SARS-CoV-2: Comparative analysis of different RNA extraction methods

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

Keywords: Oro- nasopharyngeal swabs; RNA extraction; SARS-CoV-2; rRT-PCR.

Graphical abstract

Fig. 1

Linear regression analysis of serial 10-fold serial dilutions of two synthetic ssRNA standards for SARS-CoV-2, ranging from 2 × 10¹ to 2 × 10⁷ copies/μl. Dilutions from 2 × 10° to 2 × 10⁷ copies of (A) Australia/VIC01/2020 SARS-CoV-2 RNA and (B) EURM-019 were tested by rRT-PCR using the Allplex 2019-nCoV (E, RdRp and N genes). The same 10-fold dilutions of (C) Australia/VIC01/2020 SARS-CoV-2 RNA and (D) EURM-019 were analyzed using the US CDS panel assays for detection of SARS-CoV-2 (N gene detected by primer pairs for N1, N2 and N3). The linear regression analysis is shown only for the N1 primer pair. For each assay, the linear correlation coefficients, PCR efficiency E = 10(−1/slope), R² and slope were calculated.

Fig. 2

Quantitative comparison of RNA extraction methods. Known amounts of synthetic ssRNA standard for SARS-CoV-2 were extracted using the four described methods, both following manufacturers ‘instructions and using the optimized protocols (OPT). Samples were analyzed by rRT-PCR, using Allplex 2019-nCoV assay (E, RdRp and N genes) and US CDC rRT-PCR panel (three gene regions of the N gene, designated N1, N2, and N3). Average values calculated from three independent experiments performed in duplicate are reported. Missing bars correspond to >40 Ct for Allplex 2019-nCoV assay or negative for the US CDC panel for SARS-CoV-2. Cp, copies.

Fig. 3

Quantitative comparison of SARS-CoV-2 RNA extraction from clinical swabs. RNA was extracted from oro-nasopharyngeal swabs from six patients (PZ) using the four described methods, both following manufacturers ‘instructions and using the optimized protocols (OPT). Samples were analyzed by rRT-PCR, using Allplex 2019-nCoV assay (E, RdRp and N genes) and US CDC rRT-PCR panel (three gene regions of the N gene, designated N1, N2, and N3). Average values calculated from three independent experiments performed in duplicate are reported. Missing bars correspond to >40 Ct for Allplex 2019-nCoV assay or negative for the US CDC panel for SARS-CoV-2.