犬新孢子虫不同靶基因PCR检测方法的比较

为筛选出检测犬新孢子虫更为特异、敏感的PCR方法,分别以MAG1、SAG1和Nc5为靶基因进行PCR检测,并从敏感性、特异性和临床样本检出率等方面进行了比较。结果显示,以Nc5为靶基因的PCR方法敏感性最高,最小检测DNA量为17.8fg/μL;以MAG1为靶基因的PCR方法敏感性最低,最小检测DNA量为17.8pg/μL;而以SAG1为靶基因的PCR方法的最低检测量为178fg/μL;3种靶基因引物均扩增不出刚地弓形虫、牛瑟氏泰勒虫、猪附红细胞体等基因片段,具有较好的特异性;对28份已攻犬新孢子虫标准株的BALB/c小鼠组织样本的检测结果表明,以Nc5基因设计的引物检出率最高,为75.0%(21/28),明显高于SAG1基因的67.9%(19/28)和MAG1基因的53.6%(15/28)。本试验为新孢子虫病的诊断及流行病学调查提供了更为敏感、特异的检测技术。

Comparison of different PCR methods for the detection of Neospora caninum target gene

To find a PCR method which is more specific and sensitive to Neospora caninum,MAG1 gene,SAG1 gene and Nc5 gene of N.caninum were used as target genes and the sensitivity,specificity and detection rate of PCR of clinical samples were compared.The results showed that PCR method based on the Nc5 gene had the highest sensitivity,with minimum detectable amount as 17.8 fg/μL.While the MAG1 gene had the lowest sensitivity,with the minimum detectable amount of MAG1 gene as 17.8 pg/μL.The genomic DNA of Toxoplasma gondii,Theileria sergenti and Eperythrozoon suis could not be amplified by the three PCR methods,which indicated they had good specificity.Test on 28 tissue samples of BALB/c mouse that had been challenged by N.caninum standard strain showed that,75.0%(21/28) was identified by PCR based on the Nc5 gene,which was significantly higher than that of PCR based on the SAG1 gene 67.9%(19/28) and the MAG1 gene 53.6%(15/28).This study provided a more sensitive and specific detection approach for neosporosis and epidemiological investigation.