Gupta Shivani, Taylor Tracy, Patterson Aileen, Liang Binhua, Bullard Jared, Sandstrom Paul, Van Domselaar Gary, Ji Hezhao
Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada.
The Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada.
Biomed Res Int. 2017;2017:4979252. doi: 10.1155/2017/4979252. Epub 2017 Apr 3.
The prevalence of drug resistance (DR) mutations in people with HIV-1 infection, particularly those with low-level viremia (LLV), supports the need to improve the sensitivity of amplification methods for HIV DR genotyping in order to optimize antiretroviral regimen and facilitate HIV-1 DR surveillance and relevant research. Here we report on a fully validated PCR-based protocol that achieves consistent amplification of the protease (PR) and reverse transcriptase (RT) regions of HIV-1 gene across many HIV-1 subtypes from LLV plasma samples. HIV-spiked plasma samples from the External Quality Assurance Program Oversight Laboratory (EQAPOL), covering various HIV-1 subtypes, as well as clinical specimens were used to optimize and validate the protocol. Our results demonstrate that this protocol has a broad HIV-1 subtype coverage and viral load span with high sensitivity and reproducibility. Moreover, the protocol is robust even when plasma sample volumes are limited, the HIV viral load is unknown, and/or the HIV subtype is undetermined. Thus, the protocol is applicable for the initial amplification of the HIV-1 PR and RT genes required for subsequent genotypic DR assays.
在HIV-1感染者中，尤其是那些低水平病毒血症（LLV）患者中，耐药（DR）突变的流行情况表明，有必要提高HIV DR基因分型扩增方法的灵敏度，以优化抗逆转录病毒治疗方案，并促进HIV-1 DR监测及相关研究。在此，我们报告一种经过充分验证的基于PCR的方法，该方法能在来自LLV血浆样本的多种HIV-1亚型中，实现对HIV-1基因蛋白酶（PR）和逆转录酶（RT）区域的一致扩增。来自外部质量保证计划监督实验室（EQAPOL）的涵盖各种HIV-1亚型的加标HIV血浆样本以及临床标本被用于优化和验证该方法。我们的结果表明，该方法具有广泛的HIV-1亚型覆盖范围和病毒载量跨度，且灵敏度高、可重复性强。此外，即使血浆样本量有限、HIV病毒载量未知和/或HIV亚型未确定，该方法也很可靠。因此，该方法适用于后续基因型DR检测所需的HIV-1 PR和RT基因的初始扩增。
