DNA Isolation Kit Research Articles

Human papillomavirus DNA resides in surgical drain fluid exosomes from HPV+ oropharyngeal squamous cell carcinoma patients and can be spread to neighboring HPV-negative cells.

e18050 Background: Human papillomavirus (HPV) is the primary driver of oropharyngeal squamous cell carcinoma (OPC) in the US. Previous studies have investigated the role of plasma and saliva-based cell-free HPV DNA in OPC, yet the pathophysiological impact of exosomes in HPV+ OPC remains unknown. Exosomes are nanometer-sized membrane vesicles of endocytic origin carrying DNA, RNA and proteins, and have been shown to exist in blood, plasma, and in urine. We hypothesize that serosanguinous fluid drained from the postoperative bed, which we call “surgical drain fluid” (SDF) also contains exosomes. We further hypothesize that exosomes derived from SDF in HPV+ OPC patients contain HPV DNA that can be directly transmitted into HPV-negative cells. Methods: Twenty-six 5-mL SDF samples were collected following surgical neck dissections directly from the surgical drain; 21 from HPV+ and 5 from HPV- OPC patients. Exosomes were isolated from SDF using the Qiagen exoEasy kit. Exosome structure was characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Western blotting (WB) targeted known exosomal surface markers. Exosomes were pretreated with DNase (New England Biolabs) to cleave the surface DNA, then exosomal DNA was extracted using a cell-free DNA isolation kit (Sigma). TaqMan real-time PCR was employed to detect HPV16 copy numbers. Lastly, exosomes isolated from 3 HPV+ patients’ SDF and 1 HPV- patient’s SDF were incubated with HPV- cell lines, SCC22A and SCC25, in order to query exosome-mediated HPV transmission. Results: TEM identified exosomes as round/oval-shaped vesicles measuring between 70 and 150 nm. NTA enumerated an average of 1.5 x 1011 particles per mL of SDF. WB further confirmed exosomal identity with strong CD63, CD9, and TSG101 signal detected. Among SDF exosomes from HPV+ OPC, 81% (n = 17) had detectable HPV16 DNA by real-time PCR, while all HPV- SDF exosomes (n = 5) showed undetectable HPV16. Paired comparison of HPV16 levels within exosomes to total HPV16 in SDF revealed an average of 90% of HPV16 in SDF was contained within exosomes. In an in vitro assay, SCC22A and SCC25 cells treated with HPV16 + exosomes became real-time PCR-positive for HPV16, while cells treated with HPV16- exosomes remained HPV16 negative. Conclusions: This study demonstrates that SDF, a novel post-surgical biofluid analyte, is enriched for HPV-containing exosomes in HPV+ OPC patients. We also found that exosomes from HPV+ OPC patients can transmit HPV to HPV-negative cells, suggesting a novel mode of virus-mediated spread of disease.

Comparison of potential biomarker, ACTN3 rs1815739 polymorphism, for athletic performance of Turkish athletes.

α-Actinin-3 is one of the key components of the Z-line structure of sarcomeres and also have importance in muscle cell signaling processes. Therefore, α-Actinin-3 gene rs1815739 polymorphism is one of the most analysed potential genetic biomarkers in sports genetic studies. We aimed to evaluate the genotypic and allelic distribution of α-Actinin-3 gene rs1815739 polymorphism in Turkish athletes. For this purpose, a total of 131 athletes (39 cyclists, 34 sprinters and distance athletes, 33 volleyball players, 15 bodybuilders and 10 ironmen) and 89 sedentary individuals were enrolled in the study. Genomic DNA from venous blood were isolated by using the PureLink DNA isolation kit by following the manufacturers' instructions. The α-Actinin-3 rs1815739 genotyping was carried out by Real-time PCR using the commercially provided Taqman Genotyping Assay. The statistical evaluations were assessed by the chi-square testing using the GraphPad InStat statistical package. Results showed that cyclists, ironmen and volleyball players showed statistically significant differences in terms of the genotype when compared to the controls. The OR of having the dominant trait (RR genotype vs. RX+XX combined) was 0.5 (95%CI: 0.28-0.91; P= 0.02) which was statistically significant, while the OR of having the recessive trait (XX genotype vs. RR+RX combined) was 3.76 (95%CI: 1.82-7.39; P=0.0002). Our findings indicated that the α-Actinin-3 gene rs1815739 RR genotype was more prevalent in the sprinters and distance athletes. In the bodybuilders, cyclists, and ironmen it was found that they were harboring the RR and RX genotype equally. According to the results, we suggest the α-Actinin-3 rs1815739 as a potential biomarker for personal training programs.

Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients.

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAXTM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.

