REAL TIME PCR

SADIA ARSHAD
TECHNOLOGIST

 PCR provide evidence for current infection.

Why REAL TIME advantageous over Manual!

REAL TIME PCR Molecular testing

3 Steps Extraction..(Ampliprep) Reverse Transcription, cDNA..(Taq Man 96) Amplification & Detection..(Taq Man 96)

Extraction in Ampliprep
Principle:
Extraction of DNA/RNA by Probe capturing method.

Reagent Kit Composition

4 Cassettes
CS1; CS2; CS3; CS4; Magnetic glass particles. Lysis reagent. Proteinase soln. & Elution buffer. IC, Mn soln. & masters mix. A- dNTPs (A,C,G,U) B- Primrers, 5 UTR region C- Flourescent labelled probe, HCV/IC D-Rec. DNA Pol, Zo5/Zo5D E-AmpErase

Magnetic Glass Particles (MGP)

Capture released DNA/RNA (beginning of Sample Prep.)

Can be separated from solution (Washing)

Set free bound DNA/RNA (end of Sample Prep.)

Lysis Buffer (LYS)

DNA/RNA are released from virus

DNA/RNA are stabilized against degradation (nucleases)

Degradation of inhibitory proteins Protease (Pase) Destabilization of cellular membranes Elution Buffer (EB) Release of DNA/RNA from MGP

DNA or RNA

Specimen Preparation ..
To Isolate DNA/RNA
Biotinylated probe, comp. sequence of 5UTR of target sequence. Avidine coated Magnetic Particles.
In SPUs (sample+IC+MP+lysis soln.+probe) Incubation Complex

Biotinylated probe

Avidine coated Magnetic Particles

Target DNA/RNA
5 UTR 3

Complex formed
5 UTR 3

SPU Sample Processing Unit

Sample tip (S-Tip)
Splash Guard Inlet Port Aerosol Barrier

Waste Chamber

60C Processing Chamber not used

37oC Processing Chamber

 Magnetic field 3 times washing Nucleic Acid + Elution Buffer transferred into k-tubes with the help of k-tips. Placed k-tubes in k-carrier & finally shifted into TaqMan 96 via docking.

K-tube transfer according to workflow

COBAS TaqMan Analyzer linked

Sample tube
COBAS TaqMan Analyzer docked

TaqMan 96

As extracted RNA is single stranded Reverse Transcription of RNA into cDNA.

 Transcription ?? DNA into mRNA Reverse Transcription RNA into cDNA

Polymerase Chain Reaction

An in vitro technique for rapidly synthesizing large quantities of a given DNA segment (that involves separating the DNA into its two complementary strands, using DNA polymerase to synthesize twostranded DNA from each single strand) and repeating the process.

Master Mix
dNTPS (dATP, dGTP, dCTP, dUTP) DNA Pol, Zo5/Zo5D Primers, 5 UTR region Buffer Mg++

PCR 3 Steps

Real time PCR.?

Amplification and Detection, simultaneously

quencher (Q) dye (long-wavelength colored dye, such as red) reduces the fluorescence from the reporter (R) dye.

 TaqMan probe binds with its specific site of the target DNA after denaturation 95c. Reaction cools(55c), then primers anneal to the DNA.

 DNA polymerase then adds nucleotides and removes the probe from the target DNA. This separates the quencher from the reporter, and allows the reporter to emit its energy. This is then quantified through Ampilink The more times the denaturing and annealing takes place, the more opportunities there are for probe to bind and, in turn, the more emitted light is detected.

Real Time PCR

Detection
Fluorescence readings send to Ampilink Software. Flags are generated when data lie outside the preset limit.

After passing all pre-checks fluorescence readings generate CT values for DNA/RNA & IC

Results Interpretation by graph

Results Interpretation

Positive---------- DETECTED Negative--------- NOT DETECTED Each Internal controls also run with each test samples.
IC results helps in the assessment of test results.

How life become easy!

Its only REAL TIME PCR give exact view of your any DNA/RNA based disease While routine diagnosis tell you the status after patient goes to tertiary level/chronicity