key: cord-0768240-t9jaspf2
authors: Toptan, Tuna; Hoehl, Sebastian; Westhaus, Sandra; Bojkova, Denisa; Berger, Annemarie; Rotter, Björn; Hoffmeier, Klaus; Ciesek, Sandra; Widera, Marek
title: Optimized qRT-PCR approach for the detection of intra- and extra-cellular SARS-CoV-2 RNAs
date: 2020-04-25
journal: bioRxiv
DOI: 10.1101/2020.04.20.052258
sha: 33dfe6cd92590c1a7f748a9e4f99319564024d90
doc_id: 768240
cord_uid: t9jaspf2

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19 which has become a global concern due to its rapid spread. Meanwhile, increased demand in testing has led to shortage of reagents, supplies, and compromised the performance of diagnostic laboratories in many countries. Both the world health organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate interpretation of the test results especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in the diagnostics as well as in research labs using a low cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infection and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

In several laboratories, non-specific PCR products in both SARS-CoV-2 negative patient samples and in the non-template control (NTC) using the WHO recommended SARS-CoV E-gene specific PCR (Corman et al., 2020) have been reported (Konrad et al., 2020) (personal communication) . In order to confirm and further characterize these non-specific products in negative samples, we carried out additional PCR-based analyses. To this end, RNA from pharyngeal swab samples ( Table 1 ) was isolated and subjected to WHO recommended qRT-PCR analysis using E (Figure 1a) and RdRP (Figure 1b) gene specific primers ( Table 2) . As positive controls we used RNA extracted from pharyngeal swabs of one asymptomatic, one mildly symptomatic passenger returning from Wuhan after initial quarantine (Figure 1, Table 1 ), (Hoehl et al., 2020) . Both viral samples were passaged on Caco2 cells as described previously (Bojkova et al., 2020) and the resulting virus containing supernatants (FFM1 and 2) were used for subsequent analysis. Four out of 48 (8.33%) tested swab samples showed a positive signal in the E-gene PCR (Figure   1a ), which were of the same size as the specific amplicons (data not shown). However, the confirmatory RdRP-gene PCR was negative for all patient samples except the positive control ( Figure   1b ). This data indicates that using the two-step WHO PCR protocol might complicate interpretation of the test results especially for cases with higher Cq-values.

Since the RdRP-gene PCR is more specific than the E-gene PCR but is also described as less sensitive (Konrad et al., 2020) , we aimed to develop a novel approach using specifically designed primer and probe pairs. For optimal primer design consensus sequences were aligned to the reference SARS-CoV-2 genome sequence and analyzed for primers binding in E, N, Orf1, M, and S regions (Supplementary Figure 1 ) that would allow SARS-CoV-2 but not SARS-CoV amplification.

For experimental confirmation, we used human SARS-CoV strain Frankfurt 1 (NC_004718) and SARS2-CoV-2 RNA samples (FFM1 and FFM2) and compared the PCR performances using serial dilutions (Supplementary Figure 1a) . Due to limited linearity, E and N gene specific primers were excluded from further analysis while S and M gene based PCRs (Table 1) , which were more sensitive Using plasmid DNA constructs that harbor the conserved SARS-CoV-2 amplicon sequences, we generated standard curves and compared the M-gene PCR (Figure 2b ) with the established WHO RdRP-gene PCR approach (Figure 2a) . Using the same dilution series of plasmid samples, the M gene based method allowed earlier detection (Figure 2c ). To determine the analytical sensitivity of the M-gene based approach, we additionally used in vitro-transcribed RNA standards and tested four replicates to determine the limit of detection (Figure 2d -f). We were able to detect a single RNA copy per reaction by M-Gene qRT-PCR. SYBR-green based melting curve analysis revealed a melting point at 80°C for all samples (Supplementary Figure 2) . To further characterize the capacity of M-gene PCR to detect high viral loads, we performed SYBR-green based PCR and quantified high loads of virus cell culture supernatants (Supplementary Figure 2d) .