Dietary Impacts on Changes in Diversity and Abundance of the Murine Microbiome During Progression and Treatment of Cancer

The microbiome has been recognized as being highly impactful for cancer outcomes, both with respect to the diversity and composition of the microbial population, as well as overall abundance. Presence or absence of certain microbial species has been correlated to improved cancer outcomes, in concert with specific treatments. However, little research has been performed to consider the reciprocal of this relationship: how different treatments, or the progression of disease, impact the microbiome. Further, though correlations have been demonstrated between dietary intake of both fats and carbohydrates and the composition and breadth of the microbiome, no studies have investigated the microbiome population as it is impacted by cancer and therapeutic treatments under different dietary regiments.This study aims to investigate how dietary patterns, in concert with progression of cancer and both surgical and therapeutic interventions, impact the diversity and abundance of the murine microbiome.A total of 116 c57BI/6 female mice (7‐8 weeks old) were randomized into four groups (n=29/group, each) one week post ovariectomy. Mice received a specific, semi‐purified diet consisting of A) low sucrose, high omega 3 fatty acid; B) low sucrose, low omega 3 fatty acid; C) high sucrose, high omega 3 fatty acid; or D) high sucrose, high omega 3 fatty acid. For high omega 3 fatty acid groups, 2% of daily calories were supplied to the mice as EPA and DHA in a 1.5:1 ratio. One week after implementation of diets all mice were inoculated with E0771 breast cancer cells. Twelve days following inoculation all mice were subject to lumpectomy. Eight days following, measurement procedures were repeated, as at baseline. Ten days following lumpectomy, half of the mice from each dietary cohort were treated with saline, while the other half were treated with a chemotherapeutic agent (9 mg/kg body weight doxorubicin and 90 mg/kg body weight cyclophosphamide). Six days later, the experiment ended, and mice were sacrificed following IACUC guidelines. Fecal samples, which had been collected at each time point noted above, were processed using QIAmp® PowerFecal® DNA isolation kit and were sent to MCIC (OSU Wooster Campus) for 16S ribosomal genetic profiling. A genomic library was generated, using previously validated primers for the V4 region. FASTQ files were filtered, trimmed, dereplicated, and denoised. Cleaned FASTQ files were then used to infer ASVs.Over the progression of cancer, there has been a consistent decrease in the alpha diversity of the microbiome. The effect of diet was mitigated by introduction of chemotherapeutic agents, though the significance of this effect is yet to be determined. While the analysis is still ongoing, it has been determined that there is a clear dietary effect which interacts with abundance. Multiple ASVs have been shown to be positively associated with high fat consumption, though inverse effects, as well as timepoint and treatment effects, have also been noted. Akkermansia muciniphila was a highly present bacterial species in all treatments, despite it usually being noted only as a minor resident of the gut.

PD31-04 LOWER URINARY TRACT MICROBIOTA DYSBIOSIS IS ASSOCIATED WITH URETHRAL FIBROSIS

You have accessJournal of UrologyCME1 May 2022PD31-04 LOWER URINARY TRACT MICROBIOTA DYSBIOSIS IS ASSOCIATED WITH URETHRAL FIBROSIS Michael Witthaus, Valmik Bhargava, Jill Buckley, and Mahadevan Rajasekaran Michael WitthausMichael Witthaus More articles by this author , Valmik BhargavaValmik Bhargava More articles by this author , Jill BuckleyJill Buckley More articles by this author , and Mahadevan RajasekaranMahadevan Rajasekaran More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002582.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cellular mechanisms of stricture progression after repeated transurethral interventions (TUI) are unclear. We hypothesize urethral injury and mechanical stretch can cause tears on the urethral epithelial cell lining leading to leaking epithelium and urine extravasation (Fig A). Urinary microbiota traverses the epithelial lining and fibrogenesis can be mediated by inflammation due to microbial metabolites. Our objective is to characterize the urinary microbiome of patients with urethral stricture disease (USD) using 16S rRNA taxonomic profiling to elucidate the clinical relevance of the urinary microbiome in USD progression. METHODS: First catch urine samples from USD patients and healthy volunteers were collected and frozen to −80°C. Prior to analysis, samples were thawed at 2-8°C and centrifuged at 4°C for 15 mins to form a pellet. The pellet was then used for bacterial genomic DNA extraction using the PowerMag Soil DNA Isolation Kit. PCR amplification of the V3 and V4 regions of the 16S rRNA gene was performed. Illumina sequencing adapters and unique dual-index barcodes were added by PCR. Samples were then pooled and sequenced with paired-end 150 base pair reads (PE150) on an Illumina MiSeq Instrument. Data was analyzed using Qiime, which was used to determine alpha and beta diversity metrics per sample and to determine which microbes were presented differentially between the two cohorts. RESULTS: Our results are summarized in Fig B-D. Visualizing the specific microbes using a relative abundance bar plot (Fig B), the microbiome of healthy urine was more diverse than patients with USD. A divergence between the two groups supported distinct microbiomes, and a significant difference between patients with USD and controls was observed with beta diversity analysis (Fig C, D). The findings were indicative of the overall composition of microbial communities in each patient population. CONCLUSIONS: Our findings suggest a potential role for urinary microbiota alterations in developing an inflammatory environment and consequent fibrosis progression in USD. Future studies will determine specific urinary microbes between the two cohorts and their relevance to USD. Further potential for targeting specific microbes using next-generation probiotics to correct the urinary microbiota dysbiosis may be beneficial in preventing stricture progression. Source of Funding: UCSD Academic Senate © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e542 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Witthaus More articles by this author Valmik Bhargava More articles by this author Jill Buckley More articles by this author Mahadevan Rajasekaran More articles by this author Expand All Advertisement PDF DownloadLoading ...

