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

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

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.

Advantages:

Low-cost with minimal equipment needed
Rapid process with actionable time scale
Quantitative measurement of viral load/RNA copy numbers
Early detection prior to seroconversion
Suitable for use in resource poor settings, in the clinic, and at home

Applications:

Monitor viral load in response to treatment
Detection of infection in neonates against background of maternal antibody
Combine with point-of-care plasma separation device developed by Bau lab
Use in resource-poor regions without trained staff, laboratory facilities, or electricity
Applicable for incorporation within multiplexed assays to detect co-infections

Example time course assay for fluorescence-based detection of HIV subtypes A and B using developed RT-LAMP primers. In (A), view of smart cup for minimally-instrumented, point-of-care molecular diagnostics, and in (B), a photograph of the smart cup equipped with smartphone and inset of integrated microfluidic chip with isothermal amplification reactors for nucleic acid extraction and amplification.