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pathogen dna enrichment

A S Varela-Stokes, S H Park, J V Stokes, N A Gavron, S I Lee, G M Moraru, S C Ricke
Our objective of this study was to explore the bacterial microbiome in fresh or fresh-frozen adult Amblyomma maculatum (Gulf Coast ticks) using extracts enriched for microbial DNA. We collected 100 questing adult A. maculatum, surface disinfected them, and extracted DNA from individual ticks collected the same day or after storage at -80 °C. Because only extracts with microbial DNA concentrations above 2 ng/μL were considered suitable for individual analysis, we expected fewer samples to meet these requirements...
March 1, 2018: Ticks and Tick-borne Diseases
Alejandro Garrido-Maestu, Sarah Azinheiro, Joana Carvalho, Marta Prado
Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes...
August 2018: Food Microbiology
Xin-Jun Du, Yu-Xuan Zang, Hai-Bin Liu, Ping Li, Shuo Wang
Listeria monocytogenes is an important food-borne pathogenic bacterium that causes human disease, resulting in economic losses worldwide. The current detection methods for L. monocytogenes are not well suited for direct field testing because they involve complicated, time-consuming operations. A simple, efficient method is vital for L. monocytogenes detection. In this study, we combined isothermal recombinase polymerase amplification (RPA) with a lateral flow (LF) strip to rapidly and reliably detect L. monocytogenes...
March 10, 2018: Journal of Food Science
Saud H AlDubayan, Marios Giannakis, Nathanael D Moore, G Celine Han, Brendan Reardon, Tsuyoshi Hamada, Xinmeng Jasmine Mu, Reiko Nishihara, Zhirong Qian, Li Liu, Matthew B Yurgelun, Sapna Syngal, Levi A Garraway, Shuji Ogino, Charles S Fuchs, Eliezer M Van Allen
Colorectal cancer (CRC) heritability has been estimated to be around 30%. However, mutations in the known CRC-susceptibility genes explain CRC risk in fewer than 10% of affected individuals. Germline mutations in DNA-repair genes (DRGs) have recently been reported in CRC, but their contribution to CRC risk is largely unknown. We evaluated the gene-level germline mutation enrichment of 40 DRGs in 680 unselected CRC individuals and 27,728 ancestry-matched cancer-free adults. Significant findings were then examined in independent cohorts of 1,661 unselected CRC individuals and 1,456 individuals with early-onset CRC...
February 21, 2018: American Journal of Human Genetics
Zhi-Luo Deng, Helena Sztajer, Michael Jarek, Sabin Bhuju, Irene Wagner-Döbler
Periodontitis is a worldwide prevalent oral disease which results from dysbiosis of the periodontal microbiome. Some of the most active microbial players, e.g., Porphyromonas gingivalis, Treponema denticola , and Fusobacterium nucleatum , have extensively been studied in the laboratory, but it is unclear to which extend these findings can be transferred to in vivo conditions. Here we show that the transcriptional profiles of P. gingivalis, T. denticola , and F. nucleatum in the periodontal niche are distinct from those in single laboratory culture and exhibit functional similarities...
2018: Frontiers in Microbiology
Liding Zhang, Qiujiang Wei, Qinqin Han, Qiang Chen, Wenlin Tai, Jinyang Zhang, Yuzhu Song, Xueshan Xia
Shigella is an important human food-borne zoonosis bacterial pathogen, and can cause clinically severe diarrhea. There is an urgent need to develop a specific, sensitive, and rapid methodology for detection of this pathogen. In this study, loop-mediated isothermal amplification (LAMP) combined with magnetic immunocapture assay (IC-LAMP) was first developed for the detection of Shigella in pure culture, artificial milk, and clinical stool samples. This method exhibited a detection limit of 8.7 CFU/mL. Compared with polymerase chain reaction, IC-LAMP is sensitive, specific, and reliable for monitoring Shigella...
2018: Frontiers in Microbiology
Kelsi Penewit, Elizabeth A Holmes, Kathyrn McLean, Mingxin Ren, Adam Waalkes, Stephen J Salipante
Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132 , derived from Enterococcus faecalis , as being capable of integrating single-stranded DNA oligonucleotides into the S...
