key: cord-0956067-xi7hio0z authors: Merindol, Natacha; Pépin, Geneviève; Marchand, Caroline; Rheault, Marylène; Peterson, Christine; Poirier, André; Houle, Claudia; Germain, Hugo; Danylo, Alexis title: SARS-CoV-2 detection by direct rRT-PCR without RNA extraction date: 2020-05-07 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104423 sha: ea4468adb43a7fd8e2d95f2277a6fdba6ddaaf35 doc_id: 956067 cord_uid: xi7hio0z Rapid and reliable screening of SARS-CoV-2 is fundamental to assess viral spread and limit the pandemic we are facing. In this study, we compared direct rRT-PCR method (without RNA extraction) using SeeGene AllplexTM 2019-nCoV rRT-PCR with the RealStar® SARS-CoV-2 rRT-PCR kit (Altona Diagnostics). Furthermore, we assessed the impact of swab storage media composition on PCR efficiency. We show that SeeGene and Altona's assays provide similar efficiency. Importantly, we provide evidence that RNA extraction can be successfully bypassed when samples are stored in UTM medium or in molecular water but not when samples are stored in saline solution and in Hanks medium. Background Viral detection is key to isolate positive patients and stop viral transmission (1) (2) (3) (4) (5) (6) in our battle against the 2020 SARS-CoV-2 pandemic. However, there are many challenges associated with ramping up testing capacity, including shortage in the chain of supplies for extraction reagents (7) . This situation called for alternatives protocols to ensure the continuity of testing in laboratories. The standard procedure for testing SARS-CoV-2 from oro-nasopharyngeal swabs in the clinical laboratory of the Centre Intégré Universitaire de Santé et Services Sociaux de la Mauricie et Centre du Québec (CIUSSS-MCQ) is based on RNA extraction followed by viral genes detection with a multiplex assay using the Altona RealStar ® SARS-CoV-2 rRT-PCR Kit RUO (thereafter Altona). Due to an increased demand in SARS-CoV-2 screenings, alternative protocols with similar sensitivity were needed (8) (9) (10) (11) . Thus, we compared sensitivity from Health Canada approved SeeGene Allplex TM 2019-nCoV rRT-QPCR Assay (thereafter SeeGene) and Altona, with and without RNA extraction. Samples originated from remaining swabs media and RNA extracts from symptomatic individuals that had been previously screened using the CIUSS-MCQ routine method. Hence, samples were declared negative or positive before these experiments. Specimens Swab from the oro and naso-pharyngeal area from symptomatic individuals were placed in 2 ml of UTM (Remel RE12569), or Hanks medium (0,14M NaCl; 5mM KCl , 0,4 mM MgSO4, 0,3 mM Na2HPO4 , 0,4mM KH2PO4, 1 mM CaCl2 and 6 mM glucose from Fisher; and Phenol Red 1% from Sigma), molecular water (Fisher bioreagents), or saline (BioMérieux). Specimens were inactivated at 95°C for 5 min and stored at 4°C. RNA was extracted using Abbott mSample Preparation Systems DNA kit on m2000sp instrument (Abbott) . rRT-PCR protocol was automated: prepared on the Abbott m2000sp and detected on the m2000rt using the Altona kit (Altona diagnostics). Ct values from FAM (E gene), Cy5 (S gene) and Joe/HEX (internal control) were acquired. SeeGene was performed as per the manufacturer's instruction. Ct from FAM (E gene), Cal Red 610 (RDRP gene), Quasar 670 (N gene) and HEX (internal control) were acquired. Samples were considered positive when a signal was detected at any Ct for any gene. A patient was considered negative if the internal control was amplified but not the viral genes. A specimen was considered invalid when there was no amplification of the internal control. Detection of SARS-CoV-2 with SeeGene compared to standard procedure. Altona's kit was missed using SeeGene's kit ( Figure 1 ). The swab storage media for respiratory virus detection can have important impact on the rRT-PCR efficiency, when this media is used directly for PCR reaction. To assess the feasibility of direct rRT-PCR using different media we performed the following experiments. During this study, some specimens were collected in UTM medium during routine screening. Seventeen of these swabs tested positive following standard procedure stored at -80°C, were used for direct rRT-QPCR in two independent experiments. 