Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification

Program for Appropriate Technology in Health, Seattle, Washington, USA.
mBio (Impact Factor: 6.79). 02/2013; 4(2). DOI: 10.1128/mBio.00135-13
Source: PubMed


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. IMPORTANCE 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.

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Available from: Lorraine Lillis, Apr 07, 2014
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    • "Under the optimal temperature (37 Ce42 C), the RPA reaction progresses rapidly, resulting in rapid amplification of target DNA from just a few copies to detectable levels, in typically less than 30 min [8]. In the literature, RPA has been applied to detect different DNA [9] [10] and RNA [11] [12]. However, it has not been used to detect any shrimp pathogen. "
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    • "Reverse transcriptase, can be also included in RPA reactions to facilitate RPA from RNA targets such as Middle East Respiratory Syndrome Coronavirus or Rift Valley Fever [23], [24]. Recently RPA was been demonstrated to be highly sensitive for the detection of HIV proviral DNA [22] and in this work we have investigated the use of RPA to detect Mycobacterium tuberculosis DNA from patients presenting with suspected pulmonary TB. "
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