Department of Blood Cell Research, Sanquin Research, VU University Medical Center, Amsterdam, The Netherlands.
Transfusion. 2010 Jun;50(6):1352-8. doi: 10.1111/j.1537-2995.2009.02580.x. Epub 2010 Jan 22.
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.
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.
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.
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.
通过检测核糖体 RNA（rRNA）而不是 DNA，实时聚合酶链反应（PCR）检测血小板浓缩物（PC）中细菌的灵敏度得到提高。该实时逆转录-PCR（RT-PCR）检测与 BacT/ALERT 培养系统（bioMérieux）进行了比较，以确定其用于常规筛查 PC 细菌污染的价值。
通过将细菌的连续稀释液混入 PC 来确定检测的灵敏度。使用基于细菌保守 16S rRNA 基因的通用引物和探针组进行 RNA 扩增，采用实时 RT-PCR。将低细菌滴度的四种不同细菌混入常规制备的血浆 PC 中，比较实时 RT-PCR 与 BacT/ALERT。对于 BacT/ALERT，直接在混入后取样。对于实时 RT-PCR，在 7 天储存期间每天取样。
RNA 检测将 PCR 检测的灵敏度提高了至少 10 倍。当将低细菌滴度的 PC 混入时，实时 RT-PCR 在 48 小时后可检测到所有阳性样本。BacT/ALERT 在 24 小时内几乎对所有样本均呈阳性。然而，BacT/ALERT 中的一些阳性 PC 样本仍为阴性。
通过检测 rRNA 提高了 PCR 检测的灵敏度。在与 BacT/ALERT 系统进行培养相比，通过晚期取样进行 PCR 检测具有优势。在储存期间或输血前不久对 PC 进行实时 RT-PCR 检测可以替代培养方法。