February 20, 2018: MBio
Muhsin Aydin, Jacqueline Carter-Conger, Ning Gao, David F Gilmore, Steven C Ricke, Soohyoun Ahn
Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions...
February 19, 2018: Analytical and Bioanalytical Chemistry
Yiming Lin, Mingya Han, Zhenzhu Zheng, Weihua Lin, Ke Yu, Qingliu Fu
OBJECTIVE To detect potential mutations of GCDH gene in five patients with glutaric acidemia type I (GA-I). METHODS Genomic DNA was extracted from peripheral blood samples from the patients. The 11 exons and their flanking sequences of the GCDH gene were amplified with PCR and subjected to direct sequencing. RESULTS Four mutations of the GCDH gene were identified among the patients, which included c.532G>A (p.G178R), c.533G>A (p.G178E), c.106_107delAC (p.Q37fs*5) and c.1244-2A>C. Among these, c.1244-2A>C was the most common, while c...
February 10, 2018: Zhonghua Yi Xue Yi Chuan Xue za Zhi, Zhonghua Yixue Yichuanxue Zazhi, Chinese Journal of Medical Genetics
Chung-Hsing Chen, Shih Sheng Jiang, I-Shou Chang, Hui-Ju Wen, Chien-Wen Sun, Shu-Li Wang
BACKGROUND: Phthalic acid esters are ubiquitous and antiandrogenic, and may cause systemic effects in humans, particularly with in utero exposure. Epigenetic modification, such as DNA methylation, has been hypothesized to be an important mechanism that mediates certain biological processes and pathogenic effects of in utero phthalate exposure. OBJECTIVE: The aim of this study was to examine the association between genome-wide DNA methylation at birth and prenatal exposure to phthalate...
January 19, 2018: Environmental Research
Milena Jakimovska, Ivana Maleva Kostovska, Katerina Popovska-Jankovic, Katerina Kubelka-Sabit, Mitko Karadjozov, Liljana Stojanovska, Andreja Arsovski, Snezhana Smichkoska, Emilija Lazarova, Maja Jakimovska Dimitrovska, Dijana Plaseska-Karanfilska
PURPOSE: We aimed to establish the spectrum of BRCA1/2 mutations among the breast cancer (BC) patients from the Republic of Macedonia. METHODS: We used targeted next-generation sequencing (NGS), Sanger DNA sequencing, and multiplex ligation probe amplification analysis (MLPA) to search for point mutations and deletions/duplications involving BRCA1 and BRCA2-coding regions. RESULTS: We have analyzed a total of 313 BC patients, enriched for family history of cancer, early age of onset and bilateral and/or triple negative (TN) BC...
January 15, 2018: Breast Cancer Research and Treatment
Michael E Donaldson, Yessica Rico, Karsten Hueffer, Halie M Rando, Anna V Kukekova, Christopher J Kyle
Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High-throughput sequencing (HTS) technologies, such as genotype-by-sequencing (GBS), facilitate expanded explorations of genomic variation among populations...
January 2018: Ecology and Evolution
Zoe Patterson Ross, Jennifer Klunk, Gino Fornaciari, Valentina Giuffra, Sebastian Duchêne, Ana T Duggan, Debi Poinar, Mark W Douglas, John-Sebastian Eden, Edward C Holmes, Hendrik N Poinar
Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles...
January 2018: PLoS Pathogens
Dao Thi Thuy Nguyen, Eun Yeong Lee, Bonhan Koo, Choong Eun Jin, Tae Yoon Lee, Yong Shin
Rapid and sensitive detection of low amounts of pathogen in large samples is needed for early diagnosis and treatment of patients and surveillance of pathogen. In this study, we report a microfluidic platform for detection of low pathogen levels in a large sample volume that couples an Magainin 1 based microfluidic platform for pathogen enrichment and a recombinase polymerase amplification (RPA) sensor for simultaneous pathogenic DNA amplification and detection in a label-free and real-time manner. Magainin 1 is used as a pathogen enrichment agent with a herringbone microfluidic chip...
