Download Bayer Diagnostics Branched DNA – HIV-1

bDNA Assay and HIV RNA
Quantification
Molecular Diagnostics
Spring 2004
Gary , Qi
Simple sample preparation
• Centrifugation
– Centrifuge whole blood to isolated plasma; 500 g for
15 min
– Centrifuge plasma to pellet the virus; 23,500 g for 1
hour
• Buffer contains…
– Lithium laurel sulfate – detergent to dissolve cell
membrane to release genomic material
– Proteinase K – remove protein material and isolate
RNA
– Target probes
• Incubate at 53ºC for 20 minutes
bDNA assay
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Load sample to microwells coated with capture
probes
Incubate at 53ºC over night and wash
Add bDNA amplifier molecules
Incubate at 53ºC and wash
Added alkaline phosphatase labeled probes
Incubate at 53ºC and wash
Add chemiluminescence substrate (dioxetane)
Measure light emission with luminometer
Schematic outline of bDNA Assay
Standard Curve
• Light emission is directly proportional to the amount of
viral RNA present in the plasma sample
• Determine actual RNA concentration from standard curve
Branched DNA assay
• A signal amplification assay utilizing nucleic acid
hybridization
• Specificity
– target probes
• Complementary to the RNA template
• Complementary to capture probes that coated the
microwells
• Complementary to the bDNA amplifier molecules
– Alkaline phosphatase is bound to oligonucelotide probe
that is complementary to the branches on the bDNA
– The alkaline phosphatase will only emit light in the
presence of dioxetane substrates
Branched DNA assay (continue…)
• Sensitivity
– More than 80 different target probes available to bind with
in the specific region to avoid false negative results cause
by mutations
– Each bDNA can bind to many enzyme linked probes
– Dioxetane substrate can detect 100fg of RNA
– Using signal amplification the assay can measure RNA
level as low as 500 copies/mL of plasma
Branched DNA assay (continue…)
• Controls
– A known positive control, for quality of all
reagents
– A known negative control, for possible RNA
template contamination
– samples with known RNA concentration to
generate the standard curve
• Dynamic range: 104 to 1.6 x 106 RNA
equivalent/mL of plasma
Current application of bDNA
• Evaluate plasma viral RNA level
– HIV is a RNA virus
– Viral coat stability depends on viral RNA
– Absolute dependency of presence of viral RNA
for the infection
– The assay can quantify plasma level of the
virus through out all stages of the infection by
measuring the level of viral RNA present
Stages of HIV Infection
• The virus enters a period of clinical latency as infection
progressed
• Total viral load (i.e. the measurable quantity of virus in the body)
remains in low fluctuating concentrations
• Hard to measure plasma viral level and changes of the level
Efficiency of HIV RNA assay (bDNA)
• Under most circumstances a 2.8 fold change in viral load is
statistically significant when using bDNA
• With special precaution, this value can be decreased to
only 1.4-fold change in viral loads
• Can be used to test the effective of various drugs for
treatment of the HIV infection
• Monitor the response of the immune system to the drug
therapy and evaluate the efficiency of drug therapy
• Monitor the progress of the infection
– Need to increase/decrease drug dose?
– A new drug needed?
Why is bDNA the perfect assay?
• Familiar 96 well-microwells format
• Easy sample preparation
• No nucleic acid extraction, purification, or
amplification
• Does not need to worry about contamination
• Chemiluminescent assay has long shelf life
Highly sensitive and specific
• PCR-based tests use a pair of primers and a single probe
• With bDNA, more than 80 nucleic acid probes target area
within the highly conserved pol region of the HIV genome
to avoid false negative results cause to mutations
Less cost and labor intensive
Linearity of the standard curve
Reproducibility
• bDNA assay is highly reproducible
• Good reproducibility ensures consistency
Bayer® System 340 (bDNA
Analyzer)
bDNA System 340 automatic analyzer
• A single instrument that successfully automates the
incubation, washing, reading,and data processing
• It provides precise, accurate, and reproducible
quantification of HIV.
Compare to other HIV assays…
• Quantification by PCR can monitor the therapeutic
administration but the assay is labor intensive and
results lacks consistency between different labs
and technicians.
• ELISA with p24 capture assay is easy to do and
consistent but there is no 100% accurate
correlation between p24 antigen level and level of
viral replication present and increase in p24 is not
necessarily a positive clinical outcome.
Perfection of bDNA assay
• The assay combines all the good-qualities and
diminish all those imperfect-qualities of ELISA, PCR,
and evaluation of CD4 + T cell level
• Viral RNA level measured by bDNA does increase
with decrease in level of CD4+ T cells.
• The assay easy to do and give consistent results like
the ELISA
• Like PCR the assay is able to demonstrate the
presence of HIV replication and monitor the effects of
drug therapy has on viral replication.
Additional application of dDNA
signal amplification assay
• Hepatitis C virus RNA quantitation
• Hepatitis B virus RNA quantitation
• Quantify insulin preRNA and mRNA in
determining the stimulation of insulin synthesis by
glucose
• Analysis of adipose differentiation by measuring
the amount of lectin, the product of the obese
gene, mRNA present