Simple amplification-based assay: a nucleic acid-based point-of-care platform for HIV-1 testing.

Background: A new nucleic acid-based assay (simple amplification-based assay [SAMBA]) for rapid visual detection of human immunodeficiency virus-type 1 (HIV-1) by dipstick is described. The assay was designed to be simple, stable, robust, self-contained, and capable of detecting a broad spectrum of HIV-1 subtypes and recombinant forms.

Methods: The performance of the SAMBA HIV-1 test (amplification and detection chemistry) was evaluated using the World Health Organization HIV-1 RNA Genotype Reference Panel, with clinical samples representing various viral subtypes and recombinant forms common in sub-Saharan Africa.

Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings.

Currently available nucleic acid testing (NAT)-based assays are complex and time-consuming, and they require expensive instrumentation and dedicated laboratory spaces for sample preparation as well as for amplification and detection of the nucleic acid target. Reagents required for these tests are also expensive and must be transported and stored refrigerated or frozen. These characteristics have limited the use of such assays for point-of-care (POC) testing, especially in resource-poor settings.

Simultaneous visual detection of multiple viral amplicons by dipstick assay.

A sensitive, simple, and instrument-independent method for the visual detection and identification of multiple nucleic acid amplicons by dipstick has been developed. This method is based on nucleic acid hybridization on the dipstick membrane and a signal amplification system to allow visual detection. With hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) as model analytes, it is demonstrated that the visual dipstick test combined with multiplex reverse transcription (RT)-PCR for the amplification of viral nucleic acid provides a specific and sensitive detection method.