马驽巴贝斯虫二温式、荧光PCR检测方法的建立及其初步应用

发布时间：2018-03-31 11:53

本文选题：马驽巴贝斯虫　切入点：二温式PCR　出处：《新疆农业大学》2015年硕士论文

【摘要】：马驽巴贝斯虫病病原为驽巴贝斯虫,该病是一种经由媒介蜱虫传播的严重影响马属动物健康的血液原虫病,被世界卫生组织(OIE)列为法定报告检疫疫病,在我国该病被列为二类动物疫病。该病呈全球性发生,患马呈现发热、贫血、黄疸、血红蛋白尿,急性病例死亡,亚急性、慢性病例终身成为带虫宿主,造成本土马与引进马互相感染、反复发病,给马养殖业造成重大损失。目前尚无该病的有效治疗药物及疫苗,故研发新型检测技术是有效的防控途径,可及时检测、监控和综合防治,有利于马产业的健康发展。(Ⅰ)根据GenBank中的驽巴贝斯虫(Babesia caballi)Bc48基因序列设计了一对特异性引物,建立检测驽巴贝斯虫的二温式PCR方法。该方法能够特异地扩增155 bp的片段,与双芽巴贝斯虫、环形泰勒虫、马泰勒虫、瑟氏泰勒虫均无交叉反应,能检测到的最低DNA拷贝数为5.17×102 copies/μL。应用所建立的方法检测了采集于和静县焉耆马匹样品(N=82),结果显示,马驽巴贝斯虫感染率69.51%(57/82),与血液涂片检查结果37.8%(31/82)相比,其检出率更高。该二温式PCR检测方法具有较高的特异性和敏感性,可用于马驽巴贝斯虫的早期诊断。(Ⅱ)根据Bc48保守部分基因序列设计合成特异性引物和MGB荧光探针,建立了驽巴贝斯虫病的MGB FQ-PCR检测方法。对所建立的FQ-PCR检测方法进行特异性、灵敏性和重复性试验,并分别用FQ-PCR检测方法和普通PCR方法对临床样品进行检测对比。结果显示,该方法灵敏度高,最小检出量为5.17×101copies/μL的标准质粒,比常规PCR灵敏100倍;建立的FQ-PCR检测方法标准曲线的循环阈值与模板浓度具有良好的线性关系,相关系数为0.9996,重复性好,组内与组间重复性试验的变异系数分别为0.7%和0.42%,均小于1%;该方法能特异性检测驽巴贝斯虫,而对马泰勒虫、双芽巴贝斯虫、环形泰勒虫、瑟氏泰勒虫等病原检测结果均为阴性。用建立的MGB-FQ PCR和常规PCR分别对90份临床样品进行检测,MGB-FQ PCR和常规PCR检出率分别为53%和30%。本次建立的驽巴贝斯虫病MGB-FQ PCR检测方法特异性强、灵敏度高、重复性好,可用于马驽巴贝斯虫病临床样品诊断、流行病学调查,将为蜱传马梨形虫病的检测提供新的方法。
[Abstract]:The pathogen of cabal Babes's disease is Babes caballi, a blood protozoan disease that is transmitted by ticks and seriously affects the health of equine animals. It is listed as a reported quarantine epidemic by the World Health Organization (WHO). The disease is classified as a second class animal disease in China. The disease occurs globally, with horses presenting with fever, anemia, jaundice, hemoglobinuria, acute death, subacute, chronic cases and life-long host of the parasite. As a result of mutual infection between local and imported horses, recurrent diseases have caused great losses to the horse breeding industry. At present, there are no effective drugs and vaccines for the treatment of the disease. Therefore, the development of new detection techniques is an effective way to prevent and control the disease and can be detected in a timely manner. Monitoring and integrated control are beneficial to the healthy development of horse industry. (I) A pair of specific primers were designed according to the caballi)Bc48 gene sequence of Babes caballi in GenBank. A two-temperature PCR method was established for the detection of Babes caballi. The method was able to amplify a 155bp fragment with no cross reaction with the double bud Babes worm, ringworm Taylor worm, Maltaire worm and Taylor serpentine. The lowest copy number of DNA detected was 5.17 脳 10 ~ 2 copies/ 渭 L. the results showed that the infection rate of Babes caballi was 69.511and 57 / 82, compared with the results of blood smear 37.8 / 82). This two-temperature PCR detection method has high specificity and sensitivity, and can be used in the early diagnosis of Babes caballi. (II) according to the conserved gene sequence of Bc48, specific primers and MGB fluorescent probes were designed and synthesized. A MGB FQ-PCR method was established for the detection of Babes caballi disease. The specificity, sensitivity and reproducibility of the established FQ-PCR detection method were tested, and the clinical samples were detected and compared by FQ-PCR detection method and common PCR method. The sensitivity of the method is high, the minimum detection amount is 5.17 脳 101copies/ 渭 L, and the sensitivity is 100 times higher than that of conventional PCR. The standard curve of FQ-PCR has a good linear relationship with the concentration of template, the correlation coefficient is 0.9996, and the reproducibility is good. The coefficients of variation of repeatability test were 0.7% and 0.42%, respectively, which were less than 1. The method could be used to detect Babes caballi specifically, but to MarTaylor's, Babes's double bud, Taylor's annulus. The detectable rates of MGB-FQ PCR and PCR in 90 clinical samples were 53% and 30%, respectively. The specificity of MGB-FQ PCR assay for Babes's caballi was strong. It has high sensitivity and good reproducibility. It can be used in the diagnosis and epidemiological investigation of caballi Babes's disease. It will provide a new method for the detection of tick-transmitted piridiasis.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.21

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