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A reverse transcription loop-mediated isothermal amplification method to quantify multiple HIV subtypes

Description:

A rapid, quantitative point-of-care diagnostics method for viral load detection of HIV-1 clades A, B, C, D, and G

 

 

Inventor

Haim H. Bau, Professor of Mechanical Engineering and Applied Mechanics

Changchun Liu, Research Assistant Professor, Mechanical Engineering and Applied Mechanics

Frederic D. Bushman, Professor of Microbiology

 

Problem

HIV infection remains an international health crisis, where it is especially acute in sub-Saharan Africa, despite the development of antiretroviral therapy.  High sensitivity, point-of-care clinical diagnostic tests for detecting and quantifying HIV viral load are lacking in development, particularly for resource-limited settings. Existing technologies are immunoassay-based, which are not quantitative and do not allow for early detection prior to seroconversion or in neonates.  Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) amplifies cDNA. The amplicons can be detected by various methods, including intercalating fluorescent dye. 

 

Solution

Researchers in the Bushman and Bau labs have improved existing HIV RT-LAMP assays to detect HIV subtypes A, B, C, D, and G, of which A and C are prevalent in Africa and India.  A bioinformatics study identified highly conserved sequences within the HIV genome, followed by the design and optimization of primers, targeting the HIV integrase coding region.  The work culminated in a rapid quantitative assay, with clinical samples currently being tested.  The fluorescence-based DNA detection time is less than 20 minutes for amplifying more than 5,000 copies of nucleic acid.  In combination with a microfluidic diagnostic chip, a smart cup, consisting of a Thermos cup, a 3D printed cup lid, chip holder, and smartphone holder, designed by the Bau lab can be employed to detect the amplified nucleic acids in a minimally-instrumented format.