【作者】 张颖；

【导师】 胡敬东； 兰邹然；

【作者基本信息】 山东农业大学 ， 兽医（专业学位）， 2024， 硕士

【摘要】 布鲁氏菌病(简称“布病”)是世界公认的危害严重的人畜共患病之一,该病主要造成家畜生殖系统损害,人可通过密切接触患病动物及其产品受到传染。在我国被列为乙类传染病,对畜牧业和公共卫生构成了严重威胁。疫苗免疫是我国家畜布病防控的重要策略,在布鲁氏菌疫苗的使用中,牛种布鲁氏菌A19疫苗最为有效且使用较为广泛,主要用于牛的免疫。具有分子诊断标记的A19-△Vir B12基因缺失疫苗株的保护力与亲本株A19无显著差异,但毒力弱于亲本A19,且通过Vir B12基因鉴别诊断可区分疫苗株与自然感染株,该疫苗已于2021年正式上市推广。A19-△Vir B12基因缺失疫苗自推广以来,尚无核酸定量鉴别诊断Vir B12基因缺失疫苗株的微滴式数字PCR方法。微滴式数字PCR是一种高精度的核酸分子定量技术,相较于普通荧光定量PCR,具有灵敏度高、可定量的优势。本研究以布鲁氏菌Ⅳ型分泌系统Vir B8基因设计引物鉴别布鲁氏菌属,并以牛布鲁菌A19-△Vir B12缺失疫苗缺失的Vir B12基因序列作为诊断标记,旨在建立区分牛布鲁氏菌A19-△Vir B12基因缺失疫苗株与自然感染株的双重微滴式数字PCR方法,为今后快速、准确鉴别诊断基因缺失疫苗免疫牛与野毒感染牛提供了较为可靠的快速诊断方法。通过引物浓度、探针浓度、退火温度、降温速率等条件的优化,灵敏性、特异性及重复性检测,本研究建立了牛种布鲁氏菌病A19-△Vir B12基因缺失疫苗双重微滴式数字PCR鉴别诊断方法,并应用此方法进行了疫苗鉴别及临床样品检测,具体结果如下:所建立的方法中Vir B8的引物浓度为600 n M、探针浓度为300 n M,Vir B12的引物浓度为500 n M、探针浓度为200 n M,二者的退火温度和降温速率分别为54℃和2℃/s。该方法对Vir B8标准品质粒在拷贝数2.13×10~4copies/μL～2.13×10~0copies/μL范围内能百分百检出,且检测下限为2.13×10~0copies/μL。Vir B12标准品质粒在拷贝数2.26×10~4copies/μL～2.26×10~0copies/μL范围内能百分百检出,且检测下限为2.26×10~0copies/μL,所建立的标准曲线相关系数分别为0.9991和0.9999,线性良好。与大肠杆菌、沙门氏菌、牛结核分支杆菌、链球菌无交叉反应,特异性良好。其组间和组内重复试验变异系数分别为7.1%和7.9%,表明重复性较高。应用该方法对布鲁氏菌病活疫苗(A19-△vir B12株)、布鲁氏菌病活疫苗(A19株)、布鲁氏菌病基因缺失活疫苗(M5-90△26株)与布鲁氏菌病活疫苗(S2株)进行检测,能够鉴别出A19-△vir B12基因缺失疫苗株,从而为该疫苗的鉴别诊断提供了有力的技术支撑。同时应用该方法与荧光定量方法检测了200份临床样品,荧光定量检测结果为阳性的样品共48份,建立的双重微滴式数字PCR检测结果为阳性的样品共53份,说明微滴式数字PCR对于阳性样本的检出率要高于荧光定量方法。将双重微滴式数字PCR检测为阳性,而荧光定量检测为阴性的同一样品进行比较,结果表明双重微滴式数字PCR检测更灵敏,仍能检测到微量拷贝数,检测结果呈阳性,说明双重微滴式数字PCR方法能够检测较低浓度的核酸样品,其灵敏度高于荧光定量PCR,更适合低浓度核酸样品检测。更多还原

