Diagnostic Testing for SARS-CoV-2: How governments, regulators and the laboratory-diagnostics industry are responding to the challenge


In this article we examine how standard polymerase chain reaction (PCR) based testing for the novel coronavirus (SARS-CoV-2) works, explore the new CRISPR-based tests under development and the new rapid point-of-care tests being rolled out, and consider the initiatives by governments and medical device regulators to fast-track the availability of SARS-CoV-2 diagnostic tests.

Diagnostic testing capacity has emerged as a key limitation of our ability to contain SARS-CoV-2, the novel coronavirus responsible for COVID-19 disease. Regulatory barriers and shortages of test kits have impeded effective scale-up of SARS-CoV-2 testing. In response to this health crisis the World Health Organization (WHO) has a simple message for all countries: “test, test, test”.[1] Now governments, regulatory agencies and laboratory-diagnostic and biotechnology companies are responding with multiple new SARS-CoV-2 tests being fast-tracked for use, and production of test kits and reagents being dramatically scaled-up globally.

The identification of symptomatic and asymptomatic at-risk individuals is of immediate importance to enable early case detection and contact-tracing. In these circumstances, diagnostic testing is an essential tool not only to clinical care, but also to tracking and containing the disease in the community.[2] In order to achieve this end, reliable test kits, reagents and laboratory capacity must be readily available. Below, we discuss how these challenges are starting to be met.

Current strategies and the need for scaled-up testing capability and capacity

As a preliminary consideration, there is a distinction between testing for public health surveillance and testing for clinical care. Clinical testing is aimed at diagnosis of individuals with symptoms for the purpose of clinical care, together with a secondary purpose of quarantining and contact-tracing. Surveillance testing is broader community testing for individuals with risk factors such as overseas travel and in high-risk settings such as aged-care facilities, Indigenous communities and for essential-service and healthcare workers. Community surveillance can also occur more generally for early symptoms such as a raised body temperature.

One difficulty with surveillance testing is the prolonged incubation period of SARS-Cov-2, demonstrated to be a mean of 5 days (with a range of between 1 to 14 days),[3] with the effect that a point in time negative result may not rule out infection. Standard molecular testing (known as qRT-PCR, explained further below) is also limited by the type of sample taken (such as a nasopharyngeal swab) as the virus may only be detectable within the lungs. Once an infection is resolved and the virus cleared, the test will also be negative. Detection of past infection instead requires serological blood testing.

It is well documented that testing capacity for SARS-CoV-2 is lacking worldwide, and particularly so in the US and parts of Europe where the disease is spreading rapidly through asymptomatic carriers and individuals with only mild non-specific symptoms. At a WHO forum convened to identify research gaps and priorities for COVID-19 in February, the first of the eight immediate needs identified was for rapid point-of-care diagnostics, recognising the urgent need for accurate and standardised tests which can be deployed in community settings.[4]

Government and regulatory-agency interventions

In Australia, pathology diagnostic tests are regulated by the Therapeutic Goods Administration (TGA) as in-vitro diagnostic medical devices (IVDs). In response to the urgent need to make testing widely available, the Australian Federal Parliament on 22 March 2020 enacted emergency exemptions under the Therapeutic Goods Act 1989 (Cth) permitting the importation, manufacture and supply of SARS-CoV-2 IVDs without prior TGA assessment, for use only by accredited laboratories.[5] Specifically, this exempts SARS-Cov-2 diagnostic tests from regulations including compliance with the TGA’s essential principles for manufacture, conformity assessment certification, and requirement for inclusion on the Australian Register of Therapeutic Goods (ARTG) until 31 January 2021.[6]

While Australia currently has one of the highest rates of per capita SARS-CoV-2 testing, readily accessible community testing is still lacking. The Federal Government has announced a comprehensive AU$2.4 billion funding-package to address COVID-19, which includes Medicare-funded and bulk-billed pathology test for SARS-CoV-2, and AU$2.6 million in research funding to the Peter Doherty Institute for research into the development of improved SARS-CoV-2 diagnostics testing protocols, and for the post-market assessment of the new rapid point-of-care tests.[7]

