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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.
Highlights
The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide
Is not known if IGEPAL-630 affects SARS-CoV-2 RNA integrity or if this detergent inhibits activity of enzymes included in the room temperature (RT)-PCR COVID detection kits approved by Centers for Disease Control and Prevention (CDC)
Initially we tested amplification of viral RNA spiked in a viral Lysis Amplification Buffer [0.25% IGEPAL, 150 mM NaCL, Tris 10 mM, BSA 1X] using Federal Drug Administration (FDA) aproved primers/probes for detection of nucleocapside genes N1 and N2
Summary
The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. 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). The RT-PCR primers described in the CDC protocol showed high sensitivity (600–1200 viral genome copies/mL) and specificity[7] so are routinely used in many laboratories around the world. This assay requires RNA purification and several purification kits have been validated. We used IGEPAL to prepare a viral lysis-amplification buffer (vLAB) and demonstrated that this buffer is suitable for the qualitative detection of SARS-CoV-2 by dRT-PCR in clinical samples following the CDC protocol
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