【作者】 王丽；

【导师】 宫鹏涛；

【作者基本信息】 吉林大学 ， 兽医硕士（专业学位）， 2024， 硕士

【摘要】 新孢子虫(Neospora caninum,N.caninum)是一种专性细胞内寄生的顶复门原虫,牛、羊等动物为中间宿主,犬科动物为终末宿主。新孢子虫病主要引起妊娠母畜流产、死胎、新生犊牛的神经系统疾病,是导致牛繁殖失败的重要病因,每年对养殖业造成的经济损失超过4亿美元。隐孢子虫(Cryptosporidium)是一类寄生于人和多种动物胃肠道的机会性致病原虫,免疫功能正常的个体多表现为自限性腹泻;免疫低下或免疫缺陷的宿主多表现为顽固性腹泻,重者甚至危及生命。微小隐孢子虫(Cryptosporidium parvum,C.parvum)是重要的人兽共患的隐孢子虫虫种,是引起新生犊牛腹泻的重要病因;感染的犊牛可能会增加人群爆发隐孢子虫病的风险,对公共卫生安全造成潜在威胁。目前,及时发现新孢子虫、微小隐孢子虫感染,采取相应的措施在疫病防控中的作用显得尤为重要。传统的诊断方法存在一定的局限性,如低灵敏性和特异性、依赖经验丰富的技术人员和昂贵的仪器设备等,无法满足非实验室环境下的现场快速诊断。因此,建立准确、快速、灵敏的检测方法,是预防和控制新孢子虫病、隐孢子虫病的前提和关键。CRISPR/Cas系统是一种强大的基因编辑工具,近年来,部分Cas蛋白被发现具有独特的反式切割功能,在核酸诊断领域具有广阔的发展前景。搭载等温扩增技术开发的诊断工具,可突破实验室条件的限制,使现场诊断成为可能。重组酶聚合酶扩增(Recombinase polymerase amplification,RPA)在37-42℃恒温条件下即可实现目标核酸的指数式扩增。本研究基于CRISPR/Cas12a系统和RPA技术,与荧光信号以及侧流层析试纸条输出方式相结合,建立新孢子虫、微小隐孢子虫的检测方法。(1)Cas12a重组蛋白的表达纯化及功能鉴定:构建p GEX-4T-1-Cas12a质粒后,在E.coli表达体系中通过诱导表达、纯化等手段获得大小约为169.7 KDa的Cas12a重组蛋白;利用荧光报告分子验证纯化的Cas12a蛋白具有反式切割活性,可以非特异性剪切体系中存在的荧光报告分子,释放FAM荧光信号。(2)新孢子虫RPA-CRISPR/Cas12a检测方法的建立与初步应用:选择新孢子虫Nc5基因作为检测靶标,设计和筛选NC-1为最佳Target位点,F3和R2为最佳PRA引物。建立的新孢子虫RPA-CRISPR/Cas12a检测方法特异性良好,荧光检测方法的最低检测限为1个速殖子/m L,侧流层析试纸条法的最低检测限为10个速殖子/m L,具有良好的敏感性;利用本方法检测32份牛胎盘组织和70份犬粪便样本,结果显示新孢子虫阳性率分别为59.4%(19/32)和8.6%(6/70),与巢式PCR的复核结果一致。(3)微小隐孢子虫RPA-CRISPR/Cas12a检测方法的建立与初步应用:选择微小孢子虫SSU r RNA基因作为检测靶标,设计筛选SSU-1为最佳Target位点,F1和R3为最佳PRA引物;建立的微小隐孢子虫RPA-CRISPR/Cas12a检测方法特异性良好;荧光检测方法和侧流层析试纸条法的最低检测限均为10个卵囊/m L,具有良好的敏感性;利用本方法检测70份牛粪便样本,结果显示微小隐孢子虫阳性率为15.7%(11/70),与巢式PCR的复核结果一致。综上所述,本研究建立的新孢子虫、微小隐孢子虫RPA-CRISPR/Cas12a检测方法,在37℃恒温条件下即可完成,无需复杂的仪器设备,应用于临床样品检测时均表现出较好的准确性,可通过蓝光仪和侧流层析试纸条两种方式实现可视化判读,为新孢子虫、隐孢子虫病的诊断提供新策略。更多还原

