Introduction: 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.
Materials and methods: 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.
Results: 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.
Conclusions: 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.
Keywords: AIDS; HIV-1; Molecular diagnostics; RT-PCR.
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