微生物学技术

Studying large genomic regions at the molecular level requires access to the DNA representing such sites. The availability of large and stable clones derived from target genomic regions is essential for detailed analysis such as sequencing. Since its introduction in 1992, the bacterial ...

The ultimate goal of the Human Genome Project is to establish the DNA sequence of human and model organism genomes as the critical first step in understanding disease, development and evolution. To accomplish this goal and a broad spectrum of applications requires integration of genome sequ ...

Analysis of complex genomes includes characterization of complete large-insert genomic libraries comprising several hundreds of thousands of clones. Conventional methods to screen large-insert clone libraries for specific clones within a defined chromosomal interval ...

This chapter describes a nonradioactive, agarose gel-based, high-throughput DNA restriction digest fingerprinting methodology first described by Marra et al. (1) for use in the construction of high-resolution physical maps from low-copynumber, large-insert clones. The proc ...

Amplified fragment-length polymorphism analysis (AFLP) has been shown to be a suitable method for subtyping of bacteria belonging to the genus Campylobacter. Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid ...

Environmental monitoring and public health risk assessments require methods that are rapid and quantitative with defined sensitivity and specificity thresholds. Although several molecular techniques have been developed to rapidly detect bacteria in complex matrices, the ...

The human pathogenic Legionella bacteria are found ubiquitously in natural and human-made aquatic environments as residents in biofilms, where close interactions with other microorganisms like protozoa are possible. Nosocomial legionellosis already has been linked freq ...

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an economically significant veterinary pathogen that causes Johne’s disease in cattle and sheep. There is a critical need for improved diagnostic tests to detect M. paratuberculosis infection in these ...

A microimmunofluorescence technique for the diagnosis of Q fever is described. Although this method is useful for serological diagnosis of Q fever, some technical difficulties are associated with it. First, the test antigens must be produced by a cell culture method in a level-3 biohazard fa ...

Isolation of Mycobacterium bovis from suspected cases of bovine tuberculosis demands laborious and time-consuming procedures. Also, direct PCR procedures on tissue samples show poor sensitivity, whereas radiometric and fluorescence-based identification procedures ...

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined on ...

In this chapter, a protocol called on-chip polymerase chain reaction (PCR) is presented for the deoxyribonucleic acid (DNA) microarray-based detection of bacterial target sequences. On-chip PCR combines, in a single step, the conventional amplification of a target with a simultaneo ...

Direct deoxyribonucleic acid (DNA)-based detection methods are crucial for future environmental monitoring and clinical diagnosis. In this chapter, we provide an overview of of the various sample preparation approaches for bacteria for direct analyses (i.e., without culturing) ...

The polymerase chain reaction (PCR) is a fundamental part of modern molecular biology. Fluorescence-based PCR methods also are now available, which enable rapid, specific, and sensitive assays for the amplification and analysis of deoxyribonucleic acid (DNA). These methods are perf ...

The Naval Research Laboratory has developed an array-based biosensor system capable of detecting multiple pathogenic and toxic species in complex matrices. Sandwich fluoroimmunoassays are performed on the surface of a patterned microscope slide that acts as an optical waveguid ...

The rapid and accurate identification of methicillin-resistant Staphylococcus aureus (MRSA) is of great importance for the affected patient, the involved ward, and the microbiological laboratory. Resistance to methicillin is encoded by the mecA gene in S. aureus. Because routine l ...

Escherichia coli O157:H7 is a highly virulent pathogen that causes severe food poisoning in humans. Many outbreaks involving this pathogen have been linked to minced beef. A protocol is presented here to detect E. coli O157:H7 in minced beef. The method consists of an enrichment step in modified tr ...

Use of high copy number bacterial RNA offers several advantages in a diagnostics context compared with current deoxyribonucleic acid-based assays. The opportunity to only detect viable cells by targeting labile RNA transcripts may create an opportunity for “real-time” monitoring ...

Quencher extension is a novel single-step closed tube real-time method to quantify single nucleotide polymorphisms (SNPs) in combination with primer extension. A probe with a 5′-reporter is single-base extended with a dideoxy nucleotide containing a quencher if the target SNP allele is ...

The membrane glycoproteins (Gn and Gc) of viruses in the family Bunyaviridae form projections on the virion envelope and are involved in virus entry and eliciting protective immunity. The glycoproteins are modified by N-linked glycosylation and accumulate in the Golgi complex where vi ...