ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.
Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , COVID-19 , Colorimetry/methods , Colorimetry/statistics & numerical data , Coronavirus Infections/epidemiology , Coronavirus Nucleocapsid Proteins , Humans , Molecular Diagnostic Techniques/statistics & numerical data , Nucleic Acid Amplification Techniques/statistics & numerical data , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Pneumonia, Viral/epidemiology , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Seq , SARS-CoV-2 , Sensitivity and Specificity , Translational Research, BiomedicalABSTRACT
Ageing is a feature of nearly all known organisms and, by its connection to survival, appears to trade off with fecundity. However, in some organisms such as in queens of social insects, this negative relation appears reversed and individuals live long and reproduce much. Since new experimental techniques, transcriptomes and genomes of many social insects have recently become available, a comparison of these data in a phylogenetic framework becomes feasible. This allows the study of general trends, species specific oddities and evolutionary dynamics of the molecular properties and changes which underlie ageing, fecundity and the reversal of this negative association. In the framework of social insect evolution, we review the most important recent insights, computational methods, their applications and data resources which are available.
