PCR Laboratory Set-up.

• The arrangement or design of a PCR laboratory is of great

importance in maintaining a high standard of performance.

• As a minimum you need three dedicated areas (minimum

requirement).

The reagent preparation area (1).The specimen

preparation area (2). The PCR and detection area (3).

• If more space is available, it may be used to good

advantage.
Minimum layout requirements for a basic PCR laboratory

LAB 3
Amplification Analysis
laboratory laboratory OUT
Area Area

LAB 2 LAB 1
Extraction
Area
Master mix
Area
IN
 Ideal physical arrangement for a PCR laboratory

LAB 3 LAB 4 LAB 5

Offices
RT-PCR PCR/Ampli Analysis
Laboratory Laboratory Laboratory
General Area

Area Area Area

LAB 6
Nested-PCR
Laboratory
Area
LAB 2 LAB 1
Extraction Master-mix

Offices
Laboratory Laboratory
Area Area
 Ideal physical arrangement for a real-time PCR laboratory

LAB 3 LAB 4 LAB 5

Offices
Real-time PCR Real-time PCR Analysis
Robotic extraction
Laboratory Laboratory Laboratory
General Area

Area Area Area

Store

LAB 2 LAB 1
Extraction Master-mix

Offices
Laboratory Laboratory
Area Area
 Reagent Preparation - Area 1

• The first steps in the PCR procedure starts in Area 1.

•Here, master mix (amplification reagents) reagents and primers are

prepared along with controls.

•Prepared master mix and controls are then added to the PCR reaction
tubes.

•If you can divide this area into two spaces so as to keep the control
DNA away from the master mixes.
 Equipment required in Area 1
If using one room, a dead-air box with an optional ultraviolet
(UV) light or, if using two rooms a clean, dedicated
area.
• Freezer/Fridge and ice-boxes.
• Dedicated micropipettes with plugged (aerosol-barrier) tips
(25 µL, 50 µL, and 100 µL) or
• Dedicated positive-displacement pipette and tips. A repeat
pipettor (optional).
• Perkin-Elmer GeneAmpTM PCR System reagents (Taq Pol,
buffer etc), MicroAmpTM consumables (tubes, caps,
base, tray, and retainer).
• Microcentrifuge.
• Lab coat
• Gloves
 Important considerations applicable to Area 1

With PCR diagnostics, always work in a one-way direction from Area

1 to Area 2 to Area 3 in order to avoid carryover contamination from
amplified products.

• Area 1 should be located as far away from Area 3 as possible.

• Area 1 should not be in close proximity to the centrifuge area (Area 2)
to avoid possible aerosol contamination.
• Clean, dedicated area working surface must be decontaminated prior
to use with 10% bleach solution (freshly made daily) followed by
a 70% ethanol rinse or equivalent.
• If a UV box is used, for your own safety, you must turn off the
ultraviolet light in the box before placing your arms and hands
into the working area.
• Master mix and controls are to be prepared in this area.
 Important considerations applicable to Area 1 (cont.)
•Prepared master mix and controls will be added to PCR reaction
tubes and capped in this area.
• Dedicated pipettes with plugged (aerosol-barrier) tips or
positive-displacement tips, or a repeat pipettor are required in
Area 1 and should not be transferred to either Area 2 or
Area 3.
• Lab coats designated for wear in Areas 1 and 2 should never be
worn into Area 3 to avoid carryover contamination of
amplified product from Area 3 back to Area 1 or Area 2:-
two coats may be used, disposable coats may be worn over a
cloth coat
• Gloves must be worn at all times for your safety as well as for
control of contamination from one area to another.
• Gloves are to be changed at each of the three work areas.
• Gloves worn in Area 3 must never be worn in Area 1 or Area 2.
 DNA/RNA Extraction - Area 2
Specimens are prepared in this area and are added to PCR tubes with
master mix. Specimens are now considered amplification-ready.

