Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.
Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.
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DOI：
10.1128/mbio.00135-13
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发表时间：
2013-04-02
期刊：
影响因子：
6.4
通讯作者：
Overbaugh J
中科院分区：
文献类型：
--
作者：
Boyle DS;Lehman DA;Lillis L;Peterson D;Singhal M;Armes N;Parker M;Piepenburg O;Overbaugh J
婴儿感染人类免疫缺陷病毒1型（HIV-1）的早期诊断和治疗可大大降低死亡率。然而，目前的婴儿艾滋病毒-1诊断不能在护理点可靠地进行，往往延误治疗并损害其疗效。酶聚合酶扩增（RPA）是一种新型技术，是HIV-1诊断的理想选择，因为它在恒定温度下在<20分钟内扩增靶DNA，而不需要复杂的热循环设备。在这里，我们测试了63种HIV-1特异性引物和探针组合，并确定了两种RPA检测试剂，它们靶向HIV-1基因组的不同区域（长末端重复序列[LTR]和pol），并且可以通过使用荧光检测和侧流条带检测可靠地检测3个拷贝的前病毒DNA。这些pol和LTR引物分别扩增了98.6%和93%的不同HIV-1变异体。这是第一个等温检测的例子，一致地检测所有主要的HIV-1全球亚型。婴儿HIV-1感染的诊断不能依赖于成人使用的基于抗体的检测，因为母体HIV-1抗体会从母亲转移到孩子身上。因此，婴儿诊断依赖于病毒本身的检测。然而，目前的婴儿HIV-1诊断方法需要具有复杂设备的实验室环境。在这里，我们描述了HIV-1诊断的婴儿，可以在农村卫生诊所的护理点进行的初步发展。我们利用一种可以在25至42 ° C范围内的孵育温度下扩增和检测HIV-1 DNA的方法，无需热循环设备。由于全球范围内HIV-1毒株的高度多样性，HIV-1诊断方法的开发具有挑战性。在这里，我们展示了这种方法检测全球流行的主要HIV-1毒株。
Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes. Diagnosis of HIV-1 infection in infants cannot rely on the antibody-based tests used in adults because of the transfer of maternal HIV-1 antibodies from mother to child. Therefore, infant diagnostics rely on detection of the virus itself. However, current infant HIV-1 diagnostic methods require a laboratory setting with complex equipment. Here we describe the initial development of an HIV-1 diagnostic for infants that may be performed at the point of care in rural health clinics. We utilize a method that can amplify and detect HIV-1 DNA at an incubation temperature within the range of 25 to 42°C, eliminating the need for thermocycling equipment. HIV-1 diagnostics are challenging to develop due to the high diversity seen in HIV-1 strains worldwide. Here we show that this method detects the major HIV-1 strains circulating globally.