基于ENO基因的猪附红细胞体病 PCR-ELISA检测方法的建立  

Establishment of PCR-ELISA detection method for Mycoplasma suis based on ENO gene

作　　者：相思宇 倪婷婷[1] 薛书江[1] XIANG Siyu;NI Tingting;XUE Shujiang(Agricultural College of Yanbian University,Yanji Jilin 133002,China)

机构地区：[1]延边大学农学院

出　　处：《延边大学农学学报》2019年第2期68-73,共6页Agricultural Science Journal of Yanbian University

基　　金：国家自然科学基金(31460657);吉林省教育厅“十三五”科学技术项目(JJKH20191134KJ)

摘　　要：为建立猪附红细胞体病的PCR-ELISA检测方法,试验根据猪附红细胞体ENO基因序列,设计合成1对分别标记生物素(BIOT)和地高辛(DIG)的引物,进行PCR-ELISA检测。通过对包被液的种类和浓度、结合抗体的反应条件、封闭条件、PCR产物的稀释度及显色时间等条件进行优化,确定阴性、阳性判定的阈值,并进行特异性、敏感性及重复性验证。结果表明:该方法与猪链球菌、猪大肠杆菌、猪肺炎支原体、弓形虫等均无交叉反应;敏感性比常规PCR高10倍,最低检出量为1.77×10^6拷贝/μL;对40份猪血液样品的,阳性检出率为22.5%。该试验建立的方法具有特异、敏感、安全等优点,将为猪附红细胞体病的临床诊断提供更准确、实用的检测方法。To establish a PCR-ELISA detection method for Mycoplasma suis , a pair of primers labeled with biotin (BIOT) and digoxin (DIG) were designed and synthesized for PCR-ELISA detection in this study based on the sequence of ENO gene of Mycoplasma suis .By optimizing the types and concentrations of coating solution, reaction conditions of binding antibody, blocking conditions, dilution degree of PCR products and developing time, the threshold of negative and positive determination was determined and the sensitivity, specificity and repeatability were verified. The results showed that this method had no cross reaction with Streptococcus suis , Escherichia coli, Mycoplasma suis, Toxoplasma gondii and so on, and the sensitivity was 10 times higher than that of conventional PCR, and the lowest detectable amount was 1.77×10^6 copies/μL. At the same time, the detection result of 40 pig blood samples showed that the positive detection rate was 22.5%. The method established in this study has the advantages of specificity, sensitivity and safety, and it will provide a more accurate and practical detection method for the clinical diagnosis of Mycoplasma suis .

关 键 词：猪附红细胞体 ENO基因 PCR-ELISA 方法建立 临床应用 

分 类 号：S858.28[农业科学—临床兽医学]