用于检测HIV-1逆转录酶耐药性的基于彩色编码微珠检测法的开发与定制

Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.

作者信息

Gu Lijun, Kawana-Tachikawa Ai, Shiino Teiichiro, Nakamura Hitomi, Koga Michiko, Kikuchi Tadashi, Adachi Eisuke, Koibuchi Tomohiko, Ishida Takaomi, Gao George F, Matsushita Masaki, Sugiura Wataru, Iwamoto Aikichi, Hosoya Noriaki

机构信息

Research Center for Asian Infectious Diseases, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan; Japan-China Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P. R. China.

Division of Infectious Diseases, Advanced Clinical Research Center, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan.

出版信息

PLoS One. 2014 Oct 14;9(10):e109823. doi: 10.1371/journal.pone.0109823. eCollection 2014.

DOI:10.1371/journal.pone.0109823

PMID:25314293

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4196989/

Abstract

BACKGROUND

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.

METHODS

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.

RESULTS

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%).

CONCLUSIONS

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.

摘要

背景

HIV-1的耐药性（DR）可通过基因型或表型进行检测。虽然测序是基因型耐药性检测（GRT）的金标准，但针对负责耐药性的密码子的高通量GRT可能更适合流行病学研究和公共卫生研究。

方法

我们使用一个日本数据库来设计和合成序列特异性寡核苷酸探针（SSOP），用于检测B亚型HIV-1逆转录酶区域的野生型序列和6种耐药性突变。我们将SSOP与Luminex 100 xMAP系统的微珠偶联，并开发了一种基于聚合酶链反应（PCR）-SSOP-Luminex方法的GRT。

结果

设计并合成了16种寡核苷酸探针，用于区分6个位点的野生型序列中的耐药性突变，并使用同基因质粒确认了它们的敏感性和特异性。然后，使用来自未接受过治疗的患者或治疗失败患者的74份血浆标本，将PCR-SSOP-Luminex耐药性检测与直接测序进行比较。在大多数标本中，PCR-SSOP-Luminex耐药性检测的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%），M184为63/73（86.3%），T215为68/73（93.2%）。有许多标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份标本具有由G2748A编码的D67N突变。我们为K65额外合成了14种寡核苷酸探针，K65位点的敏感性从43/74（58.1%）提高到68/7（91.9%）。