Equipment required in Area 2

• Biological cabinet, flow cabinet, dead area space.
• Standard clinical tabletop centrifuge and/or microcentrifuge (max RCF 16,000g).
• Dedicated micropipettes with plugged (aerosol-barrier) tips (25µl, 50µl, & 100 µl) or
• Dedicated positive-displacement pipette and tips.
• Repeat pipettor (optional).
• Balance/Scale.
•Spectrophotometer.
• Vortex mixer
• Dry-heat temperature block.
• Sterile calibrated transfer pipettes
• All reagents and chemicals.
• All consumables.
• Lab coat (different coat to Area 1).
•Gloves (new pair).
 Important considerations applicable to Area 2
• Working surface in cabinet must be decontaminated prior to specimen preparation
with 10% bleach solution (freshly made daily) followed by a 70% ethanol
rinse or equivalent.
• Cabinet must be on 30 minutes before specimen preparation procedure.
• All pipettors, pipettes, bulbs, and other equipment used in specimen preparation
should be kept in this cabinet at all times.
• Specimens must be stored separately from reagents-two separate refrigerators are
required.
• Dry baths are preferred over water baths water baths can contaminate specimens
through seepage into poorly stoppered tubes.
• A set of pipettes should be dedicated exclusively to be used for specimen preparation
• To avoid specimen-to-specimen contamination, plugged (aerosol-barrier) pipette tips
or positive-displacement tips, must be used in Area 2.
• Lab coats designated for wear in Areas 2 should never be worn into Area 3 or 1 to
avoid carryover contamination.
• Gloves must be worn at all times for your safety as well as for control of
contamination from one area to another gloves are to be changed at each of the
three work areas.
 Amplification (PCR) and Detection - Area 3

• The final steps in the procedure takes place in Area 3.

• Here, amplification-ready specimens undergo PCR and the PCR

products (amplicons) of this PCR are then detected.

• This area should also be divided into two areas if possible.

• Amplification area

• Analyses area.
 Equipment required in Area 3

Perkin-Elmer GeneAmpTM PCR Systems 9600/9700/2400/2700 Thermal Cyclers or

equivalent.
• Dedicated micropipettes with plugged (aerosol-barrier) tips (25 µL and 100 µL) or
dedicated positive-displacement pipette and tips
• Multichannel pipettor, Repeat pipettor, Nonplugged pipette tips (all optional)
• Electrophoreses apparatus and power supplies.
• Microcentrifuge
• Disposable reagent reservoirs
• Incubator (37°C ± 2°C) with or without CO2
• Microwell plate reader, Microwell plate washer. Key for microwell strip removal.
• UV trans-illuminator
• Gel documentation system (computer hardware/software & printer)
• Lab coat (different from the one worn in Areas 1 and 2)
• Gloves (new pair)
• Electrophoresis apparatus and documentation system.
• Balance/Scale and microwave oven
 Important considerations applicable to Area 3

•Area 3 should be kept as far away as possible from Areas 1 and 2 to avoid aerosol
contamination.
• Incubator temperature should be kept stable.
• Traffic in and out of incubator should be reduced to a minimum.
• Area 3 pipettes should never be used in Areas 1 or 2.
• Pipettes with plugged (aerosol-barrier) tips are used for pipetting denaturing solution
into PCR tubes and denatured amplified product into microwell plates.
• Non-plugged tips may be used for all other reagent additions a pipette contaminated
with this highly concentrated product could cause false-positive results
plugged tips therefore are required to prevent this potential carryover
contamination.
• As this is a one-way workflow, the lab coat worn in Area 3 must never be worn in
Areas 1 and 2.
• Gloves must be worn at all times for your safety as well as for control of
contamination from one area to another. Gloves are to be changed at each of
the three work areas. Gloves worn in Area 3 must never be worn in Area 1 or Area 2.
 Establishment of a PCR assay

The selection of a PCR assay must be based on factors including scientific and
international acceptance, cost, available resources, nature of the intended use,
sensitivity and specificity, number of tests to be done and availability of standard
reagents.

Development and optimization of the assay must then be performed and should
include a series of experimental procedures and the evaluation of the data
generated. Analysis should determine a fixed protocol for use, the nature and
number of controls required and specifications required of reagents and equipment.

The optimisation of the PCR should be followed by evaluation of the test. This will
include a period of testing samples with known histories.
 Validation of the assay

Validation establishes the performance standards of the assay (e.g.,

sensitivity, specificity, accuracy, precision, positive/negative cut-
off criteria etc.), using appropriate statistical methods.

