tetano
Editor, Senior Moderator
PLoS One
. 2021 Feb 26;16(2):e0247792.
doi: 10.1371/journal.pone.0247792. eCollection 2021.
Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR
Valeria Genoud[SUP] 1 [/SUP], Martin Stortz[SUP] 2 3 [/SUP], Ariel Waisman[SUP] 4 [/SUP], Bruno G Berardino[SUP] 1 3 [/SUP], Paula Verneri[SUP] 3 [/SUP], Virginia Dansey[SUP] 1 5 [/SUP], Melina Salvatori[SUP] 6 [/SUP], Federico Remes Lenicov[SUP] 6 [/SUP], Valeria Levi[SUP] 1 3 [/SUP]
Affiliations
Abstract
Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.
. 2021 Feb 26;16(2):e0247792.
doi: 10.1371/journal.pone.0247792. eCollection 2021.
Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR
Valeria Genoud[SUP] 1 [/SUP], Martin Stortz[SUP] 2 3 [/SUP], Ariel Waisman[SUP] 4 [/SUP], Bruno G Berardino[SUP] 1 3 [/SUP], Paula Verneri[SUP] 3 [/SUP], Virginia Dansey[SUP] 1 5 [/SUP], Melina Salvatori[SUP] 6 [/SUP], Federico Remes Lenicov[SUP] 6 [/SUP], Valeria Levi[SUP] 1 3 [/SUP]
Affiliations
- PMID: 33635936
- DOI: 10.1371/journal.pone.0247792
Abstract
Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.