Workshop Real time PCR 12. & 13. February 2011

Tutorial 1:

PCRReal Time PCRFTD Kits (FTD Respiratory PLUS)

Principle of PCR

1. Denaturation

2. Hybridisation

3. Elongation

•Template (DNA),

which should be copied

• Primer= sequence

specific nucleotides

•Enzym Polymerase

•Nukleotids

• Buffer

Principle of PCR

1

2

3

Endpointanalysis

1.Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive2. Linearity stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting. 3. Plateau: No more product accumulates due to exhaustion of reagents and enzyme

„Classical PCR“

• Amplification and detection are two independent steps

• Amplification => thermocycler

• Detection => Gel

UV visualisation

PCR

Agarosis - gel electrophoresis

3-4 hours

Real time PCR

Real time PCR is.....

The ability to monitor or visualize accumulation of PCR products

using fluorescence

•Amplification and detection are combined in one analytical step•Amplification => thermocycler•Detection => thermocycler

AmplificationVisualization

< 2 Stunden

Why Real-Time PCR?

Rapid cycling times (1,5 hour)

High sample throughput (~200 samples/day)

Low contamination risk (sealed reactions)

Very sensitive (3pg or 1 genome eq of DNA)

Broad dynamic range (10 - 1010 copies)

Reproducible (CV < 2.0 %)

Allows quantitation of results

Software driven operation

Real-time Requirements…

Real Time PCR is the ability to monitor or visualize accumulation of PCR products using fluorescence

1.Reporter : A fluorescent reporter where increase in fluorescence must be proportional to the increased amount of DNA (amplicon)

2.Instrument to excite & detect reporter and be able to perform analysis

3.Analysis : Method to discriminate positive and negative results and quantification.

PCR equipment ....a few machines

Rotorgene 6000-6 chanels

Rotorgene 3000-4 Chanels

ABI 7500-5 Chanels

Reporter methodsIntercalating Dyes : SYBR® Green I

Hybridization Probes

Hydrolyzable probes : Taqman®

Displaceable probes : Molecular Beacon®

FRET probes : LC640, LC705

Labeled Primers : Amplifluor® primers

Mix primer/probe : ScorpionsTM

Intercalating Dyes : SYBR® Green I

• Easy

– Use with existing PCR primers

• Non-specific

– Detects all double stranded products, including primer dimers

– Need to optimize carefully so no nonspecific amplification

• OK for presence / absence detection but not great for quantitative measurements

• Problem

- Primer Dimer Artefact:Non-sequence specific fluorescent intercalating agent quantify all double stand DNA including non-specific amplification and primer-dimer complex

- Another problem is that longer amplicons create a stronger signal (if combined with other factors) this may cause saturation

Taqman®

The probe is labbeled with:

R= Reporter

Q= Quenscher

Taqman®

The probe is labbeled with:

R= Reporter= Fluorophore

molecule that emits light of a certain wavelength after having absorbed light of a specific, but different wavelength first

the emission wavelength is always higher than the absorption wavelength

a lot of different fluorophores are available: FAM, VIC, Hex, Ned, Rox, Cy5......

Q= Quencher

molecule that accepts energy from a fluorophore in the form of light and dissipates this energy either in the form of light or heat

different fluorophores are available: MGB, BBQ, Tamra.....

The probe with reporter and

quencher anneal with the target

DNA-singlestrand

The probe is intact, there is no

signal

Then the primer bind and elongate

the strand

The labeled probe is seperated and

demaged

Taqman®

The polymerase then carries out the

extension of the primer and replicates

the template to which the TaqMan® is

bound.

The 5' exonuclease activity of the

polymerase cleaves the probe, releasing

the reporter molecule away from the

close vicinity of the quencher.

The fluorescence intensity of the reporter

dye, as a result increases.

This process repeats in every cycle and

does not interfere with the accumulation

of PCR product.

Taqman®

Multiplexing

Multiplex-PCR is a rapid method of detecting

multiple targets in a single reaction

• PCR reagent mix contains multiple sets of primers

and probes. Any one of these may be amplified

during the PCR

• Primer/probe sets to multiple organisms

• Primer/probe sets to multiple target genes in the

same organism

Major advantage is the reduction in test processing

time

FTD FLU

Influenza A

Influenza B

A(H1N1) swl

Internal

Control

Multiplex PCR detects and differentiates in one tube:

Real time PCR-Multiplexing

Fast-track Diagnostics

FTD/Glasgow synergies

• Glasgow designs tests, uses them routinely, finds out what

doesn’t work so well, re-evaluates

• Transfers to FTD

• FTD converts into kit format, does intra- inter-test

variability, evaluates on multiple commercial machines etc

and embeds in quality system

• FTD CE marks

• Glasgow updates FTD as changes introduced

Widest range of

clinically important

menus

Once you know

how to do one test,

you can do them all

Same extraction

protocol

One cycler

program for all tests

6 tube multiplex PCR for detection of 25 respiratory pathogens

1. Multiplex: Influenza A, Influenza B, Rhinovirus (detecting also new subtype C) and A(H1N1)swine

2. Multiplex: Coronavirus NL 63, 229E, OC43, HKU3. Multiplex: Parainfluenza 2, 3, 4, Internal control4. Multiplex: Parainfluenza 1, Human metapneumovirus A and B,

