Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral lysis-amplification buffer prepared with IGEPAL-630

Abstract

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.

Figure 1

PCR efficiency of SARS-CoV-2 RNA in vLAB. PCR efficiency (E) for N1 and N2 gene was assessed using a duplicate tenfold dilution series of SARS-CoV-2 viral RNA. Linear regression was performed to obtain the slope and R2. The percentage efficiency was calculated from the slope using the formula E = 100 × (− 1 + 10 − 1/slope). vLAB virus Lysis Amplification Buffer.

Figure 2

Immunofluorescence images of Vero E6 cells infected with mNG-SARS-CoV-2 diluted in vLAB. Immunofluorescence images of Vero E6 cells infected with stable mNeonGreen (mNG)-SARS-CoV-2 diluted or not in vLAB. Vero E6 Cells monolayers were analyzed 3 days post inoculation (3 dpi). Green cytoplasmic fluorescence is indicative of viral replication. (A) Pass1, stable mNeonGreen (mNG)-SARS-CoV-2 virus was diluted in vLAB and then incubated 10 and 20 min at room temperature (RT). Positive control, cells infected with mNG SARS-CoV-2 were diluted only with cell culture media; Negative control = only cell culture media. (B) Passage 2nd, supernatants from P1 (10 µL) were used to infect Vero E6 cells.

Figure 3

PCR efficiency of SARS-CoV-2 RNA in vLAB at 65 °C and 95 °C. PCR efficiency (E) for N1 and N2 gene was assessed using a duplicate tenfold dilution series of SARS-CoV-2 viral RNA.