实时荧光PCR检测血小板制品中常见细菌方法的建立

The establishment of real-time PCR method for the detection of common bacteria in platelet products

目的建立一种快速灵敏便于开展的血小板制品中常见细菌的实时荧光PCR检测方法,并评价其在血小板制品细菌检测中的应用效果。方法在NCBI中查找血小板中八种常见细菌的特异性基因序列,分别设计出保守区域的引物和探针,将设计好的引物在基因库中进行比对,确认无交叉再进行合成。将合成好的引物和探针通过扩增血小板常见细菌实验,确定最佳引物和探针,同时确定最佳扩增条件,最终建立完整的血小板制品细菌检测的PCR方法。结果通过多种引物的实验,确定了实验方案,本实验中共有8组特异性引物,最终实现金黄色葡萄球菌可以检到10 CFU/mL,铜绿假单胞菌可以检到10 CFU/mL,蜡状芽孢杆菌可以检到10 CFU/mL,鲍曼不动杆菌可以检到10 CFU/mL,奇异变形杆菌可以检到20 CFU/mL,白色念珠菌可以检到10 CFU/mL,白喉杆菌可以检到30CFU/mL,肺炎克雷伯菌可以检到20 CFU/mL。整个检测过程从血小板制品取样到核酸的提取,再到扩增结束,需要4—5 h。结论本方法能够快速准确地检测出血小板制品中的常见细菌,但也存在一定的局限性,对于常见菌以外的细菌无法检出,因此,需要加大样本检测量来验证本方法的有效性。

Objective：To establish a rapid, sensitive and convenient method for the detection of common bacteria in platelet products by real-time PCR, and to evaluate the performance of this method in the detection of platelet bacteria. Methods Firstly, the specific gene sequences of eight common bacteria in platelets products were acquired via NCBI database. Primers and probes in the conserved regions were designed separately. The designed primers were then compared in the gene bank to confirm that there was no crossover and synthesis. The synthesized primers and probes were used to amplify the bacteria to determine the best primers and probes as well as the best amplification conditions. Finally, a complete PCR method for detecting platelet products bacteria was established. Results By a serious of primer experiments, the experimental protocol was generated. In this experiment, there were eight groups of specific primers. Finally, 10 CFU/mL could be detected in Staphylococcus aureus, and 10 CFU/mL could be detected in Pseudomonas aeruginosa, Bacillus can be detected lO CFU/mL, Acinetobacter baumannii can he detected 10 CFU/mL, Proteus mirabilis can be detected 20 CFU/mL, Candida albieans can be detected lO CFU/mL, diphtheria can be detected Up to 30 CFU/mL, K.pneumoniae can detect 20 CFU/mL. The whole detection process takes 4--5 hours. Conclusion The method can detect common bacteria in platelet products swiftly and accurately. However, it does show certain limitations. The method only can detect the eight common bacteria, but it＇s no way to detect other rather rare ones. Therefore, it＇s necessary to expand the sample pool for further improvement.