First report of halo blight of hop (Humulus lupulus) caused by Diaporthe humulicola in Quebec, Canada.

Halo blight of hop caused by Diaporthe humulicola has recently been reported in Michigan and Connecticut (Higgins et al. 2021, Allan-Perkins et al 2020). In August 2020 growers in Quebec, Canada reported necrotic foliar lesions and desiccation of the hop strobile (cone) on Chinook and Nugget cultivars. The foliar lesions were dry concentric circles with a chlorotic halo surrounding the lesions; no pycnidia were observed on leaves or cones. Up to 100% of the infected bract tissue was dry and easily shattered, the grower estimated that more than 90% of the plants in the hopyard exhibited symptoms. Twenty-six isolates were obtained from surface-sterilized leaf and cone tissue by plating the leading edge of lesions on potato dextrose agar. Fungal isolates were hyphal tipped and were incubated at 22°C with a 12 h photoperiod. After 21-days, all cultures were white to beige with pycnidia. DNA was extracted from cultures using the MagMAX Plant DNA Isolation Kit (Applied Biosystems, Foster City, CA). DNA amplification of a representative isolate (CD6C) was performed with primers ITS1/ITS4 (White et al. 1990) for the internal transcribed spacer (ITS), CYLH3F/H3-1b (Glass and Donaldson 1995) for histone 3 (HIS), and Ef1728f/EF1-986R (Carbone and Kohn 1999) for translation elongation factor 1-α (TEF). Amplification primers were used for bidirectional Sanger sequencing, reads were assembled using Geneious Prime (Biomatters, New Zealand), and identified using NCBI BLAST. BLAST results showed that the sequences for TEF, ITS, and HIS all had 100% pairwise identity to Diaporthe sp. 1-MI (MT909101, MT909099, MT909093, OK001342, MZ934713, OK001341). Futhermore, BLAST results showed that ITS and HIS have 100% pairwise identity D. humulicola (MN152929, MN180214). The TEF sequence also had 99.7% pairwise identity to D. humulicola (MN180209). Koch's postulates were conducted by inoculating six 3-mo-old 'Chinook' plants with conidia harvested from 28-day-old cultures and spraying 50 ml of inoculum (6 x 105 conidia/ml) or water to each plant. Plants were then stored in a greenhouse at 100% relative humidity at 22°C with a 14-h photo period. Lesions appeared on the adaxial side of the leaf after 21 days. D. humulicola was re-isolated from all infected leaf tissue, but not from any water inoculated plants and identified by conidial morphology using descriptions from Higgins et al. (2021). So far, Diaporthe sp. 1-MI appears to be synonymous with Diaporthe humulicola, but currently two names are being utilized (i.e. Diaporthe leaf spot and halo blight). In Higgins et al., (2021) it was proposed that the name halo blight might be more appropriate because disease symptoms are not confined to the leaves and cause significant blighting of cones. Halo blight caused by D. humulicola appears widespread in Michigan and Canada and may become an issue in other eastern North American growing regions with humid conditions.

The First Complete Genome Resource of a Ralstonia solanacearum Phage UAM5 from Colombia.

The First Complete Genome Resource of a Ralstonia solanacearum Phage UAM5 from Colombia.

