贝氏贝诺孢子虫巢式PCR检测方法的建立

Establishment of Nested PCR Method for Besnoitia besnoiti

根据Gen Bank上发表的贝氏贝诺孢子虫核糖体内转录间隔区(ITS)序列,在传统PCR方法的基础上设计一对特异性内引物,提取病牛血液中贝氏贝诺孢子虫基因组DNA,建立了贝氏贝诺孢子虫巢氏PCR检测方法并进行临床样品检测。结果表明,建立的巢式PCR检测方法能检测到贝氏贝诺孢子虫DNA的最低浓度为24.3 ag·μL-1,刚地弓形虫、犬新孢子虫、牛巴贝斯虫、环形泰勒虫、边缘无浆体DNA检测均为阴性。196份临床血液样本,检测出3份阳性,阳性率为1.53%。由此可见,研究所建立的巢氏PCR检测方法具有较高的特异性和敏感性,可用于牛贝诺孢子虫病的早期临床诊断。

Based on the ITS r DNA sequence of Besnoitia besnoiti available in Gen Bank,two specific PCR primers were designed.Total genomic DNA for B. besnoiti was isolated from blood samples of the sick dairy cow,and the nested PCR method for B. besnoiti was established to detect clinical samples. The results showed that using the nested PCR method,the minimum detectable concentration was 24.3 ag·μL-1,and Toxoplasma gondii,Neospora caninum,Babesia bovis,Theileria annulata and Anaplasmataceae marginal pathogens were negative. The positive rate of B. besnoiti was 1.53% in 196 blood samples. The nested PCR method in this study had high sensitivity and specificity and could be used for early diagnosis of Bovine besnoitiosis.