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Updated: May 12, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification
David S Boyle1, Dara A Lehman, Lorraine Lillis
1Program for Appropriate Technology in Health, Seattle, WA, USA. dboyle@path.org
Insights
Developing rapid point-of-care diagnostics for infant human immunodeficiency virus type 1 (HIV-1) is crucial. This study presents novel Recombinase Polymerase Amplification (RPA) assays for early HIV-1 detection in infants, enabling faster treatment.
Area of Science:
- Molecular Biology
- Virology
- Diagnostics
Background:
- Early diagnosis and treatment of infant human immunodeficiency virus type 1 (HIV-1) significantly reduce mortality.
- Current diagnostic methods for infant HIV-1 are not suitable for point-of-care settings, leading to treatment delays.
- Recombinase Polymerase Amplification (RPA) offers a rapid, isothermal DNA amplification method ideal for point-of-care diagnostics.
Purpose of the Study:
- To develop and validate novel Recombinase Polymerase Amplification (RPA) assays for the early point-of-care diagnosis of infant HIV-1 infection.
- To identify RPA primer and probe combinations effective against diverse HIV-1 global subtypes.
Main Methods:
- Screened 63 HIV-1-specific primer and probe combinations for RPA assays.
- Targeted distinct regions of the HIV-1 genome: long terminal repeat (LTR) and pol.
- Evaluated assay sensitivity using fluorescence detection and lateral-flow strip detection.
Main Results:
- Identified two RPA assays targeting LTR and pol regions with high sensitivity, detecting as few as 3 copies of proviral DNA.
- The pol and LTR primer sets demonstrated high coverage of diverse HIV-1 variants, amplifying 98.6% and 93%, respectively.
- Achieved reliable detection of major HIV-1 global subtypes using an isothermal amplification method.
Conclusions:
- Developed the first isothermal assay capable of consistently detecting all major HIV-1 global subtypes for infant diagnosis.
- The novel RPA assays show promise for rapid, point-of-care HIV-1 diagnostics, potentially improving treatment outcomes in infants.
- This advancement addresses a critical need for accessible and efficient infant HIV-1 testing in resource-limited settings.
Abstract:
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.
