INFECTION DIAGNOSTICS :: CROSS-CONTAMINATION
Preventing cross-contamination in a molecular laboratory
By Jeanne Rhea-McManus, PhD, MBA, DABCC, NRCC P
olymerase Chain Reaction (PCR) is a standard laboratory technique used to amplify a specific sequence
of DNA many times over. This technique has become an important tool in clinical laboratories for the detection of infectious pathogens that do not grow in convention- al culture media.1
powerful techniques2
PCR-based methods are ; with the ability to
detect a single molecule of DNA, disease can be detected at extremely low levels. Such sensitivity, however, makes PCR highly susceptible to cross-contamina- tion, particularly from exogenous DNA sources. The most common sources of contamination include sample to sample transfer and amplicon carryover.3 The consequences of contamination can
be severe and include erroneous results, extensive investigation to identify the source of contamination, cleanup leading to laboratory downtime and delays, as well as loss of credibility. Implementation of safeguards and strict adherence to robust protocols such as those summarized below is often sufficient to ensure that cross- contamination in the molecular laboratory is a rare event.
Laboratory Construction The layout for a molecular laboratory should be designed to allow for areas dedicated for sample and reagent prepa- ration and for amplification reaction and detection activities (e.g., agarose gel elec- trophoresis, when necessary).4
Ideally, a
molecular laboratory would be designed with three or four separate rooms, de-
pendent on the PCR method used.5 At
a minimum, it is recommended to have two separate rooms defined as a pre-PCR/ clean room where sample preparation steps such as DNA/RNA extraction and PCR preparation occur, and a post-PCR/ dirty room where PCR reactions and other post-PCR analyses are performed.5
In sce-
narios where space does not permit dedi- cated rooms, separate benches should be identified for pre-PCR activities (reagent preparation, sample preparation) and post-PCR activities (PCR amplification, PCR analysis). As an additional control when all PCR steps are performed in the same room, timetables can be established where pre-PCR and post-PCR steps are performed at different times of the day.4 Reagent Preparation room.5
The cleanest
of all spaces used for PCR, this room is dedicated to activities such as prepara- tion and aliquoting of reagent stocks. In addition, reaction mixes may also be prepared here, excluding the addition of the extracted nucleic acids from the clinical sample. To reduce the possibility of con- tamination, extracted DNA/RNA and/or PCR products should never be present in the reagent preparation room. Sample preparation room.5
Because it
is important to keep PCR products away from where clinical sample extraction occurs, this room is generally split into two areas: one dedicated to DNA and/or RNA extraction activities and one dedicated to nucleic acid addition to the PCR reaction mixtures. The reverse transcriptase step of reverse transcription (RT)-PCR is per-
R eag en t P rep arati on R oom S amp le P rep arati on R oom P C R Amp li fi c ati on R oom P C R An aly si s R oom
formed in this room, as well as the addition of DNA or cDNA, and positive controls to the PCR reaction mixture. Ideally isolated nucleic acids are added to PCR reaction mixes in a laminar flow hood. PCR amplification room.5
PCR in-
struments (i.e., thermocyclers) used for amplification steps are kept in this room. Additional analysis is not required for real time PCR, and tubes containing RT-PCR products should never be opened. Post-PCR analysis.5
When agarose gel
electrophoresis is required, it should occur in a dedicated room/area as it will be contaminated with amplicons from the PCR reaction. A laminar flow hood is recommended for any steps requiring the opening of tubes containing amplicons. To control for contamination, it is also criti- cal that personnel performing post-PCR analysis not return to pre-PCR work areas in the same day.
Engineering Controls Air flow.6
The reagent preparation room
should have positive air pressure to keep contaminants out. In contrast, the post-PCR analysis room should be under negative air pressure to keep amplicon contaminants in. When possible, the air handlers for the different PCR areas should be connected to separate air ducts, with each leading to a separate location for exhaust. Air lock doors. Ideally, a vestibule pro- vides the main access point to the clean room and is constructed with two or more doors. The interlock function allows only one door to be open at a time. By prevent- ing both doors form being open simultaneously, air and airborne particulates are unable to enter or exit the airlock, thus reduc- ing the opportunity for cross-contamination.
Laboratory Operation UnidirectionaI.5
Dedicated PPE Dressing Area Dedicated PPE Dressing Area Dedicated PPE Dressing Area Dedicated PPE Dressing Area All steps
Ideal layout for a molecular laboratory to reduce contamination events. This layout ensures separation of clean reagents and equipment from exog- enous DNA sources (i.e., samples and/or amplicon containing material).
40 MAY 2022
MLO-ONLINE.COM
should occur sequentially moving from clean room to dirty room. For example, PCR protocols should begin in the reagent preparation room, move to the sample preparation room, and end in the PCR amplification or post-PCR analysis room.
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