Real Time PCR Technology

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Real Time PCR Technology Reagents

• Dr. Tehseen Qayum
• General Manager

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Diagnostic tools

• Two classes of assays are used in the
• Diagnosis and management of Hepatitis
• infection
• Serologic Assays that detect specific antibody or
  Antigens (Rapid ICT devices and ELISA)
• Molecular Assays that detect viral nucleic acid
  (DNA/RNA Qualitative and Quantitative detection)

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Recommendations for HCV RNA testing

• Patients with a positive anti-HCV test (Class I,
  Level B)
• Patients for whom antiviral treatment is being
  considered, using a sensitive quantitative assay
  (Class I, Level A)
• Patients with unexplained liver disease whose
  anti-HCV test is negative and who are
  immuno-compromised or suspected of having acute
  HCV infection (Class I, Level B).

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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 2- to
  6-month intervals.
• In responders, repeat HCV RNA testing 6 months
  after stopping

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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 90s

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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
  

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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

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Why Molecular Dx?

• Need an accurate and timely
• diagnosis
• Important for initiating the proper
• treatment
• Important for preventing the spread of a
  contagious disease

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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

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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

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Problems with Agarose Gels

• Carcinogenic
• Poor precision
• Low sensitivity
• Low resolution
• Non-automated
• Short dynamic range lt 2 logs
• Size-based discrimination only
• Results are not expressed as numbers
• Ethidium bromide staining is not quantitative

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qPCR Chemistry

• Primer
• Short (Often lt 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

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Detection Chemistry

• DNA binding agents
• Intercalating method SYBRGreen I
• Fluorescent dyes
• Hydrolysis Probe
• - TaqMan probe, Molecular Beacon
• Hybridization Probe
• - Dual oligo FRET probes
• Primer based Probe
• - Scorpion

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The Market

• International IVD gt US 40B
• Molecular diagnostics US 3B (7.5)
• Diabetes Care US 9.8B (24.5)
• Near Patient Testing US 4B (10)
• Immuno Chemistry US 14B (35)
• Clinical Chemistry US 6B (15)
• Other IVD US 3.2B (8)
• (Manual Micro /Blood Culture US 1.1m)
• Pakistan IVD US 75m
  (Molecular Diagnostics US 6m i.e. 8 of IVD)

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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

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Global IVD

• The genetic testing market will be the fastest
  growing market at a CAGR of 6.7 during 20092016
  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

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Worldwide Molecularbiology players

• 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
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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

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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)
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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

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Molecular Biology Reagents

• Sacace (Extraction Amplification)
• Artus-Qiagen/BioGen
• RoboScreen/Analytica Jena
• Roche
• Home Brewed kits

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qPCR Players Worldwide

• ABI
• Roche Diagnostics
• Abbott Laboratories
• Cepheid
• QIAGEN N.V,/ Corbett RotorGene 3000/6000
• Biorad
• Others
• ABI is the Market leader

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qPCR Players PK
Principal Instruments Number of Systems
Corbett Rotor-gene (3000/6000) 86
Biorad CFX-96, Mini Opticon, IQ-5, I-cycler, Chromo 4 55 (9,20,19,6,1)
Cepheid Smart cycler II 46
Roche Light Cycler/Amplicor 22
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qPCR Players PK
Principal Instruments Number of Systems
ABI 7000 series 12
BioFlux Line Gene 6
Abbott m2000 6
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qPCR Applications

• Clinical Diagnostics
• Bacterial/ Viral pathogen detection
• Absolute pathogen quantification
• Drug therapy efficacy / drug monitoring
• Differential gene expression
• RNA validation
• SNP Genotyping

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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 patients samples
• Viral/bacterial pathogens

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USP of Smart Cycler

1. One of the fastest
2. Robust tubes
3. 4-color detection
4. Random Access
5. Flexibility
6. Transportable
7. Competitive pricing

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One of the fastest

• Temperature ramps

• Heating (Ceramic Heaters) 10/sec
• Cooling (air) 2.5/sec

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One of the fastest

• Independent reading of each tube
  (I-Core) Unique Feature

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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
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4-color detection

• Smart Cycler 4 color detection
• - 4 LEDs for excitation
• - 4 photodiodes for detection

Simultaneous Amplification and Detection Unique
Feature
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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
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Molecular Biology Reagents

• Sacace offers complete module of Extraction
  Amplification
• Internal control (recombinant RNA-containing-struc
  ture) extracted with the sample
• Kits can be optimized against almost all systems

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Details Available on Request

• 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

• 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

and many more
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• Thanks