Application of qualitative and quantitative real-time PCR, direct sequencing, and terminal restriction fragment length polymorphism analysis for detection and identification of polymicrobial 16S rRNA genes in ascites

J Clin Microbiol. 2014 May;52(5):1754-7. doi: 10.1128/JCM.00552-14. Epub 2014 Mar 12.

Abstract

Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct sequencing were applied for rapid detection and identification of bacterial DNA (bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a mean of 3.24 log copies ml(-1). Direct sequencing of PCR products revealed 62% mixed chromatograms predominantly belonging to Gram-positive bacteria. Terminal restriction fragment length polymorphism (T-RFLP) results of a sample subset confirmed sequence data showing polymicrobial DNA contents in 67% of bactDNA-positive ascites samples.

MeSH terms

  • Ascites / diagnosis*
  • Ascites / microbiology*
  • DNA, Bacterial / genetics
  • DNA, Ribosomal / genetics
  • Genes, rRNA / genetics
  • Humans
  • Polymerase Chain Reaction / methods
  • Polymorphism, Restriction Fragment Length / genetics*
  • RNA, Ribosomal, 16S / genetics*
  • Real-Time Polymerase Chain Reaction / methods
  • Sequence Analysis, DNA / methods

Substances

  • DNA, Bacterial
  • DNA, Ribosomal
  • RNA, Ribosomal, 16S