Detection and identification of pathogens by use of the polymerase chain reaction
Abstract
The reductionistic approach of using the polymerase chain reaction (PCR) for detecting pathogens was demonstrated by using examples from three major groups of organisms: viruses, bacteria, and protists. Amplification primers were selected from the sequence of the St. Louis encephalitis virus RNA genome. Random hexamers were found to be much better than specific downstream primers for priming the reverse transcriptase reaction. Two restriction endonuclease digestions confirmed the identity of the amplified fragments. One primer pair, which amplified a 161 base pair (bp) fragment, was tested in a blinded experiment on seven samples of homogenized mosquitoes. The PCR assay correctly identified the infected sample. Amplification of the internal transcribed spacer (ITS) between the 16S and the 23S rDNA and digestion of the amplification products with endonucleases were used to distinguish different isolates of Neisseria meningitidis. Several isolates from an outbreak of meningococcal disease in east-central Illinois were analyzed; results indicated that a Decatur, Illinois isolate was not clonally related to the isolates from Champaign-Urbana, Illinois. Amplification of a 295-bp fragment from a region within the 16S rDNA specifically identified a single N. meningitidis-positive sample among five clinical swabs. No amplification was found for two swabs which were positive for commensal species of Neisseria, as determined by standard microbiological techniques. Regions within the 18S rDNA and across the 18S-5.8S rDNA ITS1 of Acanthamoeba were shown to distinguish different strains of Acanthamoeba. Secondary amplification products from one primer pair showed a high correlation with pathogenicity; a secondary product was cloned and sequenced, and selected primers from the sequence were able to distinguish pathogenic from nonpathogenic strains. Restriction endonuclease analyses of the amplification products were used to estimate Acanthamoeba phylogeny. The utility of the PCR for detecting and identifying pathogens was demonstrated in this study. However, the eventual niche for the PCR in diagnosis, molecular epidemiology, and taxonomy remains to be seen.
Degree
Ph.D.
Advisors
McLaughlin, Purdue University.
Subject Area
Microbiology|Molecular biology
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