多重PCR检测肠源性脓毒症常见病原菌的方法构建及初步临床应用  

Establishment and preliminary clinical application of multiplex qPCR for detection of common pathogenic bacteria in gut-derived sepsis

作　　者：向崛 刘长峰[1] 施新萍[2] 张永乐[2] 张竝[1] XIANG Jue LIU Chang-feng SHI Xin-ping ZHANG Yong-le ZHANG Bing(Tongde Hospital of Zhejiang Province Affiliated to Zhejiang University of Chinese Medicine, Hangzhou, Zhejiang 310012, China)

机构地区：[1]浙江中医药大学附属浙江省立同德医院肝胆外科,浙江杭州310012 [2]浙江省立同德医院检验科,浙江杭州310012

出　　处：《中华医院感染学杂志》2017年第7期1466-1469,共4页Chinese Journal of Nosocomiology

基　　金：浙江省科技厅公益项目(2013C33197)

摘　　要：目的构建多重PCR反应体系检测肠源性脓毒症常见病原菌的快速可靠的方法,并初步探索其临床价值。方法选择2013年6月-2015年7月医院42例肠源性脓毒症患者,随机分为多重PCR组及对照组,各21例;根据10种肠源性脓毒症常见病原菌(以下简称目标细菌)的16sRNA特异性片段设计互补序列的引物和探针;优化多重PCR反应体系并进行特异性实验及浓度梯度敏感性实验。结果多重PCR体系检测10种目标细菌的标准菌株均呈现阳性结果;浓度梯度敏感性实验提示本项目设计的多重PCR反应体系可检测的最小细菌浓度为1×103 CFU/ml,最适宜检测浓度范围1×103~1×106 CFU/ml;多重PCR组检出率显著高于对照组,多重PCR组患者SIRS持续时间、抗菌药物费用与对照组比较,差异均有统计学意义(P<0.05)。结论本项目建立的多重PCR反应体系具备高通量、精准检测脓毒症患者感染的常见病原菌的能力;初步的临床研究提示该方法在指导肠源性脓毒症患者的抗微生物治疗中有一定的应用价值。OBJECTIVE To establish a rapid and reliable multiplex qPCR for bacterial identification in patients suf- fering gut-derived sepsis and evaluate its value of clinical application. METHODS From Jun. 2013 to Jul. 2015, 42 patients with gut-derived sepsis were selected, and randomly divided into multiplex qPCR group and control group, with 21 cases in each group. Primers and probes with complementary sequences from specific 16sRNA of 10 popular gut-derived bacteria （abbreviated as targeted bacterium） were designed and synthesized. The reactive system of this method was optimized to assure its specificity and sensitivity. RESULTS Totally 10 species of stand- ardized bacteria were positive in multiplex qPCR test. Ten times of repeated experiments revealed that the mini- mum detectable bacterial concentration was 1 × 10^3 cfu/mL, and the most appropriate detective concentration were 1 × 10^3 CFU/mL-1× 10^6 CFU/mL. The detection positive rate of multiplex qPCR group was significantly higher than that of qontrol group（P〈0.05）, and consecutive time of SIRS and antibiotics expense between multiplex qPCR group and control group were significantly different （P〈0.05）. CONCLUSION The established multiplex qPCR assay with high sensitivity, specificity and broad detecting range, is a rapid and accurate technique in the de- tection of bacterial pathogens of gut-derived sepsis. Preliminary clinical research illuminated that multiplex qPCR might be a potential method in guiding anti-infective therapy in gut-derived sepsis.

关 键 词：肠源性脓毒症 抗微生物治疗 细菌DNA 多重PCR 

分 类 号：R378[医药卫生—病原生物学]