A sensitive and simple RT-LAMP assay for sarbecovirus screening in bats

Abstract

ABSTRACT The availability of simple, inexpensive assays for coronavirus (CoV) detection is critical for conducting animal surveillance studies to prevent new zoonotic epidemics. We have previously developed a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for diagnosing coronavirus disease 2019 (COVID-19) in humans using respiratory samples. In this study, we improved and extended the application of this assay to detect other sarbecoviruses in bats. Twenty-four and twenty-one out of 838 oral and alimentary samples collected during 2017–2019 were sarbecovirus-positive by quantitative reverse transcription PCR (qRT-PCR) and our colorimetric RT-LAMP assay, respectively. PCR and Sanger sequencing of the partial spike (S) gene in the S1 subunit region and RNA-dependent RNA polymerase gene of the 21 sarbecovirus-positive samples showed that they were all closely related to bat SARS-related coronavirus HKU3. A green fluorescent nucleic acid stain, SYTO9, was also added for real-time quantification and evaluated in the colorimetric RT-LAMP assay. We observed a positive correlation (Spearman’s rank correlation coefficient of 0.77, P < 0.0001) between the time to positivity in the colorimetric RT-LAMP assay and cycle threshold (Ct) values in qRT-PCR assay, suggesting that our assay allows quantitative analysis of viral load in samples. This easily performed, highly sensitive, and specific colorimetric RT-LAMP assay could facilitate mass screening for sarbecoviruses in bats and other animal populations. It will be particularly useful for field studies without sophisticated laboratory equipment and expertise. IMPORTANCE We report the application of a colorimetric and fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to facilitate mass screening for sarbecoviruses in bats. The assay was evaluated using a total of 838 oral and alimentary samples from bats and demonstrated comparable sensitivity and specificity to quantitative reverse transcription PCR (qRT-PCR), with a simple setup. The addition of SYTO9, a fluorescent nucleic acid stain, also allows for quantitative analysis. The scalability and simplicity of the assay are believed to contribute to improving preparedness for detecting emerging coronaviruses by applying it to field studies and surveillance.

Keywords

• RT-LAMP
• sarbecovirus
• bat screening
• coronavirus
• SYTO9
• molecular detection