RT-qPCR

See our quick YouTube guide to SARS-CoV-2 RT-qPCR (also available with French and Portuguese subtitles on the PANDORA-ID-NET YouTube channel).

Protocol for Qualitative detection of SARS Coronavirus-2 (SARS-CoV-2) by reverse transcription quantitative PCR assay (University of Tubingen).

Extraction of viral RNA

It is important to ensure extraction of viral RNA is done under the appropriate health and safety conditions in a laboratory.

Current WHO guidelines state this should be done in a minimum of BSL2 laboratory, inside a microbiological safety cabinet with the appropriate personnel protective equipment.

There are a number of different viral RNA extraction kits available, although you may find, depending on your country, that some are more available than others. Whilst we are aware that this may be the only option for some smaller laboratories, we don’t recommend manual extraction, due to the increased exposure risk to laboratory workers.

You will need an internal control (e.g. human housekeeping gene such as Ribonuclease P) to ensure your extraction procedure is working properly.

The following links to a publication comparing Automated and Manual Nucleic Acid Extraction Methods for Detection of Enterovirus RNA.

Nucleic Acid quantification

Quantification of your nucleic acid after extraction is important for the validation and verification of your diagnostic assays. There are a number of different kits available using different technologies.

The following links to a publication of a comparative analysis of three methods used for RNA quantitation.

Qubit RNA HS users guide  |  Aglient Tapestation RNA quick guide

RT-qPCR – how to choose your assay

There are many different RT-qPCR kits that can be used for diagnosing COVID-19. The one(s) you choose may depend on the availability and/or type of thermocycler you have. There are commercial kits (these will have been validated by the company already and are widely used, but may be difficult to procure due to demand) and in-house kits (these will likely require some validation and optimisation steps before use, but are more likely to be available and enable flexibility).

At the beginning of the outbreak, WHO supported access to COVID-19 in-house PCR protocols assays by posting them online on the WHO website. Protocols that have been shared can be accessed here. Being listed in this document does not imply any endorsement or validation by WHO. These protocol postings online are not being updated and may not reflect subsequent refinements of the assays.

FIND have updated the SARS-CoV-2 molecular tests that it is independently evaluating.

What genes to target?

Commercial kits come with primers included, but if you are planning to use an in-house assay, primers have been designed for the following genes:

Gene name

Description

Used in the following protocols (WHO summary):

Potential issues

RdRp

RNA-225 dependent RNA polymerase

Institut Pasteur Paris (France) (2 targets) Charité (Germany)

Has been shown that some SARS-CoV-2 viruses have mutations in the primer regions of this gene and may not be picked up by RT-qPCR, giving a false negative

E

Envelope protein

Charité (Germany)

These primers also target SARS-CoV-1 and therefore is not specific to the current COVID-19 SARS-CoV-2 virus. However, as SARS-CoV-1 is currently not thought to be circulating, a positive result from this gene can be assumed to be SARS-CoV-2

S

Spike protein

National Institute of Infectious Diseases (Japan)

 May have limited sensitivity.

N

Nucleocapsid protein

China CDC (China),

US CDC (USA) (2-3 targets)

Charité (Germany)

HKU (Hong Kong SAR)

National Institute of Health (Thailand)

Commonly used in WHO recommended assays. Has been shown that some SARS-CoV-2 viruses have mutations in the primer regions of this gene and may not be picked up by RT-qPCR, giving a false negative

ORF1ab

Open reading frame 1a and b 226, includes the RdRp; RNA-dependent RNA polymerase

China CDC (China)

HKU (Hong Kong SAR) (ORF1b-nsp14)

Considered less sensitive than other targets.

NB don’t forget to also order primers for the internal control (human housekeeping gene, e.g. RNaseP) to ensure that your extraction has worked. If you are using a commercial kit, check which primers are part of it.

Of interest to note that sequencing suggests that there are mutations in the binding sites for RT-qPCR primers.

Validation of RT-qPCR

Integrated DNA research has provided tips on how to validate your qPCR.

Guidelines for validation of qualitative qPCR  |  Checklist for Optimization and Validation of Real-Time PCR Assays

Bio Rad qPCR assay design and optimisation

Pooled PCR might be of use in surveillance studies, where the infection rate is relatively low, e.g. in the community.