Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation.
Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation.
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DOI：
10.1371/journal.pone.0145962
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Lutz BR
中科院分区：
文献类型：
--
作者：
Panpradist N;Beck IA;Chung MH;Kiarie JN;Frenkel LM;Lutz BR
人类免疫缺陷病毒（HIV）是一种慢性感染，可通过抗逆转录病毒治疗（ART）加以控制。然而，在终身抗逆转录病毒治疗期间，次优病毒抑制期可以选择HIV耐药（DR）变体。耐药病毒的传播可降低或取消抗逆转录病毒治疗的疗效。因此，建议在开始治疗之前对个体进行耐药性检测，以确保抗逆转录病毒治疗有效。在发生大多数艾滋病毒感染的低资源环境中，需要敏感和廉价的艾滋病毒基因分型方法。寡核苷酸连接试验（OLA）是一种检测HIV pol耐药突变的灵敏点突变试验。目前的OLA包括从样品到分析的四个主要步骤：(1)裂解和/或核酸提取，(2)HIV RNA或DNA扩增，(3)设计用于检测赋予HIV耐药性的单核苷酸突变的寡核苷酸探针的连接，以及(4)通过寡核苷酸表面捕获，变性和检测（CDD）进行分析。这些步骤的相对复杂性限制了其在资源有限的实验室中的采用。在这里，我们描述了一种将2.5小时的印版格式CDD简化为45分钟的纸质格式CDD，从而消除了对印版阅读器的需要。对26份血样中4个HIV-1 DR密码子（K103N、Y181C、M184V和G190A）的突变分析表明，纸制CDD与平板CDD的突变信号与总信号之比具有较强的相关性。所描述的分析使OLA更容易在低资源实验室中执行。
Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.