tetano
Editor, Senior Moderator
J Biomol Tech
. 2021 Sep;32(3):158-171.
doi: 10.7171/jbt.21-3203-011.
Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment
Lena M Diaz[SUP] 1 [/SUP], Brandon E Johnson[SUP] 2 [/SUP], Daniel M Jenkins[SUP] 1 [/SUP]
Affiliations
Abstract
Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1× (50 genomes/reaction) or 2× (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.
Keywords: COVID-19; RNA detection; RT-LAMP; diagnostics; near patient.
. 2021 Sep;32(3):158-171.
doi: 10.7171/jbt.21-3203-011.
Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared with endpoint assessment
Lena M Diaz[SUP] 1 [/SUP], Brandon E Johnson[SUP] 2 [/SUP], Daniel M Jenkins[SUP] 1 [/SUP]
Affiliations
- PMID: 35027873
- PMCID: PMC8730521
- DOI: 10.7171/jbt.21-3203-011
Abstract
Controlling the course of the Coronavirus Disease 2019 (COVID-19) pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current Food and Drug Administration (FDA)-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop-mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than 2 h to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS-CoV-2 at either 1× (50 genomes/reaction) or 2× (100 genomes/reaction) of the LOD. Importantly, the quantitative method was based on dynamic optical changes during the reaction and was able to correctly classify samples that were misclassified by endpoint observation of color.
Keywords: COVID-19; RNA detection; RT-LAMP; diagnostics; near patient.