摘要
为建立牛新孢子虫准确快速检测方法,本研究根据犬新孢子虫NcSAG1基因、Nc5基因和NcSRS2基因,以及弓形虫GRA6基因和牛性腺基因,设计合成相应的5对引物和5条芯片探针,以牛性腺基因为内标监控基因,进行芯片杂交试验,经反应条件的优化,建立了含监控内标,可对新孢子虫3个基因进行同步检测,并可与弓形虫GRA6基因进行区分检测的基因芯片检测方法.该方法对新孢子虫的最低检测限分别为102拷贝/μL,并且具有良好的特异性和重复性.利用建立的方法对157例临床样品进行检测,与普通PCR检测结果符合率为99.3 ％～98％.该方法的建立为新孢子虫等多病原高通量检测方法的研究奠定基础.
In order to establish a sensitive and rapid detection method for bovine Neospora caninum, a gene microarray was developed with specific primers designed according to NcSAG1 gene, Nc5 gene, NcSRS2 gene of N.caninuln, GRA6 gene of Toxoplasma gondii and bovine prolactin gene, respectively. The gene chip was able to detect three genes of N.caninum and distinguish from T.gondii GRA6 gene simultaneously. In addition, the bovine prolactin gene was used as internal control to monitor the detection quality. This method was specificity, sensitivity and repeatability with a detection limit of 102 copies for each reaction. Moreover, a total of 157 clinical samples were tested by both the gene chip and traditional PCR method and the concordance rate of the two methods 99.3% to 98%. The results provided a technical basis for developing high throughput detection methods on simultaneous detections of other pathogens.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2014年第9期715-719,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家质量监督检验检疫总局科研项目(2011IK011
2011IK017)
关键词
牛新孢子虫
多基因
内标
基因芯片
bovine Neospora caninum
multiplex gene
internal control
gene microarray
