Current Protocols in Microbiology最新文献

Listeria monocytogenes causes foodborne disease in humans that ranges in severity from mild, self-limiting gastroenteritis to life-threatening systemic infections of the blood, brain, or placenta. The most commonly used animal model of listeriosis is intravenous infection of mice. This systemic model is highly reproducible, and thus, useful for studying cell-mediated immune responses against an intracellular bacterial pathogen, but it completely bypasses the gastrointestinal phase of L. monocytogenes infection. Intragastric inoculation of L. monocytogenes produces more variable results and may cause direct bloodstream invasion in some animals. The foodborne transmission model described here does not require specialized skills to perform and results in infections that more closely mimic human disease. This natural feeding model can be used to study both the host- and pathogen-derived factors that govern susceptibility or resistance to orally acquired L. monocytogenes. Curr. Protoc. Microbiol. 31:9B.3.1-9B.3.16. © 2013 by John Wiley & Sons, Inc.

Streptococcus pyogenes (the Group A Streptococcus, GAS) is a Gram-positive bacterium responsible for a wide spectrum of diseases ranging from mild superficial infections (pharyngitis, impetigo) to severe, often life-threatening invasive diseases (necrotizing fasciitis, streptococcal toxic shock syndrome) in humans. This unit describes molecular techniques for the genetic manipulation of S. pyogenes with detailed protocols for transformation, gene disruption, allelic exchange, transposon mutagenesis, and genetic complementation. Curr. Protoc. Microbiol. 30:9D.3.1-9D.3.29. © 2013 by John Wiley & Sons, Inc.

The importance of laboratory mice in experimental microbiology and biomedical research is indisputable, and their care and handling is a contributing factor to the quality of the science resulting from their use. This unit provides guidelines for practical housing, handling, and the methodology for sample collection to ensure animal health and minimize variable experimental parameters. Curr. Protoc. Microbiol. 31:A.3N.1-A.3N.18. © 2013 by John Wiley & Sons, Inc.

Streptococcus pyogenes is a Gram-positive bacterium that strictly infects humans. It is the causative agent of a broad spectrum of diseases accounting for millions of infections and at least 517,000 deaths each year worldwide. It is a nutritionally fastidious organism that ferments sugars to produce lactic acid and has strict requirements for growth. To aid in the study of this organism, this unit describes the growth and maintenance of S. pyogenes. Curr. Protoc. Microbiol. 30:9D.2.1-9D.2.13. © 2013 by John Wiley & Sons, Inc.

DNA immunization was discovered in early 1990s, and its use has been expanded from vaccine studies to a broader range of biomedical research areas, such as the generation of high-quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation, and gene gun. In addition, several common considerations related to DNA immunization are discussed. Curr. Protoc. Microbiol. 31:18.3.1-18.3.24. ©2013 by John Wiley & Sons, Inc.

Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) provides a means to investigate RNA structure with better resolution and higher throughput than has been possible with traditional methods. We present several protocols, which are based on a variety of previously published methods and were adapted and optimized for the analysis of poliovirus RNA in the Andino laboratory. These include methods for nondenaturing RNA extraction, RNA modification and primer extension, and data processing in ShapeFinder. Curr. Protoc. Microbiol. 30:15H.3.1-15H.3.12. © 2013 by John Wiley & Sons, Inc.

Complex regulation of viral transcription patterns and DNA replication levels is a feature of many DNA viruses. This is especially true for those viruses which establish latent or persistent infections (e.g., herpesviruses, papillomaviruses, polyomaviruses, or adenovirus), as long-term persistence often requires adaptation of gene expression programs and/or replication levels to the cellular milieu. A key factor in the control of such processes is the establishment of a specific chromatin state on promoters or replication origins, which in turn will determine whether or not the underlying DNA is accessible for other factors that mediate downstream processes. Chromatin immunoprecipitation (ChIP) is a powerful technique to investigate viral chromatin, in particular to study binding patterns of modified histones, transcription factors or other DNA-/chromatin-binding proteins that regulate the viral lifecycle. Here, we provide protocols that are suitable for performing ChIP-PCR and ChIP-Seq studies on chromatin of large and small viral genomes.© 2016 by John Wiley & Sons, Inc.

This protocol describes a method for the rapid detection of leptospiral DNA in environmental water. In summary, the DNA is extracted from water samples and tested in a TaqMan-based real-time quantitative polymerase chain reaction (qPCR) for the presence of lipl32, a gene that is present only in pathogenic Leptospira spp. The gene target used in this assay is important in that it only detects pathogenic leptospires and not the saprophytic leptospires that may be present in environmental samples. © 2017 by John Wiley & Sons, Inc.