December 28, 2017: Analytical Biochemistry
Xiaofan Yu, Fang Chen, Ronghui Wang, Yanbin Li
The rapid detection of foodborne pathogens is critical to ensure food safety. The objective of this study is to select aptamers specifically bound to Escherichia coli O157:H7 using the whole-bacterium SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and apply the selected aptamer to a QCM (quartz crystal microbalance) sensor for rapid and sensitive detection of target bacteria. A total of 19 rounds of selection against live E. coli O157:H7 and 6 rounds of counter selection against a mixture of Staphylococcus aureus, Listeria monocytogenes, and Salmonella Typhimurium, were performed...
December 12, 2017: Journal of Biotechnology
Jun Cui, Pinsan Xu, Jun Meng, Jingbin Li, Ning Jiang, Yushi Luan
SpWRKY3 was identified as a resistance gene to Phytophthora infestans from Solanum pimpinellifolium L3708 and its transgenic tomato showed a significant resistance to P. infestans. This finding reveals the potential application of SpWRKY3 in future molecular breeding. Transcription factors (TFs) play crucial roles in the plant response to various pathogens. In this present study, we used comparative transcriptome analysis of tomatoes inoculated with and without Phytophthora infestans to identify 1103 differentially expressed genes...
April 2018: TAG. Theoretical and Applied Genetics. Theoretische und Angewandte Genetik
Alexander Yitbarek, J Scott Weese, Tamiru Negash Alkie, John Parkinson, Shayan Sharif
The impact of low pathogenic influenza viruses such as subtype H9N2, which infect the respiratory and the gastrointestinal tracts of chickens, on microbial composition are not known. Twenty-day-old specific pathogen-free chickens were assigned to two treatment groups, control (uninfected) and H9N2-infected (challenged via the oral-nasal route). Fecal genomic DNA was extracted, and the V3-V4 regions of the 16S rRNA gene were sequenced using the Illumina Miseq® platform. Sequences were curated using Mothur as described in the MiSeq SOP...
January 1, 2018: FEMS Microbiology Ecology
Julia L Drewes, James R White, Christine M Dejea, Payam Fathi, Thevambiga Iyadorai, Jamuna Vadivelu, April C Roslani, Elizabeth C Wick, Emmanuel F Mongodin, Mun Fai Loke, Kumar Thulasi, Han Ming Gan, Khean Lee Goh, Hoong Yin Chong, Sandip Kumar, Jane W Wanyiri, Cynthia L Sears
Colorectal cancer (CRC) remains the third most common cancer worldwide, with a growing incidence among young adults. Multiple studies have presented associations between the gut microbiome and CRC, suggesting a link with cancer risk. Although CRC microbiome studies continue to profile larger patient cohorts with increasingly economical and rapid DNA sequencing platforms, few common associations with CRC have been identified, in part due to limitations in taxonomic resolution and differences in analysis methodologies...
2017: NPJ Biofilms and Microbiomes
Sardar Karash, Rohana Liyanage, Abdullah Qassab, Jackson O Lay, Young Min Kwon
Salmonella is an intracellular pathogen infecting a wide range of hosts and can survive in macrophages. An essential mechanism used by macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2 O2 . For Tn-seq, a saturated Tn5 insertion library was grown in vitro under either 2.5 (H2 O2 L) or 3.5 mM H2 O2 (H2 O2 H). We identified two sets of overlapping genes required for resistance of S...
December 6, 2017: Scientific Reports
Ji-Yeon Hyeon, Shaoting Li, David A Mann, Shaokang Zhang, Zhen Li, Yi Chen, Xiangyu Deng
Metagenomics analysis of food samples promises isolation-independent detection and subtyping of foodborne bacterial pathogens in a single workflow. Selective concentration of Salmonella genomic DNA through immunomagnetic separation (IMS) and multiple displacement amplification (MDA) were shown to shorten culture enrichment of Salmonella -spiked raw chicken breast samples by over 12 hours while permitting serotyping and high-fidelity single nucleotide polymorphisms (SNP) typing of the pathogen using short shotgun sequencing reads...
December 1, 2017: Applied and Environmental Microbiology
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