Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay
Abstract 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, an...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-11-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-47645-0 |
| _version_ | 1797452999621083136 |
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| author | Minhee Kang Eunjung Jeong Ji-Yeon Kim Sun Ae Yun Mi-Ae Jang Ja-Hyun Jang Tae Yeul Kim Hee Jae Huh Nam Yong Lee |
| author_facet | Minhee Kang Eunjung Jeong Ji-Yeon Kim Sun Ae Yun Mi-Ae Jang Ja-Hyun Jang Tae Yeul Kim Hee Jae Huh Nam Yong Lee |
| author_sort | Minhee Kang |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-09T15:16:31Z |
| format | Article |
| id | doaj.art-60a68dab0ff048519b9f8955731f4aef |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-09T15:16:31Z |
| publishDate | 2023-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-60a68dab0ff048519b9f8955731f4aef2023-11-26T13:03:31ZengNature PortfolioScientific Reports2045-23222023-11-011311910.1038/s41598-023-47645-0Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assayMinhee Kang0Eunjung Jeong1Ji-Yeon Kim2Sun Ae Yun3Mi-Ae Jang4Ja-Hyun Jang5Tae Yeul Kim6Hee Jae Huh7Nam Yong Lee8Smart Healthcare Research Institute, Biomedical Engineering Research Center, Samsung Medical CenterSmart Healthcare Research Institute, Biomedical Engineering Research Center, Samsung Medical CenterSamsung Biomedical Research Institute, Center for Clinical Medicine, Samsung Medical CenterSamsung Biomedical Research Institute, Center for Clinical Medicine, Samsung Medical CenterDepartment of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of MedicineDepartment of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of MedicineDepartment of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of MedicineDepartment of Medical Device Management and Research, Samsung Advanced Institute for Health Sciences & Technology, Sungkyunkwan UniversityDepartment of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of MedicineAbstract 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.https://doi.org/10.1038/s41598-023-47645-0 |
| spellingShingle | Minhee Kang Eunjung Jeong Ji-Yeon Kim Sun Ae Yun Mi-Ae Jang Ja-Hyun Jang Tae Yeul Kim Hee Jae Huh Nam Yong Lee Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay Scientific Reports |
| title | Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay |
| title_full | Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay |
| title_fullStr | Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay |
| title_full_unstemmed | Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay |
| title_short | Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay |
| title_sort | optimization of extraction free protocols for sars cov 2 detection using a commercial rrt pcr assay |
| url | https://doi.org/10.1038/s41598-023-47645-0 |
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