Species specific identification of bacteria after amplification with a single primer pair complementary to highly conserved regions of 16S rDNA

K. M. McCabe, Y. H. Zhang, B. L. Huang, Su-Ting Terry Li, G. Khan, E. R B McCabe

Research output: Contribution to journalArticle

Abstract

Molecular genetic testing for bacterial DNA will reduce time and cost for identification of pathogens, improve the care of patients with infections, and permit molecular triage of sepsis. A single primer pair designed for highly conserved regions specific to the bacterial 16S ribosomal RNA coding sequence (rDNA) can amplify a wide variety of organisms. The highly conserved rDNA primer regions flank a domain that varies between bacterial species. The purpose of this study was to determine whether the amplification products could be used to identify individual bacteria by species specific hybridization. We examined a variety of bacterial 16S rDNA sequences in the GenEMBL, a non-overlapping nucleotide sequence database, using FastA and PileUp (GCG, Madison, WI). Sequences 19-21 bp in length were identified that were specific for individual species. Polymerase chain reaction (PCR) amplification of IBS rDNA from a number of different bacteria was performed using a previously reported primer pair, 8FPL and p806R. Dot blots were prepared with the amplification products and hybridized with radiolabeled species specific probes for gram positive and gram negative organisms. Using this approach we were able to identify individual PCR products generated with a single primer pair. We conclude that individual species can be identified after amplification of bacterial DNA with a single PCR primer pair complementary to sequences conserved among all bacteria. Therefore, after initial molecular triage, if a patient's sample shows positive amplification indicating the presence of bacteria, those amplification products can be tested by species specific hybridization for rapid identification of the organism causing the infection. Just as we have shown that the initial triage amplification can be automated, we would propose that the species specific hybridization also could be automated.

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume44
Issue number1
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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