Validation can include a comparison with other methods, with

reference standards, collaborative studies with other laboratories
using the same test procedure, experimental challenge studies or
reproduction of data from accepted standard methods or a
reputable publication
 Validation
• Specificity is the ability of the assay to exclusively detect the agent of
interest.
• Sensitivity is described as the ability to precisely detect small quantities
of the test agent

Stage 1 validation
Involves the development of the assay performing a feasibility study to
determine whether the assay can detect a range of agents (e.g., virus
concentrations, virus serotypes/genotypes), without background activity.

This stage include all aspects of development and optimisation of the

test, including the identification of the test, the determination of the
target template(s), the sequence determination of the primers and the test
conditions and criteria.
 Stage 2 validation
Involves the evaluation of the test against a panel of known positive
and negative template samples to determine the analytical sensitivity
and specificity.

Stage 3 validation
This involves the determination of diagnostic sensitivity and
specificity, I.e. involving field samples in 2 parallel tests (classical vs
PCR). .

Diagnostic sensitivity (DS) and specificity (DP)

DS determines the proportion of known infected reference animals testing positive
[TP/(TP+FN)], while DP determines the proportion of uninfected reference animals
that test negative [TN/(TN+FP)]. (TP = true positive; FN = false negative; TN =
true negative; FP = false positive).
DS - how many false negatives you get.
DP - how many false positives you get
 Stage 4 validation
Entails the continued monitoring of the validity of assay
performance in the field by calculating the predictive value of
positive or negative results based on estimates of pathogen
prevalence in the target animal population. This can only be done
satisfactorily if a diagnostic sensitivity and specificity data (stage
3) are available.
Stage 5 validation
Involves the maintenance of validation criteria using internal
quality controls. Frequent monitoring for repeatability and
accuracy are needed. The OIE also recommends biannual ring-
testing to determine reproducibility between laboratories, although
annual testing is also described.
 Quality assurance programme or accreditation

The intention of accreditation or the formal recognition of a quality assurance

programme by an independent third party, is to ensure that GLP, QA (QC -
IQC/EQC) is in place within the diagnostic laboratory. Specific standards are used
(e.g. ISO 17025) in accordance with the requirements of the inspecting body (e.g.,
the South African National Laboratory Accreditation Service).

Requirements will include a quality policy, a quality manual, procedures for

evaluating and reviewing new and exciting test methods and protocols, the use of
blind quality assurance within and between laboratories, monitoring of equipment,
ongoing training, an audit programme, regular review of the quality system and
documentation and records of all quality activities and evaluations. Standard
operating procedures (SOP’s) will be required for not only the test methods used,
but also for the calibration and maintenance of instrumentation and equipment
used.
 Proficiency testing

Proficiency testing is the means used to determine the capability

of a laboratory to perform the assay and effectively detect the
agent (internal proficiency testing).

Such testing will also contribute to ensuring that within or

between laboratories performing routine diagnostic services, a
specific assay is performed according to established international
standards (external proficiency testing
 PCR Controls
Positive control samples
These should include:
• Specimen material with a high pathogen concentration.
• Specimen material with a low concentration (preferably the lowest limit
routinely detected, or preferably the lowest amount of detectable target
agent present in sample material by conventional means and by reported
PCR data.
• Specimen material with expected pathogen concentration in an infected case (not
essential)
• Extracted positive DNA/RNA can be used only if the above are not available.

In the case of a proficiency testing process, specimen material spiked with a

dilution series of virus (e.g., 10-1 to 10-10) should also be included. Virus titre
would have been established by means of a golden standard assay.
 Negative control samples

• A negative control consisting of all reagents except extracted sample DNA

or RNA - this should confirm absence of amplicon or agent itself or
other cross reactive entities in reagents.
• A negative specimen control sample (allantoic fluid, buffer, tissue), if
available, which will undergo nucleic acid extraction to confirm
absence of contamination in the extraction procedure.
• Extracted nucleic acid derived from pathogen-free sample material obtained
previously (e.g., tissue or other medium normally submitted,
including tissue, blood, buffer) - should confirm that no non-
specific binding to virus-free specimen DNA/RNA has taken place
(eukaryotic and prokaryotic genomic DNA or RNA present in
sample). Not essential.
• In the case of RT-PCR, an additional negative control consisting of all
reagents as well as the extracted sample nucleic acid, but without
reverse transcriptase included, this should confirm absence of
DNA or amplicon contaminants in both reagents and test material.