Okay.

Who knew my previous post on rapid antigen tests would do so well. 😅

So while riding this brief increase in traffic, I’ll follow it up with posts describing the theory behind a COVID-19 PCR test.

But given how large a topic this is, I’ve broken it up into sections.

Part 1 will describe how your sample goes from a swab to pure genetic material that can then be analysed using a PCR.

This part is called sample extraction.

Okay, so the previous post covered how to take swabs (briefly).

When the swab comes to the lab, the first thing you need to do is to inactivate the virus and extract the genetic material from it.

The swab is going to be covered in gunk from your body (again- ya grub!), and leaving that gunk will wreak havoc on the PCR later because the reaction can get ‘clogged up’ from all this extra material.

So the best way to proceed is to purify your genetic material from your sample.

And we have to do this safely.

(Skip ahead if you just want to know the actual process, rather than the equipment)

Enter- the Biosafety Cabinet (BSC).

Also called ‘the Hood’. 🕶

I won’t go into the exact detail of how it works, but essentially these BSCs use airflow and filters to draw air into the cabinet from the front- but never the other way around.

The ‘dirty’ air inside the cabinet is pumped out through a HEPA filter, so that nothing sinister can escape. It all gets trapped in the filter. You also sanitise in between uses by exposing the workspace to UV for at least 20 minutes.

This means that, with correct usage, the user is protected from any infectious materials that might be handled inside the cabinet.

For COVID-19, a class II BSC (BSCII) is sufficient for use during the extraction process. You can also get BSCIs and BSCIIIs depending on how infectious or harmful your materials are.

Inside your BSCII workstation, you’ll need the following items.

• Some micropipettes

• Some tip boxes for said micropipettes

• (Assuming we’re dealing with COVID) An RNA extraction kit

It should go without saying, but if your genetic material is DNA, then you’d use a DNA extraction kit. 😅

• Some tubes to store your samples in (both for short term while doing the extraction, and long term for storage)

• Tube racks to hold all your stuff

• A waste discard of some sort
  • This can be a autoclavable plastic bag, a container, a tube- ideally something large enough to easily fill with waste without having to constantly touch it
  • It’s good to lay some sort of absorbent pad in it as you’ll be handling potentially infectious liquid/s

And then there’s a bunch of optional items to have inside the BSCII. If you don’t have it inside the BSCII, you’ll at least need it on a nearby bench in the same workroom.

• A microcentrifuge

This helps you spin the tubes during the extraction process- the speeds can be around 17,100 x g, which… it’s fast. 😂😅 If it can pellet small bits of protein in suspension, you can imagine what would happen to you if you were inside it.

• A vortex mixer

For some reason, of all the things that blow people’s minds when they first go into a lab- this is in the top three.

It just helps us mix liquids very quickly. You can use the dial to adjust the speed. You can have it auto or manual… I dunno- it just blows students’ minds. 🤯

But each time we add a liquid into a tube, we always vortex for a few seconds to ensure that the liquids have mixed evenly. Saves us trying to mix it by hand.

• A tabletop minicentrifuge

These don’t spin anywhere near as fast as the microcentrifuges, but they do help you quickly pool liquid down to the bottom of the tube. Every time we open a tube, if there’s liquid at the top of the lid, it could potentially contaminate our fingers and hands, or our equipment. We don’t want to get potentially infectious material all over ourselves or risk cross-contamination (see below), so it’s good practice to centrifuge and pool all liquids down to the bottom of the tube- away from the lids.

• A large waste bin for all of these things.
  • Not a puncturable plastic bag- a large bin lined with two layers of autoclave bags
  • The waste gets collected and once the bag is full, it can be taken down and autoclaved
  • After autoclaving, all the waste ends up in landfill (science is not eco-friendly)
• Some sort of disinfectant for the occasional spill
  • We’re not perfect, and sometimes spills do occur
  • Usually we just use 70%+ ethanol to dowse the spill and then wipe it up
• And finally- a 4 degrees Celsius fridge and a -20 degrees Celsius freezer
  • Long term sample storage will mean you need a -80 degrees Celsius freezer as well

Now, on top of all of that lab equipment, you also need some basic personal protective equipment (PPE).

You’ll need:

• A lab gown/coat (disposable or something that can be disinfected upon washing)
• Safety glasses
• Disposable gloves
• Closed toe shoes

Okay! Still with me? I think we’re about ready to start the whole process. 😅

The extraction process relies heavily on having a manufactured kit. I’ve only used QIAGEN kits myself, but you can even make the reagents and buffers yourself- only the yield (i.e. how much good quality genetic material you extract) might drop. You’ll still need the other non-liquid consumables from the kit, too. You can’t make those yourself.

