Download HIV GENOTYPE ASSAY

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
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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 DNA
Amplicons 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 (1335). 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.