Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.
Betacoronavirus
COVID-19
COVID-19 Testing
Clinical Laboratory Techniques
/ methods
Coronavirus Infections
/ diagnosis
DNA Primers
/ genetics
Humans
Pandemics
Pneumonia, Viral
/ diagnosis
RNA, Viral
/ analysis
Real-Time Polymerase Chain Reaction
/ methods
Respiratory System
/ virology
SARS-CoV-2
Sensitivity and Specificity
Specimen Handling
/ methods
Time Factors
COVID-19
Crude specimen
Direct RT-qPCR
No RNA extraction
Respiratory materials
SARS-CoV-2
Journal
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology
ISSN: 1873-5967
Titre abrégé: J Clin Virol
Pays: Netherlands
ID NLM: 9815671
Informations de publication
Date de publication:
Sep 2020
Sep 2020
Historique:
received:
12
07
2020
accepted:
03
08
2020
pubmed:
17
8
2020
medline:
6
10
2020
entrez:
16
8
2020
Statut:
ppublish
Résumé
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. 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. 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. 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. 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.
Sections du résumé
BACKGROUND
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
OBJECTIVE
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
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
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
CONCLUSIONS
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.
Identifiants
pubmed: 32795959
pii: S1386-6532(20)30321-8
doi: 10.1016/j.jcv.2020.104579
pmc: PMC7405857
pii:
doi:
Substances chimiques
DNA Primers
0
RNA, Viral
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
104579Informations de copyright
Copyright © 2020 Elsevier B.V. All rights reserved.
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