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Development of a reverse transcription–polymerase chain reaction assay for eubacterial RNA detection in platelet concentrates
Author(s) -
Rood Ineke G.H.,
Pettersson Annika,
Savelkoul Paul H.M.,
De Korte Dirk
Publication year - 2010
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/j.1537-2995.2009.02580.x
Subject(s) - serial dilution , real time polymerase chain reaction , bacteria , reverse transcription polymerase chain reaction , polymerase chain reaction , microbiology and biotechnology , biology , primer (cosmetics) , assay sensitivity , 16s ribosomal rna , ribosomal rna , reverse transcriptase , rna extraction , rna , chemistry , gene , messenger rna , medicine , biochemistry , genetics , alternative medicine , organic chemistry , pathology
BACKGROUND: The sensitivity of a real‐time polymerase chain reaction (PCR) assay detecting bacteria in platelet concentrates (PCs) was improved by detection of ribosomal RNA (rRNA) in addition to DNA. The real‐time reverse transcription–PCR (RT‐PCR) assay was compared with the BacT/ALERT culturing system (bioMérieux) to determine its value for routine screening of PCs for bacterial contamination. STUDY DESIGN AND METHODS: The sensitivity of the assay was determined by spiking PCs with serial dilutions of bacteria. RNA amplification was performed with real‐time RT‐PCR, using a universal primer and probe set based on the conserved 16S rRNA gene of bacteria. Routinely prepared PCs in plasma were spiked with low bacterial titers of four different bacteria to compare the real‐time RT‐PCR with the BacT/ALERT. For the BacT/ALERT, samples were taken directly after spiking. For the real‐time RT‐PCR, samples were taken daily during 7 days of storage. RESULTS: RNA detection improved the sensitivity of the PCR assay at least 10‐fold. When PCs were spiked with low bacterial titers, all positive samples were detected by the real‐time RT‐PCR after 48 hours. The BacT/ALERT became positive for almost all samples within 24 hours. However, some positive PC samples remained negative in the BacT/ALERT. CONCLUSION: The sensitivity of the PCR assay was improved by detection of rRNA. A spiking study demonstrated the advantage of late sampling for PCR testing compared to early sampling for culturing with the BacT/ALERT system. A real‐time RT‐PCR assay that is performed on PCs during storage or shortly before transfusion can be a good alternative to culturing methods.