Genetic Variation of the CYP2C9 Genetic of Minangkabau as a Predictor of Side Effect Providing Indications of Non-steroidal Anti-inflammatory Drugs

BACKGROUND: Non-steroidal Anti-inflammatory Drugs (NSAID) activity showed a varied response based on the genetic polymorphism of the CYP29C enzyme. Metabolism of some NSAIDs such as celecoxib, diclofenac, and ibuprofen is highly dependent on CYP2C9. This enzyme has several variants of the heterozygous and homozygous genotypes such as CYP2C9 *2/2 and CYP2C9 *1/*3 which differ for each population. Genetic testing for specific variations of this allele will help determine whether the drug is a valid pharmacotherapeutic option for the patient or not. AIM: This study aims to analyze the genetic variation of the CYP2C9 gene from the Minangkabau ethnic, West Sumatra, Indonesia, which can later be used as a determinant of selecting the right NSAID with optimal effectiveness and to minimalize side effects. MATERIAL AND METHODS: Respondents of this study were users of NSAID drugs obtained from several hospitals in Padang City and were of Minang ethnicity. Blood samples taken from patients were stored in EDTA tubes and DNA was isolated using a genomic DNA isolation kit, DNAzol® Genomic DNA Kits (Thermofischer Scientific). PCR primer and sequencing were designed using Primer Blast (NCBI) software. The synthesized primer was purified by HPLC. DNA fragment application was carried out using the PCR method. The amplicon DNA was purified and prepared as much as 500 ng for sequencing using Illumina’s Next Generation Sequencing method. This analysis was performed with the help of SPSS software. RESULTS: When analyzing the CYP2C9 gene on Primer F23, 44 people were found to be homozygous for the normal allele (AA genotype), and 11 people were heterozygous (GT genotype), 45 people were normal allele (TT genotype), five people were heterozygous (CG genotype), 49 people were normal allele (AA genotype), and five people were heterozygous (GA genotype). CONCLUSION: This study concluded that relationship between CYP29C genetic variation and NSAID drug metabolism is found at the genotypic frequency rs229837 and rs1934969.

Differences in the gut microbiome composition of Korean children and adult samples based on different DNA isolation kits.

Recent studies have revealed that the composition of human gut microbiota varies according to region, race, age, diet, living environment, and sampling and DNA extraction method. The purpose of this study was to broaden our understanding of the intestinal microbial composition of Koreans by conducting a 16S rRNA amplicon sequencing on 78 Korean samples composed of adults, children, normal and obese groups. We compared the microbiome composition and diversity of these groups at different levels including the phylum and genus level using two different stool DNA extraction kits of QIAamp® PowerFecal® DNA Kit (Qiagen, Hilden, Germany) and CT Max Fecal DNA Kit (Ct bio, Korea). We found that Ct bio (Ct) kit recovered higher DNA yields and OTUs than QIAamp® PowerFecal® DNA Kit (Qia). The Ct kit, which adopted more rigorous bead beating method, detected the most Gram-positive (G+) bacteria, Firmicutes, at the Phylum level, whereas the Qia kit, which used a less rigorous cell lysis method, found the most Gram-negative (G-) bacteria, Bacteroidetes. The Firmicutes-to-Bacteroidetes (F/B) ratio showed no significant difference between the obese and the normal groups of same kit; however, they were significantly different with two different kits. There was a difference in the intestinal flora between healthy Korean adults and children. The taxa that differed significantly between the adults and children were Bacteroides, Bifidobacterium, Prevotella, and Subdoligranulum. There was no significant difference in the intestinal flora between the normal weight group and the obese group in adults and children, respectively. This is probably because the difference in body mass index (BMI) between the sample groups collected in this study is statistically significant, but it is not large enough to show a clear difference in the flora. Therefore, these results should be interpreted with caution while considering the BMI values and Korean obesity criterion together.

Selection and Optimization of High-Yielding DNA Isolation Protocol for Quantitative Analyses of Methanogenic Archaea.

Methanogenic archaea are a functionally important component of the intestinal microbiota of humans and animals, participating in the utilization of detrimental hydrogen produced during gut fermentation. Despite this, archaeal DNA has rarely been found in intestinal microbiome analyses, which prompts the need to optimize detecting procedures of these microorganisms, including the DNA isolation step. Three commercially available kits for DNA isolation and one extra purification kit that removes PCR inhibitors were evaluated on chicken droppings. In addition, different variants of mechanical lysis and a double elution were tested to ensure the maximum efficiency of DNA isolation from archaea as well as bacteria. A quantitative real-time PCR was used to monitor the optimization progress. As a result, the combination of the selected Genomic Mini AX Bacteria+ kit with a 2-min-long sonication by ultrasonic probe and enzymatic pretreatment gave excellent extraction efficiency rates for DNA of methanogenic archaea (an approximate 50-fold increase compared to the standard enzymatic lysis described by the producer) and, at the same time, provided optimal protection of DNA extracted from bacteria susceptible to enzymatic lysis. The presented results indicate that the optimized protocol allows for highly efficient extraction of total DNA, which is well-suited for quantitative microbial analyses by real-time PCR.

