SARS-CoV-2 detection by direct rRT-PCR without RNA extraction

Abstract

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.

Keywords: COVID19; Coronavirus; Direct rRTPCR; RNA extraction; SARS-CoV-2; Virus detection.

Fig. 1

Detection of SARS-CoV-2 with SeeGene compared to standard procedure. Data are presented as scattered dot plots with means. Each dot represents 1 C_t value of 65 reactions, each corresponding to a specimen. Purified RNA from 65 SARS-CoV2⁺ specimens as validated by standard procedure (Altona, S) were amplified using SeeGene (SeeGene_RNA). The most sensitive targets, i.e. positive in the highest number of specimens, are shown (S for Altona and N for SeeGene). The grey dot represents one sample detected at C_t = 35.6 during standard procedure and missed using SeeGene.

Fig. 2

Detection of SARS-CoV-2 from swabs stored in Hanks medium. Data are depicted as scattered dot plots with means. Each dot represents 1 C_t value corresponding to 1 specimen. 94 SARS-CoV2⁺ specimens as validated by standard procedure (Altona, S, n = 94) stored in Hanks medium at −80 °C were used for direct rRT-PCR using SeeGene (No Extr_N, n = 65/94). RNA isolated from these specimens and stored at −80 °C were separately used as template for SeeGene rRT-PCR (RNA_N, n = 30/30). The most sensitive target is shown. 29 positive specimens were not detected by direct rRT-PCR (30,85%).

Fig. 3

Detection of SARS-CoV-2 using direct rRT-PCR from swabs stored in UTM medium (without RNA extraction). Results are plotted as bar with mean graphs and scattered dot plots. Each dot represents one reaction corresponding to one specimen. A. Seventeen SARS-CoV-2⁺ swabs collected and stored in UTM at −80 °C were randomly chosen and directly amplified (UTM) or amplified from RNA eluates (RNA). ‘n’ stands for the number of SARS-CoV-2⁺ specimens. Results for all three targets amplified by SeeGene multiplex assay are shown. The N gene is successfully detected in all the samples. B. Five positive specimens stored in UTM at −80 °C were randomly chosen, thawed and kept at 4 °C for 3 days. Levels of SARS-CoV-2 RNA was assessed at each time point. All 5 samples were detected by amplification of the N gene at all timepoint.

Fig. 4

Detection of SARS-CoV-2 in saline or molecular water samples. Results are plotted as bar graphs with standard deviation. A. Each dot represents one reaction corresponding to one specimen. One hundredth dilution of RNA purified from 5 positive specimens with high C_t values were performed in molecular water and in saline water. All 5 diluted samples were detected, however, samples diluted in saline water required 10 more C_t 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. C_t 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’.

Fig. 5

Detection of SARS-CoV-2 testing using specimens collected in molecular water stored at −80C. Results are plotted as box and violin graph with range and histograms of distribution. A. 39 samples with a declared positive status collected and stored in water at −80 °C were used for RNA extraction and amplification, or for direct amplification using Altona’s kit. Using RNA or directly the wab as a template, 27 were detected post-thawing. Internal control was amplified efficiently from both types of template, but C_t values were significantly higher when swabs were used as direct templates. 12 samples could not be detected post-thawing. Their C_t mean before freezing was significantly higher than the C_t mean of samples that could be amplified (25,69 vs 33,7; p < 0.0001; unpaired t-test). B. The same 39 positive swabs were used for RNA extraction and amplification or direct amplification using SeeGene’s kit. Using RNA or swabs as templates, 32 were detected post-thawing. Internal controls were successfully amplified using both RNA and swabs as templates. 7 samples were lost upon thawing and the C_t mean of these samples before freezing was significantly higher compared to the initial C_t mean of samples that were successfully (25,97 vs 35,68; p < 0.0001; unpaired t-test).