The kits automatically come with the spin columns and collection tubes that you’ll need to isolate your genetic material.

Basically there’s two tube materials. One sits on the outside, and it’s called the collection tube because it ‘collects’ the liquid at the bottom.

The second tube sits internal to the collection tube, and is called the spin column. This smaller tube contains a filter (shown in white in the figure above), and this filter traps and binds genetic material (RNA) in your sample. All the other debris gets washed through during the extraction process. Once you’ve done a few washes to remove any other debris, you can collect your genetic material at the bottom (elution), and hopefully you have a relatively clean bit of purified genetic material to use for your PCR.

Let’s go through the overall steps involved in the extraction process, and what each step is doing.

Lysis (virus inactivation)

It’s going to be a bit of a problem if the virus remains active (i.e. infectious) throughout this whole process- so the first thing we need to do is to break it open and ‘inactivate’ it, so that it’s no longer infectious/harmful to us.

I mentioned in my previous post that the best way to break open a virus is to add detergents.

Ignoring the antibodies for a second, but the same theory applies here for the extraction process.

Now that the virus is safely inactivated, we can move onto the next step.

Adding sample to the filter (binding), and then doing all the wash steps

The first thing we need to do is to add the sample to the spin column, so that we can filter out the genetic material from the rest of the viral material and human gunk.

You pipette the sample into the spin column, and as the sample passes through the filter, the genetic material becomes embedded in the filter material, while the rest of the gunk passes through and ends up in the collection tube. Once you discard the collection tube with the waste, you can replace it with a fresh one. Neat, right?

Now, don’t ask me exactly how the filter binds to the genetic material. That’s beyond my general understanding and it’s also just… too much information. 😅 It’s basic chemistry, essentially, but it’s too complicated for this post.

Anyway-

If we waited for the filtration to happen by gravity- we’d be here forever.

So to speed this process up, we use the microcentrifuges to spin the tubes. The centrifugal force will push the liquid through the filter and into the collection tube in a matter of… well- a minute. That’s all it takes to sufficiently pass a sample through the filter in the spin column.

To ensure that there’s no more gunk left in the filter, we also perform a bunch of washes using an ethanol-based solution. We just add it to the spin column, centrifuge the whole thing, discard the waste from the collection tube, and then repeat.

The thing to be really careful here is to avoid touching any of the liquid with your fingers, because it might be infectious.

But most importantly– You also don’t want to get samples on your fingers, because if you’re working with multiple different samples, you could cross-contaminate them. If you get a little bit of sample A on your fingers, then handle sample B, then your sample B because sample A+B! If sample A had virus, but sample B didn’t- now it will, because you’ve contaminated it.

The most important thing to be careful of throughout this entire process is contamination.

You just need to be aware of what you’re touching and where you’re touching them. You need very dextrous/steady hands, that’s all. Just a matter of practice.

And if all else fails, just change gloves, change pipette tips, or decontaminate using ethanol. Clear your head, and start again.

Once the washes are done (usually a two stage process), we always spin again one more time, with no addition of liquids, just to dry the filter of any excess ethanol.

Elution (i.e. harvesting the purified genetic material from the filter)

At this point in the process- the genetic material is sitting out of solution, bound to the filter.

To remove it, we need to bring the material back into solution, and this can be done fairly easily.

You can add enzyme free water, as in, water free from anything that might break down your genetic material.

Or you can add the elution buffer that comes with the kit. This is more likely to preserve your genetic material the best.

Either way, it’s as simple as adding it to the column. Letting the liquid sit with the filter for a minute or two, and then spinning the tube once again- making sure you now have a tube for storing your sample, not just a waste collection tube. Definitely don’t forget to change tubes!!

If you’re going to use the sample straight away (within the following few hours), then keeping it in the fridge is fine. But if you’re going to leave it for a few days, the -20 freezer is better.

For long term storage, you should keep it at -80. Just to be safe.

Now, all of these basic steps are used when you have a small number of samples.

But what if you have to process thousands upon thousands of samples per day?

Well- you can automate the whole thing by using machines.

This robot here will do all of that process on its own. All you need to do is to set up the samples, plates, lysis buffers, wash buffers, and elution buffers beforehand.

At such an industrial scale- you’ll no longer be using individual tubes that you hold in your hands. You’ll be using plates that have hundreds of small wells that act as the tubes.

But these machines are expensive- so unless you have the money and resources, it’s difficult to obtain.

They still work on the same principles, though.

Hope you liked part 1 – extraction!

Part 2 will explore how this extracted genetic material is then prepped for a PCR. Stay tuned!

Categories: General

Tagged as: #PCRTest COVID19Aus COVIDTesting LabLife RNAExtraction scicomm STEM

ABugsLife

A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