Similarly, in the US the Food and Drug Administration (FDA) has, as of the date of this publication, provided Emergency Use Authorizations (EUAs) to 20 SARS-CoV-2 tests, including those by Abbott Laboratories, Roche Molecular Systems and ThermoFisher Scientific.[8] This follows the US Federal Government’s declaration of a national emergency,[9] allowing the US Federal Emergency Management Agency to deploy support and provide disaster funds to US state and local governments, including US$50 billion in funding to fight the disease.[10] The manufacturing capacity for SARS-CoV-2 diagnostic testing in the US is expected to be significantly scaled-up as a result of these measures.

SARS-CoV-2 Testing

Diagnostic testing for viral pathogens can be by molecular means to identify viral genetic material (nucleic acid – DNA or RNA) or by serological testing to identify antibodies directed against the virus (Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies). IgM antibodies are produced as a first response to a new infection providing short-term protection. They increase for several weeks and then decline as IgG production begins, with specific IgG antibodies forming the basis of long-term protection against viral pathogens.

Molecular testing by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is now well established as the gold-standard in testing and is highly sensitive (capable of detecting genetic material from a single viral particle) and specific (capable of distinguishing between similar strains).

Standard qRT-PCR

Diagnostic testing for RNA viruses such as SARS-CoV-2 are routinely performed by qRT-PCR. The PCR reaction alone only amplifies DNA. RT-PCR testing works by first converting viral RNA to its complimentary DNA (cDNA), amplifying the cDNA by standard PCR, and then detecting specific target DNA sequences unique to the virus by fluorescent-labelled probe.[11] Testing encompasses a number of steps and takes at least 4-6 hours in the laboratory, with final results taking up to several days were there is a back log of testing to be performed in the laboratory:[12]

  • A nasopharyngeal swab is used to collect secretions from the back of the nose or throat.
  • The swab is placed into viral transport media and sent to the lab for testing.
  • In the lab the sample is mixed with reagents that release the viral RNA from its capsule, allowing the viral RNA to be isolated.
  • The conversion of RNA into cDNA is facilitated by combining the RNA with deoxyribonucleotides, primers and other reagents. Primers anneal to the RNA strand and provide the reverse transcriptase enzyme with a starting point for DNA synthesis.
  • The cDNA is then used as the template for PCR amplification.
  • The amplified cDNA is labelled with a fluorescent marker that is detected by the real-time PCR machine, which quantifies the amount of fluorescence detected.
  • This value of fluorescence is called the Ct number, and is inversely proportional to the amount of cDNA. Typically Ct values are analysed relative to a ‘housekeeping’ gene to determine whether viral sequences are present in the sample.

The qRT-PCR test is performed in many laboratories worldwide for a variety of different viral pathogens. Since the sequencing of the SARS-CoV-2 genome, many companies have customised their qRT-PCR tests for SARS-CoV-2, using different primers designed to bind to differing target viral genetic sequences.[13]

Serological testing

Serological testing is performed on blood samples analysed by enzyme immunoassay or lateral flow devices and allows for the detection of IgM and IgG antibodies directed against the virus. Viral antibodies take 5 to 7 days to become detectable, making serological testing of more limited use for the diagnosis of acute infection. Antibody tests are also prone to ‘cross-reactivity’ with other similar antibodies (such as antibodies produced by similar strains of seasonal coronavirus causing the common cold), making the test less reliable.[14] Serological testing is however of use in testing for evidence of past resolved infection, and may be used in combination with molecular testing to detect evidence of both current and past infection.

