tetano
Editor, Senior Moderator
Mol Biotechnol
. 2024 Sep 24.
doi: 10.1007/s12033-024-01275-7. Online ahead of print. Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects
Faezeh Mahmoudi[SUP] 1 [/SUP], Davod Jafari[SUP] 1 [/SUP], Seyedeh Mona Mousavi Esfahani[SUP] 1 [/SUP], Arshad Hoseini[SUP] 1 [/SUP], Mahmood Barati[SUP] 2 [/SUP], Neda Saraygord-Afshari[SUP] 3 [/SUP]
Affiliations
Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60 °C), assay runtime (60 min), primers, MgSO[SUB]4[/SUB] (6 mM), dNTPs (1 mM), LAMP Buffer (1.2 mM Tris-HCl), KCl (50 mM), pH (8), and phenol red (10 mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with C[SUB]t[/SUB] values up to 29, while the RT-qLAMP assay identified up to C[SUB]t[/SUB] = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to C[SUB]t[/SUB] ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.
Keywords: Diagnostic virology; RT-qLAMP; RT-qPCR; SARS-CoV-2.
. 2024 Sep 24.
doi: 10.1007/s12033-024-01275-7. Online ahead of print. Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects
Faezeh Mahmoudi[SUP] 1 [/SUP], Davod Jafari[SUP] 1 [/SUP], Seyedeh Mona Mousavi Esfahani[SUP] 1 [/SUP], Arshad Hoseini[SUP] 1 [/SUP], Mahmood Barati[SUP] 2 [/SUP], Neda Saraygord-Afshari[SUP] 3 [/SUP]
Affiliations
- PMID: 39316362
- DOI: 10.1007/s12033-024-01275-7
Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60 °C), assay runtime (60 min), primers, MgSO[SUB]4[/SUB] (6 mM), dNTPs (1 mM), LAMP Buffer (1.2 mM Tris-HCl), KCl (50 mM), pH (8), and phenol red (10 mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with C[SUB]t[/SUB] values up to 29, while the RT-qLAMP assay identified up to C[SUB]t[/SUB] = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to C[SUB]t[/SUB] ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.
Keywords: Diagnostic virology; RT-qLAMP; RT-qPCR; SARS-CoV-2.