Development of nucleic acid extraction-free one-step real-time RT-PCR for diagnosis of SARS-CoV-2 infection
Introduction: The aim of this study is to develop a one-step real-time PCR assay for SARS-CoV-2 detection. The study was designed to circumvent the routine RNA isolation step and to optimize a lysis buffer and parameters for direct quantitative PCR. Methodology: A lysis solution was prepared usin...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
The Journal of Infection in Developing Countries
2025-06-01
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| Series: | Journal of Infection in Developing Countries |
| Subjects: | |
| Online Access: | https://www.jidc.org/index.php/journal/article/view/18079 |
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| Summary: | Introduction: The aim of this study is to develop a one-step real-time PCR assay for SARS-CoV-2 detection. The study was designed to circumvent the routine RNA isolation step and to optimize a lysis buffer and parameters for direct quantitative PCR.
Methodology: A lysis solution was prepared using Tween-20, Triton X-100, EDTA, and Tris buffer (pH 7.4). Various parameters including the use of detergent combinations, V/V ratios and usage of carrier molecules were standardized to achieve the optimal amplification curve and Ct values of SARS-CoV-2 gene for improving the routine diagnostics procedures.
Results: Adding carrier molecules [Poly(A), glycogen, and linear polyacrylamide] to the lysis solution significantly improved real-time reverse-transcription PCR (rtRT-PCR) efficacy. Poly(A) was the most effective of all carriers. The diagnostic potential of this Poly(A) solution was demonstrated using 150 patient swabs infected with SARS-CoV-2 and 200 uninfected swab samples, and the sensitivity of the rtRT-PCR diagnostic test was estimated to be 98.6 (95% CI: 96.0, 101.17, p < 0.001) for group 1; Ct ≤ 25 and 87.2 (95% CI: 80.2, 94.0, p < 0.001) for group 2; Ct ≥ 26–30, with excellent accuracy (0.9 < AUC < 1.0), and 100% specificity.
Conclusions: Our finding imply that this strategy is feasible, and it may contribute to the development of a rapid, less laborious, and economical rtRT-PCR test for dealing with the SARS-COV-2 disease in the pandemic area.
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| ISSN: | 1972-2680 |