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Contamination is a tough issue that concerns almost every clinical laboratory and research lab. LAMP is a simple molecular test, requiring little hands-on work and some expertise, but that doesn’t mean contamination doesn’t exist there either! As a matter of fact, in low tech testing settings and especially when using LAMP, contamination must be taken extremely seriously. An ounce of prevention early can save you a ton of trouble later on! In this article, we’ll go over how such contamination can arise, what to look out for and how to prevent it in testing setups and also what to do once you notice you have some contamination problems.
LAMP, just like PCR and other nucleic acid amplification methods, works by making more copies of a specific segment of DNA based on a template of the same nucleic acid sequence. Billions of these copies are generated in a single, tiny reaction, leading to a fluorescent or colorful result that we can interpret. These copies of an original template that are generated through amplification are called amplicons, they’re small DNA molecules, chemically exceptionally stable, much more than the RNA of a virus for example. In a true positive test, the DNA amplification kicks off based on the presence of a template sequence in the sample, the viral RNA. If, for some reason, a small amount of amplicons from previous finished reactions gets into the reaction tubes from whatever source, you will get a false positive reaction that wasn’t triggered by the presence of a template sequence from the virus, but from an amplicon. In this case, you essentially can’t separate a true positive from a false positive and your results become unreliable and useless.
While DNA amplicons don’t have legs and can’t jump into reactions that you are preparing on a bench, they can linger on surfaces, be present in reagents, cling to pipettes and your gloves and most of all be in the air in the form of an aerosol. There’s several kinds of danger happening here and you want to make sure you always think about how risky any given action or behavior is when it comes down to contamination, you want to be taking sensible preventative measures and avoid the worst practices that make running into a problem only a matter of time.
A small, low tech LAMP lab and a high-throughput lab have lots of differences. When it comes to contamination though, key things have to be observed even in the most basic testing setups. We’ve established that DNA being generated in these LAMP tests can act as a template for further false amplification, and that’s the key source of contamination. The more reactions you run, the higher the risk is, essentially. Luckily, there’s a few things you can do to substantially decrease the risk.
This is an aspect where a lot of people (including us!) make mistakes out of carelessness. There's two risks you are working with when testing: risks coming from the samples, that you can mitigate by wearing PPE and inactivating the samples in a safety hood, and then risks coming from the finished, non-infectious reactions. If you handle these reactions without any concern, they become a major source of contamination and false positives for your further work. You want to make sure you take out your strips or plates from the incubator after amplification only while wearing gloves, take a picture and record results soon, do not store the finished reactions and dispose of them immediately after you're done with them. Never opening reactions is intuitive, but there can be amplicons on the sides of the tubes or plates, or around the incubator. After handling reactions, trash your gloves as well. Never bring finished reactions onto the workspace for preparing reactions. One could almost say, handle your finished reactions as if they were radioactive, they're not dangerous for your health but can be catastrophic for your testing setup.
is the key precaution. You should have at least two workspaces, one where you prepare LAMP master mixes and pipette reactions, and another one where you actually incubate the reactions and read out and note down the results. The first one is called a pre-amplification workspace, while the second one is, predictably, a post-amplification workspace. You should try to have them separated as much as possible, ideally they are in two different rooms, and you don't carry over things from the pre-amplification workspace into the post amplification one. Clinical labs usually operate on a so-called "three room rule", one room for sample preparation (inactivation, extraction, infectious work), second room for mastermix preparation, and a third one for amplification. Of course, not everyone can have that, but you should have two workbenches at the minimum.
Wear gloves in the lab, even when not handling anything dangerous. It's about protecting yourself, and keeping your workspaces clean. When going from a post-amplificaiton to a pre-amplification area, trash your gloves and take new ones. Before handling fresh reagents that you use to make a mastermix, trash your gloves and take new ones. If you're interrupted during your work and you're not sure if you should change your gloves, be on the safe side, trash your gloves and take new ones. It's fast and cheap, compared to having failed reactions and spending days on troubleshooting.
Beware that ethanol, while being excellent at most other things, does absolutely nothing to prevent DNA contamination. For that, you have to use household bleach, active ingredient sodium hypochlorite. Household bleach is a 5 % solution of sodium hypochlorite and you want to dilute it 1:10, to a total of 0.5 % solution of sodium hypochlorite, for use as a cleaning agent. Dilute the bleach with water, and prepare a fresh dilution every few days - in dilute solutions, the sodium hypochlorite breaks down after a few days, so fresh is always better. Spraying bleach on your bench and letting it do its work for 10 - 20 minutes is most efficient, but for routine cleaning a thorough wiping down of all surfaces with a bleach is also good.
Pipetting skills are a factor as well. Pipetting accurately and preferably using filter tips can significantly increase the quality of your work and decrease the chances of contamination or false positives. Sensitivity and specificity of a molecular test is determined in part by the skill of the person performing in. Practice and following the advice of experienced technicians pays off in this regard.
If you see that your negative control has turned positive, contamination is a certainty. Higher than normal positive rates in recent samples that are negative upon retesting in a different lab are also a clear sign that contamination is present. A contamination problem can be building up for weeks before tipping over into noticeable symptoms, and even then it takes a keen eye and making connections to realize that contamination is what you’re dealing with.
Once you do spot a problem, don’t put it off. You want to find out the following things as soon as possible:
Getting rid of a present contamination is much harder than taking preventative measures. But it’s possible. A key item is to find the source of contamination, and get rid of it. Then, a 10% bleach solution becomes your best friend to solve the problem.
Work in a well ventilated environment and wear a mask and a lab coat – bleach is nasty, the fumes are irritating and the droplets can bleach your clothes. Avoid bleaching aluminium surfaces.
For maximum effectiveness, mix the bleach fresh before every use and leave the bleach on surfaces for 10 minutes. Wipe down your reagents, door handles, pipettes and machines with bleach on tissue paper. If contamination is coming from the pipettes, find out if it’s possible to disassemble the pipettes (from the documentation or by contacting the manufacturer) and if so, soak the barrels of the pipettes in bleach overnight. Ideally, incorporate a bleach-cleaning step into your daily or weekly routine, depending on the severity of the problem. Persistence is key when getting rid of contamination.
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