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Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing
BACKGROUND: The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate fals...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10593008/ https://www.ncbi.nlm.nih.gov/pubmed/37873363 http://dx.doi.org/10.1101/2023.10.03.23296477 |
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author | Sritong, Navaporn Ngo, Winston Wei Ejendal, Karin F. K. Linnes, Jacqueline C. |
author_facet | Sritong, Navaporn Ngo, Winston Wei Ejendal, Karin F. K. Linnes, Jacqueline C. |
author_sort | Sritong, Navaporn |
collection | PubMed |
description | BACKGROUND: The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked. In some cases, the IACs were run spatially, complicating assay workflow. Therefore, the multiplex assay for pathogen and IAC is needed. RESULTS: We developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for saliva samples, a non-invasive and simple collected specimen for POC NAATs. The ORF1ab gene of SARS-CoV-2 was used as a target and a human 18S ribosomal RNA in human saliva was employed as an IAC to ensure clinical reliability of the RT-LAMP assay. The optimized assay could detect SARS-CoV-2 viral particles down to 100 copies/μL of saliva within 30 minutes without RNA extraction. The duplex RT-LAMP for SARS-CoV-2 and IAC is successfully amplified in the same reaction without cross-reactivity. The valid results were easily visualized in triple-line lateral flow immunoassay, in which two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test line) represent valid positive results. This duplex assay demonstrated a clinical sensitivity of 95%, specificity of 100%, and accuracy of 96% in 30 clinical saliva samples. SIGNIFICANCE: IACs play a crucial role in ensuring user confidence with respect to the accuracy and reliability of at-home and POC molecular diagnostics. We demonstrated the multiplex capability of SARS-COV-2 and human18S ribosomal RNA RT-LAMP without complicating assay design. This generic platform can be extended in a similar manner to include human18S ribosomal RNA IACs into different clinical sample matrices. |
format | Online Article Text |
id | pubmed-10593008 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
spelling | pubmed-105930082023-10-24 Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing Sritong, Navaporn Ngo, Winston Wei Ejendal, Karin F. K. Linnes, Jacqueline C. medRxiv Article BACKGROUND: The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked. In some cases, the IACs were run spatially, complicating assay workflow. Therefore, the multiplex assay for pathogen and IAC is needed. RESULTS: We developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for saliva samples, a non-invasive and simple collected specimen for POC NAATs. The ORF1ab gene of SARS-CoV-2 was used as a target and a human 18S ribosomal RNA in human saliva was employed as an IAC to ensure clinical reliability of the RT-LAMP assay. The optimized assay could detect SARS-CoV-2 viral particles down to 100 copies/μL of saliva within 30 minutes without RNA extraction. The duplex RT-LAMP for SARS-CoV-2 and IAC is successfully amplified in the same reaction without cross-reactivity. The valid results were easily visualized in triple-line lateral flow immunoassay, in which two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test line) represent valid positive results. This duplex assay demonstrated a clinical sensitivity of 95%, specificity of 100%, and accuracy of 96% in 30 clinical saliva samples. SIGNIFICANCE: IACs play a crucial role in ensuring user confidence with respect to the accuracy and reliability of at-home and POC molecular diagnostics. We demonstrated the multiplex capability of SARS-COV-2 and human18S ribosomal RNA RT-LAMP without complicating assay design. This generic platform can be extended in a similar manner to include human18S ribosomal RNA IACs into different clinical sample matrices. Cold Spring Harbor Laboratory 2023-10-03 /pmc/articles/PMC10593008/ /pubmed/37873363 http://dx.doi.org/10.1101/2023.10.03.23296477 Text en https://creativecommons.org/licenses/by-nd/4.0/This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, and only so long as attribution is given to the creator. The license allows for commercial use. |
spellingShingle | Article Sritong, Navaporn Ngo, Winston Wei Ejendal, Karin F. K. Linnes, Jacqueline C. Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing |
title | Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing |
title_full | Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing |
title_fullStr | Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing |
title_full_unstemmed | Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing |
title_short | Development of an Integrated Sample Amplification Control for Salivary Point-of-Care Pathogen Testing |
title_sort | development of an integrated sample amplification control for salivary point-of-care pathogen testing |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10593008/ https://www.ncbi.nlm.nih.gov/pubmed/37873363 http://dx.doi.org/10.1101/2023.10.03.23296477 |
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