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The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.

Nucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80–8.23% and is stable at 4 °C for 1 week and at −70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.

Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control. No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit. The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.

SARS-CoV-2 in vitro transcribed RNA reference materials (RM), UME RM 2019 and UME RM 2020, were produced by Scientific and Technological Research Council of Turkey (TUBITAK), National Metrology Institute (UME), to be used as a quality control material for SARS-CoV-2 measurements, in liquid-frozen and lyophilized forms, respectively. These RNA RMs include ten internationally recommended SARS-CoV-2 target gene fragments (Pasteur-RdRp-IP2, Pasteur-RdRp-IP4, Charite-E, Charite-RdRp, CDC-N1, CDC-N2, China CDC-ORF1ab, China CDC-N, Hong Kong-ORF1b, and Hong Kong-N) for virus detection and one human gene fragment (RNase P) as an internal control. Two different platforms, RT-qPCR and RT-ddPCR, were used to characterize UME RM 2019 (UME RM 2020 was only characterized with RT-qPCR). The homogeneity studies were evaluated by RT-qPCR. According to these results, it has been shown that both reference materials are homogeneous for intended use. Short-term studies were also conducted similarly for mimicking transport conditions and UME RM 2020, which is produced in lyophilized form, unlike other reference materials available in the market, provides convenience for users by ensuring that the reference material remains stable for 17 days even at 45 °C temperature. The lyophilized formulation of the reference material had greater stability which would allow it to be shipped without cooling items. The development of such RNA reference materials provides quality control for existing and newly designed RNA-based virus detection tests and it helps the prevention and control of epidemics.

DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms.

RV Armored RNA constructed in the laboratory was shown to be resistant to RNase and DNase digestion. It could remain stable at 4 °C for at least 60 days, and was much more stable than RV strain JR23 in normal human plasma. Therefore it could be used as stable, well-characterized positive control and standard for quantitative RT-PCR assay.

Abstract Background The ongoing outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a challenge for worldwide public health. A reliable laboratory assay is essential both to confirm suspected patients and to exclude patients infected with other respiratory viruses, thereby facilitating the control of global outbreak scenarios. Content In this review, we focus on the genomic, transmission, and clinical characteristics of SARS-CoV-2, and comprehensively summarize the principles and related details of assays for SARS-CoV-2. We also explore the quality assurance measures for these assays. Summary SARS-CoV-2 has some unique gene sequences and specific transmission and clinical features that can inform the conduct of molecular and serological assays in many aspects, including the design of primers, the selection of specimens, and testing strategies at different disease stages. Appropriate quality assurance measures for molecular and serological assays are needed to maintain testing proficiency. Because serological assays have the potential to identify later stages of the infection and to confirm highly suspected cases with negative molecular assay results, a combination of these two assays is needed to achieve a reliable capacity to detect SARS-CoV-2.

On 21 February 2020, a resident of the municipality of Vo’, a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection 1 . This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province 2 . In response, the regional authorities imposed the lockdown of the whole municipality for 14 days 3 . Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo’ at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1–3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8–1.8%). Notably, 42.5% (95% CI: 31.5–54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9–9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections ( P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon–Mann–Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures. The authors describe the prevalence of SARS-CoV-2 infection, viral load and the frequency of symptomatic versus asymptomatic and presymptomatic infection in an Italian town, before and after a strict 14-day lockdown.