PCR诊断牛新孢子虫病的初步应用  

Preliminary Application of PCR-based Assay for the Detection of Neospora caninum in Bovine Aborted Fetus

作　　者：王春仁[1] 翟延庆[1] 赵兴存[2] 谭秋菊[1] 陈佳[1] 陈爱华[1] 王宇[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]中华人民共和国福建出入境检验检疫局,福州350001

出　　处：《中国寄生虫学与寄生虫病杂志》2009年第2期140-143,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：大庆市科技局攻关项目(SGG2005-032)~~

摘　　要：目的以犬新孢子虫Nc-5基因为目的基因的PCR诊断方法,检测奶牛流产的胎牛脑组织的新孢子虫。方法根据GenBank发布的新孢子虫特异性基因片段Nc-5基因序列,设计1对特异性引物,以犬新孢子虫(Neospora caninum)标准株DNA为模板,PCR扩增Nc-5基因,与pMD18-T载体连接,转化大肠埃希菌JM109,获得阳性重组质粒pMD18-T-Nc-5,并测序。以环形泰勒虫、牛巴贝斯虫、刚地弓形虫、杜氏利什曼原虫以及犬新孢子虫标准株DNA为模板进行扩增以验证PCR的特异性,采用紫外分光光度计测定犬新孢子虫标准株DNA浓度和纯度,取高纯度的DNA样品用灭菌水稀释,分别取不同量的DNA进行PCR扩增,确定PCR方法的敏感性;利用该方法对32份奶牛流产的胎牛脑组织样品进行检测,同时,对其中23份流产的母牛血样进行ELISA血清学检测(作为对照),以评价犬新孢子虫PCR方法的检测效果。结果克隆的犬新孢子虫标准株目的基因大小为350bp,与GenBank(AY459289)中Nc-5基因序列一致性为98%,建立的犬新孢子虫PCR方法与环形泰勒虫、牛巴贝斯虫、刚地弓形虫和杜氏利什曼原虫均无交叉反应,最低能检测犬新孢子虫DNA3.125pg,检测流产胎牛脑组织阳性率为18.8%(6/32)。ELISA检验流产母牛血样抗体阳性率为17.4%(4/23),这4份阳性样品与其流产胎牛PCR检测结果均为阳性。结论建立的犬新孢子虫PCR诊断方法用于检测奶牛流产的胎牛脑组织新孢子虫的感染效果较好。Objective To establish a PCR diagnostic method based on Nc-5 gene of Neospora caninum,for being used to detect Neospora in brain tissues of bovine aborted fetus. Methods Specific primers were designed and synthesized based on the reported Nc-5 gene of N. caninum（GenBank Accession No. AY459289）. Using genomic DNA from N.caninum as templates, Nc-5 gene was amplified by PCR. The PCR product was cloned into pMD18-T vector, transformed into Escherichia coli JM109 and then sequenced. To evaluate the specificity of the PCR, genomic DNA of Theileria annulata,Babesia bovis,Toxoplasma gondii,Leishmania donovani and standard strain of N. caninum were used as a template in the PCR. For determining the detection limit of amplification procedure, PCR was run on a dilution series of genomic DNA from N. caninum（1.562 5-200 ng/ml）. Brain tissue samples of 32 aborted fetuses were detected by PCR-based assay, and 23 blood samples from mothers were tested by ELISA. Results The amplified DNA fragment （350 bp）had a high identity of 98% with the Nc-5 gene sequence of N. caninum（GenBank Accession No. AY459289）. The PCR was specific for N. caninum and allowed the detection of 3.125 pg DNA of the parasite, while no amplification occurred with the other four species of protozoa. PCR-based assay and ELISA showed a positive rate of 18.8%（6/32） and 17.4%（4/23）of the samples tested, respectively. Moreover,all the 4 antibody positive samples showed PCR positive. There is no significant difference between the two assays（P〉0.05）. Conclusion PCR diagnostic method is promising in detecting Neospora infection in brain tissues of aborted bovine.

关 键 词：犬新孢子虫 Nc-5基因 PCR扩增 胎牛 

分 类 号：R382.3[医药卫生—医学寄生虫学]