多重PCR检测肠源性脓毒症常见致病菌的方法构建及初步临床应用

发布时间：2019-06-12 05:54

【摘要】：多重PCR检测肠源性脓毒症常见致病菌的方法构建及初步临床应用目的构建检测肠源性脓毒症常见病原菌的快速的、可靠的多重PCR反应体系,并初步探索其临床价值。方法根据10种肠源性脓毒症常见致病菌(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。将42例肠源性脓毒症患者随机分为多重PCR检测指导治疗组(以下简称多重PCR组)和经验用药及血培养指导组(以下简称对照组),观察两组患者抗菌药物使用时间、抗菌药物总费用、总住院费用、SIRS持续时间以及各种并发症发生率和死亡率。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反应体系的最小可检测细菌浓度为1×103cfu/ml,最适宜检测浓度范围1×103cfu/ml～1×106cfu/ml。多重PCR组检出率(81.8%)显著高于对照组(10%),两种检验方法阳性率差异有统计学意义(P0.05)。多重PCR组患者SIRS持续时间、抗生素费用与对照组的差异有统计学意义(P0.05)。两组患者住院总时间、抗菌药物使用时间、住院总费用及并发症的发生率和死亡率的差异无统计学意义(P0.05)。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。
[Abstract]:Construction and preliminary clinical application of multiplex PCR for detection of common pathogenic bacteria in enterogenic sepsis objective to construct a rapid and reliable multiplex PCR reaction system for detection of common pathogenic bacteria in enterogenic sepsis and to explore its clinical value. Methods primers and probes of complementary sequences were designed according to the 16sRNA specific fragments of 10 common pathogens of enterogenic sepsis (hereinafter referred to as target bacteria). The multiplex PCR reaction system was optimized and the specific test and concentration gradient sensitivity test were carried out. 42 patients with enterogenic sepsis were randomly divided into multiple PCR treatment group (multiple PCR group) and empirical drug use group and blood culture guidance group (control group). The use time of antibiotics, the total cost of antibiotics, the total hospitalization cost, the duration of SIRS and the incidence and mortality of various complications were observed. Results the standard strains of 10 target bacteria detected by multiplex PCR system showed positive results. The concentration gradient sensitivity experiment indicates that the minimum detectable bacterial concentration of the multiplex PCR reaction system designed in this project is 1 脳 10 ~ 3 CFU / ml, and the most suitable detection concentration range is 1 脳 103cfu/ml~1 脳 10 ~ 6 CFU / ml. The positive rate of multiplex PCR group (81.8%) was significantly higher than that of control group (10%). There was significant difference between the two methods (P 0.05). The duration of SIRS and the cost of antibiotics in multiple PCR group were significantly different from those in control group (P 0.05). There was no significant difference in the total hospitalization time, the use time of antibiotics, the total cost of hospitalization and the incidence and mortality of complications between the two groups (P 0.05). Conclusion the multiplex PCR reaction system established in this project has the ability of high throughput and accurate detection of common pathogens in sepsis patients. Preliminary clinical studies suggest that this method has certain application value in guiding antimicrobial therapy in patients with enterogenic sepsis.
【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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