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Sci Rep
. 2023 Nov 21;13(1):20364.
doi: 10.1038/s41598-023-47645-0. Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay
Minhee Kang # 1 2 , Eunjung Jeong # 1 2 , Ji-Yeon Kim 3 , Sun Ae Yun 3 , Mi-Ae Jang 4 , Ja-Hyun Jang 4 , Tae Yeul Kim 5 , Hee Jae Huh 6 7 , Nam Yong Lee 4
Affiliations
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.
. 2023 Nov 21;13(1):20364.
doi: 10.1038/s41598-023-47645-0. Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay
Minhee Kang # 1 2 , Eunjung Jeong # 1 2 , Ji-Yeon Kim 3 , Sun Ae Yun 3 , Mi-Ae Jang 4 , Ja-Hyun Jang 4 , Tae Yeul Kim 5 , Hee Jae Huh 6 7 , Nam Yong Lee 4
Affiliations
- PMID: 37990045
- DOI: 10.1038/s41598-023-47645-0
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.