key: cord-0865817-xonsc8ru
authors: Adel, Amirouche; Djida, Ait-Ali; Hamid, Nouri; Lila, Boudrahme-Hannou; Souhil, Tliba; Abderrezak, Ghidouche; Idir, Bitam
title: TRIzol-Based RNA extraction for detection protocol for SARS-CoV-2 of the coronavirus Disease 2019
date: 2021-03-31
journal: New Microbes New Infect
DOI: 10.1016/j.nmni.2021.100874
sha: 15a3ce97c00a8b265adf918115d5bcecc0035be9
doc_id: 865817
cord_uid: xonsc8ru

The diagnostic is an important intervention for the management of the 2019 novel coronavirus (SARS-CoV-2). In the present study we developed an optimized protocol for SARS-COV- 2 RNA extraction from the surface of the respiratory mucosa with nasopharyngeal swabs and to compare the sensitivity of RNA extraction methods. Here, RNA extraction was performed using three different procedures (TRIzol, Qiamp, VMT-TRIzol) from nine positive SARS-CoV-2 patients. SARS-CoV-2 was detected by RT-PCR assay using a detection kit for SARS-CoV-2 (Sun Yat-sen University). Comparison of Real-time RT-PCR results, there were no discernible differences in detection rates when comparing the three different extraction procedures. Based on these results, TRIzol as media transport and RNA extraction method for SARS-CoV-2 detection can be a helpful alternative for laboratories facing commercial kit shortages.

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 2019 in Wuhan, Hubei Province, China. Phylogenetic analysis conducted by the China Novel Coronavirus Investigating and Research Team showed that SARS-CoV-2 falls into the genus Betacoronavirus, including coronaviruses discovered in humans and wild animals [1] . It is well established that most of the reported cases had similar clinical manifestations at the onset of illness such as fever, cough, myalgia or fatigue, headache and conjunctivitis [2, 3] . Most cases were respiratory diseases such as acute respiratory distress syndrome and multiple organ failures [4, 5] .

In view of the rapid spread of SARS-CoV-2, real time RT-PCR remains the main way for diagnosing the 2019 novel coronavirus among the many diagnostic platforms available [6, 7] . Converged evidence have demonstrated that SARS-CoV-2 RNA has been diagnosed by qRT-PCR in respiratory tract samples 1-2 days before the appearance of the first symptoms which remain detectable for 7-12 days in moderate cases, and up to 2 weeks in severe cases [8] [9] [10] . Corman et al. have been the first to validate diagnostic RT-PCR tests of SARS-related coronavirus [6] . In this work, two assays targeting the E gene (envelope) and RNA dependent RNA polymerase (RdRp) genes were selected where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing [7] . However, the N gene assay was slightly less sensitive than RdRp and E gene [7] .

We have conducted RT-PCR with a kit for 2019 novel coronavirus RNA (Sun Yatsen University). ORF1ab and N regions are highly conserved amongst sarbecoviruses and are chosen for probe and primers designs [6] . These assays have been evaluated by using a panel of positive and negative controls (ORF1ab/N) [6] . Since start of the minutes at room temperature. The sample was centrifuged for 15 minutes at 20 000 g at 4°C. The supernatant was discarded and the remaining pellet was resuspended in 1000 μL of 80% ethanol. The sample was again centrifuged for 5 minutes at 15,000 g at 4°C. The extra ethanol was removed with 200 μL pipette, then 2 μL pipette. (Pellet should be glassy in appearance). Total RNA isolated was eluted by 20 μL of nucleasefree water (Ambion). The RNA concentration and purity were determined with BioDrop (Biochrom Ltd, UK) by calculating the ratio of optical density at wavelengths of 260/280 nm and 260/230 nm. 

Statistical comparisons using one-way analysis of variance followed by Bonferroni's tests were performed and were further used to determine whether specific group mean differences were significant. The minimum α-level of significance was set at 0.05.

Data are presented as means ±SEM.

Over the last two decades, the laboratory diagnostic methods for human coronavirus infections have developed considerably [5] . The primary, and preferred, method for diagnosis is the collection of upperrespiratory samples via nasopharyngeal and oropharyngeal swabs [8, 12] . TR Izol reagent (a phenol-guanidine isothiocyanate solution) is com m only used to isolate R N A from cells and tissues [13] . M oreover, phenol and guanidinium isothiocyanate is effective at inactivating endogenous ribonucleases [13, 14] . A s expected, w e found significant differences in yields of total R N A extracted w ith different m ethods (Table 1) 

We then proceeded to determine whether these differences in yields without compromised RNA quality. Interestingly, the A260/A280 absorbance ratio for 

To assess virus replication, we performed RT-qPCR tests to detect viral RNAs in clinical samples. SARS-CoV-2 was detected by RT-PCR assay using a detection kit for 2019 novel RNA according to the manufacturer's protocol (Sun Yat-sen University). .48 respectively for both FAM and VIC assays (Fig 2A and 2B ). Our result is in perfect agreement with msanufacturer's recommendations. We then tested the effectiveness of the three different procedures of SARS-CoV-2 RNA extraction. We found no significant difference between Ct values of samples extracted with three procedures (TRIzol, QIAam, VMT -TRIzol) (Fig 3) . Consistent to what we observed by other approaches such as CTscan and rapid diagnostic test [20] . Overall, we compared three RNA extraction methods from human SARS-CoV-2 samples for the N and ORF1ab genes, reporting 9 positive results for the two marker genes with positivity confirmed by all three RNA extraction methods.

More specifically, patients were detected positive with threshold cycle value around 25.13 ± 5.17 for FAM assay and 27.09 ± 4.98 for VIC (Fig 2C, Fig3) . In the line with previous observation, the N gene assay is more sensitive than the ORF-1ab gene assay in detecting positive clinical specimens [7] . Moreover, Chu et al. have reported that the N gene assay is about 10 times was more sensitive than the ORF-1b gene assay in detecting positive clinical specimens [7] . In their model, the Orf1b assay is recommended as a confirmatory test and the N gene RT-PCR is recommended as a screening assay [7, 21] . In another study, Won et al. established a RT-PCR-based assay protocol composed of easy specimen self-collection from a subject via pharyngeal swab; TRIzol based RNA purification, and SYBR Green-based RT-PCR [11] . In agreement with our data, a recent study demonstrated that TRIzol is equally sensitive compared to the commercial available kit [11] .

In conclusion, this study contributes to the knowledge of the extraction of SARS- 

A Novel Coronavirus from Patients with Pneumonia in China

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Virological assessment of hospitalized patients with COVID-2019

A Case Series of Children With 2019 Novel Coronavirus Infection: Clinical and Epidemiological Features

COVID-19 pandemic-a focused review for clinicians. Clinical microbiology and infection : the official publication of the

Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19)

COVID-19 diagnosis and management: a comprehensive review

The single-step method of RNA

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A performed experiments

edited and revised manuscript