犬钩端螺旋体病PCR和荧光定量PCR诊断试剂盒的研制

发布时间：2018-05-09 22:02

本文选题：犬钩端螺旋体病 + 诊断　；参考：《吉林大学》2015年硕士论文

【摘要】：钩端螺旋体病（简称钩体病），是因感染具有致病性的钩体所引起的一种人兽共患病。犬钩端螺旋体病，是由常见对犬致病的犬型、黄疸出血型、塔拉索夫型等钩体所引起。犬对该病具有易感性，犬感染本病后，临床表现多样，不易进行诊断，因此需要实验室检测对本病进行辅助的鉴别诊断。常见实验室诊断方法有直接镜检，分离培养，血清学检测，动物接种等，但这些方法或不具备时效性，或缺乏准确性。相较于这些方法，聚合酶链反应（PCR）方法更省时省力，特异性和敏感性优异，并且可用于钩体病的早期诊断。本实验依据仅致病性钩端螺旋体含有的LigB基因序列的保守片段，并对常见对犬致病的钩体株的相关序列进行比对，设计了数对特异性的引物和探针，目的片段大小为124bp。对探针和引物进行筛选及反应条件与体系的优化，构建了含目的基因的质粒作为标准阳性对照，，并对质粒进行测序验证。建立了犬钩端螺旋体病PCR和荧光定量PCR诊断方法，并组装成可实现对犬钩体病进行快速高效诊断的试剂盒。对试剂盒的灵敏度、特异性、重复性等参数进行了检验，确定其保存条件和保存期,采集并检测临床犬血液和尿液样本，与显微凝集试验相比较，结果一致。近年来，宠物犬的数量不断增加，各类工作犬的数量也有上升。犬易感钩体病，影响犬自身及其主人的健康，该检测方法可推广应用于犬钩体病的早期检测，避免该病的进一步发展和扩散。钩端螺旋体的培养需要数周的时间，培养基的配制程序复杂，而诊断钩体病的金标准方法MAT，需要活体钩体才能进行诊断，因而很难得到推广和应用，而PCR方法不需要进行钩体的培养，并且相比于血清学方法，还可用于钩体病的早期诊断。在该实验中，建立的普通PCR方法检测钩体浓度的灵敏度可达到101，荧光定量PCR方法的灵敏度可达100。
[Abstract]:The leptospirosis is a kind of zoonotic disease caused by the leptospirosis caused by infection . The canine leptospirosis is caused by the common dog type , jaundice , blood type and Tarasov type . The dog has a susceptibility to the disease . The dog has a variety of clinical manifestations and is not easy to diagnose . The common laboratory diagnosis method has direct microscopic examination , separation culture , serological detection , animal vaccination , etc . The method has the advantages of saving time and effort , specificity and sensitivity , and can be used for early diagnosis of leptospirosis .

The PCR and fluorescence quantitative PCR diagnosis of leptospirosis in dogs was established . The PCR and fluorescence quantitative PCR methods were used to test the sensitivity , specificity and repeatability of the kit . The blood and urine samples were collected and tested . The results were consistent with the microscopic agglutination test .

In recent years , the number of pet dogs has increased , and the number of various kinds of working dogs has also increased . The dog is susceptible to leptospirosis and affects the health of the dog itself and its owner . The detection method can be popularized and applied to the early detection of dog leptospirosis , so as to avoid further development and diffusion of the disease .

The culture of leptospirosis requires several weeks , the preparation procedure of the culture medium is complex , and the gold standard method MAT for diagnosing leptospirosis needs a living hook body for diagnosis , so it is difficult to be popularized and applied , and the PCR method does not need to carry out early diagnosis of leptospirosis compared with serological method .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.292

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