Broadly applicable PCR restriction fragment length polymorphism method for genotyping bovine leukemia virus
Broadly applicable PCR restriction fragment length polymorphism method for genotyping bovine leukemia virus
复制标题
DOI：
10.1292/jvms.18-0603
复制
发表时间：
2019-08-01
影响因子：
1.2
通讯作者：
Kobayashi, Tomoko
中科院分区：
文献类型：
--
作者：
Nishikaku, Kohei;Ishikura, Rina;Kobayashi, Tomoko
牛白血病病毒（BLV）是引起牛淋巴瘤（EBL）的病原体。BLV在世界范围内流行，根据包膜糖蛋白（gp 51）基因的序列，已将其分为10种基因型。在这项研究中，我们提出了一个简单的和普遍适用的PCR限制性片段长度多态性（PCR-RFLP）的方法来确定BLV基因型。虽然通过先前描述的PCR-RFLP方法获得的基因分型结果与系统发育分析结果仅匹配78.96%，但我们证明了我们的PCR-RFLP方法可以通过计算机识别数据库中90.4%的序列。用20个来自EBL肿瘤组织的BLV序列和3个来自BLV感染牛血液的BLV序列对该方法进行验证，发现该方法具有较高的特异性。我们利用这种方法，以确定基因型的血液样品从18 BLV血清阳性牛在神奈川和新泻，以及12 EBL牛在日本千叶。我们的分析与改进的PCR-RFLP检测到两种基因型，基因型1和3。基因1型为主要基因型，基因3型为零星分布。该技术可作为一种可靠的系统用于筛查大量流行病学样本。
Bovine leukemia virus (BLV) is a causative agent of enzootic bovine lymphoma (EBL). BLV is prevalent worldwide, and ten genotypes have been classified based on the sequence of the envelope glycoprotein (gp51) gene. In this study, we present a simple and generally applicable PCR restriction fragment length polymorphism (PCR-RFLP) method to identify BLV genotypes. While the genotyping results obtained by previously described PCR-RFLP methods matched only 78.96% to the results of phylogenetic analysis, we demonstrated that our PCR-RFLP method can identify 90.4% of the sequences available in the database in silico. The method was validated with 20 BLV sequences from EBL tumor tissues and 3 BLV sequences from blood of BLV infected cattle, and was found to show high specificity. We utilized this method to determine genotypes of blood samples from 18 BLV seropositive cattle in Kanagawa and Niigata, as well as 12 EBL cattle in Chiba, Japan. Our analysis with the modified PCR-RFLP detected two genotypes, Genotypes 1 and 3. Genotype 1 was detected as the main genotype, while Genotype 3 was sporadically observed. This technique can be used as a reliable system for screening a large number of epidemiological samples.