Improved Glass Bead-Vortex Oscillation Method for DNA Extraction from Diatom.

To examine the effect of improving diatom DNA extraction by glass bead - vortex oscillation method. The DNeasy PowerSoil Pro kit was used as control, two plant DNA extraction kits with different principles (New Plant genomic DNA extraction kit and Plant DNA Isolation kit) and one whole blood DNA extraction kit (whole blood genomic DNA extraction kit) were selected to extract diatom DNA from lung tissue and water sample of the same drowning case. The combination of mass ratio of glass beads with different sizes and vortex oscillation time was designed, and the optimal DNA extraction conditions were selected with the addition of glass beads oscillation. The extracted products of the conventional group and the modified group were directly electrophoretic and detected by diatom specific PCR. Finally, all the extracts were quantified by qPCR, and the Ct values of different groups were statistically analyzed. When the frequency of vortex oscillation was 3 000 r/min, the optimal combination of DNA extraction was vortex oscillation for 4 min, and the mass ratio of large glass beads to small glass beads was 1∶1. The DNeasy PowerSoil Pro kit was used as a reference, and the Ct value of 10 mL water sample was greater than that of 0.5 g tissue. The Ct values of the other three kits used for plant DNA extraction decreased after the glass beads-vortex oscillation method was used, and the Ct values of the tissues before and after the improvement were statistically significant (P<0.05). The whole blood genomic DNA extraction kit used in this study could successfully extract diatom DNA, the extraction of water samples was close to DNeasy PowerSoil Pro kit, after the modified method was applied to tissue samples, the difference in Ct value was statistically significant (P<0.05). However, when the three kits were used to extract diatom DNA from water samples, Ct values before and after the improvement were only statistically significant in New Plant genomic DNA extraction kit group (P<0.05). The improved glass bead-vortex oscillation method can improve the extraction efficiency of diatom DNA from forensic materials, especially from tissue samples, by plant and blood DNA extraction kits.

Alterations of gut bacteria Akkermansia muciniphila and Faecalibacterium prausnitzii in late post-transplant period after liver transplantation

Introduction: Recent studies have shown that the intestinal microbiota can modulate certain systemic metabolic and immune responses, including liver graft function and the development of complications in patients after liver transplantation (LT). Akkermansia muciniphila (AKM) and Faecalibacterium prausnitzii (FAEP) are two of the most abundant gut commensal bacteria, with mucosa-protective and anti-inflammatory effects that are important for maintaining normal intestinal homeostasis and gut barrier function. Our objective was to quantify levels of Akkermansia muciniphila and Faecalibacterium prausnitzii in immunosuppressed patients with LT. Materials and methods: Fecal samples from 23 liver transplant patients (15 adults and 8 children) and 9 non-LT controls were examined. Bacterial DNA was isolated from the samples using the stool DNA isolation kit and the obtained DNA was analyzed with commercially available qPCR kit for AKM and FAEP. Results: We found a statistically significant decrease in the amount of AKM and FAEP compared to the control group. The median values were: for AKM 8.75 for patients and 10.25 for the control group (p = 0.030), and for FAEP 9.72 and 10.47, p = 0.003, respectively. In children after LT, this difference was also statistically significant: AKM (p = 0.051) and FAEP (p = 0.014). In contrast no statistically significant differences were found between adult patients and controls, AKM (p = 0.283) and FAEP (p = 0.056), although the amount of both bacteria showed tendency for reduction. Conclusions: In this pilot study, we found a reduction in the total amount of the two studied bacteria in transplanted patients compared to the control healthy group.

Evaluation of the reliability of SE33 locus in forensic DNA identification in Turkish Population

Forensic DNA studies is a powerful tool in criminal investigations and paternity test. In recent years, STR (Short Tandem Repeat) DNA loci are being used more and more in forensic identification all over the world. In this context, polymorphism rates and frequency of the SE33 region, which is an autosomal STR locus, is aimed to be determined about its reliability. Evaluations were made on the samples of 500 individuals who did not have close relatives. Genomic DNA isolation was carried out by DNA isolation kits (EZ-1 investigator kit and Power Plex Fusion 6C Direct kit) previously by the authorized staff of İzmir Forensic Medicine Group Presidency. SE33 STR locus amplification was achieved with the Powerplex Fusion 6C kit from the isolated DNA. In the study, homozygote genotype rate was 5.4%, heterozygote genotype rate was 94.6%, power of discrimination (PD) 0.992 and matching probability (PM) was 0.008. The lowest frequency alleles were determined as 7,10,16.3,17.2,23,24,29, 34.2,35.2,37 alleles with 0.1%. It is seen that SE33 STR locus is a strong marker in forensic identification compared to other studies in terms of power of discrimination and matching probability.

