基于微流控芯片的实时荧光定量 ＰＣＲ 技术快速检测血小板制剂细菌 污染

摘要：目的 通过微流控芯片检测平台应用 Ｔａｑｍａｎ 探针的实时定量 ＰＣＲ 技术对血小板制剂中的细菌１６Ｓ ｒＤＮＡ 进行检测，探 讨该体系在血小板细菌污染的快速检测的应用。方法 向机采血小板中人为添加一定浓度的金黄色葡萄球菌或铜绿假单胞 菌，模拟成１０２ ～１０８ ＣＦＵ／ ｍＬ 细菌污染的血液标本，经１０倍梯度稀释后进行细菌计数及微流控芯片 ＦＱ ＰＣＲ 检测细菌１６Ｓ ｒＤＮＡ，并 对检测体系进行特异性、检出限和重复性评价。结果 微流控芯片 ＦＱ ＰＣＲ 体系特异性较强，探针和引物对阳性标本均有反 应，与空白对照无反应。对污染血小板的金黄色葡萄球菌，该方法的最低检出限为 ８６５ ＣＦＵ／ ｍＬ，而对铜绿假单胞菌的最低检 出限为 ８８５ ＣＦＵ／ ｍＬ。当金黄色葡萄球菌与铜绿假单胞菌浓度分别达到最低检测限时，空白对照与各细菌组的 Ｃｔ 值之差分别 为 ４．３５±１．０１ 和 ２．０３±０．６１。各浓度菌液提取的 ＤＮＡ 进行微流控芯片 ＦＱ ＰＣＲ 扩增后的 Ｃｔ 值计算重复性指数在 ０．０１２～ ０．０５２ 范围内。结论 微流控芯片 ＦＱ ＰＣＲ 检测平台可特异、有效地检出血小板中的细菌污染，为确保输血安全提供帮助。

Microfluidic chip for rapid detection of bacterial contamination in platelet concentrates by Real-time fluorescent quantitative PCR

Objective The microfluidic chip detection platform was used to detect bacterial 16srDNA in platelet concentrates by real-time quantitative PCR with Taqman fluorescent probes, and the application of this system in the rapid detection of bacterial contamination in platelets was discussed. Methods The suspension of Staphylococcus aureus or Pseudomonas aeruginosa were artificially added to the platelets to simulate the blood samples contaminated by bacteria. While platelets without bacteria were set as blank control and sterile water was set as negative control. After 10-fold gradient dilution, the relative experiments such as bacterial counts, bacterial genomic DNA extraction, bacterial 16sDNA detection by microfluidic chip FQ-PCR were carried out. The specificity, sensitivity and repeatability of the microfluidic chip FQ-PCR detection system were evaluated. Results The probes and primers responded to the positive reference, but did not respond to blank control. For Staphylococcus aureus contamination of platelets, the sensitivity of this method was 865CFU/mL and for Pseudomonas aeruginosa, the sensitivity was 885CFU/mL. When the bacterial content of Staphylococcus aureus was 865CFU/mL, the difference of Ct value between the blank control group and bacterial group was 4.35±1.01; and for Pseudomonas aeruginosa (885CFU/mL), the difference was 2.03±0.61. The repeatability index of Ct values of bacterial with various concentrations ranged from 0.012 to 0.052. Conclusion Based on the microfluidic chip detection platform, the TaqMan probe FQ-PCR system can do well to detect bacterial contamination of platelets. It can be applied to the rapid detection of bacterial contamination before platelet transfusion when the sensitivity and repeatability of the system have been improved.