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

Author(s): Krohn, S., S. Boehm, C. Engelmann, J. Hartmann, A. Brodzinski, A. Chatzinotas, K. Zeller, D. Prywerek, I. Fetzer, T. Berg
In: Journal of Clinical Microbiology
Year: 2014
Type: Journal / article
Full reference: Krohn, S., S. Boehm, C. Engelmann, J. Hartmann, A. Brodzinski, A. Chatzinotas, K. Zeller, D. Prywerek, I. Fetzer, T. Berg. 2014. 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. Journal of Clinical Microbiology 52(5): 1754-1757.

Summary

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.

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