微生物学技术

The pneumococcus is now the most important bacterial pathogen causing pneumonia, meningitis, and otitis media. Traditional approaches to the diagnosis of Streptococcus pneumoniae as the etrologic agent causing these diseases include colony morphology, microscopy, optoch ...

The ability to identify rapidly organisms to the species, and at times subspecies level, is an important step in the treatment of bacterial infections and for monitoring the spread of microorganisms. Conventional identification of streptococci relies on the isolation and culturing of ...

Several families of short repetitive DNA sequences, widely distributed in the genome, have been identified in bacteria (1). They have an intercistronic location, are not translated, and their function is unclear, although they may be involved in transcription termination, mRNA stabil ...

The impact of molecular (nucleic acid-based) methods on the basic science of medical microbiology is undeniable. Indeed, microbiologists have been at the forefront of the molecular biology revolution that has had such a dramatic effect on our understanding of biological science. Alth ...

Pseudotype viruses are phenotypically mixed virions containing the genome or nucleocapsid of one enveloped virus and the surface or envelope (env) glycoproteins of another. This chapter will concentrate on vesicular stomatitis virus (VSV) pseudotypes retaining the VSV nucleo ...

Viral-specific antibodies of any class that bind to particular epitopes on the surface protein of a virion are capable of neutralizing the infectivity of the virion. Classical neutralization results when antibody binds to the virion and thereby prevents infection of a susceptible cell. ...

Investigation of the biological activity of viruses in vitro necessitates some means of identifying their presence within cells and assaying their activity. Since virus particles themselves are metabolically inert, their detection and quantitation is dependent on their cellu ...

Since the polymerase chain reaction (PCR) technique was developed in 1986 (1), it has found wide application in molecular biology and is now regarded as an irreplaceable tool in many laboratories. This chapter will deal with its use for the detection of virus genomes for diagnostic and experimen ...

The transfer of DNA sequences into a population of cells can rarely, if ever, be achieved with 100% efficiency. Typically, transfection of cells with the calcium phosphate method will transduce only between 0.1 and 1% of the cells with the sequences of interest (1), although some workers have achiev ...

When using recombinant retroviruses for gene transduction, it is necessary to have a measurement of the concentration of virus particles in the medium conditioned by virus-producing cells (i.e., the viral titer). In the case of viruses encoding selectable markers, this can readily be deter ...

This chapter aims to provide the reader with the experimental protocols required to produce a baculovirus containing the gene of interest, including transfection of Spodoptera frugiperda cells and the screening of progeny virus by plaque assay. In addition, an outline of the biochemic ...

Retroviral vectors are uniquely suitable for high-efficiency gene transfer to a large target cell population and this (among other things) has led to the exploration of previously impractical strategies for somatic gene therapy (1,2). Thus, for example, cultured bone marrow can be used to r ...

Baculoviruses have been used for many years as effective pest-control agents; however, their interest to molecular virologists stems from their exploitation as helper-independent viral expression vectors for the production of proteins in a eukaryotic environment. The pionee ...

The self-assembly properties of a protein encoded by the TYA gene of the yeast Ty element can be exploited to produce hybrid Ty-VLPs (virus-like particles) (1,2). There has been developed a series of expression vectors that allow the construction of Ty fusion genes containing protein coding seq ...

The synthesis of recombinant proteins or protein domains in microbial, insect, or mammalian systems is now commonplace in molecular biology laboratories. The gene or gene fragment encoding the protein of interest is inserted into a specialized expression vector, flanked by efficient ...

Chapter 22 outlines the construction of engineered, full-length poliovirus cDNAs in which the region encoding a well-characterized antigenic site has been replaced by sequences of choice. This chapter briefly describes the methods used to generate, maintain, and characterize inf ...

We have developed the very safe and efficacious live-attenuated Sabin 1 poliovirus vaccine strain as a vehicle for the presentation of defined epitopes from foreign pathogens (1–3). Precise modification of the poliovirus capsid is made possible by the application of recombinant DNA tec ...

In Chapter 20, a general procedure was described for the construction of the intermediate vectors necessary for the insertion of foreign DNA sequences into the vaccinia virus (Vv) genome. The principles and basic methodology for the isolation of recombinant vaccinia viruses will be disc ...

Vaccinia virus (Vv) is a member of the genus Orthopoxvirus, one of seven genera included in the family Poxviridae. Most of these viruses infect vertebrates (Orthopoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, and Parapoxvirus), but one genus, Entomopoxvir ...

Epstein-Barr virus (EBV) is able to immortalize human B-lymphocytes with high efficiency. This property underlies the role of EBV in a number of human diseases. First, EBV is the causative agent of infectious mononucleosis, which is a benign proliferation of B-lymphocytes (1). Second, EBV is in ...