Rapid blood sample preparation for bacterial DNA amplification

Su-Ting Terry Li, Kevin M. McCabe, Edward R B McCabe

Research output: Contribution to journalArticle

Abstract

The clinical diagnosis of septicemia remains imperfect and a blood culture typically requires two or more days to yield results. Our strategy to improve the evaluation of possible sepsis involves rapid and reliable polymerase chair reaction (PCR) amplification of bacterial DNA with a universal primer pair, each member of the pair annealing to a highly conserved sequence that is present in all bacteria examined to date. Implementation of a PCR strategy for molecular triage of sepsis (K. McCabe et al, Pediatrics 95:165-169, 1995) will require a method for rapid sample preparation that provides bacterial DNA of sufficient quality for amplification. The purpose of this project was to test a method for rapid extraction of DNA from bacteria in blood specimens and determine Its sensitivity. E. coli was serially diluted with whole blood. Bacterial DNA was prepared using Chelex-100 resin to bind heme, an inhibitor of the polymerase, then heated for 15 min. at 95°C (A.E. Lew and P.M. Desmarchelier. J. Clin. Microbiol. 32:1326-1332, 1994), and amplified with a primer pair for highly conserved regions within the 16S ribosomal RNA coding sequence. The amplification products were visualized after electrophoresis on an agarose gel containing ethidium bromide. We obtained PCR signals with 20% or less of the amplification products from as few as 84 organisms in 0.5 ml. blood. The method required 30 min. for extraction and less than 3 hrs. for amplification. No signal was observed when human blood without bacteria was prepared in this manner. We conclude that bacterial DNA can be extracted rapidly and reproducibly from intact organisms in whole blood by eliminating the component that inhibits PCR amplification. Improvement will be required to increase the sensitivity to fewer than 150-200 bacteria per milliliter. These early results move us closer to clinical implementation of molecular triage of patients with signs and symptoms of sepsis, to separate rapidly those who need immediate hospitalization and parenteral antibiotics from those who may be managed at home.

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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