Positive & negative air pressure in PCR lab

[newmanreb]

Hi there! I am with a company who is lucky enough to be expanding our operations into a new building. Our PCR laboratory will feature 3 rooms: A reagent prep room, a sample preparation room (DNA extraction) and a post-PCR laboratory. Since we are designing the building, we get to decide what air flow we need throughout this unidirectional workflow.

Now. It's clear to me that the reagent prep room, the "cleanest room", needs positive pressure to keep contaminants out. Also clear - the post-PCR room needs negative pressure to keep amplicon contaminants IN. What is not clear to me is how to treat the extraction room.

In both of my previous labs, we didn't have the luxury of having a separate room for DNA extraction. DNA extraction was considered a "pre-PCR" activity and done under positive pressure in the pre-PCR area. HOWEVER, upon further research, I've found multiple sources which say the DNA extraction room, being "dirtier" than the reagent prep room, should have negative pressure like the post-PCR room for the reason of keeping DNA inside the room and nowhere else.

This resource, on page 13, shows that the nucleic acid prep room should have negative pressure, and then on page 16 shows an example floor plan with POSITIVE pressure in the extraction room! https://www.aphl.org/programs/newborn_screening/Documents/2015_Molecular-Workshop/Molecular-Laboratory-Design-QAQC-Considerations.pdf

What pressurization would you use in a DNA extraction room: positive or negative?

I have found conflicting sources online about what to do about this. I would really appreciate any insight. For context, our rooms are separated by a shared hallway. My concern with putting negative pressure in the DNA extraction room is that we will pull in amplicon contamination. This is the room in which we will be opening our reaction tubes to add DNA template - I wouldn't want to introduce amplicon contamination at that point. Thank you for any input!

[CharonY]

it really depends on your precise extraction protocols and specimens and I have seen quite a few different setups. If the concern is more about cleanliness, one would obviously opt for positive pressure. Though sometimes the specimen are biohazards or otherwise should not escape the room (e.g. if you work through soil samples you do not want it to get elsewhere) negative pressure would be better. At the same time, it should be noted that the room configuration is more about general concerns. Specific ones can further be addressed by installing clean benches or biosafety benches or zero-flow boxes as appropriate, for example.

But generally speaking, when sensitive tests are run (such as for diagnostics) folks generally are more concerned about getting the test right (i.e. uncontaminated and clean) rather than worrying about stuff getting out. So if you run a lot of RNA samples or amplify from low abundance then most would opt for positive.