Current Protocols in Microbiology最新文献_第2页

An Exonuclease V–qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues 用核酸外切酶V-qPCR分析人乳头瘤病毒16在细胞系和组织中的基因组状态

Current Protocols in Microbiology Pub Date : 2020-10-16 DOI: 10.1002/cpmc.119

Julia E. Myers, Katarzyna Zwolinska, Martin J. Sapp, Rona S. Scott

{"title":"An Exonuclease V–qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues","authors":"Julia E. Myers, Katarzyna Zwolinska, Martin J. Sapp, Rona S. Scott","doi":"10.1002/cpmc.119","DOIUrl":"10.1002/cpmc.119","url":null,"abstract":"Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC.Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cellsSupport Protocol: Exonuclease V analysis of HPV16 genome configuration in tissuesAlternate Protocol: Determining HPV integration type or integrity of HPV episome","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.119","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38591742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 8

Laboratory Maintenance and Propagation of Freshwater Planarians 淡水涡虫的实验室维护和繁殖

Current Protocols in Microbiology Pub Date : 2020-10-15 DOI: 10.1002/cpmc.120

Makayla R. P. Dean, Elizabeth M. Duncan

引用次数: 0

Flow Cytometric Measurement of Efflux in Candida Species 念珠菌种外排的流式细胞术测定

Current Protocols in Microbiology Pub Date : 2020-10-13 DOI: 10.1002/cpmc.121

Kali R. Iyer, Nicole Robbins, Leah E. Cowen

引用次数: 2

Generation of Recombinant SARS-CoV-2 Using a Bacterial Artificial Chromosome 利用细菌人工染色体制备重组SARS-CoV-2

Current Protocols in Microbiology Pub Date : 2020-10-13 DOI: 10.1002/cpmc.126

Kevin Chiem, Chengjin Ye, Luis Martinez-Sobrido

引用次数: 16

Purification of Yeast Spores to Investigate Their Dynamics of Activation 酵母孢子的纯化及其活化动力学研究

Current Protocols in Microbiology Pub Date : 2020-10-09 DOI: 10.1002/cpmc.123

Samuel Plante, Christian R. Landry

引用次数: 4

High-Yield Purification of Giardia intestinalis Cysts from Fecal Samples 粪样中肠贾第虫囊的高效纯化

Current Protocols in Microbiology Pub Date : 2020-10-09 DOI: 10.1002/cpmc.117

Paul Ogbuigwe, Anthony B. Pita, Matthew A. Knox, Niluka Velathanthiri, David T. S. Hayman

{"title":"High-Yield Purification of Giardia intestinalis Cysts from Fecal Samples","authors":"Paul Ogbuigwe, Anthony B. Pita, Matthew A. Knox, Niluka Velathanthiri, David T. S. Hayman","doi":"10.1002/cpmc.117","DOIUrl":"10.1002/cpmc.117","url":null,"abstract":"Giardia is an enteric protozoan parasite that causes gastroenteritis in all classes of vertebrates. It is ranked among the leading causes of death in children under 5 years of age. Giardiasis affects approximately 280 million people worldwide annually, a situation exacerbated by the low availability of effective treatments and the lack of a vaccine. In addition, the parasite is difficult to manipulate in in vitro environments, which hampers the development of effective disease management strategies. This article highlights the development of a method for the purification of viable Giardia cysts from fecal samples, verified by a trypan blue dye exclusion test. This protocol produces a 10-fold increase in yield over current methods. By combining sucrose flotation with gated filtration, the protocol significantly reduces the amount of debris in the purified cysts suspension. Cyst viability is verified by a trypan blue dye exclusion test. The ability to purify large quantities of Giardia from fecal samples could advance the development of effective treatments to target this worldwide prevalent parasite. © 2020 Wiley Periodicals LLC.Basic Protocol: Purification of Giardia cysts from fecal samplesSupport Protocol: Cyst viability test","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.117","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38472881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Ixodid Tick Dissection and Tick Ex Vivo Organ Cultures for Tick-Borne Virus Research 蜱媒病毒研究中蜱的解剖和离体器官培养

Current Protocols in Microbiology Pub Date : 2020-10-08 DOI: 10.1002/cpmc.118

Jeffrey M. Grabowski, Ryan Kissinger

{"title":"Ixodid Tick Dissection and Tick Ex Vivo Organ Cultures for Tick-Borne Virus Research","authors":"Jeffrey M. Grabowski, Ryan Kissinger","doi":"10.1002/cpmc.118","DOIUrl":"10.1002/cpmc.118","url":null,"abstract":"Tick-borne viruses cause thousands of cases of disease worldwide every year. Specific countermeasures to many tick-borne viruses are not commercially available. Very little is known regarding tick-virus interactions and increasing this knowledge can lead to potential targets for countermeasure development. Virus infection of ex vivo organ cultures from ticks can provide an approach to identify susceptible cell types of tissue to infection. Additionally, these organ cultures can be used for functional genomic studies to pinpoint tick-specific genes involved in the virus lifecycle. Provided here are step-by-step procedures to set up basic tick organ cultures in combination with virus infection and/or functional genomic studies. These procedures can be adapted for future use to characterize other tick-borne pathogen infections as well as tick-specific biological processes. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Loading 96-well plates with gelfoam substrateBasic Protocol 2: Step-by-step aseptic dissection of unfed female/male Ixodes scapularis ticks for multiple organsBasic Protocol 3: Step-by-step aseptic dissection of fed female Ixodes scapularis ticks to remove salivary glandsBasic Protocol 4: Metabolic viability analyses of tick organ culturesBasic Protocol 5: Virus infection of tick organ culturesBasic Protocol 6: Functional RNA interference analyses using tick organ cultures","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.118","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38469854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 6

