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RC Technology
The RC assay utilizes PhenoSense™ technology to provide a rapid and accurate method for evaluating RC. Patient virus is evaluated for RC in the absence of drug and is compared to the replication capacity of a wild-type reference virus, and expressed as a percentage. Thus, a virus with an RC value of 50% is able to replicate only half as well as the reference.
To learn more about the technology used in the RC assay, click on the links below.
Constructing the vector —
A retroviral vector capable of measuring RC was constructed using an infectious molecular clone of HIV-1.
- The vector contains a luciferase expression cassette inserted within a deleted region of the envelope gene
- HIV-1 protease (PR) and reverse transcriptase (RT) sequences are amplified from patient plasma samples and inserted into the vector using defined restriction enzyme sites
- Vectors are prepared as libraries (or sequence pools) to capture the PR/RT sequence heterogeneity of the virus in the patient
RC Assay Process
- Recombinant viruses for RC testing are generated by introducing resistance test vector DNA into cell cultures (transfection) along with an expression vector that produces a mouse retrovirus envelope protein
- Two days after transfection, virus is harvested from cells and the amount of virus produced is evaluated
- The virus is inoculated onto new cell cultures and cells are incubated for 2 to 3 days. After normalization for the amount of input virus, luciferase activity detected in the infected cells is determined and used as a direct measure of "infectivity," [ i.e., the ability of the virus to infect target cells (dependent on protease activity) and to complete the reverse transcription and integrase steps of the life cycle (dependent on reverse transcriptase and integrase activity)]
- Relative RC is assessed by comparing the amount of luciferase activity produced by sample-derived viruses to the amount of luciferase activity produced by a well-characterized reference virus (wild-type) derived from a molecular clone of HIV-1 (NL4-3)
- RC measurements are expressed as a percent of the wild-type reference virus (e.g. 70%)
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