一种用于脓毒症快速检测的多重PCR检测体系构建

Establishment of a multiplex PCR system for rapid detection of sepsis

目的:建立一种基于多重实时荧光定量聚合酶链反应(multiple real-time fluorescence quantitative polymerase chain reaction,多重PCR)技术的检测体系,快速、特异检测脓毒症患者血液中的病原菌。方法:分析血液标本分离菌主要菌种分布,绘制脓毒症特异性病原微生物谱。同时根据保守区序列设计特异性引物,构建多重PCR检测反应体系。收集脓毒症患者静脉全血样本79例,健康体检者样本(阴性样本)40例,用所建立的检测体系进行鉴定,并与血培养方法比较。结果:多重PCR法缩短检出时间至3 h,是血培养法的1/16。多重PCR法对目标病原菌样本符合率为88%(22/25),阴性样本符合率为100%(20/20),总符合率为94%,2种方法对目标病原菌的检出差异无统计学意义(P=0.250),且一致性良好(Kappa=0.867,P<0.05)。多重PCR法与血培养法对临床诊断脓毒症样本的检出率分别为23.0%(17/74)及14.9%(11/74),2种方法间检出率差异有统计学意义(P=0.031)。结论:与经典的“金标准”血培养法相比,本项目构建的多重PCR法检测时间短、符合率好、检出率更高,可为脓毒症患者更早诊断和治疗提供可靠依据。

Objective:To establish a detection system based on multiple real-time fluorescence quantitative polymerase chain reaction(multiplex PCR)technique to rapidly and specifically detect pathogens in the blood of sepsis patients.Methods:The main strains of pathogens isolated from blood samples were analyzed,and the spectrum of pathogens specific to sepsis was drawn. Specific primers were designed according to the sequences of the conserved regions to construct a multiplex PCR detection reaction system. Blood samples of 79 sepsis patients were collected,and 40 health subjects were included as negative and health controls during the same period. Then they were identified by the established detection system,and the blood culture method was compared.Results:The detection time was shortened to 3 hours by multiplex PCR,which was 1/16 of blood culture. Compared with blood culture,the coincidence rate of multiplex PCR for target pathogen samples was 88%(22/25),the coincidence rate of negative samples was 100%(20/20),and the total coincidence rate was 94%. There was no significant difference between the two methods in the detection of target pathogens(P=0.250)with good consistency(Kappa=0.867,P<0.05). The detection rates of multiple PCR method and blood culture method for clinically diagnosed sepsis samples were 23.0%(17/74)and 14.9%(11/74),respectively. The detection rates of the two methods were statistically significant(P=0.031).Conclusion:Multiplex PCR method can provide a reliable basis for earlier diagnosis and treatment to sepsis patients. Compared with“gold standard”blood culture method,the multiplex PCR detection system constructed in the project has shorter detection time,better coincidence rate and higher detection rate,which can provide reliable evidence for the early diagnosis and treatment of sepsis patients.