Polymerase Chain Reaction PCR

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Principles of molecular biology and application
to AI molecular diagnostic
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• Extraction of RNA

• LYSIS STEP
• Fast / thorough avoid degradation of RNA
• Cell disruption Strong denaturant SDS, GITC,
  phenol
• Disruption of membrane Inactivation of RNAse
• Physical action/ Mortar and pestle/liquid
  nitrogen

PURIFICATION OF RNA
Organic solvant Phenol/Chloroform/Isoamyl
alchoohol Collect of RNA from aqueous
phase Precipitation in isopropanol of RNA Wash in
Ethanol Dry pellet resuspend in water

• Affinity chromatography ( resin column)
• RNA is retained on the resin column with
  appropriate salt concentration and pH
• Elution of RNA using buffer at low salt
  concentration

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• Extraction of viral RNA of AI virus Nucleospin
  RNA virus kit from Macherey-nagel

Chromatography affinity
Silica column permitting specific link with RNA

• Lysis step
• Lysis buffer RAV1 (GITC) RNA carrier (favoring
  the RNA anchorage to column)
• Washing step
• RAW and RAV3 Ethanolic buffers removal of
  contaminants, PCR inhibitors
• Elution step
• RNA in pre heated RNase/DNase free water

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RT-PCR Reverse transcription PCR
I. First strand reaction Conversion in cDNA
RNA extract Single strand
Use of reverse transcriptase
( Enzyme from RNA virus Moloney murine leukemia
virus)
Primer for RT Random hexamer primer Total
cDNA OligodT primer (eucaryotic RNA polyA in 3)
Total cDNA Specific primer of the RNA target
cDNA target
Obtention of a single strand of complementary DNA
from RNA
II. Second strand reaction PCR on cDNA template
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• PCR Polymerase Chain Reaction

First step Denaturation 5 minutes at 94C
Tannealing depends on primers
30-40 cycles
Final Elongation 10 minutes at 72C
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PCR
High number of copies from low template DNA
XXo x 2 n
Revelation on electrophorese gel using BET/ End
point amplification
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PCR Requirements

• MgCl2 activity of DNA polymerase.5-2.5mM
• Buffer pH 8.3-8.8
• dNTPs 20-200µM
• Primers F/R 0.1-0.5µM
• Taq DNA Polymerase 1-2.5 units
• Target DNA or cDNA ? 1 µg

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RT-PCR for diagnostic AI

• One step RT-PCR

Reverse transcription and PCR in a single
step Use of specific primers for RT and PCR
Qiagen one step RT-PCR Kit
Buffer MgCl2 Enzyme Mix Taq polymerase/RTranscrip
tase dNTP Primers 30 minutes at 50C for Reverse
transcription 15 minutes at 94C for inactivation
of reverse Transcriptase activation of Taq
polymerase Run with PCR cycles
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RT-PCR for diagnostic AI
Specific of AI type A Gene M Primers
M1/M2 Conserved region of the matrix protein
suitable for the AIV detection from poultry as
well as from pigs, horses and humans
Specific of H5 subtype Gene H5 Primers H5
Kha-1/H5 Kha-3 J3/B2a HA-875F/HA-1114R
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Gene M
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Gene H5
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• Real Time PCR/Quantitative PCR/Q RT-PCR

Measure of fluorescence after each amplification
cycle Fluorescent signal proportional to amplicon
amount
Absolute quantification Measure of relative gene
expression Plus-Minus detection (AI Q RT-PCR)
Applications
2 Fluorescent reporters Sybergreen fluorescent
intercalate Double strand DNA Taqman probe with
choice of fluorescent dye
- FAM blue - VIC green - NED black -
PET red
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(No Transcript)
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TAQMAN PROBES
Fluorescent dye (Reporter)
(Quencher)
Probe free in solution
light
Light emission
Energy transfer
Taq
Probe and primers annealing FRET
lignt
Light emission
Taq DNA polymerase encounters the probe,
deplaces and degrades it, releasing free reporter
in solution Fluorescence emission
Taq
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Real-time PCR with the TaqMan-probe
Probe can not be extended at the 3 end (dideoxy
nucleotide) Fluorescence Resonance Energy
Tranfser FRET
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CT-value

• CT cycle treshold
• CT is inversely proportional to the initial copy
  number
• The Ct value can be directly correlated to the
  starting target concentration.

CT
Standard known concentration
SlopePCR efficiency
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Q-RTPCR for the detection of the AIV A type (M
Gene)
M25
M-124
M64
Gène M
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Detection of AIV in samples field from cloacal
or tracheal swabs by Q-RTPCR (CIRAD-Montpellier)
Dilution of standards
positif control strain A/PR/8/34/H1N1 (6,3.106
EID50/ml)
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Positifs lt 35 Ct
Ct CIRAD positif control 25/-3
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RT-QPCR for the identification of the H5 subtype

• QRT-PCR
• Qiagen one step Kit RT-PCR
• Primers H5 (VLA, Weybridge, UK)
• ? H5LH1 
• ? H5RH1 
• Taqman Probe
• ? H5PRO 5(FAM ) // (BHQ1)-3

Sonde H5PRO
H5LH1
H5RH1
Cleavage site
Gène HA
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THANK YOU FOR YOUR ATTENTION