Extraction-Free Scalable Sample Preparation for the Rapid Detection of HIV in Blood Plasma

Nucleic acid amplification tests that are rapid and low-cost are needed for expanding access to point-of-care (POC) diagnostics for infectious diseases and viral load monitoring. Several companies have commercialized POC devices using rapid isothermal amplification; however, these tests use nasal swabs or saliva samples. Translating these tests to blood plasma is challenging due to clotting factors, heat- or pH-induced coagulation of human albumin, high concentrations of ribonucleases (RNases), and other sample matrix inhibitors. Crude, extraction-free sample preparation methods are promising alternatives that reduce the complexity of sample preparation. In this paper, we present an extraction-free sample preparation method for in-tube and paper-based reverse transcription recombinase polymerase amplification for the detection of HIV in blood plasma samples. We use a rapid heat treatment protocol with a reducing agent to inhibit RNases and lyse HIV virus, enabling RNA preservation into direct amplification without the need for complex RNA purification steps. We demonstrate sample treatment in 5 min and amplification within 20 min in reaction volumes ranging from 50 to 300 μL and show that large volume reactions achieve a limit of detection of 800 copies/mL (16 copies/reaction) for HIV-positive plasma in both paper and tube formats.