Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials

Abstract

Background: Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed.

Objectives: To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials.

Material and methods: Different SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene.

Results: The protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 μl of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing.

Conclusion: Direct RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.

Keywords: COVID-19; Crude specimen; Direct RT-qPCR; No RNA extraction; Respiratory materials; SARS-CoV-2.

Fig. 1

Analysis of PCR conditions for the detection of SARS-CoV-2 by direct RT-qPCR. (A) Direct RT-qPCR with the Sarbeco and the new CoV-E primers. Patient matched respiratory crude samples (n = 20) including swabs, tracheal secretion, brochoalveolare lavage and sputum were analyzed for the detection of SARS-CoV-2 by direct RT-qPCR with the Sarbeco primers and the new CoV-E primers. (B) Direct RT-qPCR with fresh and pre-frozen PCR-mixes. Patient matched respiratory crude samples (n = 8) including swabs, tracheal secretion and BAL were analyzed for the detection of SARS-CoV-2 by direct RT-qPCR with either fresh prepared master-mixes (MMX) or pre-frozen master-mixes. Dotted line: detection limit by performed qPCR cycles; *** (p ≤ 0.001); n.s. (not significant).

Fig. 2

Analysis of different sample conditions for optimization of the direct RT-qPCR protocol for the detection of SARS-CoV-2 in respiratory crude samples. For the direct comparison of different sample conditions three patients samples with RNA Ct values 16.1 (TS, blue), 23.4 (swab, red) and 26 (TS, black) were selected. Ct values are shown in difference to RNA RT-qPCR Ct values (ΔCt). Mean Ct values with SEM are indicated. TS: tracheal secretion; frozen: storage at -20 °C before sample processing; fresh: storage at 4 °C <24 h; *: selected condition for direct RT-qPCR protocol; SEM: standard error of mean.

Fig. 3

Impact of long-term storage of respiratory samples at 4 °C on SARS-CoV-2 detection by direct RT-qPCR. Patient matched respiratory samples (n = 32) including swabs, tracheal secretion, bronchoalveolare lavage and saliva were analyzed for the detection of SARS-CoV-2 by direct RT-qPCR at the timepoint of sample receipt (week 0) and 4 weeks after storage at 4 °C (week 4). Dotted line: detection limit performed qPCR cycles; ****(p ≤ 0.0001).

Fig. 4

Correlation of Ct values obtained by direct RT-qPCR versus classical RT-qPCR with extracted RNA for the detection of SARS-CoV-2. Respiratory samples (n = 91) including swabs, tracheal secretion, bronchoalveolare lavage, aspirate and saliva were analyzed for the detection of SARS-CoV-2 by RT-qPCR with crude specimen and RNA-based RT-qPCR. Ct values were correlated.