100% were detected using SeeGene and Ct means were equivalent whether or not RNA was extracted before the rRT-PCR ( Figure J o u r n a l P r e -p r o o f 3A). Next, we measured the stability of the detection in 5 additional samples by amplifying from fresh (stored at 4°C), frozen at -80 °C overnight, thawed and stored at 4°C for 3 days (T0, T24, T48 and T72) ( Figure 3B ). Ct values remained constant across the experiment and all samples could be detected at all timepoints. Our results suggest that UTM medium can be used for direct rRT-PCR and that storage at 4°C for a few days before testing does not lead to a significant loss of detection. Next, we assessed easily available and cheap storage media: saline and molecular grade water. First, we used extracted and purified RNA from five positive specimens (Ct between 12,61 and 14,22) that we diluted using saline or water and used for rRT-PCR. All five samples were detected by the N target when diluted in saline or in water. However, when samples were Thirty-nine swabs collected in water and stored at -80°C for one week, with characterized Ct values (ranging between 16,3 and 39,7) were used as a template for direct rRT-PCR and for RNA extraction and amplification, using Altona ( Figure 5A ) and SeeGene ( Figure 5B ). Twenty-seven of them could be amplified post-thawing when RNA or swabs were used with Altona's multiplex assay. Thirty-two could be detected from RNA eluates or directly from swabs with SeeGene's multiplex assay (Figure 5b ). The initial Ct mean (before freezing) of samples that were lost following thawing was significantly higher than the Ct mean of samples that could be amplified (25,7 vs 33,7; p<0.0001; unpaired t-test) using Altona and SeeGene (25,97 vs 35,68; p<0.0001; unpaired t-test). This suggests that storing specimens in water at -80°C can lead to a significant sensitivity loss for low viral load samples. Direct rRT-PCR was as efficient as RNA extraction followed by rRT-PCR to detect positive samples using both methods. In addition, Ct values of the internal control was significantly higher using direct rRT-PCR compared to rRT-PCR from purified RNA with Altona (p=0.0002, paired t-test), suggesting PCR inhibition when RNA is not purified which was not observed with SeeGene. From this experiment, we conclude that RNA extraction can be successfully skipped when swabs are collected in molecular water. However, storing samples at -80°C collected in water before screening could impact the sensitivity and a decrease detection of patients with lower viral loads. Our results suggest that: i) SeeGene and Altona kits provide similar efficiency, ii) direct rRT-PCR without RNA extraction is possible if samples are in UTM or molecular water; ii), specimens J o u r n a l P r e -p r o o f collected in water should be screened rapidly, iii) RNA extraction is necessary if samples are in saline water or Hanks medium. were performed in molecular water and in saline water. All 5 diluted samples were detected, however, samples diluted in saline water required 10 more Ct before being detected. B. Detection of SARS-CoV-2 by direct rRT-PCR from swabs stored in molecular water. Each dot represents one reaction corresponding to one dilution of the same specimen that contained RNAse inhibitor. The specimen collected in water was diluted 5 times following a serial dilution of 1:10 and used for RNA extraction and amplification using the routine procedure in comparison to SeeGene. Ct values from amplification of extracted RNA from a swab stored in water by standard procedure (Altona_RNA, S and E genes) and using SeeGene's kit (SeeGene_RNA; N, RdRP and E genes), or amplified directly from swab and dilutions in water medium (H20; N and E gene) using SeeGene's kit are plotted. 'na' means 'not applicable'. Coronaviridae Study Group of the International Committee on Taxonomy of V. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 The Laboratory Diagnosis of COVID-19 Infection: Current Issues and Challenges Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-nCoV) infected pneumonia (standard version) Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens SARS-CoV-2 Testing Comparison of Diasorin Simplexa, and CDC FDA EUA methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from individuals diagnosed with COVID-19 Limits of Detection of Six Approved RT-PCR Kits for the Novel SARS-coronavirus-2 (SARS-CoV-2) The Detection of SARS-CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and Roche cobas SARS-CoV-2 Assays Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2. Emerg Infect Dis The authors would like to thank Dr Marco Bergevin for kindly providing a calibrated CFX96 Thermal cycler machine waiting for ours to be calibrated and the SeeGene committee from the Province of Quebec for insightful discussions.