Experimental Evolution of Antifungal Resistance in Cryptococcus neoformans 新型隐球菌抗真菌耐药性的实验进化

Current Protocols in Microbiology Pub Date : 2020-09-28 DOI: 10.1002/cpmc.116

Arianne Bermas, Rebecca S. Shapiro, Jennifer Geddes-McAlister

{"title":"Experimental Evolution of Antifungal Resistance in Cryptococcus neoformans","authors":"Arianne Bermas, Rebecca S. Shapiro, Jennifer Geddes-McAlister","doi":"10.1002/cpmc.116","DOIUrl":"10.1002/cpmc.116","url":null,"abstract":"Cryptococcus neoformans, an opportunistic yeast-like fungal pathogen, has demonstrated resistance to all major classes of antifungals used to treat cryptococcal meningitis. However, combatting this fungal disease is an ongoing challenge among clinicians due to the evolution of antifungal-resistant strains. The limited availability of clinically approved antifungals has heightened the urgency to investigate the molecular mechanisms underscoring resistance. Studying how a fungal pathogen evolves to an antifungal drug in vitro using experimental evolution provides a simple, yet powerful approach to study the mechanisms of antifungal resistance. Experimental evolution involves the serial passaging of microbial populations under laboratory conditions, such that adaptive mutations can occur and be monitored in real time. This technique plays a key role in investigating the mechanisms of antifungal resistance in C. neoformans, and this can help in developing novel strategies to combat the emergence of resistance. Here, we outline how to make overnight cultures of C. neoformans and how to perform experimental evolution, and we present a spectrophotometric analysis to evaluate the evolution of antifungal resistance. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Growth and sample preparation of Cryptococcus neoformansBasic Protocol 2: Experimental evolution of antifungal resistanceBasic Protocol 3: Analyzing the evolution of antifungal resistanceBasic Protocol 4: Glycerol stock preparation","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.116","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38427680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Genetic Manipulation of Vibrio fischeri 费氏弧菌的基因操作

Current Protocols in Microbiology Pub Date : 2020-09-25 DOI: 10.1002/cpmc.115

David G. Christensen, Jovanka Tepavčević, Karen L. Visick

{"title":"Genetic Manipulation of Vibrio fischeri","authors":"David G. Christensen, Jovanka Tepavčević, Karen L. Visick","doi":"10.1002/cpmc.115","DOIUrl":"10.1002/cpmc.115","url":null,"abstract":"Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squid Euprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation of V. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenize V. fischeri with transposons. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Transformation of V. fischeri with linear DNABasic Protocol 2: Plasmid transfer into V. fischeri via conjugationSupport Protocol 1: Removing FRT-flanked antibiotic resistance cassettes from the V. fischeri genomeSupport Protocol 2: Eliminating unstable plasmids from V. fischeriAlternate Protocol 1: Introduction of exogenous DNA using a suicide plasmidAlternate Protocol 2: Site-specific transposon insertion using a suicide plasmidAlternate Protocol 3: Random transposon mutagenesis using a suicide plasmid","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.115","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38418892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7

A Simple Nematode Infection Model for Studying Candida albicans Pathogenesis 研究白色念珠菌发病机制的简单线虫感染模型

Current Protocols in Microbiology Pub Date : 2020-09-25 DOI: 10.1002/cpmc.114

Grace H. Kim, Sierra Rosiana, Natalia V. Kirienko, Rebecca S. Shapiro

{"title":"A Simple Nematode Infection Model for Studying Candida albicans Pathogenesis","authors":"Grace H. Kim, Sierra Rosiana, Natalia V. Kirienko, Rebecca S. Shapiro","doi":"10.1002/cpmc.114","DOIUrl":"10.1002/cpmc.114","url":null,"abstract":"Candida albicans is an opportunistic fungal pathogen and a model organism to study fungal pathogenesis. It exists as a harmless commensal organism and member of the healthy human microbiome, but can cause life-threatening mucosal and systemic infections. A model host to study C. albicans infection and pathogenesis is the nematode Caenorhabditis elegans. C. elegans is frequently used as a model host to study microbial-host interactions because it can be infected by many human pathogens and there are also close morphological resemblances between the intestinal cells of C. elegans and mammals, where C. albicans infections can occur. This article outlines a detailed methodology for exploiting C. elegans as a host to study C. albicans infection, including a C. elegans egg preparation protocol and an agar-based C. elegans killing protocol to monitor fungal virulence. These protocols can additionally be used to study C. albicans genetic mutants in order to further our understanding of the genes involved in pathogenesis and virulence in C. albicans and the mechanisms of host-microbe interactions. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Preparation of Caenorhabditis elegans eggsSupport Protocol 1: Freezing and recovering Caenorhabditis elegansSupport Protocol 2: Making superfood agar and OP50 platesBasic Protocol 2: Caenorhabditis elegans/Candida albicans agar killing assaySupport Protocol 3: Constructing a worm pick","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.114","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38418891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 7