Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection.

BACKGROUND: Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests.

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

Fast preparation of a long chimeric armored RNA as controls for external quality assessment for molecular detection of Zika virus.

BACKGROUND: The emergence of Zika virus demands accurate laboratory diagnostics. Nucleic acid testing is currently the definitive method for diagnosis of Zika infection. In 2016, an external quality assurance (EQA) for assessing the quality of molecular testing of Zika virus was carried out in China. METHODS: A single armored RNA encapsulating a 4942-nucleotides (nt) long specific RNA sequence of Zika virus was prepared and used as positive samples. A pre-tested EQA panel, consisting of 4 negative and 6 positive samples with different concentrations of armored RNA, was distributed to 38 laboratories that perform molecular detection of Zika virus. RESULTS: A total of 39 data sets (1 laboratory used two test kits in parallel), produced by using commercial (n=38) or laboratory developed (n=1) quantitative reverse-transcriptase PCR (qRT-PCR) kits, were received. Of these, 35 (89.7%) had correct results for all 10 samples, and 4 (10.3%) reported at least 1 error (11 in total). The testing errors were all false-negatives, highlighting the need of improvements in detecting sensitivity. CONCLUSIONS: The EQA reveals that the majority of participating laboratories are proficient in molecular testing of Zika virus.

Quality control assessment of human immunodeficiency virus type 2 (HIV-2) viral load quantification assays: results from an international collaboration on HIV-2 infection in 2006.

Human immunodeficiency virus type 2 (HIV-2) RNA quantification assays used in nine laboratories of the ACHI(E)V(2E) (A Collaboration on HIV-2 Infection) study group were evaluated. In a blinded experimental design, laboratories quantified three series of aliquots of an HIV-2 subtype A strain, each at a different theoretical viral load. Quantification varied between laboratories, and international standardization of quantification assays is strongly needed.