【作者】 李静；

【导师】 李庆阁；

【作者基本信息】 厦门大学 ， 生物学， 2018， 硕士

【摘要】 感染性腹泻(infectious diarrhea)是指由细菌、病毒、真菌以及寄生虫而引起的腹泻。由于腹泻病人的临床症状极为相似,难以根据症状鉴定出导致腹泻的病原体,使得临床医生无法拟定针对性的治疗方案。目前已经报道的文献中少有同时检测细菌和病毒的相关研究。本论文采用媒介探针结合熔解曲线分析技术,建立了一管检测12种细菌、6种病毒以及一种血清型的快速分析方法。在第一章中,第一节介绍了感染性腹泻的背景、临床表现、发病机制以及抗菌药物的治疗;第二节介绍了检测胃肠道病原体常用诊断技术;第三节介绍了多重媒介探针结合熔解曲线分析技术的原理以及胃肠道病原体靶标基因的选择。第二章为材料与方法部分包括所用的仪器与试剂、质粒的构建、引物及探针的设计、单重及多重体系的建立、灵敏度考察、特异性考察、重复性考察、混合感染识别能力考察以及临床评价。第三章介绍了本体系的性能考察以及标本的检测结果。本论文以细菌和病毒为检测对象,建立了一管5色29重体系。检测的灵敏度可以达到50拷贝/反应,部分对象可达5拷贝/反应。体系的重复性好,熔点的CV值最大为0.78%,其他均小于0.5%。本体系检测了深圳疾病预防控制中心收集的100份粪便标本以及119份临床分离株。以实时荧光PCR技术作为对照方法,病毒的检测一致率达到94%(94/100),119份临床分离株的检测结果和深圳疾控一致。另外还检测了济宁疾病预防控制中心的39份粪便标本,用商品化病毒检测试剂盒作为对照方法。病毒检测一致率达到89.7%(35/39),细菌检测结果和测序结果一致。对于不一致的病毒标本采用本实验室建立的单重实时PCR体系来验证,验证结果与本体系检测结果一致。更多还原

【Abstract】 Infectious diarrhea refers to diarrhea caused by bacteria,viruses,fungi,and parasites.Because the clinical symptoms of diarrhea patients are very similar,it is difficult to identify the pathogens leading to diarrhea according to the symptoms,which makes it impossible for clinicians to formulate targeted treatment plans.There are few studies on the simultaneous detection of bacteria and viruses in the literature that has been reported.In this paper,a rapid method for the detection of 12 kinds of bacteria and 6 kinds of viruses and a serotype was established by mediator probe melting curve analysis.The first section of the first chapter,we introduced the background,clinical manifestations,pathogenesis of infectious diarrhea and the treatment of antibacterial drugs;The second section introduced the commonly used diagnostic techniques for the detection of gastrointestinal pathogens.The third section introduced the principle of multiplex mediator probe melting curve analysis and the selection of gastrointestinal pathogen target genes.The second chapter introduced the system’s materials and methods including instruments and reagents used,the construction of plasmids,the design of primers and probes,the establishment of single and multiplex systems,sensitivity investigations,specificity investigations,repetitive investigations,the ability to identify mixed infections,and clinical evaluations.In the third chapter,we introduced the performance of the system and test results of the specimen.In this paper,a 5-color 29-plex system was established for the detection of bacteria and viruses.The detection sensitivity reaches 50 copies/reaction,and some subjects can reach 5 copies/reaction.The reproducibility of the system is good.The CV value of the melting point is the maximum 0.78%,and the others are all less than 0.5%.The system has detected 100 stool samples and 119 clinical isolates from the Shenzhen Center for Disease Control and Prevention.Using real-time fluorescence PCR as a control method,the virus detection rate was 94%(94/100).The results of the 119 clinical isolates were consistent with Shenzhen Center forDisease Control and Prevention.In addition,39 fecal specimens from the Jining Center for Disease Control and Prevention were tested and a commercial virus detection kit was used as a control method.The virus detection consistency rate reached 89.7%(35/39),and the bacterial detection results were consistent with the sequencing results.The inconsistent virus samples were verified by a uniplex real-time PCR system established in our laboratory.The verification results were consistent with the detection results of this system.更多还原