A highly sensitive aptamer-based HIV reverse transcriptase detection assay.

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Traditional radioactivity-based assays for HIV reverse transcriptase (RT) remain in use due to their reliability.
• New methods are needed to improve the sensitivity and simplicity of HIV RT detection and quantification.

Purpose of the Study:

• To develop a novel, sensitive, and simple assay for detecting and quantifying HIV RT activity.
• To utilize a high-affinity DNA aptamer for capturing and detecting RT.

Main Methods:

• A DNA aptamer with high affinity for HIV RT was immobilized on 96-well plates.
• Bound RT was detected by its ability to extend the aptamer using radiolabeled nucleotides.
• Extensive washing steps removed non-specific binding, and the radioactive signal was measured.

Main Results:

• The assay demonstrated high sensitivity, detecting as few as ~6400 RT molecules and ~100-300 virions.
• Assay linearity was observed over a range of at least 10^4.
• Both wild-type and drug-resistant HIV-1 RT, as well as HIV-2 RT, were detectable.

Conclusions:

• This aptamer-based assay offers a simple, sensitive, and reliable method for HIV RT detection and quantification.
• The assay is effective for various HIV RT forms, including drug-resistant strains.
• This approach provides a valuable alternative to traditional radioactive assays.