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Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
Published on: April 14, 2016
Sterility Testing of Stem Cell Products by Broad-Range Bacterial 16S Ribosomal DNA Polymerase Chain Reaction
Osamu Tokuno1, Akira Hayakawa2, Tomoko Yanai2
1Blood Transfusion Division, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan otokuno@cira.kyoto-u.ac.jp.
Objective:
To evaluate broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) as a rapid screening tool to detect bacterial contamination of stem-cell products.
Methods:
We performed the evaluation using whole blood spiked with serially diluted bacterial-type strains. Detection sensitivity was defined as the bacterial concentration for which all replicates were positive at each concentration (100% detection). We tested the sterility of 29 bags of autologous peripheral blood stem cell (PBSC) products harvested at our facility using the 16S rDNA PCR method.
Results:
The detection sensitivity of 16S rDNA PCR in spiked whole blood was 10¹ to 10² colony-forming units (CFU) per mL, depending on the bacterial strain. We detected no amplified 16S rDNA among the PBSCs we used in this study. The BacT/ALERT automated bacterial culture system that we used also showed no positive signals in any of the PBSCs tested.
Conclusions:
Our data indicate that bacterial 16S rDNA PCR is a useful alternative for rapid sterility testing, not only for blood products used in transfusion medicine but also for stem-cell products used in regenerative medicine.
