Transcript Real Time PCR Technology & Reagents Dr. Tehseen Qayum

Real Time PCR
Technology &
Reagents
Dr. Tehseen Qayum
General Manager
Diagnostic tools
Two classes of assays are used in the
Diagnosis and management of Hepatitis
infection:
1. Serologic Assays that detect specific
antibody or Antigens (Rapid ICT devices
and ELISA)
2. Molecular Assays that detect viral
nucleic acid (DNA/RNA Qualitative and
Quantitative detection)
Recommendations for HCV RNA testing
a) Patients with a positive anti-HCV test (Class I,
Level B)
b)Patients for whom antiviral treatment is being
considered, using a sensitive quantitative
assay (Class I, Level A)
c) Patients with unexplained liver disease whose
anti-HCV test is negative and who are immunocompromised or suspected of having acute
HCV infection (Class I, Level B).”
During & After Treatment
 At week 12, retest for HCV RNA level. If HCV
RNA is negative or has decreased by at least
two log10 units (such as from 2 million IU to
20,000 IU or from 500,000 IU to 5,000 IU or
less), continue therapy
 At 24 weeks, assess Aminotransferase levels
and HCV RNA and stop therapy
 After therapy, assess Aminotransferase at 2to 6-month intervals.
 In responders, repeat HCV RNA testing 6
months after stopping
Flash back
InVitro Nucleic Acid amplification first
described in 1971 by Kleppe
Kary Mulis postulated PCR concept in
1983
Thermal Cyclers introduced in 1986
Real Time PCR introduced in late 90’s
What is PCR
Technique to amplify the copies of a specific
region of DNA
to produce enough DNA
material to be adequately tested
Selectively amplifying a particular segment (a
single strand) of DNA which may represent a
small part of a large & complex mixture of DNA
We may call it molecular (genetic)
photocopying
Molecular Diagnostics
“Tests and methods to identify a disease or the
predisposition for a disease analyzing DNA or
RNA of an organism”
• It enables the early detection of diseases and
conditions,
the
selection
of
targeted,
individualized treatment options and the
monitoring of treatment efficacy
• It has brought to the market, cutting-edge
industry leading tests, reagents, technologies,
instrumentation, platforms, systems & software
Why Molecular Dx?
• Need an accurate and timely
diagnosis
–Important for initiating the proper
treatment
–Important for preventing the
spread of a contagious disease
Pitfalls of Conventional PCR
• Involves post-amplification processing
such as gel electrophoresis
• Interpretation of results can be highly
subjective
–Size of a band on a gel
–Intensity of a band on a gel
Pitfalls of Conventional PCR
• Quantification requires laborious
specialized techniques
• Endpoint analysis gives poor
quantification results
• Longer time to results (3-4 hours
minimum typical)
• Requires a controlled laboratory
environment for post-amplification
processing
Problems with Agarose Gels
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Carcinogenic
Poor precision
Low sensitivity
Low resolution
Non-automated
Short dynamic range < 2 logs
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide staining is not quantitative
qPCR Chemistry
Primer
• Short (Often < 50nt) Oligonucleotide sequence of DNA
• Complementary to the beginning and the end of the target DNA
sequence
• Needed to initiate the synthesis of new DNA in a PCR reaction
• Involved in AMPLIFICATION
Probe
• A single-stranded DNA with a specific base sequence
• Labeled with fluorescence dyes (TaqMan probe)
• Used to detect the complementary base sequence of target
DNA/RNA by hybridization
• Involved in DETECTION Reporter dye / Quencher dye
Detection Chemistry
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DNA binding agents
– Intercalating method: SYBR®Green I
• Fluorescent dyes
– Hydrolysis Probe
- TaqMan® probe, Molecular Beacon
– Hybridization Probe
- Dual oligo FRET probes
– Primer based Probe
- Scorpion
The Market
• International IVD > US$ 40B
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Molecular diagnostics
Diabetes Care
Near Patient Testing
Immuno Chemistry
Clinical Chemistry
Other IVD
US$ 3B
US$ 9.8B
US$ 4B
US$ 14B
US$ 6B
US$ 3.2B
(Manual Micro /Blood Culture US$ 1.1m)
• Pakistan IVD US $ 75m
(Molecular Diagnostics US $ 6m i.e. 8% of IVD)
(7.5%)
(24.5%)
(10%)
(35%)
(15%)
(8%)
Global IVD
• Forecast to grow to US $49,843.5m by
2016 at the rate of 4.3% per year
• Clinical chemistry and the
immunochemistry markets will be more
than 60% of the total revenues growing
at 4.5% and 4.7% CAGR between 2009
and 2016 to reach $16,425.3m and
$14,136.1m respectively
Global IVD
• The genetic testing market will be the
fastest growing market at a CAGR of 6.7%
during 2009–2016 to become an over the
billion dollar market
• This market will be driven by new
technological advancements, and a shift
towards more complex immunochemistry
tests to Point of Care (POC) testing
Worldwide Molecularbiology players
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Roche Diagnostics
Abbott Laboratories
ABI
Agilant Tech
bioMérieux
Cepheid
Gen-Probe Inc
QIAGEN N.V
Digene Corporation
Quest Diagnostics Inc.
