Detecting and identifying any virus depends on collecting a small sample of DNA/RNA from the patient and checking that it has the same structure as the suspected infection. Unfortunately, there is not usually enough DNA/RNA in the collected sample to get a pattern using gel electrophoresis (known as a Southern blot for DNA and a Northern blot for RNA), so it has to be ‘amplified’ by a process called the polymerase chain reaction (PCR). PCR only works on DNA, so where the genetic material is RNA (as with SARS-Cov-2) it is necessary to make an equivalent DNA by a process called reverse transcription.
PCR uses an enzyme and a soup of the nucleotides (the building blocks of both DNA) and replicates the sample over and over again until there is enough to give a meaningful analysis.
The ‘reagents’, which are getting a lot of attention are primarily these nucleotides. They are not basic chemicals, but sophisticated chemical structures that have to be manufactured in their own right. The good news is that although a large number of molecules are necessary for effective amplification, they still only make up a small mass, so a reasonably sized plant can make enough for about 1,000,000 tests in a day.
It is worth noting that it takes about 45 minutes to go through the 40 cycles of amplification needed – this would produce over a trillion copies of the original molecule of DNA.
And PCR machines cost from £2500 upwards.
'Reagents'
- Target DNA or RNA – collected using a nasal swab in the case of COVID-19
- PCR ‘primers’
PCR primers are synthetic DNA oligonucleotides of approximately 15–30 bases – they act as the start of the process of duplication
- Nucleotides – the building blocks of DNA
Deoxynucleoside triphosphates
- dATP, Deoxyadenosine triphosphate
- dCTP, Deoxycytidine triphosphate
- dGTP, Deoxyguanosine triphosphate
- dTTP, Thymidine triphosphate
- PCR buffer
This provides a suitable chemical environment for activity of DNA polymerase by keeping the pH between 8.0 and 9.5 where the enzyme works best and is often stabilised by Tris-HCl (2-Amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride. For Taq DNA polymerase, a common component in the buffer is also potassium ion (K+) from KCl, which promotes primer annealing.
- Taq polymerase
Taq polymerase is a thermostable DNA polymerase (it makes copies of DNA molecules) named after the thermophilic eubacterial microorganism Thermus aquaticus, from which it was originally isolated by Chien and co-workers in 1976. It is this enzyme that does the business, and the fact that it can tolerate higher temperatures is important for the process.
Hardware
The PCR machines are thermal cyclers. They take the target DNA up to a higher temperature so that it ‘denatures’ (the double helix uncoils), then lowers the temperature so that the polymerase can make a copy of the original DNA. It then repeats the process and each time doubles the number of molecules.
Once the target DNA has been amplified, it can be identified using gel phase electrophoresis – to give the standard spot patterns beloved of the crime dramas. We know what the pattern that identifies SARS-Cov-2 is, so a match means the original sample was from an infected patient.
Quality and Reproducibility
The early issues were, in part, due to the need for standards and quality control.
In the early stages, Public Health England (PHE) focused on establishing a small number of large laboratories where quality and reproducibility could be assured. They centralised the UK supply of hardware and reagents and were distributing on a regional basis to ensure quality control.
- Hardware
Initially, the Thermofisher thermocycler was declared as the PCR machine which should be used. Many universities donated them to be used at the Milton Keynes laboratory.
- Reagents – Taq polymerase
There are many variations of Taq with specific functions and ‘add-ons.’ It was important to standardise the specific Taq that should be used. Once again, other laboratories donated their supply. Because of the high demand, it was important to coordinate to avoid deterioration and to guarantee storage history.
Most of the reagents can be obtained through many distributing companies but there appears to be a limited number of actual manufacturers – perhaps only half a dozen.
Hoffman-LaRoche, ThermoFisher, Sigma Aldrich and Promega all supply reagents and Taq.
Most are sold as kits (with the polymerase arriving with buffer and nucleotides – this is sometimes referred to as a mastermix). These are mostly bought from distributors rather than the primary manufacturers.
- Swabs
Specific swabs need to be used in the testing and this again partly comes down to standardisation. Initially, there was a shortage of the right swabs.