tetano
Editor, Senior Moderator
Anal Chim Acta
. 2022 Apr 1;1200:339590.
doi: 10.1016/j.aca.2022.339590. Epub 2022 Feb 15.
A point-of-care SARS-CoV-2 test based on reverse transcription loop-mediated isothermal amplification without RNA extraction with diagnostic performance same as RT-PCR
Nelson Odiwuor[SUP] 1 [/SUP], Jin Xiong[SUP] 2 [/SUP], Faith Ogolla[SUP] 1 [/SUP], Wei Hong[SUP] 3 [/SUP], Xiaohong Li[SUP] 2 [/SUP], Fazal Mehmood Khan[SUP] 3 [/SUP], Nuo Wang[SUP] 2 [/SUP], Junping Yu[SUP] 2 [/SUP], Hongping Wei[SUP] 4 [/SUP]
Affiliations
Abstract
The global public health crisis and economic losses resulting from the current novel coronavirus disease (COVID-19) pandemic have been dire. The most used real-time reverse transcription polymerase chain reaction (RT-PCR) method needs expensive equipment, technical expertise, and a long turnaround time. Therefore, there is a need for a rapid, accurate, and alternative technique of diagnosis that is deployable at resource-poor settings like point-of-care. This study combines heat deactivation and a novel mechanical lysis method by bead beating for quick and simple sample preparation. Then, using an optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to target genes encoding the open reading frame 8 (ORF8), spike and nucleocapsid proteins of the novel coronavirus, SARS-CoV-2. The test results can be read simultaneously in fluorometric and colorimetric readouts within 40 min from sample collection. We also calibrated a template transfer tool to simplify sample addition into LAMP reactions when pipetting skills are needed. Most importantly, validation of the direct RT-LAMP system based on multiplexing primers S1:ORF8 in a ratio (1:0.8) using 143 patients' nasopharyngeal swab samples showed a diagnostic performance of 99.30% accuracy, with 98.81% sensitivity and 100% selectivity, compared to commercial RT-PCR kits. Since our workflow does not rely on RNA extraction and purification, the time-to-result is two times faster than other workflows with FDA emergency use authorization. Considering all its strengths: speed, simplicity, accuracy and extraction-free, the system can be useful for optimal point-of-care testing of COVID-19.
Keywords: Bead beating; COVID-19; Point-of-care; RT-LAMP; Rapid testing; SARS-CoV-2.
. 2022 Apr 1;1200:339590.
doi: 10.1016/j.aca.2022.339590. Epub 2022 Feb 15.
A point-of-care SARS-CoV-2 test based on reverse transcription loop-mediated isothermal amplification without RNA extraction with diagnostic performance same as RT-PCR
Nelson Odiwuor[SUP] 1 [/SUP], Jin Xiong[SUP] 2 [/SUP], Faith Ogolla[SUP] 1 [/SUP], Wei Hong[SUP] 3 [/SUP], Xiaohong Li[SUP] 2 [/SUP], Fazal Mehmood Khan[SUP] 3 [/SUP], Nuo Wang[SUP] 2 [/SUP], Junping Yu[SUP] 2 [/SUP], Hongping Wei[SUP] 4 [/SUP]
Affiliations
- PMID: 35256137
- DOI: 10.1016/j.aca.2022.339590
Abstract
The global public health crisis and economic losses resulting from the current novel coronavirus disease (COVID-19) pandemic have been dire. The most used real-time reverse transcription polymerase chain reaction (RT-PCR) method needs expensive equipment, technical expertise, and a long turnaround time. Therefore, there is a need for a rapid, accurate, and alternative technique of diagnosis that is deployable at resource-poor settings like point-of-care. This study combines heat deactivation and a novel mechanical lysis method by bead beating for quick and simple sample preparation. Then, using an optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to target genes encoding the open reading frame 8 (ORF8), spike and nucleocapsid proteins of the novel coronavirus, SARS-CoV-2. The test results can be read simultaneously in fluorometric and colorimetric readouts within 40 min from sample collection. We also calibrated a template transfer tool to simplify sample addition into LAMP reactions when pipetting skills are needed. Most importantly, validation of the direct RT-LAMP system based on multiplexing primers S1:ORF8 in a ratio (1:0.8) using 143 patients' nasopharyngeal swab samples showed a diagnostic performance of 99.30% accuracy, with 98.81% sensitivity and 100% selectivity, compared to commercial RT-PCR kits. Since our workflow does not rely on RNA extraction and purification, the time-to-result is two times faster than other workflows with FDA emergency use authorization. Considering all its strengths: speed, simplicity, accuracy and extraction-free, the system can be useful for optimal point-of-care testing of COVID-19.
Keywords: Bead beating; COVID-19; Point-of-care; RT-LAMP; Rapid testing; SARS-CoV-2.