Importance of UV irradiation to prevent PCR contamination
January 16th, 2018
This blog post looks at some of the benefits of UV decontamination as well as its limitations for PCR work. The principal sources of contamination are aerosolized PCR products that settle on surfaces, equipment and consumables which are then used for PCR. Cleaning flat surfaces with strong oxidizers such as NaOCl or EtOH on a routine basis is a good practice. However it is not practical or time efficient to immerse or wipe down a box of pipette tips, a tube rack, mag stirrer, or a box of gloves.
A solution to these issues is to expose all work surfaces, equipment and consumables to UV which cause adjacent pyrimidines to undergo dimerization (1, 2), eliminating nucleic acid contaminations. UV light is pervasive and covers almost all exposed surfaces (flat, round, overhanging) and its effectiveness on reducing amplicons over time is well established (3). UV irradiation is also more thorough and practical than chemical immersion for equipment and consumables.
There are two important principles to understand the efficiency and limitations of UV decontamination:
Reciprocity law: The dose of radiation to which microorganisms (including dry or aqueous Nucleic Acids) are exposed to is equal to the intensity of the radiation multiplied by the duration of exposure. Equal doses of UV radiation have the same disinfecting action. Therefore, exposure to 100uW.cm2 for 10 seconds has the same killing power as exposure to 50uW.cm2 for 20 seconds.
Inverse square law: As with visible light, the intensity of UV radiation diminishes as the square of the distance from the source. For example, 20 cm from a point source the intensity of the radiation would be only (1/2)2 or 1/4 of what it was at 10 cm, and at 40 cm it would be (1/4)2 or 1/16 of the level at 10 cm.
And of course, as with visible light, UV radiation is blocked by non-transparent objects which create shadowy areas where there is poor disinfection, or even none at all.
It is common to use standard tissue culture laminar flow hoods for UV irradiation but they often cannot deliver a high enough dosage or have poor efficiency of germicidal UV. This means there is a high chance they will not completely decontaminate surfaces, equipment and consumables used for PCR.
PCR Workstations with dual UV bulbs are designed to decontaminate any nucleic acid contamination present on the surface of equipment and consumables by delivering up to 800uW/cm2 of UV to the work surface inside the workstation, including shadowy areas, thanks to the separated dual UV source and interior reflective surfaces which create multi-directional cross-beam UV exposure. PCR Workstations are also Dead Air Boxes which protect against cross or airborne contamination by limiting exposure of experimental set-ups to open lab environments.
UV irradiation is the most efficient method to eliminate DNA contamination, especially for PCR work, but there are some limitations and it is important to use powerful light sources and highly reflective surfaces to ensure every area, piece of equipment and consumable is irradiated. In addition to cleaning with regular strong oxidizers and working in a dead air environment, appropriate UV disinfection can prevent PCR contaminations.
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