【Abstract】 Neospora caninum is an intracellular protozoan parasite,with a definitive host of canids and intermediate hosts of cattle and sheep.N.caninum is a main cause of reproductive failure in cattle,causing abortion,stillbirth,and neurological disorders in newborn animals,causing over $400 million in economic losses to the livestock industry annually.Cryptosporidium is an opportunistic pathogenic protozoan that causes diarrhea and death in humans and a variety of animals,mainly causing selflimiting diarrhea in the host;malnourished or immunocompromised hosts mostly exhibit persistent diarrhea and life-threatening diarrhea.Cryptosporidium parvum is an important Cryptosporidium species commonly associated with zoonotic cryptosporidiosis and a major cause of diarrhea and production loss in cattle.Calves infected may increase the risk of population outbreaks of cryptosporidiosis,posing a potential threat to public health safety.The role of timely diagnosis of N.caninum and C.parvum infections and the adoption of appropriate measures in the prevention and control of epidemics is now particularly important.Traditional assays have certain limitations,such as low sensitivity and specificity,reliance on experienced technicians,and expensive instrumentation,which cannot meet the need for rapid on-site diagnosis in a nonlaboratory setting.Therefore,the establishment of an accurate,rapid,and sensitive diagnostic method is a key to the prevention and control of neosporosis and cryptosporidiosis.The CRISPR/Cas is a powerful gene-editing tool,and in recent years,some Cas proteins have been found to have unique trans-cleavage activity which has a broad development prospect in the field of nucleic acid diagnosis.Diagnostic tools developed with isothermal amplification technology can break through the limitations of laboratory conditions and make on-site diagnosis possible.The RPA can achieve exponential amplification of nucleic acids at a constant temperature ranging from 37 to42°C,without the requirement of sophisticated instruments and equipment.In this study,based on the CRISPR/Cas12 a system,combined with the highly efficient amplified RPA,the results of the test were presented by fluorescence values and the lateral flow test strips to establish a simple,accurate,and sensitive on-site rapid detection method for N.caninum and C.parvum and was applied to the detection of clinical samples.The method was established to provide new technical support for the diagnosis of N.caninum and C.parvum.Expression and purification and functional validation of Cas12 a recombinant protein: after the construction of p GEX-4T-1-Cas12 a plasmid,Cas12 a recombinant protein with the size of approximately 169.7 KDa,was obtained by induced expression and purification in E.coli expression system.The Cas12 a protein was verified to have trans-cleavage activity,which can non-specifically cut tagged reporter probes nonspecifically present in the system and release fluorescent signals.Establishment and preliminary application of the N.caninum RPACRISPR/Cas12 a detection assay: The Nc5 gene of the N.caninum was selected as the detection target,NC-1 was selected as the best Target site,F3 and R2 were selected as the best PRA primer pairs.The established N.caninum RPA-CRISPR/Cas12 a assay showed good specificity,the lowest limit of detection was as low as one parasite per milliliter when employing the fluorescent reporter system,and was ten parasites per milliliter based on the LFS biosensor,showed good sensitivity.The bovine placenta tissues and canine fecal samples were detected using the established assay,and the results showed that the positive rate was respectively 59.4%(19/32)and 8.6%(6/70),which were completely consistent with that of the nested PCR assay.Establishment and preliminary application of the C.parvum RPACRISPR/Cas12 a detection assay: The SSU r RNA gene of the C.parvum was selected as the detection target,SSU-1 was selected as the optimal Target site,F1 and R3 were carried out as the optimal PRA primer pairs.The established C.parvum RPACRISPR/Cas12 a assay showed good specificity,the lowest limit of detection was as low as 10 oocysts/m L by the fluorescent reporter system and the lateral flow strip biosensor showed good sensitivity.The cattle fecal samples were detected by our assay,and the results showed that the positive rate was respectively 15.7%(11/70),which was completely consistent with that of the nested PCR assay.In conclusion,the RPA-CRISPR/Cas12 a assay for N.caninum and C.parvum was established in this study,which can be completed at 37℃,low equipment requirements and showed good accuracy when applied to clinical samples,and the results can be directly visible to the naked eye by both with a blue meter and lateral flow strip biosensor.They can provide new strategies for the diagnosis of N.caninum and C.parvum.更多还原