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. 2021 May:41:100874.
doi: 10.1016/j.nmni.2021.100874. Epub 2021 Mar 31.

TRIzol-based RNA extraction for detection protocol for SARS-CoV-2 of coronavirus disease 2019

A Amirouche  1   2 , D Ait-Ali  2   3 , H Nouri  2   4 , L Boudrahme-Hannou  3   5   6 , S Tliba  3   5   7 , A Ghidouche  2   3 , I Bitam  8   9
Affiliations

TRIzol-based RNA extraction for detection protocol for SARS-CoV-2 of coronavirus disease 2019

A Amirouche et al. New Microbes New Infect. 2021 May.

Abstract

Diagnostic testing is important for managing the 2019 novel coronavirus (SARS-CoV-2). We developed an optimized protocol for SARS-CoV-2 RNA extraction from the surface of the respiratory mucosa with nasopharyngeal swabs and compared the sensitivity of RNA extraction methods. RNA extraction was performed using three different procedures (TRIzol, QIAamp, VMT-TRIzol) from nine positive SARS-CoV-2 samples. SARS-CoV-2 was detected by real-time reverse transcriptase PCR (RT-PCR) using a detection kit for SARS-CoV-2 (Sun Yat-sen University). Compared to RT-PCR results, there were no discernible differences in detection rates when comparing the three different extraction procedures. On the basis of these results, the use of TRIzol as a transport medium and RNA extraction method for SARS-CoV-2 detection may be a helpful alternative for laboratories facing shortages of commercial testing kits.

Keywords: Diagnostic techniques; RT-PCR; SARS-CoV-2; TRIzol; throat swab.

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Figures

Fig. 1
Fig. 1
Experimental design of different RNA extraction methods from human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples. SARS-CoV-2 was confirmed by positive nasopharyngeal swab specimen at hospital admission. For each volunteer, at the same time point, two nasopharyngeal swabs were collected. The first was immediately placed in a 15 mL cone with 2 mL of TRIzol reagent. The second was immersed in viral transport medium.
Fig. 2
Fig. 2
Real-time reverse transcriptase PCR (RT-PCR) results for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19). (A) FAM (N gene) quantitation. (a) RNA extraction and transport medium used TRIzol. (b) QIAamp kit for RNA extraction. (c) Viral transport medium with TRIzol RNA extraction. (d) Positive control. (B) VIC (ORF1ab) quantitation. (a) RNA extraction and medium transport were performed with TRIzol. (b) QIAamp RNA extraction with viral transport medium. (c) TRIzol extraction with viral transport medium. (d) Positive control. (C) FAM and VIC quantitation. (a) RNA extraction and medium transport were performed with TRIzol. (b) QIAamp RNA extraction with viral transport medium. (c) TRIzol extraction with viral transport medium. (d) Positive control. Three RNA extractions were performed for each patient.
Fig. 3
Fig. 3
Determination of detection efficiency of real-time reverse transcriptase PCR (RT-PCR). Average cycle threshold (Ct) values were compared between different procedures. Abbreviations: TRIzol, TRIzol reagent as transport medium and for RNA extraction; VMT-QIAamp, viral transport medium and QIAamp kit for RNA extraction; VMT-TRIzol, viral transport medium and TRIzol for RNA extraction. Values are expressed as mean ± standard error.
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