April 1, 2020

expert comments about reagents needed for COVID-19 swab PCR testing

There have been reports about the reagent chemicals required for COVID-19 testing.

Comments sent out on Thursday 2nd April 2020

Prof Robin May, Professor of Infectious Disease and Director of the Institute of Microbiology and Infection, University of Birmingham, said:

“SARS-COV-2 is an RNA virus, which means that its genome is based not on DNA, but on RNA (a closely-related molecule).  Detecting the virus relies on specifically detecting a ‘sequence’ of RNA that is unique to this virus, converting it into DNA and then amplifying that DNA (essentially, making copies of it) via a process called PCR (polymerase chain reaction).  The conversion of RNA to DNA requires one enzyme (reverse transcriptase) and then amplifying the DNA requires another (polymerase).  Both of these enzymes are biological molecules that have to be produced (typically in bacteria), then purified, usually only in small scale.  Normally, demand for these enzymes is very steady and relatively low, so scaling up to the massive quantities that are required for COVID19 testing takes time.  In addition, it is critical to ensure ‘quality control’ of the extraordinarily large number of kits that must be produced, to minimise the risk of false positive/negatives.  Tackling all of these rate-limiting steps together to eliminate all of the bottlenecks is the primary reason why testing has been relatively slow to roll-out so far.”

Comments sent out on Wednesday 1st April 2020

Prof Stephen Baker, Professor of Molecular Microbiology, Department of Medicine, University of Cambridge, said:

“There are multiple issues with setting up new diagnostic testing capacity outside of regular diagnostic laboratories.  This is not an overnight solution.  Research laboratories have different approaches, equipment and staff.  There are multiple steps to ensure that the methods used and results that are a correctly validated, as there is nothing more dangerous at the moment than reporting incorrect negative or positive results to staff or patients.  That being said we can adapt and validate assays and make them work using our equipment that can then be rolled out to support the NHS.

“Another issue is reagents.  There is a massive demand for raw materials and commercial kits, this is not unique to the UK, and many places no longer have stock of essential reagents.  Different companies provide kits that are compatible with their machines and clearly there is a demand for extraction kits to produce nucleic acid from samples.

“Additionally, given the nature of COVID-19 the samples need to be prepared in a suitable laboratory to reduce the risk of contamination or exposure to staff.  Such facilities may be limited in places, which also may impact on capacity.  Currently, we are trying to create the capacity to assist with diagnostic testing and hopefully we should have the equipment and reagents to continue with this in the coming weeks.  However, for this to be continued and capacity increased we need a sustained supply of reagents for the whole process from swabbing through to virus detection, some guarantee from biotechnology companies that they can provide this would aid in our ability to predict how much screening can be done and for how long.”

Dr Al Edwards, School of Pharmacy, University of Reading, said:

What are these chemicals or reagents?

“There are a lot of ingredients that go into a RT-PCR virus test from a swab.  Any of these might be hard to get hold of in the current rush.  The world is seeing more of these virus tests conducted than ever before.

“This shortage could be the enzymes – called polymerase – that detect the virus.  But they could also be simply the swabs or containers for collecting samples.

“The real catch is – we are very good at making a highly accurate test fast.  But to make that test accurate, you need to keep the ingredients fixed.  So we are likely seeing a shortage of the exact ingredients used in the approved tests.  Although other versions might work it’s not easy to simply switch to a different type.  There can be tiny changes that make the test fail – for example if you use the wrong swab to take the sample, the virus might not be detected.

“Furthermore, it’s hard to square the government suggestion that centrally we are running out of chemicals, with the comment this morning from Matt Hancock that local hospitals can use spare lab space to test staff – how can they do this if there aren’t enough reagents to do tests centrally?”

Prof Lawrence Young, Professor of Molecular Oncology, Warwick Medical School, University of Warwick, said:

What are these reagents?

“The reagents are required to: (i) extract RNA from the sample and (ii) run the test itself which requires primers and probes (synthetic oligonucleotides) and two enzymes (reverse transcriptase and DNA polymerase).

Are they in short supply and if so why?

“So the test is complicated, requires many different synthetic components which probably accounts for the shortages given the unprecedented demand.

Is it the reverse transcriptase enzyme or other chemicals, or nothing?

“I think it will be a combination – RNA extraction kits, the primers and probes, the enzymes.

Why is the UK struggling with this where other countries (e.g. Germany) seem not to be?

“More joined up approach.  Relationship with the biotech industry, more German virologists producing the diagnostic test in January, the central Robert Koch Institute which is able to nationally coordinate.

Is there anything about scientific research that would explain why this is such a challenge?

“No – just requires better national coordination.  Highlights lack of investment in virology over many years.”

Dr Colin Butter, Associate Professor and Programme Leader in Bioveterinary Science, University of Lincoln, said:

“The qRT-PCR test for the virus is detailed here: https://www.fda.gov/media/134922/download

Sorry, rather technical.

“Two main elements of chemistry:

“1. The extraction of RNA from the swab sample.  Many companies make suitable kits for this.  Universities and others will have lots on the shelves.

“2. The qRT-PCR itself: Here is a quite simple description of qRT-PCR:  https://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction

The Reverse Transcriptase, Taq Polymersase, nucleotides and buffers are the “general chemicals”.   Several companies supply these as kits.  The primers and probes are the components that make the assay specific for the virus.  Again, there are several companies that make these to order.   Strictly speaking these are not antigen tests (as they measure viral RNA) but this is a pedantic point.  All of these things are ‘reagents’, but as to which are in short supply, it is not clear.

“Really important is the availability of an antigen test, a lateral flow device (like the antibody test or a pregnancy test).  One has just been announced and no doubt others will follow, see: http://news.mit.edu/2020/covid-19-diagnostic-test-prevention-0312  If accurate this is a complete game changer: rapid, point of care and less expensive than qRT-PCR.  Note that this is a viral antigen test, NOT the much discussed antibody test.

“We should therefore end up with two lateral flow tests, one for the viral antigen (have I got it) and one for the antibody (have I had it / am I now immune?).”

All our previous output on this subject can be seen at this weblink: www.sciencemediacentre.org/tag/covid-19/

Declared interests

None received.