PREVENTION OF CONTAMINATION IN PCR LABORATORY

PRESENTER- DR. VIDHYA

BATCH -3

Polymerase chain reaction or PCR is a

• simple laboratory technique

• obtain multiple copies of specific DNA fragments even from samples containing only minute quantities of DNA or RNA.

CONTAMINATION IN A PCR LAB

• Remains an issue

• Stringency measures depend on the assay being performed.

• the most stringent contamination efforts- detection of infectious agents.

Amplicon aerosol -

• single most important source of PCR product contamination

• Post-PR analysis leading to generation of aerosols of PCR amplicons

Target template contaminants

• target template can be a source of contamination

• DNA templates are more troublesome as contaminants

Regardless of the template to be detected, good laboratory practices should be followed

Approach to contamination prevention

• Kwok and Higuchi (1989)- additional guidelines for researchers using the PCR.

• Essential for successfully operating a PCR laboratory on a long-term basis to maintain a PCR lab contamination free.

PCR ACTIVITIES

• Pre-PCR activities (sample preparation and PCR preparation)

• Post-PCR activities (PCR execution and analysis)

Contamination in PCR primarily because of:

1. Generation and spread of aerosols

2. Contaminating materials present on hands, clothing or hair introduced into PCR mixes

3. Fomites – Laboratory coats, gloves, vortexers, pipettes

4. Circulating PCR amplicons (aerosols/fomites)

5. Positive control specimens

6. DNA clones used for synthesis of positive control material

CONTAMINATION CONTROL PROGRAM

• space and time separation of pre- and post-PCR activities

• use of physical aids

• use of ultraviolet (UV) light

• use of aliquoted PCR reagents

• incorporation of numerous positive and negative or blank PCRs

• use of various contamination control methods that use chemical and biochemical reactions.

Space and Time Separation

• separating the source of the amplicons i.e. post-PCR activities from the pre-PCR activities

• different areas designated for sample preparation and PCR setup -located away from the area for post-PCR analysis

In a single room,

• sample preparation- inside a laminar flow hood equipped with a UV light.

• walls of the hood should be wiped with a disinfectant before processing or preparing samples

• Establish a daily schedule for performing PCR

Laboratory Space Arrangement

• Separate rooms each with dedicated resources.

• An alternative to two-facility arrangements is Real-Time PCR methods.

• the results provided throughout the reaction- no need to open the containers, and can be discarded while still sealed.

Equipment in PCR Laboratories

• each room must have its own separate set of equipment, including pipettes, reagents, pipette tips, racks, and so forth

• should not leave the area to which they are assigned.

• should be labeled as to location and used in that location only.

• Lab coats should be dedicated for both areas

• Each area needs its own dedicated pipettes

- never exchanged between work areas

- color-coded pipettes can be used.

- pre-PCR pipettes and tips when not in use, should be stored in airtight bags to keep them clean.

• Reactions should be constructed using master mixes-

- prevent cross contamination

- template should always be added last using positive displacement tips.

Pre-PCR Activities-

• Automated better than manual

• positive displacement tips or barrier tips should be used to pipette the template (last step)

• Performing in a hood or

bio-safety cabinet

• Use of UV irradiation

Environmental Considerations-

• Air handling- to prevent contamination from the air being recirculated between the pre- and post-PCR labs

-air handlers to be separate

-air handlers for the pre- and post-PCR laboratories connected to separate air ducts, each lead to a separate location for exhaust.

-air pressure individually adjusted in each laboratory.

-slight positive pressure in pre- PCR compared to the air in the connecting hallway

-post-PCR lab, at slightly reduced pressure - prevent escape of amplicons

• UV irradiation-

- nucleic acid are sensitive to UV

- UV lights to sterilize the entire pre-PCR laboratory.

