Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
21 Nov 2023
21 Nov 2023
Historique:
received:
16
08
2023
accepted:
16
11
2023
medline:
23
11
2023
pubmed:
22
11
2023
entrez:
22
11
2023
Statut:
epublish
Résumé
In the ongoing global fight against coronavirus disease 2019 (COVID-19), the sample preparation process for real-time reverse transcription polymerase chain reaction (rRT-PCR) faces challenges due to time-consuming steps, labor-intensive procedures, contamination risks, resource demands, and environmental implications. However, optimized strategies for sample preparation have been poorly investigated, and the combination of RNase inhibitors and Proteinase K has been rarely considered. Hence, we investigated combinations of several extraction-free protocols incorporating heat treatment, sample dilution, and Proteinase K and RNase inhibitors, and validated the effectiveness using 120 SARS-CoV-2 positive and 62 negative clinical samples. Combining sample dilution and heat treatment with Proteinase K and RNase inhibitors addition exhibited the highest sensitivity (84.26%) with a mean increase in cycle threshold (Ct) value of + 3.8. Meanwhile, combined sample dilution and heat treatment exhibited a sensitivity of 79.63%, accounting for a 38% increase compared to heat treatment alone. Our findings highlight that the incorporation of Proteinase K and RNase inhibitors with sample dilution and heat treatment contributed only marginally to the improvement without yielding statistically significant differences. Sample dilution significantly impacts SARS-CoV-2 detection, and sample conditions play a crucial role in the efficiency of extraction-free methods. Our findings may provide insights for streamlining diagnostic testing, enhancing its accessibility, cost-effectiveness, and sustainability.
Identifiants
pubmed: 37990045
doi: 10.1038/s41598-023-47645-0
pii: 10.1038/s41598-023-47645-0
pmc: PMC10663557
doi:
Substances chimiques
Endopeptidase K
EC 3.4.21.64
Ribonucleases
EC 3.1.-
RNA, Viral
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
20364Subventions
Organisme : National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT)
ID : 2022M3H4A4097528
Organisme : Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Wel-fare, Republic of Korea, the Ministry of Food and Drug Safety
ID : RS-2020-KD000004
Organisme : Future Medicine 2030 Project of the Samsung Medical Center
ID : SMX1230761
Informations de copyright
© 2023. The Author(s).
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