【Abstract】 Brucellosis,commonly referred to as "Brucella disease," is one of the most hazardous zoonotic diseases globally recognized.This disease primarily affects the reproductive system of livestock,and humans can be infected through close contact with infected animals and their products.In China,it is classified as a Class B infectious disease,posing a serious threat to animal husbandry and public health.Vaccination is a crucial strategy for preventing and controlling brucellosis in domestic animals in China.Among the Brucella vaccines,the bovine Brucella A19 vaccine is the most effective and widely used,primarily for immunization of cattle.The A19-△ Vir B12 gene-deleted vaccine strain,with molecular diagnostic markers,exhibits no significant difference in vaccine efficacy compared to the parental strain A19,but has lower virulence.Additionally,it allows for the differentiation between vaccinated and naturally infected strains through Vir B12 gene discrimination.This vaccine was officially launched and promoted in 2021.However,since the introduction of the A19-△ Vir B12 gene-deleted vaccine,there has been no droplet digital PCR method for quantitative nucleic acid differential diagnosis of the Vir B12 gene-deleted vaccine strain.Droplet Digital PCR(dd PCR)is a high-precision nucleic acid quantification technology that offers advantages in sensitivity and quantitation compared to conventional fluorescent quantitative PCR.In this study,primers were designed based on the Vir B8 gene of the Brucella type IV secretion system to identify Brucella species.Using the Vir B12 gene sequence,which is deleted in the Brucella abortus A19-Δ Vir B12 vaccine strain,as a diagnostic marker,our aim was to establish a duplex dd PCR method to distinguish between the B.abortus A19-ΔVir B12 gene-deleted vaccine strain and naturally infected strains.This provides a reliable and rapid diagnostic approach for future fast and accurate differential diagnosis between gene-deleted vaccine-immunized cattle and those infected with wild-type Brucella.Through optimization of primer concentration,probe concentration,cooling rate,and other conditions,as well as sensitivity,specificity,and repeatability testing,we established a duplex dd PCR differential diagnostic method for the B.abortus A19-ΔVir B12gene-deleted vaccine.This method was applied for vaccine identification and clinical sample testing,with the following specific results:The established duplex dd PCR method utilized primer concentrations of 600 n M and probe concentrations of 300 n M for Vir B8,while for Vir B12,the primer concentration was500 n M and the probe concentration was 200 n M.The annealing temperature and cooling rate for both targets were set at 54 ℃ and 2 ℃ /s,respectively.This method achieved 100%detection for the Vir B8 standard plasmid in the range of 2.13×104 copies/μL to 2.13×100copies/μL,with a detection limit of 2.13×100 copies/μL.Similarly,for the Vir B12 standard plasmid,100% detection was achieved in the range of 2.26×104 copies/μL to 2.26×100copies/μL,with a detection limit of 2.26×100 copies/μL.The correlation coefficients of the established standard curves were 0.9991 and 0.9999 for Vir B8 and Vir B12,respectively,indicating excellent linearity.The method demonstrated good specificity,showing no crossreaction with Escherichia coli,Salmonella,Mycobacterium bovis,or Streptococcus.The coefficients of variation for inter-group and intra-group repeat tests were 7.1% and 7.9%,respectively,indicating high reproducibility.When applied to detect Brucella abortus live vaccines(A19-△vir B12 strain),Brucella abortus live vaccines(A19 strain),Brucella abortus gene-deleted live vaccines(M5-90△26 strain),and Brucella abortus live vaccines(S2 strain),the method could accurately identify the A19-△vir B12 gene-deleted vaccine strain,providing strong technical support for the differential diagnosis of this vaccine.Additionally,the method was used to test 200 clinical samples alongside fluorescent quantitative PCR.The fluorescent quantitative PCR detected 48 positive samples,while the duplex dd PCR detected53 positive samples.This suggests that duplex dd PCR has a higher detection rate for positive samples compared to fluorescent quantitative PCR.A comparison of samples that tested positive by duplex dd PCR but negative by fluorescent quantitative PCR showed that duplex dd PCR is more sensitive and can detect even trace amounts of nucleic acid,making it suitable for detecting low-concentration nucleic acid samples with higher sensitivity than fluorescent quantitative PCR.更多还原