National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan.
Vet Parasitol. 2010 Aug 27;172(1-2):65-70. doi: 10.1016/j.vetpar.2010.04.011. Epub 2010 Apr 18.
We developed and evaluated two nested polymerase chain reaction (nPCR) assays for the diagnosis of Babesia bovis infection in cattle based on two membrane protein genes from B. bovis, BBOV_IV005650 (BV5650) and BBO_IV008970 (BV8970). The specificities and sensitivities of the tests were compared with B. bovis Rhoptery associated protein 1 gene (RAP-1) nPCR. The specificity of the tests was 100% for B. bovis DNA. The sensitivities of nPCR to B. bovis from the in vitro cultured parasites were as low as 10(-8)%, 10(-6)%, and 10(-7)% parasitemia for BV5650, BV8970, and RAP-1 nPCR, respectively. The nPCR detected as little as 1 fg genomic DNA per test for BV5650 and 100fg per test for both BV8970 and RAP-1 genes. For field applications, the sensitivity was evaluated to a total of 165 field samples from Ghana, Mongolia, Brazil and Japan. The nPCR assay of BV5650 was the most sensitive for the detection of B. bovis from the field samples. The BV5650 nPCR assay provides a good diagnostic tool for laboratory diagnostic assessment of B. bovis infection in cattle worldwide.
我们基于来自牛巴贝斯虫（B. bovis）的两个膜蛋白基因（BBOV_IV005650 [BV5650] 和 BBO_IV008970 [BV8970]），开发并评估了两种巢式聚合酶链反应（nPCR）检测方法，用于诊断牛巴贝斯虫感染。与牛巴贝斯虫 Rhoptery 相关蛋白 1 基因（RAP-1）nPCR 相比，这些检测方法的特异性和敏感性得到了比较。这些检测方法对牛巴贝斯虫 DNA 的特异性为 100%。nPCR 对体外培养寄生虫的牛巴贝斯虫的敏感性分别低至 BV5650、BV8970 和 RAP-1 nPCR 的 10-8%、10-6%和 10-7%寄生虫血症。nPCR 可检测到每个测试样本中低至 1 fg 基因组 DNA 的 BV5650，以及每个测试样本中低至 100 fg 的 BV8970 和 RAP-1 基因。对于现场应用，共评估了来自加纳、蒙古、巴西和日本的 165 个现场样本的敏感性。检测 B. bovis 从现场样本的敏感性中，BV5650 nPCR 检测方法最为敏感。BV5650 nPCR 检测方法为全球范围内牛巴贝斯虫感染的实验室诊断评估提供了一种良好的诊断工具。
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