Rapid point-of-care tests

The new rapid ‘point-of-care’ tests use the same qRT-PCR method implemented through a small portable device that can be used in clinics and community-screening settings, and provide results within 5 to 45 minutes depending on the test. Examples of such rapid tests are the Cepheid Xpert Xpress,[15] which in Australia has been included on the ARTG since 22 March 2020, and Abbott’s ID-NOW[16] authorised by the FDA. Similar rapid testing for blood serology by finger prick are also being rolled out, for example VivaCheck Biotech’s VivaDiag SARS-CoV-2 IgM/IgG Rapid test[17] which has been included on the ARTG since 20 March 2020.[18]

CRISPR-Cas tests in development

Two CRISPR biotechnology specialists, Sherlock Biosciences and Mammoth Biosciences, are working with various collaborators to adapt their CRISPR platforms for SARS-CoV-2 diagnosis. Both have developed diagnostic assays using CRISPR technology for the detection of viral pathogens.

CRISPR-associated Cas-proteins developed within bacteria as an evolutionary adaptive immune mechanism to enable bacteria to fight off foreign invaders such as bacteriophages.[19] The discovery of this mechanism led to the development of the CRISPR-Cas system as technology capable of its known use as a genome-editing tool.

The technology is now also being applied to diagnostics, whereby a small segment of ‘guide’ RNA binds to a target sequence of genetic material, followed by use of a Cas12 or Cas13 nuclease for precise target location and cleavage of a ‘reporter’ molecule added to the reaction.[20] This in effect uses CRISPR’s functionality as a means of detecting unique genetic “fingerprints” of virtually any DNA or RNA sequence, in any organism.

Sherlock Biosciences has licensed CRISPR and related technology from the Broad Institute of MIT and Harvard and the Wyss Institute of Harvard[21] to develop a diagnostic test, known as SHERLOCK (Specific High-sensitivity Enzymatic Reporter Unlocking). The test detects two SARS-CoV-2 genes – the S gene and the Orf1ab gene. The test can be adapted to work on a simple paper strip test (similar to a pregnancy test), on laboratory equipment or by electrochemical readout that can be read on a mobile phone.[22]

Mammoth Biosciences has also applied CRISPR-Cas9 gene-editing technology to develop its molecular diagnostic platform called DETECTR (DNA endonuclease-targeted CRISPR trans reporter). As with the SHERLOCK test, DETECTR can be tailored to detect any DNA or RNA target, with results provided in an instrument-free and disposable format within 20 minutes. The technology can also be integrated into other products and platforms. DETECTR is now being developed for identification of the N and E SARS-CoV-2 genes.[23]

These innovations are yet to be validated for clinical use in humans but nonetheless represent a promising development in advanced clinical diagnostics

Currently approved or authorised diagnostic tests

A plethora of SARS-CoV-2 diagnostic tests have been deployed around the world. In Australia 16 SARS-CoV-2 tests have been included on the ARTG as of the date of this publication, including most notably:[24]

  • Cepheid’s Xpert® Xpress (rapid portable RT-PCR test)
  • Roche Diagnostics’ Cobas® (real time RT-PCR test)
  • Becton Dickinson’s VIASURE (real time RT-PCR test)
  • Shanghai ZJ Bio-Tech’s 2019-nCoV (real time RT-PCR test)
  • Viva Check Biotech’s VivaDiag IgM/IgG Rapid (rapid serology test)
  • Hangzhou Clongene Biotech’s COVID-19 IgG/IgM Rapid (rapid serology test)

As of the date of this publication the FDA has provided EUA authorisation to 20 SARS-CoV-2 tests, with a few notable examples not yet available in Australia listed below:[25]

  • Abbott’s ID NOW (rapid portable RT-PCR test)
  • Abbott’s Real Time SARS-CoV-2 assay (real time RT-PCR)
  • ThermoFisher Scientific’s TaqPath (real time RT-PCR)

Concluding remarks

Governments, regulatory bodies and industry are now starting to respond to the immense challenge of meeting the demand for reliable, fast and portable SARS-CoV-2 diagnostic tests in response to this global pandemic, bringing new testing technologies to fruition and scaling up the manufacture of existing tests with increasing urgency. Hopefully, this increased testing capability, together with range of other public health measures currently being implemented, can assist in reducing COVID-19 infection rates in the coming weeks and months.