Others (Sacace, Analytica Gena etc)
Market leader Roche Diagnostics 32%
Pakistan
• Pakistan IVD Rs. 6.3B CAGR 6-8%
• Molecular Diagnostics Rs. 504m i.e. 8%
of IVD
• Pathology Labs
– Total 3500 approximately
• Class A 270
• Class B 1400-1700
• Class C 1500
PCR Reagent Players
Total market approx. 45000 tests/month
Roche
15000 tests (33%)
GMS
10500 tests (23%)
PMA
8500 tests (19%)
Chemical House
3500 tests (8%)
Others
7500 (17%)
Pakistan Hepatitis Testing
• Tests per month 45000-50000/month
• Few Major Facilities for Hepatitis testing
• DUHS, Karachi
• LUMHS, Jamshoro
• AKUH, Karachi
• LNH, Karachi
• CEMB, Lahore
• QAMC, Bahawalpur
• NORI, Islamabad
• NIBGE, Faisalabad
• SKMTH, Lahore
• Shifa, Islamabad
Molecular Biology Reagents
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Sacace (Extraction & Amplification)
Artus-Qiagen/BioGen
RoboScreen/Analytica Jena
Roche
Home Brewed kits
qPCR Players Worldwide
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ABI
Roche Diagnostics
Abbott Laboratories
Cepheid
QIAGEN N.V,/ Corbett RotorGene
3000/6000
• Biorad
• Others
ABI is the Market leader
qPCR Players PK
Principal
Instruments
Number of
Systems
Corbett
Rotor-gene
(3000/6000)
86
Biorad
CFX-96, Mini Opticon,
55 (9,20,19,6,1)
IQ-5, I-cycler, Chromo 4
Cepheid
Smart cycler II
46
Roche
Light
Cycler/Amplicor
22
qPCR Players PK
Principal
Instruments
Number
of
Systems
ABI
7000 series
12
BioFlux
Line Gene
6
Abbott
m2000
6
qPCR Applications
• Clinical Diagnostics
 Bacterial/ Viral pathogen detection
• Absolute pathogen quantification
• Drug therapy efficacy / drug monitoring
• Differential gene expression
• RNA validation
• SNP Genotyping
qPCR Applications
• Structural Assay
– Uses DNA, typically genomic extractions
• Single Nucleotide Polymorphism
• Functional Assay
– Uses RNA extractions
– Uses reverse transcriptase to generate cDNA
templates
• Differential expression
• Diagnostics involving gene expression
• RNA interference
• Clinical Diagnostic Assay
– Uses DNA or RNA extracted from patient’s samples
• Viral/bacterial pathogens
USP of Smart Cycler
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One of the fastest
Robust tubes
4-color detection
Random Access
Flexibility
Transportable
Competitive pricing
One of the fastest
Temperature ramps
 Heating
(Ceramic Heaters) 10°/sec
 Cooling (air) 2.5°/sec
One of the fastest
Independent reading of each
tube (I-Core): Unique Feature
Robust tubes
Sample positioning
Tubes tightly closed. Vol.: 25 and 100 µl
no contamination
With plates, capillaires or classic PCR tubes
there is a higher risk of contamination
4-color detection
• Smart Cycler 4 color detection
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LEDs for excitation
- 4 photodiodes for detection
Simultaneous Amplification and
Detection: Unique Feature
Random Access Flexibility
• Random Access from 16 to 96
samples
Each site is completely independent
Up to 96 different protocols at the
same time
 Modular system
Unique Features
Molecular Biology Reagents
• Sacace offers complete module of
Extraction & Amplification
• Internal control (recombinant
RNA‐containing‐structure) extracted
with the sample
• Kits can be optimized against
almost all systems
Details Available on Request
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Hepatitis C Qualitative
Hepatitis C Quantitative
Hepatitis C Genotype
Hepatitis B Qualitative
Hepatitis B Quantitative
HIV Qualitative
HIV RNA Quantitative
HCV/HBV/HIV Combination
CMV Qualitative
EBV Quantitative
CMV/EBV/HHV6 Screen
HPV High Risk Screen
HPV High Risk Screen Quantitative
HPV High Risk Typing (HPV 16, 18,
31, 33, 35, 39, 45, 52, 56, 58, 59, 66)
• HPV 16/18 Quantitative
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Avian A Screening & H5N1
Chlamydia Trachomatis
Ureaplasma parvum/urealyticum
Neisseria Gonorrhoeae
Trichomonas vaginalis
Gardnerella Vaginalis
HSV I/II Real-TM
Treponema Pallidum
Bacillus Anthracis
Rubella Real-TM
Candida Albicans
Parvo virus B19
Pneumocystis (Jerovecii) Carinii
Rotavirus/Norovirus/Astrovirus
HDV Qualitative
MTB Resistance
Vibrio Cholerae
and many more……
Thanks