Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.
Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.
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开发和定制基于颜色编码微珠的 HIV-1 逆转录酶耐药性测定。
DOI：
10.1371/journal.pone.0109823
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Hosoya N
中科院分区：
文献类型：
--
作者：
Gu L;Kawana-Tachikawa A;Shiino T;Nakamura H;Koga M;Kikuchi T;Adachi E;Koibuchi T;Ishida T;Gao GF;Matsushita M;Sugiura W;Iwamoto A;Hosoya N
HIV-1 的耐药性 (DR) 可以通过基因型或表型进行检查。尽管测序是基因型耐药性检测 (GRT) 的黄金标准，但针对 DR 密码子的高通量 GRT 可能更适合流行病学研究和公共卫生研究。我们使用日本数据库设计和合成序列特异性寡核苷酸探针（SSOP），用于检测B型HIV-1逆转录酶区域的野生型序列和6个DR突变。我们将 SSOP 与 Luminex 100 xMAP 系统的微珠偶联，并开发了基于聚合酶链式反应 (PCR)-SSOP-Luminex 方法的 GRT。设计并合成了 16 个寡探针，用于在 6 个位点区分 DR 突变和野生型序列，并使用同基因质粒证实了它们的敏感性和特异性。然后使用来自未接受治疗或治疗失败的患者的 74 份血浆样本，将 PCR-SSOP-Luminex DR 测定法与直接测序进行比较。在大多数标本中，PCR-SSOP-Luminex DR 检测结果与测序结果一致：M41 为 62/74 (83.8%)，K65 为 43/74 (58.1%)，K70 为 70/74 (94.6%)，K103 为 55/73 (75.3%)，K103 为 63/73 (86.3%) M184 为 68/73 (93.2%) T215。有许多样本没有任何阳性信号，尤其是 K65。 A2723G、A2747G和C2750T的核苷酸位置分别是野生型氨基酸K65、K66和D67的常见多态性，14个样本具有G2748A编码的D67N突变。我们另外合成了 14 个 K65 寡核苷酸探针，K65 位点的敏感性从 43/74 (58.1%) 提高到 68/74 (91.9%)。我们开发了一种针对 B 型 HIV-1 DR 突变的快速高通量检测方法，可以通过合成适合流行病毒的寡核苷酸探针进行定制。该测定可能是一种有用的工具，特别是对于资源丰富和资源有限环境中的公共卫生研究。
Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research. We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method. Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%). We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.
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影响因子：
3
作者：
Jiang, Han-Liang;Zhu, Hai-Hong;Chen, Zhi
通讯作者：
Chen, Zhi
影响因子：
105.7
作者：
Cane, P;Chrystie, I;Zuckerman, M
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Zuckerman, M
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作者：
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Eshleman, Susan H.
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Claas, Eric C.;Burnham, Carey-Ann D.;Topin, Francois
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Topin, Francois
影响因子：
12.7
作者：
Mohamed, Sofiane;Ravet, Sophie;Halfon, Philippe
通讯作者：
Halfon, Philippe