and precipitation. We tested our optimized method on 38 clinical isolates from the M. avium and M. abscessus complexes, which yielded optimal quality and quantity measurements for Oxford Nanopore Technologies sequencing. We also present the efficient completion of circularized M. avium subspecies hominissuis genomes using our extraction technique and the long-read sequencing MinION platform, including the identification of a novel plasmid.ConclusionsOur optimized extraction protocol and assembly pipeline was both sufficient and efficient for genome closure. We expect that our finely-tuned extraction method will prove to be a valuable tool in long-read sequencing and completion of mycobacterial genomes going forward. Utilization of comprehensive, long-read based approaches will advance the understanding evolution and pathogenicity of NTM infections.

Q-LAMP Assays for the Detection of Botryosphaeria dothidea Causing Chinese Hickory Canker in Trunk, Water, and Air Samples

Trunk canker disease caused by Botryosphaeria dothidea with a prolonged latent infection phase poses a serious threat to Chinese hickory production. To further understand the epidemiological characteristics and develop reasonable management techniques, a quantitative loop-mediated isothermal amplification (q-LAMP) assay was developed to quantitatively monitor B. dothidea in hickory plants, water, and air samples. Specific primers were designed based on the different sites of the β-tubulin sequence between B. dothidea and other fungi commonly found on Chinese hickory. At the optimum reaction temperature of 65.9°C, this loop-mediated isothermal amplification (LAMP) assay can specifically distinguish B. dothidea from other tested fungi. The limit of detection of LAMP assays for B. dothidea was 0.001 ng/µl of pure genomic DNA and 10 spores per 1 ml of water. The q-LAMP assay enables rapid detection of B. dothidea within 60 min in hickory trunk, water in hickory forests, and spores captured on tapes. These results provide a powerful and convenient tool for monitoring B. dothidea, which could be applied widely in epidemiology, forecast, and management of tree canker disease.

First Report of Powdery Mildew Caused by Leveillula lappae on Tricholepis stewartei in Pakistan

Tricholepis stewartei C.B.Clarke ex Hook.f. is an Asian plant belonging to family Asteraceae Bercht. & J. Presl, whose members are mostly herbs and subshrubs. During the plant pathogenic survey of Ayubia National Park, district Abbottabad, Khyber Pakhtunkhwa Province, Pakistan, and its adjacent areas conducted in October 2017 to 2018, plants of T. stewartei with powdery mildew symptoms were collected to be checked for infection with powdery mildew fungus. Disease symptoms (i.e., white powdery mycelium with blackish spots immersed in these mycelium patches covering the entire leaf) were observed. Microscopic observations showed hyaline, thin, and smooth walled, 3- to 5-μm-wide hyphae. Conidiophores were erect to bent, with one following cell, and 5 to 12 × 76 to 92 μm. Conidia were formed singly, primary conidia broadly lanceolate, apically narrowed, obtuse to pointed, and 14 to 30 × 35 to 60 μm; secondary conidia were ellipsoid or cylindrical and 12 to 30 × 39 to 55 μm. Germ tubes were subterminal or near the base, short, cylindrical, straight to sinuous, apex simple. Chasmothecia were scattered, globose to subglobose, light brown to dark brown or blackish brown, and 124 to 217 μm in diameter. Peridium cells were irregularly shaped and 9 to 18 × 8 to 18 μm. Appendages were numerous, mycelioid, simple to irregularly branched, thin walled, thicker at the base, smooth, yellowish below, hyaline above (toward tip), mostly continuous, and 4 to 8 × 50 to 160 μm. Asci were numerous, stalked to sessile, 26 to 41 × 41 to 109 μm, and (1- to) 2-spored. Ascospores were subglobose, hyaline to light yellow, and 17 to 24 × 37 to 48 μm. The morphology of the asexual and sexual morphs of the powdery mildew samples found on T. stewartei in Pakistan agrees well with the concept and description of Leveillula lappae in Braun and Cook (2012). Khodaparast et al. (2010) examined the morphology of Leveillula on composites in Iran in detail and differentiated six species. Leveillula collections on a wide range of hosts, agreeing with L. lappae, were assigned to L. taurica sensu lato. To confirm morphological identification, genomic DNA was extracted directly from mycelia and chasmothecia using a Gene Jet Plant Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Waltham, MA) (Andersson et al. 2017). The internal transcribed spacer (ITS) region of the isolate was amplified using primers PMITS1 (5′-TCGGACTGGCC[T/C]AGGGAGA-3′) and PMITS2 (5′-TCACTCGCCGTTACTGAGGT-3′) as forward and reverse primers, respectively (Cunnington et al. 2003). The sequence (ITS) retrieved from this collection (568 bp) supports its identification as a species of the genus Leveillula, but a further identification to species level is not possible owing to lacking reference sequence data for L. lappae in GenBank. There is one difference between the new sequence and a sequence retrieved from L. taurica on Carthamus L. (AB667864), identity 99% (561/562), and two differences to L. duriaei on Phlomis persica Boiss. (AB667859), identity 99% (609/611) including one gap and one substitution. However, phylogenetic analyses just based on ITS data are not sufficient within Leveillula to distinguish between closely allied species (Braun and Cook 2012). Pathogenicity was confirmed by pressing a diseased leaf onto young leaves of three asymptomatic potted plants. Three noninoculated plants were used as controls. Plants were maintained in a greenhouse. Inoculated leaves developed symptoms of powdery mildew after 7 days, whereas the control plants remained symptomless. The fungus on the inoculated leaves was morphologically identical to the fungus on the original infected leaves. Furthermore, L. lappae is still a heterogeneous complex of morphologically similar Leveillula spp. on composites that is in need of phylogenetic characterization and circumscription (Braun and Cook 2012). The whole genus Leveillula, above all the L. taurica complex within this genus, requires a phylogenetic multilocus approach that is currently not available. Therefore, the present collections can currently only be assigned to L. lappae. To our knowledge, this is the first report of L. lappae on T. stewartei from Pakistan. T. stewartei is also reported as new host of L. lappae worldwide.