牛双芽巴贝斯虫巢式PCR诊断方法的建立

【目的与方法】根据Genbank上登陆的双芽巴贝斯虫HSP20基因设计两对引物,SHSP-P1与SHSP-P2和SHSP-P3与SHSP-P4,以其建立牛双芽巴贝斯虫巢式PCR快速检测方法。【结果】第一次、第二次扩增的退火温度均为57℃,分别扩增出约631和409 bp的条带,与预期的大小相符,而作为对照样本的牛巴贝斯虫、牛环形泰勒虫基因组DNA均无此扩增目的条带出现。将感染双芽巴贝斯虫的全血基因组DNA做10倍递减稀释到106倍扩增时,巢式PCR还能见到目的条带,其灵敏度相当于4.6 pg/mL全血基因组DNA。在对50份疫区牛全血DNA样本检测中,阳性检出率分别为22%(11/50)。【结论】所建立的牛双芽巴贝斯虫巢式PCR方法准确、敏感、特异,可以用于牛双芽巴贝斯虫病的快速检测和小范围的流行病学调查,具有重要的临床意义。

Development of Nested-PCR Diagnostic Method for Detection of Babesia bigemina

【Objective and Method】In order to develop the nested-PCR diagnostic method for rapidly detection of Babesia bigemina,two pairs of specific primers,SHSP-P1,SHSP-P2,SHSP-P3 and SHSP-P4 were designed according to the published gene HSP20 of Babesia bigemina in GenBank.【Result】The 1st and 2nd annealed temperatures by nested-PCR both were 57℃,and 631 bp and 409 bp of special DNA fragments were amplified from the gene HSP20,which were coincident with the expected length,while these two fragments were not found from the samples infected by Babesia bovis and Theileria Annulata.When tested blood DNAs were diluted to 106,the special DNA fragments still were found by nested-PCR assay.The sensitivity for nested-PCR was equal to find 4.6 pg DNA in 1ml tested blood.In the 50 tested blood DNA samples collected from epidemic area,the positive rate was 22%(11/50).【Conclusion】 The established method of nested-PCR assay was a precise,sensitive and specific technique,and could be used to rapidly detect Babesia bigemina and conduct and its epidemiological survey in the small scale.