牛支原体微滴式数字PCR检测方法的建立及应用  

Development and application of droplet digital PCR for detection of Mycoplasma bovis

作　　者：刘立兵 史云鹏 高雅欣 刘岳林 孙晓霞 王金凤 李睿文[2] 王建昌 LIU Li-bing;SHI Yun-peng;GAO Ya-xin;LIU Yue-lin;SUN Xiao-xia;WANG Jin-feng;LI Rui-wen;WANG Jian-chang(Technology Center of Shijiazhuang Customs,Shijiazhuang 050051,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]石家庄海关技术中心,河北石家庄050051 [2]河北农业大学动物医学院,河北保定071001

出　　处：《中国预防兽医学报》2022年第5期502-507,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：海关总署科研项目(2020HK170);农业高质量发展关键共性技术攻关专项(19226636D)。

摘　　要：为建立检测牛支原体(M. bovis)的微滴式数字PCR(ddPCR)方法,本研究以牛支原体uvrC基因为靶基因,设计特异性引物和探针,采用方阵法对ddPCR退火温度、引物和探针浓度的优化,结果显示,建立的ddPCR方法最佳退火温度为62.6℃,最佳引物和探针终浓度分别为900 nmol/μL和250 nmol/μL。利用该方法检测M. bovis、牛传染性鼻气管炎病毒、牛鼻炎病毒B型、副结核分枝杆菌、口蹄疫病毒、牛病毒性腹泻病毒和牛轮状病毒等,结果显示仅能够特异性检测到M. bovis,对其他病原均无交叉反应,特异性强;分别以10倍倍比稀释(1.2×10^(4)拷贝/μL~1.2×10^(-2)拷贝/μL)后的重组质粒标准品pMD19-T-uvrC为模板,进行敏感性试验,结果显示该ddPCR方法的检测下限为1.2拷贝/μL,是荧光定量PCR方法敏感性的10倍,该ddPCR敏感性高;对该ddPCR进行组内和组间重复性试验,结果显示其组内和组间变异系数均小于5%,重复性好。取23份来自患有乳房炎奶牛的新鲜牛奶和40份来自有呼吸道症状的牛鼻拭子样品,分别通过ddPCR、病原分离鉴定和荧光定量PCR方法检测M. bovis,结果显示,阳性率分别为42.86%(27/63)、33.33%(21/63)和33.33%(21/63),该ddPCR敏感性高于病原分离鉴定和荧光定量PCR方法,ddPCR方法与上述两种方法的符合率均为62.9%。本研究建立的ddPCR方法灵敏度高、特异性强,可对牛支原体进行定量检测,为牛支原体感染的早期监测和精准检测提供了可行技术手段。In order to establish a droplet digital PCR(ddPCR) method for detecting Mycoplasma bovis(M. bovis), the uvrC gene of M. bovis was used as target gene, the specific primers and probe were selected, the annealing temperature, primer and probe concentration in the ddPCR reaction system were optimized, and the specificity and sensitivity assay were performed.The results showed that the optimal annealing temperature was 62.6℃, the optimal primer and probe final concentrations were900nmol/μL and 250nmol/μL, respectively. The developed ddPCR exhibited specificity for M. bovis, but not for IBRV, BRBV,MAP, FMDV, BVDV and BRV, indicating high specificity. Using the recombinant plasmid pMD19-T-uvrC as the template, the detection limit of the assay was 1.2 copies/μL, which was ten times more sensitive than the real-time PCR method. The coefficient of variation within and between the repeatability test groups is less than 5%. The assay performance was evaluated by testing23 fresh milk samples from cattle with mastitis and 40 nasal swab samples from cattle with respiratory syndrome by dd PCR,isolation and identification, and a real-time PCR assay. The M. bovis positive rates were 42.86%(27/63), 33.33%(21/63) and33.33%(21/63), respectively, and the coincidence rate between the dd PCR and the above two methods was 62.90%. The dd PCR assay developed in this study demonstrated high sensitivity and specificity and could be used for precise quantitative detection of M. bovis, which is highly significant to the early monitoring and precise prevention of M. bovis infections.

关 键 词：牛支原体 uvrC基因 微滴式数字PCR 定量检测 

分 类 号：S858.23[农业科学—临床兽医学]