«Нақты уақыт» режиміндегі полимеразды тізбекті реакция әдісімен тамақ өнімдерінің түрлік фальсификациясын анықтау бойынша хаттама әзірлеу

Adulteration of meat products is becoming a serious problem all over the world, including inappropriate labeling, prohibited additives, or replacement of expensive ingredients with cheaper components. The consequences of such manipulations can be dangerous for the health of a consumer. For example, it may cause food allergies and poisoning. In this article, there was developed a protocol for the qualitative determination of the DNA of the mitochondrial genome of chicken (Gallus gallus) by the polymerase chain reaction in real-time in raw and heat-treated meat products. The study was carried out to determine mixtures of meat and meat products from chicken, turkey, horse, lamb, pork, and beef. There were prepared 11 samples of 9 types of meat and two types of plants for the study of falsification. DNA isolation kits PrepMan Ultra (Thermo Fisher, USA) were used to obtain DNA with a high concentration between 1,900 ng/ml and 140 ng/ml. The selection of special primers and fluorescent probes was based on the alignment of the sequence of the chicken mitochondrial gene (Gallus gallus) from GenBank. All circuits are aligned using Primer Designer 3.0 software and synthesized by ThermoFisher. The authors have developed a protocol to determine the specific falsification of food products by a polymerase chain reaction in "real-time" mode. The PCR protocol was tested on a collection of DNA samples to determine specificity, and PCR with different DNA content was delivered to determine sensitivity. Chicken DNA was clearly traced in the DNA matrix in the range of 0.01-10%. Studies have shown that the developed protocol is a highly sensitive and specific express method for determining chicken DNA by a polymerase chain reaction in "real-time" mode, which allows them to be used in the diagnosis of animal DNA detection.

Gender assessment of the gut microbiome in obese patients

The aim. To assess the relationship between body mass index (BMI) and gut bacteria in men and women with obesity.Materials and methods. The study included 56 overweight patients, divided into 2 groups. The first group consisted of 27 women (the average age was 62 ± 2.2 years), the second group — 29 men (the average age was 55 ± 9 years). The Quetelet index (kg / m2) was calculated for all patients. To study the gut microbiome, the method of polymerase chain reaction in real time (RT-PCR) and metagenomic sequencing were used. DNA from feces was isolated using the Express-DNA-Bio DNA isolation kit (AlkorBio, Russia). To carry out RT-PCR, a set of reagents “Colonoflor-16” (“Alfalab”, Russia) was used. For microbiome sequencing, DNA libraries were prepared using the Illumina Nextera Sample Preparation Kit with DNA primers corresponding to the V3 — V4 regions of the 16S rRNA gene. The study of fecal samples was carried out using 16S rRNA gene sequencing on the Illumina platform (MiSeq sequencer).Results. It was revealed that a higher total number of bacteria, an increased content of Bacteroides fragilis group and Faeca-libacterium prausnitzii, is statistically significantly more common in women than in men. Strong negative correlations were found between BMI and total bacterial mass, between BMI and the number of Bacteroides fragilis group among women with grade I obesity. In overweight men, a correlation was found between BMI and the Bacteroides fragilis group / Faecalibacterium prausnitzii ratio.Conclusions. The total number of bacteria, the content of Bacteroides fragilis group and Faecalibacterium prausnitzii in the gut of patients have statistically significant associations with BMI, and probably can affect the formation of metabolic disorders to a greater extent in women than in men. To clarify the identified trends and patterns in this pilot study, further study of the microbiome with a large number of patients and additional analyzes of the metagenome (16S rRNA) and metabolome, a transcriptome, allowing to control the expression of key metabolic enzymes, largely associated with the compositional features of the gut microbiocenosis, is required.

"Shining a LAMP" (Loop-Mediated Isothermal Amplification) on the Molecular Detection of Phytopathogens Phytophthora spp. and Phytophthora cactorum in Strawberry Fields.

