key: cord-0817150-n7vtbf4q authors: Fenaux, H; Limam, L; Soutiere, MP; Veillet, F; Escuret, V; Roque-Afonso, AM title: Performance of the QIAprep& Viral RNA UM Kit assay (Qiagen), an automatable method for RT-qPCR detection of SARS-CoV-2 without RNA extraction date: 2022-04-21 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2022.115700 sha: ede06c45829c5ffb84237004448bf34ffb7be76d doc_id: 817150 cord_uid: n7vtbf4q We evaluated the performance of the QIAprep& Viral RNA UM Kit (Qiagen) for SARS-CoV-2 detection. It displayed specificity and sensitivity required for SARS-CoV-2 RNA detection from swab transport media without RNA extraction. This method identifies accurately patients at risk of transmission while saving time and cost of extraction. Performance of the QIAprep& Viral RNA UM Kit assay (Qiagen), an automatable method for RT-qPCR detection of SARS-CoV-2 without RNA extraction H Fenaux 1 , L Limam 1 , MP Soutiere 1 , F Veillet 1 , V Escuret 2,3 , AM Roque-Afonso 1,* annemarie.roque@aphp.fr 1 Laboratoire de virologie, hôpital Paul Brousse, Assistance Publique -Hôpitaux de Paris, Laboratories' organization is challenged every day with frequent shortages of reagents or plastic consumables, and fluctuating but heavy daily urgent demand. The QIAprep& Viral RNA UM Kit (QIAprep) developed by Qiagen (Hilden, Germany) allows preparation and detection of viral RNA targets from viral transport media, dispensing the nucleic acids extraction step. We report here the validation of this RT-qPCR assay for SARS-CoV-2 RNA detection adapted on an automated liquid handling system. SARS-CoV-2 RNA detection was performed using QIAprep with specific primers and probes (Qiagen). This assay targets two viral genes (N1 and N2), detected with probes labelled with the same dye; the kit includes an internal control (a synthetic RNA) and a 2-target cell control (Beta-2-Microglobulin and RNase P genes), allowing monitoring of both PCR inhibition and sample quality. After heat inactivation (70°C for 10 minutes), 8µL of sample transport media were distributed on a microwell PCR plate and 2µL of lysis buffer were added. After a 2-minutes incubation at room temperature, 10µL of the beforehand prepared reaction mix were added, and the plate was loaded on a real-time PCR instrument (ViiA7, ThermoFisher). In order to be able to manage tens of samples rapidly, we automatized the method on the QiaSymphony SP/AS platform (Qiagen). The RT-qPCR reaction volume was the same but larger volumes of sample and reagents were needed to allow automated handling. An extra step was also needed because the platform has 2 modules and the first module cannot wield small volumes. Briefly, after heat inactivation, 200µL of sample were loaded on the SP module: 80µL were taken from the sample to a 1 st plate, and 20µL of lysis buffer were added. After a 2-minutes incubation, 60µL were transferred into a 2 nd plate. The AS module then distributed 10µL of the beforehand prepared reaction mix in each PCR wells of a RotorGeneQ plate and added 10µL of prepared sample. QIAprep detected SARS-CoV-2 RNA in all replicates of culture supernatants up to the 10E-6 dilution. The 10E-7 dilution was the limit of detection of both assays ( figure 1A ). SARS-CoV-2 positive clinical samples (fresh or stored frozen, n=123) were tested either with the manual or with the automated procedure. We observed no impact of freezing or type of analysis procedure on assay performance. In 5/123 (4%) samples, the cell control target was not detected. These samples had been collected in a transport medium that we determined to be not compatible with no-extraction techniques (either sampled in Cary-Blair medium or in a viral transport medium containing, among other products, guanidine isothiocyanate, chelating agent and alcohol). Among the 118 samples with interpretable results, QIAprep detected 79/79 (100%), 17/30 (56.7%), and 1/9 (11.1%) samples with Alinity m Ct <34, from 34 to 38, and > 38, respectively (Table 1; Figure 1B) . In 3/33 (9.1%) negative samples, the cell control target was not detected due to inappropriate transport medium. In the remaining 30 samples, the specificity of the QIAprep was 100%. In a context of ongoing pandemic, there is critical need for diagnostic innovation in order to get results as fast as possible and to deal with shortages. The QIAprep assay was proven a reliable method with excellent specificity and acceptable sensitivity for SARS-CoV-2 detection from nasopharyngeal swabs. Its performance was similar to that of the early developed reference method of the French NRC for Respiratory Viruses (4). Compared to the highly sensitive Alinity m assay (6), the QIAprep reliably detected samples with Ct<34, a surrogate marker of infectivity: patients with high Ct values are known to be less contagious (7) . No extraction was required with this method, which makes it faster, and to our knowledge, this is the first method to provide both an internal control and a cell control, allowing the monitoring of both PCR inhibition and sample quality. However, a limit of this absence of nucleic acid purification, with the possible persistence of PCR inhibitors, is the need for validation for each transport medium. This method can be automatized. In our hands, a round of 71 samples would take 4 hours to a trained technician, and another round can be prepared once amplification starts running for the first one. Finally, this method requires a small sample volume, even when automatized. This allows retesting, which can be advantageous for low volume samples, such as saliva, that deserves evaluation on this type of procedure. In conclusion, the QIAprep SARS-CoV-2 assay displays very good diagnostic performances, and is an easy method to set up, relatively quick, even after automatization, and relatively inexpensive for SARS-CoV-2 RNA detection from viral transport media. Depending on the laboratory's habits, it can be used as a backup in case of saturation or main method breakdown. HF, LL and AMRA designed the experiments. MPS and FV performed the experiments. VE provided the culture supernatants. HF, VE and AMRA wrote the paper. A pneumonia outbreak associated with a new coronavirus of probable bat origin The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 2 infections: practical considerations and management strategy for intensivists Journal of Clinical Medicine. Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories Verification and Validation of SARS-CoV-2 Assay Performance on the Abbott m2000 and Alinity m Systems Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards We thank Cinzia Falasca (Qiagen) for her technical help and Qiagen for providing the reagents.