An in-well direct lysis method for rapid detection of SARS-CoV-2 by real time RT-PCR in eSwab specimens
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An in-well direct lysis method for rapid detection of SARS-CoV-2 by real time RT-PCR in eSwab specimens. / Jørgensen, Rikke Lind; Pedersen, Martin Schou; Chauhan, Alisha Shazad; Andreasson, Louise Munkholm; Kristiansen, Gitte Qvist; Lisby, Jan Gorm; Rosenstierne, Maiken Worsøe; Schønning, Kristian.
I: Journal of Virological Methods, Bind 289, 114062, 03.2021.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - An in-well direct lysis method for rapid detection of SARS-CoV-2 by real time RT-PCR in eSwab specimens
AU - Jørgensen, Rikke Lind
AU - Pedersen, Martin Schou
AU - Chauhan, Alisha Shazad
AU - Andreasson, Louise Munkholm
AU - Kristiansen, Gitte Qvist
AU - Lisby, Jan Gorm
AU - Rosenstierne, Maiken Worsøe
AU - Schønning, Kristian
N1 - Publisher Copyright: © 2021 Elsevier B.V.
PY - 2021/3
Y1 - 2021/3
N2 - Background: Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification. Objectives: To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens. Study design: IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR. Results: In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08–6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR. Conclusions: Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.
AB - Background: Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification. Objectives: To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens. Study design: IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR. Results: In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08–6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR. Conclusions: Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.
KW - Crude lysate
KW - Direct PCR
KW - IGEPAL-CA-630
KW - SARS-CoV-2
U2 - 10.1016/j.jviromet.2021.114062
DO - 10.1016/j.jviromet.2021.114062
M3 - Journal article
C2 - 33428990
AN - SCOPUS:85099199858
VL - 289
JO - Journal of Virological Methods
JF - Journal of Virological Methods
SN - 0166-0934
M1 - 114062
ER -
ID: 285808786