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HIV-1 Genotypic Resistance Testing

Resistance to antiretroviral drugs occurs through specific mutations in the target viral gene and is an important factor which determines treatment outcome in HIV-infected patients. The ARI-UCSF Laboratory of Clinical Virology (LCV) uses the TRUGENE™ Genotyping assay (Siemens HealthCare Diagnostics), which provides the specific mutations that contribute to resistance and the drug susceptibility interpretation for the HIV-1 reverse transcriptase and protease genes. Synonymous mutations, polymorphisms, and unexpected mutations at resistance sites are also determined. The assay is FDA-approved for diagnostic purposes. One ml of EDTA, ACD, or PPT blood plasma is required. In addition, the laboratory performs genotypic resistance testing on HIV RNA from CSF, seminal fluid, vaginal fluids, and cellular DNA.

HIV-1 Genotypic Resistance Testing with Clinical Controls

Negative and positive specimen controls are included on each HIV-1 drug resistance testing batch for compliance with particular research protocols. The genotype results are for research use only (RUO).

HIV-1 Viral Load, Plasma, or CSF

For blood plasma or CSF HIV-1 viral load quantitation, the LCV performs the Abbott RealTime™ HIV-1 assay (Abbott Molecular). This assay is an in vitro reverse transcription-polymerase chain reaction (RT-PCR)-based test that uses the automated m2000 System for nucleic acid extraction, reagent preparation, liquid handling, and RT-PCR amplification. For each specimen, an unrelated RNA is added, independently amplified and monitored as an internal control to evaluate adequate PCR target amplification. External controls are also included in each assay batch. The dynamic range spans from 40-10,000,000 copies RNA/mL (100% probability by probit analysis), although values below 40 copies/mL can be detected (Tang et al., JVirolMethods, 2007). The assay accurately quantifies genetically diverse group M subtypes A-H, group O and group N viruses through probing of the pol integrase region of HIV-1. The test requires 1.0 mL of clarified blood (EDTA, ACD, PPT) or CSF.

Miscellaneous Sequencing

Please contact the Research Director for inquiries on viral and host gene sequencing.

Batch HIV-1 p24 ELISA

The laboratory uses the NEN HIV-1 p24 ELISA (Enzyme-Linked Immunoabsorbant Assay) for detecting and quantifying virus production in vitro. The NEN HIV-1 p24 ELISA utilizes a mouse monoclonal specific for HIV-1 p24 antigen which is coated on microtiter plates. Neutralized tissue culture supernatant is added to the coated well, and nonspecific antibody binding sites are blocked to reduce background signal. P24 molecules bind to the immobilized antibody and are detected following adequate washing by adding biotinylated human polyclonal antibody. The wells are further washed, and the polyclonal antibody is detected by adding streptavidin-horseradish peroxidase conjugate. In the final step, the substrate OPD is added, turning the positive wells yellow. The absorbance is measured spectrophotometrically, and the intensity of the signal is directly proportional to the concentration of immobilized antigen. The concentration is directly measured against a p24 antigen standard curve.

PBMC Processing

Peripheral Blood Mononuclear Cells are separated from neutrophils, granulocytes, and red blood cells in whole blood by density gradient centrifugation using Ficoll-hypaque (density 1.077). Following separation and extensive washing, cells are counted and aliquoted as desired in cryopreservation media (for viable recovery) or as dry pellets (for nucleic acid extraction).

PBMC Processing and 2nd Type Aliquoting

This processing charge applies to an additional mode of aliquoting and storage (for example, processing and storage as viable cryopreservation in DMSO and as dry PCR pellets).

Dried Blood Spots

Whole blood is spotted onto filter paper, dried, and prepared for shipping according to the research protocol's specifications.

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