Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded ribonucleic acid (RNA) virus and has been identified as the causative agent of coronavirus disease 2019 (COVID-19) disease [1
]. To avoid uncontrolled viral spread, high-throughput testing and subsequent isolation of infected individuals was advised [2
]. However, due to limited laboratory testing capacities, only symptomatic individuals could be tested in the beginning of the 2020 pandemic [3
]. On the other hand, it has been shown that a large proportion of individuals remain asymptomatic. Therefore, a clinical definition of COVID-19 is not reliable and laboratory confirmation of SARS-CoV-2 is currently advised for confirmation [4
]. As asymptomatic patients tested positive for SARS-CoV-2, the lack of test capacity resulted in a lack of knowledge of the underlying true infection risk [5
]. Since then, test capacities have been upscaled, but there is still an urgent need for reliable and cost-effective high-throughput testing methods, specifically as regulations are currently easing and loci of rapid spread need to be detected as early as possible, to avoid uncontrolled viral spread.
Mass spectrometric (MS) techniques have largely complemented or replaced traditional methods in laboratory medicine, toxicology, microbiology as well as molecular pathology and are suitable for reliable, cost-effective and rapid detection of amplified polymerase chain reaction (PCR) products [7
]. Thus, this method has a great potential to complement the current diagnostic arsenal, especially in times where a shortage of reagents may limit the application of real-time reverse transcriptase (rRT)-PCR, which is the current gold standard for the detection of SARS-CoV-2 [12
Therefore, we established and tested a MS-based test protocol for its ability to detect SARS-CoV-2 from oral or nasopharyngeal swabs and compared the results to rRT-PCR.
A total of six coronaviruses are known to cause human disease from which two strains (SARS-CoV-2 and Middle East respiratory syndrome coronavirus) are thought to be zoonotic and have been associated with more severe, potentially fatal outcomes [14
SARS-CoV-2 is an enveloped single-stranded positive-sense RNA virus and belongs to the genus of beta-Coronaviruses, is spherical in shape, about 60–140 nm in diameter and has 9–12 nm long characteristic spikes on the surface [15
]. This virus has been identified as the causative agent of COVID-19 disease after the World Health Organization Country Office was informed about cases of pneumonia of unknown etiology in Wuhan City, Hubei Province in China on 31 December 2019 [12
]. To detect infection with SARS-CoV-2 saliva, sputum, oral or nasopharyngeal swabs, stool, anal swabs, blood or urine can be analyzed and highest rates of detection have been reported for sputum, oral or nasopharyngeal swabs [16
]. However, the peak diagnostic yield might depend on the time of onset of symptoms [17
]. In this study, we tested material derived from oral and nasopharyngeal swabs.
The genome of a typical CoV contains a 5′ untranslated region (UTR); a conserved replicase domain (ORF1ab including the RdRp gene); four genes S, E, M and N to encode structural proteins spike, envelope, membrane, and nucleocapsid proteins; a 3′ UTR and several unidentified non-structural ORFs, which are potential targets for their detection [18
]. These gene regions may be used to identify infection with SARS-CoV-2.
Interestingly, recommendations regarding which gene regions should be targeted to identify the virus are highly variable. For example, in the US, three genes targeting the N-gene are recommended for emergency testing; in Germany, the Charité recommends the identification of the E gene for screening and identification of the RdRp gene for confirmation [12
]. The latter recommendation is based on the fact that the region of the E gene is commonly shared between SARS-CoV-2 and other bat or human related SARS viruses, and the RdRP sequence is specific for SARS-CoV-2 [12
]. Generally, two to three targets need to be detected to qualify as a reliable result [18
]. Moreover, an internal quality control is currently recommended, which is also in the MS detection kit [18
The analysis of rRT-PCR may be affected by false-negative and false-positive results. In this regard, it has been shown that multiple testing in the course of disease can improve the rate of viral detection [4
]. One study found that in 21.4% of patients, a positive detection of SARS-CoV-2 could only be achieved after two consecutive negative results [21
]. Interestingly, 16.7% of patients with typical features of COVID-19 pneumonia on a computer tomography scan were negative by rRT-PCR; half of these patients became positive after the second test, and half of the remaining patients became positive after the third test [4
]. It was found that the rRT-PCR results from several tests at different time points were variable from the same patients during the course of diagnosis and treatment [19
]. This might explain the fact that only about 50% of clinically confirmed cases are confirmed by a positive rRT-PCR result. As a consequence, a negative rRT-PCR should not lead to the false assumption that the patient is not infected by SARS-CoV-2, and clinical parameters need to be considered [20
]. To avoid false-negative results, due to testing failures, standardized collection, transport conditions, storage, extraction and amplification procedures of patient material are needed. Interestingly, thermal inactivation reduced the detectable amount of SARS-CoV-2 in rRT-PCR runs and would be expected also for our MS test. On the other hand, inactivation by guanidinium-based lysis exhibited less effects [23
Besides rRT-PCR (gold standard), alternative techniques such as isothermal amplification methods and MS are available for the detection of viruses [24
]. MS has been successfully used for the detection of viral DNA, RNA or proteins in previous studies [26
]. The method is fast with a turnaround time of max. 1 day and therefore suitable for routine use [29
]. However, to the best of our knowledge there have been no reports on the application of MS for the detection of SARS-CoV-2 to date. We show that (i) rRT-PCR is the fastest method to detect SARS-CoV-2, (ii) hands-on-time is comparable between rRT-PCR and the MS method and (iii) results are concordant between both assays.