一种新的实时荧光定量PCR方法对血小板制品细菌污染检测效果的评价

Evaluation of detection effect on platelet bacterial contamination using a new in-house real-time PCR system

目的建立一种自主研发的针对细菌16 S r DNA基因的实时荧光定量PCR(Real-time PCR)方法并评价该方法对浓缩血小板制品中细菌污染检测的效果。方法设计16S r DNA基因保守区引物,构建SYBR Green Real-time PCR反应体系;然后分别将大肠杆菌、金黄色葡萄球菌、表皮葡萄球菌、蜡样芽孢杆菌和绿脓杆菌以初始浓度为1CFU/m L、10 CFU/m L和100 CFU/m L接种到浓缩血小板中,经22℃保存7 d后,用实时定量荧光PCR方法进行细菌检测。结果细菌污染后的血小板在常规保存条件下,最长保存期<7 d,不同接种浓度的细菌生长情况的变化趋势基本一致,所有种类的细菌均在d 1、2表现出迅猛的增殖高峰,d 3以后增殖趋于平缓。结论该Real-time PCR检测体系可定量地检测出血小板的细菌污染的情况,可适用于血小板输注前的快速细菌污染检测。

Objective To establish an in-house real-time PCR assay to detect bacterial 16 S r DNA and to evaluate the effect of the detection system performed on different bacterial contaminated platelets. Methods Primers from conservative region of 16 s r DNA were designed to establish a SYBR Green based real time PCR system. Platelets inoculated with 5 transfusion-relevant species of bacteria( Escherichia coli,Bacillus cereus,Staphylococcus epidermidis,Pseudomonas aeruginosa,and Staphylococcus aureus) at concentrations of 1,10,and 100 colony-forming units( CFU) per m L were stored at 22°C for7 days,respectively. Then the real-time PCR system was used to detect the presence of bacteria in samples prepared from those contaminated platelets. Results After contamination,the bacterial content in platelets changed day by day during the storing period of 7 days under conventional storing conditions. All types of bacteria showed rapid population expansion on day1 and day 2,and the multiplication tended to slow down after day 3. Conclusion A real-time PCR system was established and could quantitatively detect the bacterial content of platelets. As a result,this method could be used to detect the bacterial contamination of platelets before transfusion.