Phytopathogenic microorganisms belonging to the genus Phytophthora have been recognized many times as causal agents of diseases that lower the yield of many plants important for agriculture. Meanwhile, Phytophthora cactorum causes crown rot and leather rot of berry fruits, mainly strawberries. However, widely-applied culture-based methods used for the detection of pathogens are time-consuming and often inaccurate. What is more, molecular techniques require costly equipment. Here we show a rapid and effective detection method for the aforementioned targets, deploying a simple molecular biology technique, Loop-Mediated Isothermal Amplification (LAMP). We optimized assays to amplify the translation elongation factor 1-α (EF1a) gene for two targets: Phytophthora spp. And Phytophthora cactorum. We optimized the LAMP on pure strains of the pathogens, isolated from organic plantations of strawberry, and successfully validated the assay on biological material from the environment including soil samples, rhizosphere, shoots and roots of strawberry, and with SYBR Green. Our results demonstrate that a simple and reliable molecular detection method, that requires only a thermoblock and simple DNA isolation kit, can be successfully applied to detect pathogens that are difficult to separate from the field. We anticipate our findings to be a starting point for developing easier and faster modifications of the isothermal detection methods and which can be applied directly in the plantation, in particular with the use of freeze-dried reagents and chemistry, allowing observation of the results with the naked eye.

Investigation of FMS-Like Tyrosine Kinase 3 Mutation Frequency in Myelodysplastic Syndrome

Introduction: FMS-Like Tyrosine Kinase Class 3 (FLT3) mutations harbor poor prognosis, high relapse, and decreased overall survival in acute myeloblastic leukemia (AML). This mutation is also known to be demonstrated in myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia and acute lymphoblastic leukemia. This study included 94 MDS-diagnosed patients and we tried to investigate FLT3 mutation frequency (as tyrosine kinase domain-TKD and internal tandem duplication-ITD).&#x0D; Materials and Methods: Polymerase chain reaction (PCR), restriction fragment length polymorphism, and agarose-gel electrophoresis methods were used to analyze the mutation. The blood samples were collected in K3-EDTA tubes, and total DNA was isolated using genomic DNA isolation kits (GeneMark, Cat No: DP023P). For the detection of FLT3-ITD mutation, PCR was performed to amplify a 330- base pair fragment of exons 11 and 12 of FLT3 using FAM (Carboxyfluorescein)-labeled ITD-11F and HEX (Hexachloro-Fluorescein )-labeled ITD-12R primers in a thermal cycler (Eppendorf). Similarly, to detect D835 mutation, a 115- bp region of exon 17 of the FLT3 gene region was amplified using primers.&#x0D; Results: One patient was found FLT3-ITD positive (1.1%). The patient was 64-year-old and diagnosed with MDS-excess blast type 2 according to the World Health Organisation 2016 myeloid neoplasm classification. He transformed to AML within 19 months and subsequently died after 1 month. No patient with tyrosine kinase domain mutation was detected.&#x0D; Conclusion: FLT3 mutation is considered a significant parameter to define prognosis in AML. The routine workup of FLT3 screening and the potential of targeting FLT3 inhibition for high-risk MDS may be taken into consideration in the future.

Association of bitter and sweet taste gene receptor polymorphisms with dental caries formation

Abstract Objectives The aim of the study is to analyze the association of different bitter and sweet gene receptor polymorphisms and bitter and sweet food consumption on formation of dental caries in Turkish adult population. Methods This study included 205 adults whose detailed intraoral health examination was completed and decayed, missing, filled teeth (DMFT) index values were recorded. A mini questionnaire was applied to assess the consumption of bitter and sweet food. A venous blood sample from each participant was collected in Ethylenediamine tetraacetic acid (EDTA) tubes. Further, DNA samples were isolated from the blood samples by utilizing a DNA isolation kit, which were stored at +4 °C prior to the analysis. Taste receptor type 2 member 38 (TAS2R38; rs10246939, rs713598, rs1726866), Taste receptor type 1 member 2 (TAS1R2; rs35874116, rs9701796), and Taste receptor type 1 member 3 (TAS1R3; rs307355) gene polymorphisms were detected using real-time polymerase chain reaction (PCR). Results There was no statistically significant association between the TAS2R38, TAS1R2, and TAS1R3 gene polymorphism and the DMFT index (p&gt;0.05). No significant difference was found between the consumption of bitter and sweet food and the DMFT index (p&gt;0.05). Conclusions TAS2R38 (rs10246939, rs713598, rs1726866), TAS1R2 (rs35874116, rs9701796), and TAS1R3 (rs307355) gene polymorphism may not be associated with the formation of dental caries in the Turkish adult population.

