HIV GENOTYPE ASSAY

Anabelia Perez, MLT (ASCP)

Molecular Technologist

August 6, 2008

CLINICAL REASON

The ViroSeq HIV-1 Genotype Assay is clinically used:

Detect HIV genomic mutations that confer resistance to specific types of antiretroviral drugs

To aid in monitoring and treating HIV infections.

PRINICPLE

The ViroSeq HIV-1 Genotyping System is based on six major processes:

Sample Preparation Reverse Transcription (RT) Polymerase Chain Reaction (PCR) Cycle Sequencing Automated Sequence Detection Software Analysis

SAMPLE PREPARATION

Isolate HIV-1 viral RNA from 0.5mL of EDTA human plasma

1 hour centrifuge at 4°C HIV virus particles in pellet are disrupted

with Viral Lysis Buffer Precipitation with Isopropanol Purification with 70% Ethanol RNA is dried and resuspended with

Diluent

REVERSE TRANSCRIPTION

The ViroSeq HIV-1 Genotype Assay amplifies 1.8 Kb region of the HIV-1 pol gene that spans the entire protease gene and approximately two-thirds of the reverse transcriptase (RT) gene.

Single-stranded complementary DNA is generated.

POLYMERASE CHAIN REACTION

PCR Reaction(40 cycles for PCR step)

1st step: 93°C for 20 sec to denature DNA into single strands

2nd step: 64°C for 45 sec to anneal primer 3rd step: 66°C for 3 min to extend primer to

create double-stranded DNAAmplicons are created and heated to 72° C for 10

min to allow final extension and then cooled down to 4°C infinity for next procedure

CYCLE SEQUENCING

Cycle Sequencing has 4 steps:

PCR purification- removes unincorporated dNTPs & primers DNA quantitation- gel electrophoresis Cycle sequencing- 7 primers (4 forward & 3 reverse) to sequence

entire region Protease (codon 1-99) and two-thirds RT region (1-335). Big Dye Terminator chemistry is used to permit a resolution of 600 bases on the 3100 Genetic Analyzer

Sequence Purification-removes unincorporated Big terminators from samples so they do not interfere with sample sequencing & analysis. Method: Centri-Sep 96 column spin plates are used at CPL. (Cost effective). Purified cycle sequence reactions are resuspensed in Hi Hi formamide.

AUTOMATED SEQUENCE DETECTION

• 3100 Genetic Analyzer- ABI Prism automated sequencers detect fluorescence from different dye terminators that are used to identify the A C G T bases. Each dye emits light at a different wavelength when excited by an argon ion laser. All four bases are then detected & distinguished in a single capillary.

• Sequencing involves creating an electrical flow of ions from negative to positive thru POP-6 medium which involves smaller-size fragments migrating faster than larger-size fragments thru field.

• Each fragment passes by the laser read region & it is excited by the laser

• Fluorescence is detected by a CCD camera & converted to a sequence basecall by the Sequence Analysis software

• Resulting File contains the sequence information for each of the 7 primers in each sample

SUMMARY & EXPLANATION

The ViroSeq HIV-1 Genotyping System detects mutations in the RT and protease regions of the pol gene and provides the physician with a report indicating genetic evidence of viral resistance. It is a complete system that provides reagents for viral RNA isolation from plasma, RT-PCR, and sequencing. The entire protease gene and two-thirds of the Rt gene are amplified to generate a 1.8 kb amplicon. The amplicon is used as a sequencing template for seven primers that generate an approx. 1.3 kb consensus sequence. The software compares the consensus sequence with a reference, HXB-2, to determine mutations present In the sample. Finally, the ViroSeq software uses a proprietary algorithm to analyze the mutations and generate a drug resistance report.