脓毒症常见病原菌多重实时PCR检测方法的建立  

Establishment of a multiplex RT-PCR assay for common pathogens causing sepsis

作　　者：郑皓 李文革 杨海英 吴媛 车洁 陈小萍 卢金星 ZHENG Hao LI Wen-ge YANG Hai-ying WU Yuan CHE Jie CHEN Xiao-ping LU Jin-xing(State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and Prevention, Beijing 102206, China Beijing Applied Biological Technologies Co. , Ltd. Beijing 102206, China)

机构地区：[1]中国疾病预防控制中心传染病预防控制国家重点实验室感染性疾病诊治协同创新中心,北京102206 [2]北京卓诚惠生生物科技股份有限公司,北京102206

出　　处：《疾病监测》2017年第9期752-756,共5页Disease Surveillance

基　　金：国家科技支撑计划(No.2012BAI11B05)~~

摘　　要：目的建立可同时检测脓毒症患者血液中肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌、凝固酶阴性葡萄球菌、金黄色葡萄球菌和白假丝酵母菌的多重实时PCR方法。方法根据各病原菌基因组保守序列设计引物和探针,建立多重实时PCR检测方法。对建立的多重实时PCR检测方法进行特异性、敏感性和灵敏度分析,并使用该法对112份脓毒症血培养阳性标本进行验证。结果成功建立脓毒症常见病原菌的多重实时PCR检测方法。此方法特异性、敏感性良好,灵敏度达10-5ng/μl,与标准检测方法(血培养加生化鉴定)相比,缩短了20 h。112份临床血培养阳性标本中,目标病原菌83例,多重实时PCR检出80例阳性;非目标病原菌29例,多重实时PCR检测均为阴性。检测结果显示,该方法特异性达100%,敏感性达96.39%。结论该多重实时PCR检测方法具有快速、高效、灵敏、特异的优点,可用于临床重症监护室患者血液常见感染病原菌的诊断。Objective To establish a multiplex RT-PCR assay for the simultaneous detection of Klebsiella pneumoniae,Escherichia coli, Pseudomonas aeruginosa, Bauman acinetobacter, Coagulase negative staphylococcus, Staphylococcus aureus and Candida albicans in the blood of sepsis patients. Methods Primers and probes were designed according to the conserved sequence of the pathogenic bacteria or fungi to establish a multiplex RT-PCR assay. To analyze the sensitivity and specificity of the multiplex RT-PCR assay established,112 sepsis positive blood specimens（ blood culture and biochemical identification） were tested by using the assay. Results A multiplex RT-PCR assay of common pathogenic bacteria and fungi causing sepsis was established successfully. This method had good specificity and sensitivity,the detection sensitivity was10^-5ng/μl. Compared with the standard detection method（ blood culture plus biochemical identification）,20 hours were reduced. Among 83 target pathogens,80 were detected to be positive by multiplex RT-PCR. Among 29 non target pathogens,no positive results were obtained. The specificity and sensitivity were 100% and 96. 39% respectively. Conclusion The multiplex RT-PCR assay is rapid,efficient,sensitive and specific,which could be applied in the detection of common pathogens in the blood of ICU patients.

关 键 词：脓毒症 肺炎克雷伯菌 大肠埃希菌 铜绿假单胞菌 鲍曼不动杆菌 凝固酶阴性葡萄球菌 金黄色葡萄球菌 白假丝酵母菌 多重实时PCR 

分 类 号：R440[医药卫生—诊断学] R459.7[医药卫生—临床医学]