A high-throughput detection method for the clonality of Human T-cell leukemia virus type-1-infected cells in vivo
A high-throughput detection method for the clonality of Human T-cell leukemia virus type-1-infected cells in vivo
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DOI：
10.1007/s12185-020-02935-5
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发表时间：
2020-07-29
影响因子：
2.1
通讯作者：
Yanagihara, Katsunori
中科院分区：
文献类型：
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作者：
Saito, Masumichi;Hasegawa, Hiroo;Yanagihara, Katsunori
据报道，约有1000万至2000万人类t细胞白血病病毒1型（HTLV-1）感染的携带者，其中约5%的携带者发展为成人t细胞白血病/淋巴瘤（ATL），具有典型的预后不良。在日本，Southern blotting长期以来一直被常规用于检测体内克隆扩增的ATL细胞，并作为ATL的确诊性诊断试验。然而，目前在临床实践中需要替代Southern印迹的方法，如灵敏、定量和快速的分析方法。在这项研究中，我们开发了一种高通量的方法，称为快速扩增整合位点（RAIS），可以在4小时内扩增htlv -1整合片段，并在bb0.16%的感染细胞中检测到整合位点。此外，我们利用RAIS产品建立了一种新的HTLV-1克隆性定量方法，并通过与下一代测序在克隆性方面的比较，证实了该定量方法的有效性。因此，我们认为RAIS具有很高的潜力，可作为htlv -1感染细胞克隆分析的常规分子验证试验。
Approximately 10-20 million of Human T-cell leukemia virus type-1 (HTLV-1)-infected carriers have been previously reported, and approximately 5% of these carriers develop adult T-cell leukemia/lymphoma (ATL) with a characteristic poor prognosis. In Japan, Southern blotting has long been routinely performed for detection of clonally expanded ATL cells in vivo, and as a confirmatory diagnostic test for ATL. However, alternative methods to Southern blotting, such as sensitive, quantitative, and rapid analytical methods, are currently required in clinical practice. In this study, we developed a high-throughput method called rapid amplification of integration site (RAIS) that could amplify HTLV-1-integrated fragments within 4 h and detect the integration sites in > 0.16% of infected cells. Furthermore, we established a novel quantification method for HTLV-1 clonality using Sanger sequencing with RAIS products, and the validity of the quantification method was confirmed by comparing it with next-generation sequencing in terms of the clonality. Thus, we believe that RAIS has a high potential for use as an alternative routine molecular confirmatory test for the clonality analysis of HTLV-1-infected cells.