Construction of a DNA amplification assay for detection ofLegionella species in clinical samples

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping, and in the forensic identification of individuals. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro from as little as a single molecule of DNA. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.

In the summer of 1976, a mysterious epidemic of fatal respiratory disease in Philadelphia launched an intensive investigation that resulted in the definition of a new family of pathogenic bacteria, the Legionellaceae. In retrospect, members of the family had been isolated from clinical specimens as early as 1943. Unsolved epidemics of acute respiratory disease dating to the 1950s were subsequently attributed to the newly described pathogens. In the intervening years, the Legionellaceae have been firmly established as important causes of sporadic and epidemic respiratory disease. The sources of the infecting bacteria are environmental, and geographic variation in the frequency of infection has been documented. Airborne dissemination of bacteria from cooling towers and evaporative condensers has been responsible for some epidemics, but potable water systems are perhaps more important sources. The mode of transmission from drinking water is unclear. The Legionellaceae are gram-negative, facultative, intracellular pathogens. The resident alveolar macrophage, usually an effective antibacterial defense, is the primary site of growth. Cell-mediated immunity appears to be the most important immunological defense; the role of humoral immunity is less clear. Erythromycin remains the antibiotic of choice for therapy of infected patients, but identification and eradication of environmental sources are also essential for the control of infection.