Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective.
The study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive c...
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| Format: | Article |
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Public Library of Science (PLoS)
2022-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0276464&type=printable |
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| author | Md Walid Hossain Mohabbat Hossain Khalid Arafath Subarna Sayed Ety Md Mahade Hasan Shetu Mazbahul Kabir Farjana Akther Noor Kaiissar Mannoor |
| author_facet | Md Walid Hossain Mohabbat Hossain Khalid Arafath Subarna Sayed Ety Md Mahade Hasan Shetu Mazbahul Kabir Farjana Akther Noor Kaiissar Mannoor |
| author_sort | Md Walid Hossain |
| collection | DOAJ |
| description | The study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive control and synthetic version of human RP was done on Applied Biosystems (ABI) QuantStudioTM5 (conventional), ABI 7500 Fast Dx (designated), and CFX96 Touch Real Time Detection System, Bio-Rad (conventional). Finally, a performance evaluation of Fast PCR was performed in terms of sensitivity, specificity, and precision. For a 40-cycle PCR with optimized Fast PCR protocols on QuantStudioTM5, ABI 7500 Fast Dx, and CFX96 Touch (conventional), standard/regular versus Fast PCR run times (min) were 84 vs. 49, 96 vs. 48, and 103 vs. 61, thereby saving 35, 48, and 43 min, respectively. For each thermal cycler, Standard and Fast PCR generated identical shapes of fluorescence curves, Ct values, and (3) R2 (0.95 to 0.99) for 5 10-log dilution panels of each positive control. The fast PCR approach generated results with 100% sensitivity and specificity. Median test comparisons between standard PCR and Fast PCR Cts of COVID-19 samples did not produce significance (p>0.5), suggesting that Fast PCR and Standard PCR were comparable. Also, the median and mean of each target had closely-related values, further suggesting that the two approaches were comparable. That is, there is an equivalency between Conventional and Fast PCR instruments for detection of COVID-19. |
| format | Article |
| id | doaj-art-df80a7d17fa649d89ba0df58b667a927 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2022-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-df80a7d17fa649d89ba0df58b667a9272025-08-20T03:16:35ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-011710e027646410.1371/journal.pone.0276464Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective.Md Walid HossainMohabbat HossainKhalid ArafathSubarna Sayed EtyMd Mahade Hasan ShetuMazbahul KabirFarjana Akther NoorKaiissar MannoorThe study aimed to shorten multiplex RT-PCR run time for detection of SARS CoV-2 N1 and N2 sequences and human RNase P (RP) sequence as internal mRNA control using conventional and designated real time thermal cycler systems. Optimization of Fast PCR protocol using plasmid-based N1 and N2 positive control and synthetic version of human RP was done on Applied Biosystems (ABI) QuantStudioTM5 (conventional), ABI 7500 Fast Dx (designated), and CFX96 Touch Real Time Detection System, Bio-Rad (conventional). Finally, a performance evaluation of Fast PCR was performed in terms of sensitivity, specificity, and precision. For a 40-cycle PCR with optimized Fast PCR protocols on QuantStudioTM5, ABI 7500 Fast Dx, and CFX96 Touch (conventional), standard/regular versus Fast PCR run times (min) were 84 vs. 49, 96 vs. 48, and 103 vs. 61, thereby saving 35, 48, and 43 min, respectively. For each thermal cycler, Standard and Fast PCR generated identical shapes of fluorescence curves, Ct values, and (3) R2 (0.95 to 0.99) for 5 10-log dilution panels of each positive control. The fast PCR approach generated results with 100% sensitivity and specificity. Median test comparisons between standard PCR and Fast PCR Cts of COVID-19 samples did not produce significance (p>0.5), suggesting that Fast PCR and Standard PCR were comparable. Also, the median and mean of each target had closely-related values, further suggesting that the two approaches were comparable. That is, there is an equivalency between Conventional and Fast PCR instruments for detection of COVID-19.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0276464&type=printable |
| spellingShingle | Md Walid Hossain Mohabbat Hossain Khalid Arafath Subarna Sayed Ety Md Mahade Hasan Shetu Mazbahul Kabir Farjana Akther Noor Kaiissar Mannoor Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective. PLoS ONE |
| title | Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective. |
| title_full | Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective. |
| title_fullStr | Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective. |
| title_full_unstemmed | Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective. |
| title_short | Real-Time fast PCR amplification using designated and conventional real time thermal cycler systems: COVID-19 perspective. |
| title_sort | real time fast pcr amplification using designated and conventional real time thermal cycler systems covid 19 perspective |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0276464&type=printable |
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