In order to confirm that the M-gene specific PCR primers and probes cover all known isolates and thus enable detection, an alignment of published SARS-CoV-2 full-length isolates known at the time of submission (n = 165) and viral isolates sequenced during this study was carried out (Figure 3,   Supplementary Figure 3) . Both forward and reverse primers had a 100% identity and only one isolate of 165 (0.006%) had a mismatch to the consensus sequence C/T) at position 18 of the DNA probe ( Figure 3) . However, this mismatch would not affect the detection, but possibly reduce the sensitivity but not prevent binding of the probe. In conclusion, our M-gene based qRT-PCR detection of SARS-CoV-2 RNA was at least as specific as the RdRP PCR recommended for confirmation by the WHO, but showed a significantly higher sensitivity. Importantly, unspecific signals as observed in the E-gen PCR were not detected. 

In order to validate our method, we re-tested clinically relevant samples that have been qualitatively tested positive for SARS-CoV-2 RNA during routine diagnostics (Supplementary Table 1 ). WHO recommended RdRP primer pairs were used for confirmation. As negative controls we included 8 negative samples (Figure 1, Supplementary Table 1 ). As described above, unspecific E-gene amplicons were detected in a test kit specific manner possibly due to the reagents used (Konrad et al., 2020) . Therefore, we additionally compared the performances of two research kits (New England In conclusion, non-specific PCR products and limited sensitivity pose a major problem using the WHO protocol for SARS-CoV-2 detection and could lead to numerous unnecessary confirmation tests. Our newly developed PCR protocol is suitable to save time and resources since pre-screening is no longer necessary. Thus, this approach might be used as a cost-effective alternative to the E-and RdRPbased protocol. 

Countermeasures against COVID-19 depend on testing with the highest sensitivity and specificity possible. We and others (Konrad et al., 2020 ) (personal communication) determined certain drawbacks in the two-step PCR protocol recommended by the WHO as unspecific signals for E-gene PCR may arise due to combination of several factors including primer dimers, unspecific binding of the primers and probes, RT-PCR kit and thermocycler dependent differences. In commercially available kits, primers and probes, as well as the buffers and enzyme quantities and properties have been matched to one another to reduce unspecific amplification (Konrad et al., 2020) . In times of increasing reagent shortages, however, a simple protocol should be available that can be quickly adapted with universal test kits (including research kits) and is easy to evaluate would be beneficial.

In this study, we evaluated several primer and probe pairs designed in silico and identified M-gene specific primer and probe (Table 2) to be highly versatile for detection of SARS-CoV-2 RNA in a large selection of samples. We analyzed samples from patients presented with different symptoms, viral loads and degree of infection (Table 1 and Supplementary Table 1 ) and compared isolates with varying demographic characteristics originating from several infection clusters (Table 1) .

Interestingly, via sequence comparison based on the phylogenetic analysis we were able to show that isolate FFM5 with unknown origin shows a high degree of relationship to the FFM1 isolate from China indicating the same origin (Figure 3) . Anyway, the SARS-CoV-2 M-gene PCR was able to detect SARS-CoV-2 from all clusters.

In a direct comparison of the RdRP PCR recommended by the WHO and the M-Gen PCR developed in this study, the M-Gen PCR was superior in terms of sensitivity using multiple test kits. Since we used primers that specifically bind SARS-CoV-2 RNA, this was to be expected. The reduced sensitivity of the WHO recommended RdRP-PCR may be explained by the presence of wobble pairs (Table 2) in the primer and probe sequences (Corman et al., 2020) . Nevertheless, in a situation of crisis, where the availability of reagents, equipment as well as labor of qualified personnel are limited, a reliable and convenient one-step PCR protocol with optimum sensitivity would be beneficial.

Our comparison analysis for M-and RdRP-gene PCR were consistent and reproducible for detecting RNA from progeny virus particles and also intracellular viral mRNAs extracted from infected Caco2 and Vero cells even if some of the subgenomic viral mRNAs do not harbor the target sequence (Kim et al., 2020) . We also have shown that the method is also suitable for inexpensive SYBR-green based PCR assays (Supplementary figure 2d) . Thus, M gene PCR allows the quantification of very low and very high viral loads. For time point analysis, Caco2 cells were infected with SARS-CoV-2 (0.01 MOI) in MEM media supplemented with 1% FCS, at 37°C, 5% CO2. Cells were harvested for RNA extraction 0, 3, 6, 12

and 24 h post infection using TRI Reagent (Sigma). Following chloroform extraction and isopropanol precipitation, RNA was dissolved in nuclease free water and treated with DNase I (Qiagen) for 15 min at 37°C. DNase I treated RNA was subjected to RT-PCR analysis. 

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