Development of a novel multiplex digital PCR-based method for the detection of HTLV-1 proviral deletion

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods Pub Date : 2024-11-21 DOI:10.1016/j.jviromet.2024.115071

Kou Hiraga , Kenta Tezuka , Koh Nagata , Ki-Ryang Koh , Hitomi Nakamura , Yasuko Sagara , Rieko Sobata , Masahiro Satake , Michikazu Tanio , Hiroo Hasegawa , Masumichi Saito , Kiyonori Miura , Takuo Mizukami , Isao Hamaguchi , Madoka Kuramitsu

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引用次数: 0

Abstract

The human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, integrates into host DNA and causes adult T-cell leukemia/lymphoma (ATL) in some individuals. Two types of defective proviruses, Type 1 and Type 2, are often observed in ATL cells. Here, we developed a 3-plex digital PCR (dPCR) method to detect HTLV-1 proviral deletions by comparing the ratios of copy numbers quantified using specific primer-probes for the LTR, pol, and pX regions. We analyzed HTLV-1-positive asymptomatic carriers (ACs) and AC samples at high risk for developing ATL due to high proviral load (ATL high-risk (HR) ACs) using dPCR. Deletions were identified in 11.8 % (4/34, all Type 1) of ACs and 33.3 % (7/21, Type 1:1, Type 2:6) of ATL HR ACs. dPCR analysis revealed that in three ATL samples, all exhibited Type 1 defective characteristics, and two showed extremely low ratios in the pol region. Clonality analysis of these two samples revealed high monoclonality, indicating monoclonal expansion of ATL cells with defective proviruses. These findings demonstrate that our method effectively detects defective proviruses in both ACs and ATL, providing a valuable tool for understanding the genomic characteristics of proviruses in these conditions.

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开发基于多重数字 PCR 的新型方法，用于检测 HTLV-1 前病毒缺失。

人类 T 细胞白血病病毒 1 型（HTLV-1）是一种逆转录病毒，可整合到宿主 DNA 中，并在某些人体内引发成人 T 细胞白血病/淋巴瘤（ATL）。在 ATL 细胞中经常可以观察到两种类型的缺陷前病毒，即 1 型和 2 型。在这里，我们开发了一种三重数字 PCR（dPCR）方法，通过比较使用 LTR、pol 和 pX 区域的特异引物探针量化的拷贝数比率来检测 HTLV-1 前病毒缺失。我们使用 dPCR 分析了 HTLV-1 阳性无症状携带者（AC）和因高病毒载量而有可能发展成 ATL 的高风险 AC 样本（ATL 高风险（HR）AC）。dPCR 分析显示，在三个 ATL 样本中，所有样本都表现出 1 型缺陷特征，其中两个样本在 pol 区域表现出极低的比率。对这两个样本进行的克隆性分析表明，其单克隆性很高，表明具有缺陷前病毒的 ATL 细胞是单克隆扩增的。这些研究结果表明，我们的方法能有效检测出 AC 和 ATL 中的缺陷前病毒，为了解这些情况下前病毒的基因组特征提供了宝贵的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of virological methods 医学-病毒学

CiteScore

5.80

自引率

0.00%

发文量

209

审稿时长

41 days

期刊介绍： The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.