微生物学技术

Malaria represents the world’s greatest public health problem in terms of number of people affected, and the levels of morbidity and mortality. The protozoan malaria parasites (Plasmodium spp.) are transmitted by infected female mosquitoes when feeding on blood. Parasites soon enter l ...

Over the last decade, our knowledge of the parasites causing malaria has been transformed by molecular biology. We now have an exten- sive body of data concerning many of the proteins and the genes encoding them, but much less is understood about their cellular functions. The localization of paras ...

Immunofluorescence, as the name suggests, involves the visualization of proteins and structures within cells using antibodies as fluorescent probes. It has proven to be an extremely valuable technique for several reasons: 1. Vast numbers of cells can be processed and observed in a singl

Immunoassay using antibodies bound to a solid phase was first described in 1954 when D. W. Talmage and coworkers used a crude form of radioimmunoassay (RIA) to estimate the total antibody content of serum (1). Various technological developments, notably the coupling of enzymes to antibodies, ...

Affinity chromatography is one of the most convenient methods of purifying biological molecules, with many wide ranging applications (1, 2). The main prerequisite is to have a ligand that specifically binds to the target molecule. This ligand must also have two key properties other than exqu ...

The mature schistosome has an extraordinary capacity to survive within the blood of its definitive host despite the presence of protective immune responses. Such protective responses can be stimulated by infection itself, vaccination with highly irradiated cercariae, or by indiv ...

In the field of parasitology, isozymes are mainly used to characterize and distinguish “strains.” For such a difficult purpose, isozyme data definitely have to be analyzed in genetic terms. Relying on a purely empirical, phenetic reading of the gels amounts to missing about 90% of the informati ...

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separates proteins by their iso-electric points (IEPs) and electro-phoretic mobilities (1). In the first dimension, a sample is applied to an iso-electric focusing (IEF) acrylamide gel. Within this the con stituent pr ...

DNA transfection is the introduction of molecular constructs into parasites in a manner permitting the expression of encoded genes. Transfection is an exceedingly powerful technique for analyzing gen etic regulatory elements and gene function, and is widely used in molecular biolo ...

Physical mapping of protozoan chromosomes has been transformed in recent years by the introduction of pulsed field gradient electrophor- esis (PFGE) (1). The chromosomes, which otherwise are too small to be detected in condensed form or mapped by conventional in situ hybridization meth ...

One of the most powerful ways of identifying genes is to utilize oligonucleotide probes. A prerequisite for this approach is the exist ence of a minimal amount of protein sequence information, either from the parasite protein itself (see Chapter 33) or from homologous proteins of other organi ...

This chapter essentially combines a variety of techniques already described in other chapters to allow a rapid characterization or con firmation of λ expression clones. Antibodies used to screen expres sion libraries (see Chapter 21) may have been raised against affinity purified anti ...

The many and various species of Leishmania are responsible for a broad spectrum of human and animal diseases known collectively as the leishmaniases. They are widely distributed in the warmer parts of the world and transmitted by the bite of infected female phlebotomine sandflies. The life cy ...

Over the last decade major advances in cloning technology have markedly improved the ability to construct clone libraries expressing foreign proteins and to screen them to identify clones encoding any desired gene product. A number of types of libraries, expression vec- tors, and screeni ...

The phylogeny of a large number of parasite species is still entirely based on morphological and physiological features. However, the reliability of such criteria becomes highly questionable, especially for species exhibiting a low level of organismal complexity. The advent of mode ...

Nucleic acid hybridization probes have a wide range of applica tions for the detection, identification, and quantification of microor ganisms, from environmental studies to medical diagnoses (1,2). They offer unique advantages in terms of sensitivity and specificity, with their po ...

The relationship between host and parasite is a dynamic one with both groups undergoing genetic change to maximize reproductive success. The short generation time and high reproductive capacity of the malaria parasite provides scope for extensive mutation within the parasite geno ...

The polymerase chain reaction (PCR) allows the specific amplifica tion of either RNA or DNA nucleotide sequences (1,2). The hallmarks of this technique are specificity, sensitivity, and speed. The specific ity of the reaction is a result of the requirement of DNA polymerases for a primer that is ex ...

Detection of malaria parasites to as low a level as one organism is essential for epidemiological surveillance and effectiveness of thera peutic treatments. Classical detection of the parasites has always relied on microscopic examination, a method that is relatively simple and inex ...

Parasitic protozoa, generally, present as highly disperse popula tions with complex life cycles. The ability to attain and sustain this diversity and complexity resides in the DNA of the cell. Although there is a great deal of activity directed toward analysis of parasitic protozoan DNA at the m ...