Filtration-assisted magnetofluidic cartridge platform for HIV RNA detection from blood

Abstract

The ability to detect and quantify HIV RNA in blood is essential to sensitive detection of infections and monitoring viremia throughout treatment. Current options for point-of-care HIV diagnosis (i.e. lateral...

Sensitive and Quantitative Point-of-Care HIV Viral Load Quantification from Blood Using a Power-Free Plasma Separation and Portable Magnetofluidic Polymerase Chain Reaction Instrument

“…However, inside enclosed cartridges, air-drying is not amenable since magnetic beads are immersed in either aqueous phase or oil phase throughout the process. Nucleic acid extraction based on ChargeSwitch chemistry (Thermo Fisher) does not require alcohol washing and was used in cartridges by us and other groups. ,,− However, for samples with a high content of protein, ChargeSwitch chemistry showed a lower recovery than silica-coated magnetic beads and chaotropic salts chemistry. , We and several groups reported the integration of silica-coated magnetic beads and chaotropic salts extraction chemistry into enclosed cartridges by replacing alcohol-based washing with PEG-based washing, GuHCl-based washing, − or rapid water-based washing …”

“…The magnetofluidic cartridge was designed and assembled, as previously described . Briefly, the cartridge was designed with one sample well that can hold up to 500 μL of sample solution, a washing well that can hold up to 50 μL of washing solution, and a PCR well that can hold 7.5 μL of RT-PCR reaction mix solution.…”

“…Several efforts have been made to develop such a POC HIV VL test. ,− For example, Kadimisetty et al reported a three-dimensional (3D)-printed sample concentrator for plasma separation, HIV virus lysis, and HIV nucleic acid enrichment at the POC . However, the samples had to be transported to centralized laboratories for manual washing and VL quantification using benchtop PCR machines.…”

“…Among techniques for quantifying nucleic acid copy number in a single bulk reaction, real-time RT-PCR has better quantitative accuracy than isothermal techniques. , Nevertheless, real-time RT-PCR requires bulky and expensive instruments due to the need of thermal cycling and fluorescence readout . Several research groups have attempted to develop low-cost and portable real-time RT-PCR instruments for applications at the POC. − We previously reported a filtration-assisted HIV VL quantification method based upon a portable magnetofluidic real-time RT-PCR platform with a LOD of 1000 copies using 10 μL of whole blood, i.e., 100,000 copies/mL of blood and a good quantitative accuracy . However, the WHO defines ≥1000 HIV RNA copies/mL as the threshold for virological failure, while some other public health authorities use lower thresholds (e.g., ≥200 copies/mL). , Therefore, to be able to detect virological failure, a platform must achieve a LOD of 1000 HIV RNA copies/mL or lower.…”

“…43−47 We previously reported a filtration-assisted HIV VL quantification method based upon a portable magnetofluidic real-time RT-PCR platform with a LOD of 1000 copies using 10 μL of whole blood, i.e., 100,000 copies/mL of blood and a good quantitative accuracy. 40 However, the WHO defines ≥1000 HIV RNA copies/mL as the threshold for virological failure, while some other public health authorities use lower thresholds (e.g., ≥200 copies/mL). 48,49 Therefore, to be able to detect virological failure, a platform must achieve a LOD of 1000 HIV RNA copies/mL or lower.…”