Investigating The GMO Existence in Chips and Breakfast Cereals Marketed in Turkey

In this research, processed or low processed samples containing corn or corn products (corn semolina, flour, etc.) and soybean were randomly collected from the market, and 25 products in total (chips, nuts, cereals, flour) were analyzed for genetic modification using DNA based detection method, the polymerase chain reaction. First, homogenization of the samples was performed. Then DNA isolation was done by using Cetyl Trimethyl Ammonium Bromide (CTAB) and Roche High Pure DNA Isolation Kit. Since the Roche High Pure DNA Isolation Kit gave better results, the analysis was completed with this method. After DNA isolation, the detection of the Lectin gene, Zein gene, CaMV 35S Promoter and NOS Terminator regions was performed by conventional PCR. Zein gene determination was done for searching and proving corn presence and similarly, Lectin gene determination was done for searching and proving soybean presence in the samples by conventional PCR. GMO3/GMO4 and Zein3/Zein4 primer pairs were used for Lectin and Zein gene determination, respectively. The amplification of DNA was observed in agarose gel electrophoresis. Lectin or Zein genes were detected in 17 samples while these genes were not detected in 8 samples. Samples, in which Lectin or Zein gene was detected were scanned for 35S promoter or NOS terminator. 35S-3/35S-6 and tNOS2F/tNOS2R primer pairs were used for scanning 35S Promoter and NOS Terminator, respectively. To observe possible contamination in the mix sterilized deionized water was used and 0% Bt-11 and 0% GTS 40-3-2 were used as a negative control, 5% Bt-11 and 10% GTS-40-3-2 were used as a positive control. All of the 25 samples did not provide enough DNA with the required quality. This result was considered to be sourced by the applications (frying, extruding, pressing etc.) that samples had been exposed to during processes. Neither 35S Promotor nor NOS Terminator was determined from any of the samples.

MP03-09 DESCRIBING THE URINARY MICROBIOME IN PRE-OPERATIVE URINE SPECIMENS OF LICHEN SCLEROSUS INDUCED AND NON-LICHEN SCLEROSUS INDUCED URETHRAL STRICTURE DISEASE

You have accessJournal of UrologyTrauma/Reconstruction/Diversion: Urethral Reconstruction (including Stricture, Diverticulum) I (MP03)1 Sep 2021MP03-09 DESCRIBING THE URINARY MICROBIOME IN PRE-OPERATIVE URINE SPECIMENS OF LICHEN SCLEROSUS INDUCED AND NON-LICHEN SCLEROSUS INDUCED URETHRAL STRICTURE DISEASE Amanda Sherman, Travis Sullivan, Harjivan Kohli, Eric Burks, Kimberly Rieger-Christ, and Alex J Vanni Amanda ShermanAmanda Sherman More articles by this author , Travis SullivanTravis Sullivan More articles by this author , Harjivan KohliHarjivan Kohli More articles by this author , Eric BurksEric Burks More articles by this author , Kimberly Rieger-ChristKimberly Rieger-Christ More articles by this author , and Alex J VanniAlex J Vanni More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001964.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lichen sclerosus (LS) is a chronic inflammatory condition that can affect both the penile skin and urethral epithelium in men. Over time, severe impairment of sexual and urinary function can result, from preputial adhesions and phimosis, skin tearing with erections, acquired buried penis, and urethral stricture disease (USD). The pathophysiology of LS USD is poorly understood, with a heterogeneous presentation and disease severity. This study seeks to describe differences in the urinary microbiome of patients with pathologically confirmed LS USD vs non-LS USD. METHODS: An IRB approved protocol of men with USD was performed. Pre-operative clean catch voided urine was collected in 34 men with pathologically confirmed LS USD and non-LS USD. Bacterial genomic DNA was extracted using the PowerMag Soil DNA Isolation Kit. 16S rRNA gene sequencing was performed using the MiSeq platform for paired-end sequencing. Gene sequences were clustered into Operational Taxonomic Units (OTUs) at a similarity cutoff value of 97%. Taxonomic identities were assigned to each using the scikit-learn classifier and an optimized, variable region-specific version of the SILVA Database. RESULTS: Sufficient bacterial DNA for analysis was obtained from 19 patients (10 non-LS USD and 9 LS USD). Significant differences in alpha diversity (within sample variance) and beta diversity (between sample variance) were observed (p <0.05) between the two cohorts. Likewise, differences in taxonomic profiles and differential abundance of taxa (Phylum, Class, Order, Family, Genus) were noted between the cohorts. CONCLUSIONS: LS USD have significant alterations in diversity and differential abundance of urine microbiota compared to non-LS USD controls. While further validation in a larger sample is necessary, this finding could be used to guide further investigation into the role of bacteria and the urinary microbiome in LS USD pathogenesis, severity of presentation, and stricture recurrence. Source of Funding: Support for this project generously provided by The Ellison Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e24-e24 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Amanda Sherman More articles by this author Travis Sullivan More articles by this author Harjivan Kohli More articles by this author Eric Burks More articles by this author Kimberly Rieger-Christ More articles by this author Alex J Vanni More articles by this author Expand All Advertisement Loading ...