One of the great advantages of PCR is its exquisite sensitivity. Starting with small fragments of nucleic acid (primers or oligonucleotides), more than 10 million copies of RNA or DNA molecules can be synthesized from a few copies of target sequence after only 32 cycles of amplification.1 The sensitive nature of PCR allows scientists to extract and amplify DNA from raw samples to obtain useful DNA profiles. While this level of sensitivity works in a lab’s favor, it can also create problems if care is not taken to avoid contamination with other templates and amplicons (amplified DNA product from a previous amplification) that may be present in the laboratory environment. These contamination issues can result in the wrong template being amplified, i.e., false positive results.
Here we discuss ten ways to control or minimize contamination in a molecular laboratory.
1. Laboratory Construction
Contamination prevention starts with the construction or set-up of a PCR laboratory. At a minimum, two areas should be designated for PCR testing: Pre- and Post-PCR. One room or area should be designated specifically for Pre-PCR. Optimally, this room should be further divided into two areas, PCR master mix preparation and sample preparation/addition to master mix.
- Master mix preparation can be separated from sample addition by using a Dead Air Box (DAB) with UV light. The DAB houses consumables and dedicated small equipment (mini-centrifuge, vortex, pipettors, tips, and tubes) needed for master mix preparation.
- Sample preparation may involve a manual or automated extraction. Consumables and small equipment (mini-centrifuge, vortex, pipettors, tips, and tubes, etc.) needed for sample preparation could either be on the bench top, in another DAB, or in a Biosafety Cabinet (BSC) depending on the type of samples with which you are working. This area would also be used for adding sample to your PCR master mix.
- There should be one dedicated refrigerator/freezer for kit storage and another for storage of samples.
A second room (Post-PCR) should be established for post amplification steps and analysis. This room should be physically separated from the pre-PCR room. The post-PCR room is where the thermal cyclers for amplification and any instrumentation needed for post-PCR analysis (e.g. Luminex® 100/200™, MAGPIX®) should be located. Ideally, a DAB would be used in this area for any steps that require manipulating open tubes after PCR amplification. The DAB should house consumables and small equipment (mini-centrifuge, vortex, pipettors, tips, and tubes) needed for post-PCR preparation.
2. Environmental Control
These two areas (Pre-PCR and Post-PCR) should have independent environmental control and not use common ductwork for air conditioning. Moreover, both rooms should be equipped with air-lock doors. Note: If physical barriers or separate rooms cannot be established for Pre-PCR and Post-PCR work, all efforts should be taken to set up the work areas as far apart as possible. Lab techs should treat these two work areas as if they are in separate rooms. Care should be taken to wear different personal protective equipment (PPE) in each area.
3. Unidirectional Workflow
The workflow of a molecular lab should continue in one direction only, i.e. Pre-PCR > Post-PCR. PCR master mix reagents and samples that may contain templates for PCR should be prepared in the pre-PCR room only.
Tubes that have undergone amplification in the post-PCR room contain amplicons (amplified template) and should never, under any circumstances, be opened or introduced in the pre-PCR room. Amplicons can serve as template for future PCR reactions and therefore could easily contaminate PCR or sample preparation reagents, consumables, or equipment.
This means that consumables and PPE (lab coats, gloves, goggles, etc.) that have been introduced into the post-PCR room should never be placed back in the pre-PCR room without thorough decontamination.
When moving from room to room, a lab tech must remember to change PPE. Ideally, technologists who have worked in Post-PCR should not go back and work in Pre-PCR. If one must go against the unidirectional workflow, care should be taken to change PPE.
4. Dedicated Consumables and Equipment
Another way that we can minimize contamination in a PCR laboratory is by using consumables and equipment dedicated to each room. Each room and/or work area should have its own centrifuge, vortexers, pipettors, gloves, coats, etc. For example, never “borrow” a post-PCR pipettor for use in a pre-PCR room without thoroughly decontaminating the pipettor first.
5. Use of Aerosol-Resistant Pipettes
When pipetting samples, even the most seasoned technician can create aerosols without the proper pipetting technique and pipette tips. Aerosols can lead to cross-contamination from sample-to-sample. Aerosol-resistant pipette tips have a barrier, which acts as a seal when exposed to potential liquid contaminants, trapping them inside the barrier. This protects the pipettes from any liquid contaminants.
6. Pipetting Technique
In any molecular assay, proper pipetting technique is critical to the performance and quality of your results. Moreover, correct pipetting technique can minimize contamination between samples that can lead to false positive results. Proper pipetting technique ensures that the accurate volume is aspirated and dispensed and avoids splashing when dispensing liquid. Open and close all sample tubes and reaction plates carefully so samples don’t splash out. Spinning tubes/plates before opening can prevent aerosols when opening tubes. Also remember to always keep reactions and components capped whenever possible. One of the most efficient ways to prevent cross-contamination is through the use of a good pipetting technique.
7. Frequently Changing Gloves
A lab technician should always wear fresh gloves when working in a PCR area. Change gloves frequently, especially if you suspect they have become soiled with solutions containing template DNA.
8. Aseptic Cleaning Technique
Proper aseptic cleaning should be carried out periodically before and after PCR work. This applies to all work surfaces including bench tops, pipettors, fridge/freezer handles, and any other touch points. We recommend wiping down and soaking these surfaces using 10-15% (0.5-1% Sodium Hypochlorite) bleach, made fresh daily.2 After fifteen minutes, use a DI water-dampened paper towel to remove bleach residue. This can be followed by a 70% alcohol dampened paper towel to help quickly dry the surfaces. In addition, dunking used racks in fresh 10% bleach after use for at least 10 minutes, then rinsing and air-drying overnight can prevent further contamination.
9.Wipe Tests
Periodic wipe tests should be implemented as a standard procedure to proactively monitor the laboratory environment for contamination before it becomes an issue. At a minimum, wipe tests should be performed on a monthly basis. This frequency should be increased if any contamination is suspected. Instructions on how to perform the wipe test can be found on page 21 of the package insert for our xTAG GPP assay. This is recommended by the College of American Pathologists (MIC.64850 Sample/Amplicon Contamination).
10. Positivity Rate Monitoring
It is standard practice to include a positive control to assure proper performance of extraction and amplification and functionality of the reagents. A no-template control (NTC) is used to check for the absence of contamination in the reagents, consumables, and environment. In addition to positive and negative controls, laboratories should establish a standard procedure to monitor positivity rate. Any sudden increase in positive specimens for which a cause cannot be determined (i.e. seasonal outbreaks), should be investigated. This is recommended by the College of American Pathologists (MOL.20550 Test Result Statistics).
We’ve discussed methods to eliminate contaminants and offered tips to prevent contamination in the future. By diligently following these recommendations, you can prevent contamination from ever becoming a problem in your laboratory.
For more tips and tricks, take a look at our Support Resources and our Customer Center.
References
- Pelt-Verkuil EV, van Belkum A, Hays JP. Principles and Technical Aspects of PCR Amplification. Springer Netherlands Publishing, 2008. ISBN: 978-1-4020-6240-7 (Print) 978-1-4020-6241-4 (Online).
- Collecting, preserving and shipping specimens for the diagnosis of avian influenza A (H5N1) virus infection. Guide for field operations. World Health Organization (Internet). Cited 2014 Sep. Available from: http://www.who.int/csr/publications/surveillance/Annex7.pdf.