Conventional culture method and qPCR using 16S rDNA for tissue bank: a comparison using a model of cardiac tissue contamination

J Med Microbiol. 2018 Nov;67(11):1571-1575. doi: 10.1099/jmm.0.000837. Epub 2018 Sep 12.

Abstract

Real-time polymerase chain reaction (qPCR) using 16S rDNA is an alternative to conventional culture-based tests. The aim of this study was to compare the conventional culture method with qPCR using 16S rDNA in a model of cardiac tissue contamination. Samples of cardiac tissue for artificial contamination with Escherichia coli and control samples were submitted for DNA extraction, which was conducted by selective and alkaline lysis and purification steps. A standard curve for 16S rDNA was constructed to determine the efficiency and analytical sensitivity of the assay in concentrations from 106 to 102 c.f.u. ml-1 using TaqMan Master Mix. 16S rDNA was detected in all contaminated samples; however, it was not detected in the the final washing step solution of the sample with a bioburden of 102 c.f.u. ml-1. Using qPCR is a potential alternative to conventional culture for microbiological safety testing of allograft tissues for biobanking, reducing the time and labour input required.

Keywords: 16S rDNA; cardiovascular tissues; conventional culture; real-time PCR; tissue bank.

Publication types

  • Comparative Study

MeSH terms

  • Bacterial Infections / microbiology
  • Bacterial Infections / prevention & control*
  • Bacteriological Techniques / methods*
  • DNA, Bacterial / genetics
  • DNA, Ribosomal / genetics*
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Escherichia coli / isolation & purification*
  • Heart / microbiology*
  • Heart Transplantation
  • Humans
  • Myocardium / chemistry
  • RNA, Ribosomal, 16S / genetics
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Tissue Banks

Substances

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