Albertoni G, Andrade S S, Araújo P R B, Carvalho F O, Girão M J B C, Barreto J A
Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil.
Transfus Med. 2011 Dec;21(6):408-16. doi: 10.1111/j.1365-3148.2011.01105.x. Epub 2011 Sep 5.
The performance of a bacterial 16S ribosomal DNA real-time polymerase chain reaction (PCR) assay was evaluated and validated with an automated culture system to determine its use for screening of platelet concentrates (PCs).
PCs were spiked with suspensions of Escherichia coli, Serratia marcescens, Staphylococcus epidermidis and St. aureus at 1, 10, and 100 colony-forming units (CFUs) mL and stored for 5 days. DNA amplification was performed using real-time PCR. The BacT/ALERT was used as a reference method and samples were inoculated into an aerobic culture bottle; for the PCR assay, aliquots were drawn from all (spiked) PCs on days 0 to 5 of storage.
Real-time PCR detected only the gram-positive bacteria in PCs spiked with low bacterial titres (1 CFU mL) after 48 h; however, it was able to detect all positive samples in PCs spiked with 10 CFU mL of either gram-positive or gram-negative bacteria after 48 h. In addition, real-time PCR detected all positive samples in PCs spiked with high gram-positive bacterial titres (100 CFU mL) after 24 h. On the other hand, the BacT/ALERT system showed positive results in all samples within 24 h.
The BacT/ALERT method is more sensitive and should continue to be the gold standard for identifying bacterial contaminations in blood samples. The real-time PCR approach can be used for the screening of PCs for microbial detection before they are released from blood centres or shortly before they are used in blood transfusion, and thus allow an extended shelf life of the platelets.
评估并验证了细菌16S核糖体DNA实时聚合酶链反应（PCR）检测法与自动化培养系统的性能，以确定其用于血小板浓缩物（PCs）筛查的用途。
将大肠杆菌、粘质沙雷氏菌、表皮葡萄球菌和金黄色葡萄球菌的悬浮液分别以1、10和100个菌落形成单位（CFU）/mL的浓度加入到PCs中，并储存5天。使用实时PCR进行DNA扩增。以BacT/ALERT作为参考方法，将样本接种到需氧培养瓶中；对于PCR检测，在储存的第0至5天从所有（加样的）PCs中抽取等分试样。
实时PCR在48小时后仅检测到低细菌滴度（1 CFU/mL）加样的PCs中的革兰氏阳性菌；然而，它能够在48小时后检测到加样10 CFU/mL革兰氏阳性或革兰氏阴性菌的PCs中的所有阳性样本。此外，实时PCR在24小时后检测到加样高革兰氏阳性菌滴度（100 CFU/mL）的PCs中的所有阳性样本。另一方面，BacT/ALERT系统在24小时内所有样本中均显示阳性结果。
BacT/ALERT方法更敏感，应继续作为鉴定血液样本中细菌污染的金标准。实时PCR方法可用于在PCs从血液中心放行前或在输血前不久对其进行微生物检测筛查，从而延长血小板的保质期。