[1] World Economic Forum, 17 March 2020. “The World Health Organization has called on countries to ‘test, test, test’ for coronavirus – this is why” (https://www.weforum.org/agenda/2020/03/coronavirus-covid-19-testing-disease/, accessed 29 March 2020).

[2] Hellewell J et al, 28 February 2020. Centre for the Mathematical Modelling of Infectious Diseases COVID-19 Working Group. – “Feasibility of controlling COVID-19 outbreaks by isolation of cases and contacts”. Lancet Global Health. February 2020; S2214-109X(20)30074-7. (https://www.thelancet.com/journals/langlo/article/PIIS2214-109X(20)30074-7/fulltext, , accessed 29 March 2020).

[3] Lauer SA et al, 10 March 2020. “The Incubation Period of Coronavirus Disease 2019 (COVID-19) From Publicly Reported Confirmed Cases: Estimation and Application”. Annals of Intern Medicine. 2020. (https://annals.org/aim/fullarticle/2762808/incubation-period-coronavirus-disease-2019-covid-19-from-publicly-reported, accessed 29 March 2020).

[4] WHO, 11-12 February 2020. “COVID 19 Public Health Emergency of International Concern (PHEIC), Global research and innovation forum: towards a research roadmap”. (https://www.who.int/blueprint/priority-diseases/key-action/Global_Research_Forum_FINAL_VERSION_for_web_14_feb_2020.pdf?ua=1, accessed 29 March 2020).

[5] Therapeutic Goods (Medical Devices—Accredited Pathology Laboratories) (COVID-19 Emergency) Exemption 2020 (Cth) (https://www.legislation.gov.au/Details/F2020N00032, accessed 29 March 2020).

[6] As above, note 5.

[7] PM.gov.au Media Release, 11 March 2020. “$2.4 Billion Health Plan to fight COVID-19”. (https://www.pm.gov.au/media/24-billion-health-plan-fight-covid-19, accessed 29 March 2020); Health.gov.au Media Release, 21 March 2020. “$2.6 million for coronavirus research, including a new simpler Australian pathology test” (https://www.health.gov.au/ministers/the-hon-greg-hunt-mp/media/26-million-for-coronavirus-research-including-a-new-simpler-australian-pathology-test, accessed 29 March 2020). ).

[8] FDA, 28 March 2020. “Emergency Use Authorizations” (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-authorizations#covid19ivd, accessed 30 March 2020).

[9] Whitehouse.gov, 13 March 2020. “Proclamation – Proclamation on Declaring a National Emergency Concerning the Novel Coronavirus Disease (COVID-19) Outbreak”. (https://www.whitehouse.gov/presidential-actions/proclamation-declaring-national-emergency-concerning-novel-coronavirus-disease-covid-19-outbreak/, accessed 29 March 2020).

[10] Reuters, 14 March 2020. “Trump declares coronavirus national emergency, says he will most likely be tested”. (https://www.reuters.com/article/us-health-coronavirus-usa-emergency/trump-declares-coronavirus-national-emergency-says-he-will-most-likely-be-tested-idUSKBN2102G3, accessed 29 March 2020).

[11] BioSistemika, 4 July 2017. “Real-Time PCR (qPCR) Technology Basics”. (https://biosistemika.com/blog/qpcr-technology-basics/, accessed 29 March 2020).

