Download HCV PCR

HCV PCR
Hepatitis C Virus by Polymerase Chain
Reaction
By
Henrietta Orji
July 31st, 2010
Objectives
The
timeAmpliprep
PCR
technology
utilizes
dualis oligonucleotide
HCVreal
COBAS
isacid
highly
asymptomatic
/ COBAS
and
Taqman
it affects
people
sensitive,
of
all
Nucleic
HCV
PCR
test
testing
is
performed
for
HCV
is
to
performed
determine
using
the
response
thefluids,
fully
Transmission
of
HCV
is
through
blood
and
body
probes
for
Probe
Capture
technology
during
ages,
reliable,
races,
fast,
and
has
ethnicity
low
cross
leading
contamination,
to chronic
liver
and /
automated
to and
instrument
and
duration
called
of
antiviral
the
COBAS
therapy.
Ampliprep
also
through
sexual
contact.
amplification, while
flourescence
emission is used for
increased
productivity.
COBASdisease.
Taqman
96
detection of HCV RNA.
Objectives
• HCV is highly asymptomatic and it affects people of all ages,
races, and ethnicity leading to chronic liver disease
• Transmission of HCV is through blood and body fluids, and also
through sexual contact
• Nuclueic acid testing for HCV is performed using the fully
automated instrument called the COBAS Ampliprep / COBAS
Taqman 96
• HCV PCR test is performed to determine response to and
duration of antiviral therapy
• COBAS Ampliprep / COBAS Taqman is sensitive, reliable, fast, has
low cross contamination, and increased productivity
• The real time PCR technology utilizes dual oligonucleotide
probes for Probe Capture technology during amplification, while
fluorescence emission is used for detection of HCV RNA
Any of THEM could
have the Hepatitis C
Virus…
Clinical Reasons for
the Test
• Aids in the management of HCV – infected
individuals
a. Monitoring the rate of virological
response
b. Determining the duration of therapy
COBAS Ampliprep / COBAS
Taqman 96 Analyzer
• Fully automated instrument
• Improves laboratory work
flow
• Leads to increased
productivity
• Robust, sensitive, and
reliable
Principles of the Procedure
• The principles are based on:
• Automated sample preparation (extraction)
• Automated reverse transcription, PCR
amplification, and detection
• Quantitation
Automated Sample Preparation
Automated Sample Preparation
Utilizes the Probe Capture Technology
•Specimen Preparation is based on the following key principles:
•Load of 850µL serum or plasma specimen
•Lysis with chaotrope (a reagent that causes a change in protein
conformation to release nucleic acid) at an elevated
temperature in presence of Quantification Standard (QS) or
Internal Control (IC)
•Hybridization of biotinylated oligonucleotide probes to target
•Capture of biotinylated probe: target by streptavidin magnetic
particle
•Magnetic removal and washing of particles
•Resuspension of particles
Automated Reverse Transcription
• Extracted specimens are added to the K tubes
(amplification tubes)
• Heat mixture
• Downstream primer anneal specifically to the
HCV target RNA and to the HCV QS RNA
• In the presence of Mn2+ and excess dNTPs the
ZO5 polymerase extends the annealed primer
forming a DNA strand complimentary to the
RNA target (cDNA)
PCR Amplification
• PCR Amplification can be
divided into Target
Amplification and Selective
Amplification.
• The amplification takes
place in the Thermocycler
that is inside the COBAS
Taqman 96 analyzer.
Target Amplification
• The steps for amplification
include:
Denaturation:
Thermocycler hits the mixture at
90°C to denature the RNA: cDNA
hybrid.
Specific primer target sequence is
exposed
PCR Amplification, cont’d
• Annealing:
Here the primer anneals
to the target DNA at a
temperature of 40 – 65°C
•Extension:
The enzyme thermos specie DNA
polymerase (ZO5), in the presence
of Mn2+ and excess dNTPs extend
the annealed primer along the
target template to produce a
double-stranded DNA molecule
called an amplicon.
PCR Amplification, cont’d
• The picture shows what
happens at the end of
the first PCR cycle –
results in two copies of
target sequence
•This cycle is then repeated several times.
•The cycle only occurs in the region of HCV
genome between the primers
PCR Amplification, cont’d
• Selective Amplification
• The enzyme AmpErase (Uracil-N-glycosylase)
catalyzes and destroys all the DNA containing
deoxyuridine and also removes nonspecific
product formed after initial activation of the
mastermix by Mn2+
– Naturally occuring DNA does not contain
deoxyuridine
– Deoxyuridine is in master mix and DNA containing
it is removed before amplification
Detection of PCR Product
• Instrument utilizes real time PCR technology
• Uses dual labeled fluorescent probes
• Probes monitor the emission intensity of fluorescent
reporter dyes
• Fluorescent reporter dyes are released during the
amplification process
• Amplification of HCV RNA & HCV QS RNA are measured
independently at different wavelengths
• This process is repeated for the designated number of
cycles
• The higher the HCV titre of a specimen, the earlier the
fluorescence of the reporter dye of the HCV rises above the
baseline fluorescence level
Limitations of the Procedure
•
The COBAS Ampliprep / COBAS Taqman 96 comes with its own limitations, which
include:
– Only human serum or plasma collected in EDTA anticoagulant is suited for the
test.
– Only personnel trained in PCR technique can use this instrument.
– Reliable results are dependent on adequate specimen collection, transport,
storage, and processing procedure.
– The quantity of HCV RNA is dependent on the number of virus particles present in
the specimen and may be affected by specimen collection methods and patient
factors.
– Mutation in the region of the viral genome covered by the test primers and or
probe may result in the under-quantitation of or failure to detect the presence of
the virus.
– The AmpErase enzyme reduces the risk of amplicon contamination; but
contamination from HCV positive controls and clinical specimens can be avoided
only by good laboratory practices and adherence to procedures.
– Switching from one assay that performs HCV RNA quantitation to the COBAS
Ampliprep / COBAS Taqman 96 HCV test requires users to perform method
correlation studies in order to quantify assay differences.
Causes of Erroneous Results
• Putting reagents in the wrong cassettes
• Using expired reagents
• Mixing reagents with different lot numbers /
different cassetes
• Using controls from different lots / different kits
• Using specimen other than serum or EDTA
plasma
• Using contaminated reagents