猪附红细胞体PCR-微孔板杂交-酶免疫检测方法的研究  

Study on PCR-Microtiter Plate Hybridization Assay for Detection of Eperythrozoon suis

作　　者：张浩吉[1] 谢明权[2] 张健騑[2] 蒲文珺[1] 覃宗华[2] 蔡建平[2] 王政富[1] 顾万军[1] 

机构地区：[1]佛山科学技术学院动物医学系,佛山528231 [2]广东省农科院兽医研究所,广州510460

出　　处：《畜牧兽医学报》2007年第2期184-189,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广东省农业攻关计划项目(2005B20201008);广东省自然科学基金项目(020388)

摘　　要：以猪附红细胞体和多种血营养菌16S rRNA基因的保守区设计合成引物HM-f/HM-r,在反向引物的5′端用生物素标记,以猪附红细胞体广东株16S rRNA基因的可变区序列设计种特异性寡核苷酸探针,探针的5′端用地高辛标记,成功地建立了猪附红细胞体PCR-微孔板杂交-酶免疫检测方法。通过测定不同浓度PCR产物对应的微孔板杂交-酶反应显色OD值,用SPSS统计软件进行回归分析,得到回归方程为y=2.1012-0.4492×logx,其相关系数R为0.977,显示对未知样品可进行定量检测。该方法与猪的多种细菌性病原、猫血巴尔通氏体CA株和E.wenyonii无交叉反应,其敏感性比常规PCR琼脂糖电泳检测提高了近100倍。用建立的方法对38份临床样品进行检测,结果有12份检测结果为阳性。A pair of primers,HM-f and HM-r,was designed in the conserved regions in 16S rRNA gene of E. suis and other haemotriphic bacteria, the reverse primer was labeled with biotin at the 5＇end. Meanwhile, A E. suis species-specific oligonucleotide probe was also designed in variable regions in 16S rRNA gene of E. suis Guangdong strain,and the probe was labeled with digoxigenin at the 5＇end. The E. suis species-specific PCR-microtiter plate hybridization assay was successfully developed. The relationship between the amount of PCR products and OD values was demonstrated experimentally by testing the OD values of microtiter plate hybridization assay after a series dilution of known amplifitied products. Using SPSS program analysis a standard curve was defined by the equation y=2. 101 2-0. 449 2X log（x） with R= 0. 977. Quantitative detection of clinical samples were undertaken by camparing the OD value with the standard curve to estimate the level of parasitemia. The sensitivity of E. suis PCR-microtiter plate hybridization assay was 100 times higher than that demonstrated by PCR-agarose gel electrophoreses. There were no cross-reaction with a variety of bacteritic contagions in pig and haemotriphic bacteria, such as H. fells-CA strain and E. wenyonii. Using E. suis PCR-microtiter plate hybridization assay, the 38 blood samples from different populations were successfully detected and 12 samples were positive with E. suis infection, others were negative.

关 键 词：猪附红细胞体 PCR 微孔板杂交 检测 

分 类 号：S854.4[农业科学—临床兽医学] S858.285[农业科学—兽医学]