Bocavirus, Mycoplasma pneumoniae5. Multiplex: Respiratory syncytial viruses A and B, adenovirus,

parechovirus, enterovirus6. Multiplex: Chlamydia pneumoniae, Streptococcus pneumoniae,

Haemophilus influenzae B, Staphylococcus aureus

September 2010:

- Moraxella catarrhalis

- Haemophilus all types

- Klebsiella

- Bordetella pertussis

- Legionella

- Pneumocystis jiroveci

FTD Respiratory PLUS

1 2 3 4 5 6 7 8

Color Label Contents Tubes for 12 reactions

Yellow Flu Rhino PP Primer/probe mix for Influenza A, B; H1N1 and Rhino

8 x

Red COR PP Primer/probe mix for Corona 43, 229, 63 and HKU

8 x

Blue Para 2/3/4 Primer/probe mix for Para2, 3, 4 and IC

8 x

Purple Bo-MP-Pf1 PP

Primer/probe mix for Boca, HMPV A&B, M. pneumoniae and Para1

8 x

Green RsEPA Primer/probe mix for RSV A&B, entero, parecho and adeno

8 x

Yellow RespBac PP Primer/probe mix for S.aureus, S.pneumococcus, HiB, C. pneumoniae

8 x

Red Resp PC Postive control pool for FluRhino PP, COR PP, Para2/3/4 PP, Bo-MP-Pf1 PP and RsEPA PP

8 x

Red Resp Bac PC Positive control pool for RespBac PP 8 x

Blue IC Internal control 8 x

Black NC Negative control 8 x

THE CONTROLS

The three required controls are included in the kit

• Positive control is required to check if the application works i.e. if the

pathogen is detected by the reagents. (FTD : plasmid pools for all pathogens)

• Negative control is important to prove that the application provides negative

results if no pathogen is present in the sample (wrong positive results or “false

positives”). (FTD : negative control is extractable)

• Internal control checks for wrong negative results or “false negatives”. This

may be due to inhibition by extraneous substances present in stool or due to

human error. (FTD: RNA or DNA viruses from plant or animals in denaturant

agent)

All three controls are of prime importance and only if all three control reactions

work properly, you can be sure that your results are reliable.

Process :Documentation

• Swabs, tissue, cells, blood, saliva…….

depends on the pathogen, that you want detect

Process :THE RAW MATERIAL

This test is for use with extracted RNA and DNA from respiratory samples(throat/nasal swabs, bronchoalveolare lavage, sputum) of human origin.

2. Extract 12 samples and the NC.We recommend a starting volume for the extraction of 300 ul and an elution volume of 75 ul.

1. Thaw one negative control (NC) and oneinternal control (IC)

Extraction room

Enzyme Mastermix room

Main lab

PCR machines room

3. Add 3 ul internal control (IC) directly to the lysis buffer of eachextraction4. Do not extract positive controls

Process: Extraction

Process: Preparation

of the PP mixes

Extraction room

Enzyme Mastermix room

Main lab

PCR machines room

1. Thaw reagents for the reaction: Flu/Rhino PP, COR PP, Para2/3/4 PP,Bo/Mp/Pf1 PP, RS/EPA PP, RespBac PP, PC, RespBacPC and also theenzyme and the mastermix of AgPath-ID™ One-Step RT-PCR Kit

2. Pipette the required amount of the “2xRT PCR buffer” and “25x RT-PCRenzyme mix“(AgPath-ID™ One-Step RT-PCR Kit) to the PP mixes

Process: Preparation of the plate

Extraction room

Enzyme Mastermix room

Main lab

PCR machines room

1. Pipette 15 Pl of each PP mix with the “2xRT PCR buffer” and the “25xRT-PCR enzyme mix” in the wells

2. Add 10 Pl of the extracted samples, theextracted negative control and thepositive control

Universal thermal cycling protocol

Reverse Transcriptase

Activation of the

PolymeraseDenaturation

Annealing

Depends on the enzyme that you useDepends on the primer and probes that

you use

Setting of the thermocycler

Thank you….

Stool: viruses, parasites and bacteria3 kits one sample

Pathogens Two tube mulitplex:

norovirus G1/G2,

astrovirus, rotavirus

and adenovirus

One tube multiplex:

entamoeba

histolytica,

cryptosporidium sp.

and giardia sp.

Two tube multiplex:

Shigella ssp. (EIEC),

Yersinia enterocolitica,

Campylobacter jejuni,

C.coli, E.coli (EHEC

including Verotoxin 1

or 2), Salmonella

enterica, Clostridium

difficile (Toxin B)

FTD Bacterial gastroenteritis

speciesFTD

total

positive negative

salm culture

In-house

positive 56 /210

negative / 154

shig culture

In-house

positive 4 /210

negative 1*a 205

cdiff culture

In-house

positive 27 /210

negative 2*b 181

yers culture

In-house

positive 6 /210

negative / 204

campy culture

In-house

positive 113 /286

negative 1*c 172

EHEC vtx+ culture

In-house

positive 15 /286

negative 2*d 269

Tested against a range of salmonella species