[12] Sheridan C, 23 March 2020. “Fast, portable tests come online to curb coronavirus pandemic”. Nature Biotechnology News article. (https://www.nature.com/articles/d41587-020-00010-2, accessed 29 March 2020); Dharmaraj, S (ThermoFisher Scientific). “The Basics: RT-PCR”. (https://www.thermofisher.com/au/en/home/references/ambion-tech-support/rtpcr-analysis/general-articles/rt–pcr-the-basics.html, accessed 29 March 2020); Sharfstein JM et al, 9 March 2020. “Diagnostic Testing for the Novel Coronavirus”. Journal of the American Medical Association (JAMA). (https://jamanetwork.com/journals/jama/fullarticle/2762951, accessed 29 March 2020); Corman V et al, 23 January 2020. “Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR”. Eurosurveillance, 2020;25(3). (https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.3.2000045, accessed 29 March 2020); Tang Yi-Wei et al, 1 November 1997. “Molecular diagnostics of infectious diseases”. Clinical Chemistry, 1997:43(11),2021-2038. (https://academic.oup.com/clinchem/article/43/11/2021/5640827, accessed 29 March 2020).

[13] Nature News Explainer, 23 March 2020. “Coronavirus tests: researchers chase new diagnostics to fight the pandemic” . (https://www.nature.com/articles/d41586-020-00827-6, accessed 29 March 2020).

[14] Department of Health, 22 March 2020. “PHLN statement on point-of-care serology testing for SARS-CoV-2 (the virus that causes COVID-19”. (https://www.health.gov.au/resources/publications/phln-statement-on-point-of-care-serology-testing-for-sars-cov-2-the-virus-that-causes-covid-19, accessed 28 March 2020).

[15] Cepheid. “Xpert Xpress SARS-CoV-2 has received FDA Emergency Use Authorization”. (https://www.cepheid.com/coronavirus, accessed 29 March 2020).

[16] Abbott. “Abbott launches molecular point-of-care test to detect novel coronavirus in as little as five minutes”. (https://abbott.mediaroom.com/2020-03-27-Abbott-Launches-Molecular-Point-of-Care-Test-to-Detect-Novel-Coronavirus-in-as-Little-as-Five-Minutes, accessed 29 March 2020).

[17] Viva Chek. “VivaDiag SARS-CoV-2 IgM/IgG Rapid Test”. (https://www.vivachek.com/vivachek/English/prods/prod-covid19.html, accessed 29 March 2020).

[18] tga.gov.au, 28 March 2020. “COVID-19 diagnostic tests included on the ARTG for legal supply in Australia”. (https://www.tga.gov.au/covid-19-diagnostic-tests-included-artg-legal-supply-australia, accessed 30 March 2020).

[19] Hille, F et al, 8 March 2018. “The Biology of CRISPR-Cas: Backward and Forward”. Cell. 2018;172(6):1239-1259. (https://www.cell.com/cell/fulltext/S0092-8674(17)31383-1?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867417313831%3Fshowall%3Dtrue, accessed 29 March 2020).

[20] Chiu C, 13 June 2018. “Cutting-Edge Infectious Disease Diagnostics with CRISPR”. Cell Host & Microbe. 2018;23(6):702-704. (https://www.cell.com/cell-host-microbe/fulltext/S1931-3128(18)30270-1?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1931312818302701%3Fshowall%3Dtrue, accessed 29 March 2020).

[21] Broad Institute of MIT and Harvard News Feature, 15 March 2020. “Enabling coronavirus detection using CRISPR-Cas13: Open-access SHERLOCK research protocols and design resources”. (https://www.broadinstitute.org/news/enabling-coronavirus-detection-using-crispr-cas13-open-access-sherlock-research-protocols-and, accessed 29 March 2020).

[22] Sherlock Biosciences. “Better diagnostic testing should be elementary”. (https://sherlock.bio/technology/, accessed 29 March 2020).

[23] Mammoth Biosciences. “The CRISPR-based detection platform”. (https://mammoth.bio/diagnostics/, accessed 29 March 2020); Mammoth Biosciences, 2 March 2020. “A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR”. (https://mammoth.bio/wp-content/uploads/2020/03/Mammoth-Biosciences-A-protocol-for-rapid-detection-of-SARS-CoV-2-using-CRISPR-diagnostics-DETECTR.pdf, accessed 29 March 2020).

[24] As above, note 18.

[25] As above, note 8.

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