- Accompanied with a ventilation system to remove the UV generated ozone

- Precipitation of oxidation products on the glass needs to removed

every month

• Protective clothing-

- Dedicated post-PCR lab coat

- Extreme cases- disposable gowns and boots

• Adhesive paper at lab entrances-

- Prevents trace amounts of dust and debris from entering

- expensive

Sterilization of Reagents-

• single most critical agent reagent is water

• filtering it through two 0.45μ nitrocellulose filters (very high binding capacity for nucleic acid and proteins).

• reagents and solid items destined for the pre-PCR lab should be autoclaved separately.

• large potential reservoir of contaminating DNA- should be autoclaved separately from any material that will enter the prePCR lab.

Contamination Control

(1) methods that use physical means

-to prevent dispersion of PCR amplicons

(2) methods that exploit chemical reactions to render the amplicons incapable of serving as templates in a new round of PCR.

most successful PCR laboratories use a spectrum of these methods to effectively control contamination

1. Physical Methods

• physical barrier approaches- prevent the reintroduction of small amounts of a contaminating aerosolized sample into the next sample

• positive displacement or barrier pipette tips

• recommended in the prePCR areas where samples are processed and template nucleic acids, isolated and purified.

• necessary and cost-effective in the post-PCR lab as there is already a large amount of amplicon present.

• adjunct - use of a laminar flow hood / biological safety cabinet –

- less chance of an external source of PCR amplicon contaminating the samples and reagents

2. Chemical Methods -

UV photolinking-

• can be used in both pre-PCR and post-PCR setting.

• UV light of 254nm – cross-links adjacent pyrimidines on a DNA strand (Gordon and Haseltine 1982).

• reaction is very fast and effective for bigger amplicons (>700 bp).

• Smaller amplicons - harder to inactivate (fewer adjacent pyrimidines).

• Once cross-linked the pyrimidine dimers cannot be excised DNA polymerase is sterically blocked and DNA cannot completely denature synthesis reaction is effectively halted.

• most often used in a pre-PCR setting.

• All the items are placed inside the cabinet and then illuminated before the PCRs are assembled.

1. safety concern about exposure to UV light.

2. the photoreaction favors thymidine over cytidine by about 10:1

- amplicons that are AT-rich are more efficiently disabled than AT-poor sequences.

3. short amplicons are not well controlled.

The approach is nevertheless effective and should be used when possible.

Uracil-DNA-glycosylase (UDG)-

-effective at destroying PCR amplicons when vigorously used for sample preparation (Longo et al. 1990;Thornton et al. 1992).

- pre-PCR step- dTTP substituted with dUTP, and UDG is included in the reaction mix.

- All other reaction components remain same.

TO SUMMARIZE…

• Compulsory wearing of a laboratory coat and disposable latex/ neoprene gloves and changing the laboratory coat and disposing the gloves upon moving from one area to the next in the PCR

• Maintaining strict unidirectional flow of material and personnel

• Keeping all bottles and reaction tubes sealed for as long as possible.

• Using as few manipulations as possible.

• Avoiding vigorous and excessive vortexing, pipetting and spillages in the PCR areas.

• Centrifuging vials containing specimens, nucleic acids, PCR mix ingredients and PCR amplicons, before opening them.

• Aliquoting reagents into single-use volumes

• Always perform No-Template-Control (NTC) reactions to check for the absence of contamination.

• Autoclaving /incinerating contaminated utensils (glassware, reaction tubes, pipette tips, buffers etc.)

• Cleaning the lab benches periodically with 1N HCl or expose the workstations overnight to UV light.

• Working within an easily-cleaned Biological Safety Cabinet or PCR workstation, preferably one with a built-in UV source.

• substitution of dTTP in PCR mix by dUPT and using UDG in the subsequent reactions.

In the worst case scenario of PCR contamination it is advisable to:

• Shut down the offending PCR laboratory

• Discard all the working solutions

• Thoroughly decontaminate all work surfaces, laboratory coats, pipettes etc.

• Order new supplies of PCR reagents

• Mifflin TE. Setting up a PCR laboratory. www.biosupplynet.com/pdf/01

• Establishment of PCR laboratory in developing countries. World Health Organization (WHO), 2011