Burchard Paul R, Abou Tayoun Ahmad N, Scherer Axel, Tsongalis Gregory J
Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States; Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH, United States.
Department of Electrical Engineering, California Institute of Technology, Pasadena, CA, United States.
Exp Mol Pathol. 2014 Aug;97(1):111-5. doi: 10.1016/j.yexmp.2014.06.005. Epub 2014 Jun 16.
The CDC estimates that there are currently over 1million people living with human immunodeficiency virus (HIV-1) in the United States, with new cases increasing by approximately 50,000 each year. HIV-1 consists of four distinct groups: the major M group, and the rare N, O, and P groups, each comprising of various subtypes. Without proper care, HIV-1 can lead to cardiovascular, kidney, and liver diseases, cancer, and rapid progression into acquired immune deficiency syndrome (AIDS). Here, we describe a novel, rapid, and highly sensitive assay for the detection of HIV-1 using intercalating dye based RT-PCR and melt curve analysis.
We designed an RT-PCR assay for the detection of the major M subtypes in addition to the rare (O, N, and P) HIV-1 groups, as well as an extraction/RT-PCR control, using melt curve analysis. Viral RNA was extracted using the automated Qiagen EZ1 robotic system (Qiagen, Valencia, CA). To establish the limit of detection (LOD) for this assay, we diluted the AcroMetrix HIV-1 panel (LifeTechnologies, Grand Island, NY) to concentrations ranging from 25 to 500 copies/ml. Armored RNA BCR/ABL b3/a2 (Asuragen, Austin, Texas) was used as our extraction and RT-PCR control. Specificity and accuracy were assessed by testing plasma specimens from 48 anonymized patients negative for HIV-1.
This assay has a turnaround time of less than 2.5h and has a limit of detection of 50 copies/ml of plasma. Our assay also demonstrated 100% concordance with 53 previously quantified plasma patient specimens, including 48 negative samples and 5 positive samples. HIV-1 and our extraction/RT-PCR control were consistently identified at 79 °C and 82.5 °C, respectively.
We developed a comprehensive, easy to use assay for the detection of HIV-1 in human plasma. Our assay combines a rapid and cost-effective method for molecular diagnostics with the versatility necessary for widespread laboratory use. These performance characteristics make this HIV-1 detection assay highly suitable for use in a clinical laboratory.
美国疾病控制与预防中心估计，目前美国有超过100万人感染了人类免疫缺陷病毒1型（HIV-1），且每年新增病例约增加50000例。HIV-1由四个不同的组组成：主要的M组以及罕见的N、O和P组，每组都包含各种亚型。若未得到妥善治疗，HIV-1可导致心血管疾病、肾脏疾病、肝脏疾病、癌症，并迅速发展为获得性免疫缺陷综合征（AIDS）。在此，我们描述了一种使用嵌入染料的逆转录聚合酶链反应（RT-PCR）和熔解曲线分析来检测HIV-1的新型、快速且高度灵敏的检测方法。
我们设计了一种RT-PCR检测方法，用于检测主要的M亚型以及罕见的（O、N和P）HIV-1组，同时使用熔解曲线分析设计了一种提取/RT-PCR对照。使用自动化的Qiagen EZ1机器人系统（Qiagen，加利福尼亚州瓦伦西亚）提取病毒RNA。为确定该检测方法的检测限（LOD），我们将AcroMetrix HIV-1标准品（LifeTechnologies，纽约州大岛）稀释至25至500拷贝/毫升的浓度范围。使用装甲RNA BCR/ABL b3/a2（Asuragen，得克萨斯州奥斯汀）作为我们的提取和RT-PCR对照。通过检测48例HIV-1阴性匿名患者的血浆标本评估特异性和准确性。
该检测方法的周转时间少于2.5小时，血浆检测限为50拷贝/毫升。我们的检测方法还与53份先前定量的血浆患者标本（包括48份阴性样本和5份阳性样本）显示出100%的一致性。HIV-1和我们的提取/RT-PCR对照分别在79°C和82.5°C时被一致鉴定出来。
我们开发了一种用于检测人血浆中HIV-1的全面、易于使用的检测方法。我们的检测方法将分子诊断的快速且经济高效的方法与广泛实验室使用所需的多功能性相结合。这些性能特征使这种HIV-1检测方法非常适合在临床实验室中使用。
