PCR 5 Lab Setup

8/6/2019 PCR 5 Lab Setup

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PCR Laboratory Set-up.

The arrangement or design of a PCR laboratory is of greatimportance in maintaining a high standard of performance.

As a minimum you need three dedicated areas (minimumrequirement).

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.


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AmplificationlaboratoryArea

AnalysislaboratoryArea

LAB 3

LAB 2

Extraction

Area

LAB 1

Master mix

Area

OUT

IN

Minimum layout requirements for a basic PCR laboratory


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Ideal physical arrangement for a PCR laboratory

LAB 3RT-PCRLaboratoryArea

LAB 4PCR/AmpliLaboratoryArea

LAB 5

AnalysisLaboratoryArea

LAB 2ExtractionLaboratory

Area

LAB 1Master-mixLaboratory

Area

O

ffices

Offices

Gene

ralArea

LAB 6Nested-PCRLaboratoryArea


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Ideal physical arrangement for a real-time PCR laboratory

LAB 3Real-time PCRRobotic extraction

LaboratoryArea

LAB 4

LaboratoryArea

LAB 5Analysis

LaboratoryArea

LAB 2ExtractionLaboratory

Area

LAB 1Master-mixLaboratory

Area

Offices

Office

s

Gene

ralArea

Store

Real-time PCR


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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 reactiontubes.

If you can divide this area into two spaces so as to keep the controlDNA away from the master mixes.


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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 L, 50 L, and 100 L) 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


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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 offthe

ultraviolet light in the box beforeplacing your arms and hands

into the working area. Master mix and controls are to be prepared in this area.


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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 orArea 3.

Lab coats designated for wear in Areas 1 and 2 should never be

worn into Area 3 to avoid carryover contamination ofamplified 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.


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DNA/RNA Extraction - Area 2Specimens are prepared in this area and are added to PCR tubes with

master mix. Specimens are now considered amplification-ready.


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 (25l, 50l, & 100 l) 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).


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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 beworn 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 thethree work areas.


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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.


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

equivalent.

Dedicated micropipettes with plugged (aerosol-barrier) tips (25 L and 100 L) 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 (37C 2C) with or without CO

2

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


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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 inAreas 1 and 2.

Gloves must beworn 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.


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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 willinclude a period of testing samples with known histories.


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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


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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 thetarget template(s), the sequence determination of the primers and the test

conditions and criteria.

Validation


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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


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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.


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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 (SOPs) will be required for not only the test methods used,

but also for the calibration and maintenance of instrumentation and equipment

used.


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Proficiency testing

Proficiency testing is the means used to determine the capability

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

Such testing will also contribute to ensuring that within or

between laboratories performing routine diagnostic services, aspecific assay is performed according to established international

standards (external proficiency testing


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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.


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Negative control samples

A negative control consisting of all reagents except extracted sample DNAor 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 confirmabsence 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 andtest material.

PCR 5 Lab Setup

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